US20230227584A1

US20230227584A1 - Bispecific antibodies comprising a modified c-terminal crossfab fragment

Info

Publication number: US20230227584A1
Application number: US17/937,440
Authority: US
Inventors: Maria Amann; Ali BRANSI; Peter Bruenker; Janine FAIGLE; Sabine Imhof-Jung; Roland Staack; Joerg ZIELONKA
Original assignee: Hoffmann La Roche Inc
Current assignee: F Hoffmann La Roche AG; Hoffmann La Roche Inc
Priority date: 2020-04-01
Filing date: 2022-09-30
Publication date: 2023-07-20
Also published as: US20220025046A1; AU2021247502A1; PE20221869A1; WO2021198335A1; IL294922A; TW202144437A; CA3169601A1; EP4126946A1; CO2022013898A2; MX2022012188A; CN115768473A; CR20220479A; WO2021198333A1; JP2023520684A; BR112022019531A2; TWI810541B; CN115362174A; EP4126041A1; US11780919B2; KR20220150935A

Abstract

The present invention relates to bispecific antibodies comprising a modified C-terminal crossfab fragment that have reduced or no reactivity against preexisting antidrug antibodies.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No. PCT/EP2021/058439, filed Mar. 31, 2021, which claims the benefit of priority to European Patent Application No. 20167624.4, filed Apr. 1, 2020, which are incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 20, 2022, is named P36817-US_SL.xml, and is 206,870 bytes in size.

FIELD OF THE INVENTION

The invention relates relates to bispecific antibodies comprising a modified C-terminal crossfab fragment that have reduced or no reactivity against preexisting antidrug antibodies.

BACKGROUND

Antibodies are produced by B cells in two ways: (i) randomly, and (ii) in response to a foreign antigen within the body. Initially, one B cell produces one specific kind of antibody. In either case, the B cell is allowed to proliferate or is killed off through a process called clonal deletion. Normally, the immune system is able to recognize and ignore the body’s own healthy proteins, cells, and tissues, and to not overreact to non-threatening substances in the environment, such as foods. However, most healthy individuals have autoreactive B cells that recognize “self”-antigens. Several types of auto-antibodies have been characterized including those directed against immunoglobulins. The best characterized anti-immunoglobulin auto-antibodies are those directed against the Fc region of IgG, known as rheumatoid factor, or those directed against the variable regions, known as anti-idiotype auto-antibodies. Another class of auto-antibodies are those that bind specifically to proteolytically exposed neoepitopes containing C-terminal amino acid residues in either the upper or lower hinge region. These anti-drug antibodies (ADA) or anti-hinge auto-antibodies (AHA) have been detected in several studies. Interestingly, pre-existing ADA titers vary from donor to donor and may represent past and current exposure to the neoepitopes.
Classical antibodies (IgG) are composed of two antigen binding fragments (Fab) that are fused via a flexible hinge region to the Fc region. While the Fabs are responsible for recognition and binding to the antigen, the Fc region mediates effector function by engagement with Fcγ receptors and confers long serum half-life by binding to FcRn. A number of proteases are known to cleave the intact antibody in the hinge region, for instance in the lower hinge region to produce F(ab′)₂ molecules. Production of proteases against the antibody hinge region has been determined as a mechanism by which pathogens and tumor cells attempt to evade the host immune response. However, resulting C-terminal neoepitopes are eventually recognized by the immune system and AHA are generated. Based on the individual history with infection and inflammation, healthy donors thus possess varying pre-existing ADA. The ADA can act as surrogate Fc and restore effector function of the otherwise proteolytically inactivated antibody, but may introduce potential safety concerns in case effector function is not wanted or in case the therapeutical antigen binding molecules have other formats than the classical antibody structure. These molecules may have structures wherein C-terminal neoepitopes may be regularly present. Kim et al, MABS 2016, 8, 1536-1547, describe the problem with pre-existing AHA for antigen binding fragments (Fab) and (Fab′)₂ which are attractive therapeutic formats when a short systemic half-life and an effector-silent molecule are desired. They could show that by engineering the C-terminal residues in the upper hinge region minimal reactivity toward pre-existing ADA could be observed.
The immune suppressive microenvironment in certain tumors is high in co-inhibitory signals, e.g. PD-L1, but lacks sufficient expression of tumor necrosis factor receptor (TNFR) superfamily ligands such OX40 ligand or GITR ligand. OX40 (CD134; TNFRSF4) and GITR is a member of the tumor-necrosis factor (TNF) receptor superfamily that is transiently expressed by T cells upon engagement of the T-cell receptor (TCR). OX40 engagement modulates bi-directionally the interaction of T cells with OX40L⁺ antigen presenting cells (e.g. B cells, dendritic cells (DCs), monocytes). In the context of TCR engagement, OX40 provides co-stimulatory signals predominantly to CD4⁺, but also to CD8⁺ effector T cells, resulting in enhanced proliferation, survival, and effector function (e.g. cytokine secretion). Conversely, OX40 signaling leads to functional inhibition and loss of regulatory T cells. OX40 agonism counterbalances TGF-β effects, (e.g. impedes FoxP3 induction) and lowers IL-10 secretion. In murine tumor models, OX40 engagement by an agonist anti-OX40 antibody can promote anti-tumor T-cell responses, tumor shrinkage and reproducible abscopal effects. Monotherapy efficacy of OX40 agonists was in general low, but strong anti-tumor efficacy was achieved in combination with immunogenic treatments (chemotherapy, radiation and vaccination), check point inhibitors (PD-1, CTLA-4) and other costimulatory agonists such as 4-1BB, ICOS or GITR.
Fibroblast activation protein-α (FAP) is a serine protease highly expressed on the cell surface of cancer-associated stroma cells of > 90% of human epithelial malignancies, on reticular fibroblasts, which are in the T cell priming zones of the lymph nodes, and can be found on activated fibroblasts in normal tissues. High prevalence in various cancer indications allows its usage as targeting moiety for drugs that should accumulate within the tumor environment.
One means to restore TNF receptor co-stimulation specifically in the tumor microenvironment are bispecific antibodies comprised of at least one antigen binding domain for fibroblast activating protein (FAP) in the tumor stroma, and antigen binding domain capable of specific binding to a TNF receptor. For example, such bispecific antibodies have been described in WO 2017/055398 A2 and WO 2017/060144 A1. Crosslinking and surface immobilization of such bispecific molecules by cell surface FAP creates a highly agonistic matrix for OX40 positive T cells, where it supports NFκB mediated effector functions and can replace ligation by OX40 Ligand. High FAP expression is reported for a plethora of human tumor indications, either on tumor cells themselves or on immune suppressive cancer associated fibroblasts (CAFs). There is thus a need for improved targeted bispecific antibodies with excellent pharmacological properties such as better shelf-life, less immunogenicity and with less unspecific interactions such as hypersensitivity reactions or uncontrolled cytokine release.

SUMMARY OF THE INVENTION

This invention relates to bispecific antibodies comprising a modified C-terminal crossfab fragment with improved properties. In particular, these molecules are devoid of neoepitopes that react with anti-drug antibodies (ADA), they have reduced or no reactivity against preexisting anti-drug antibodies (ADA).
In one aspect, the invention provides a bispecific antigen binding molecule comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association; wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit,
(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit,
(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the cross-fab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).

In one aspect, the bispecific antigen binding molecule has reduced or no reactivity towards pre-existing anti-drug antibodies (ADA).
In one aspect, the bispecific antigen binding molecule comprises a third Fab fragment capable of specific binding to a second antigen, wherein Fab fragment is a crossfab fragment and wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence of EPKSCS (SEQ ID NO:1).
In another aspect, the bispecific antigen binding molecule comprises a third Fab fragment capable of specific binding to a second antigen, wherein Fab fragment is a crossfab fragment and wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence of EPKSCG (SEQ ID NO:2).
In a further aspect, the bispecific antigen binding molecule comprises a third Fab fragment capable of specific binding to a second antigen, wherein Fab fragment is a crossfab fragment and wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence selected from the group consisting of EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5). In one particular aspect, the bispecific antigen binding molecule comprises a third Fab fragment capable of specific binding to a second antigen, wherein Fab fragment is a crossfab fragment and wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence of EPKSCD (SEQ ID NO:3).
In another aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the bispecific antigen binding molecule binds bivalently to the first antigen and monovalently to the second antigen.
In one aspect, the bispecific antigen binding molecule comprises

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain of a Fab fragment capable of specific binding to the first antigen and a Fc domain subunit,
(b) two light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and
(c) a cross-fab fragment capable of specific binding to the second antigen comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain is connected to the C-terminus of one of the two heavy chains of (a).

In one further aspect, provided is the bispecific antigen binding molecule as defined herein before, wherein the antigen binding molecule comprises a fourth Fab fragment capable of specific binding to the first antigen, wherein the fourth Fab fragment is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen.
In another aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the bispecific antigen binding molecule binds trivalently to the first antigen and monovalently to the second antigen.
In one aspect, the bispecific antigen binding molecule comprises

(a) a heavy chain comprising a VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,
(b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen and a Fc domain subunit,
(c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and
(d) a light chain comprising a VL and CH1 domain of the third Fab fragment capable of specific binding to the second antigen.

In another aspect, the bispecific antigen binding molecule comprises

(a) a heavy chain comprising a VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit,
(b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen and a Fc region subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc domain subunit, optionally via a peptide linker,
(c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and
(d) a light chain comprising a VL and CH1 domain of a Fab fragment capable of specific binding to the second antigen.

In one further aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the antigen binding molecule comprises a fifth Fab fragment capable of specific binding to the first antigen, wherein the fifth Fab fragment is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the second Fab fragment capable of specific binding to the first antigen.
In another aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the bispecific antigen binding molecule binds tetravalently to the first antigen and monovalently to the second antigen.
In one aspect, the bispecific antigen binding comprises

(a) a heavy chain comprising a VH-CH1 chain of a first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,
(b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen fused at its N-terminus to the VH-CH1 chain of the fifth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit,
(c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and
(d) a light chain comprising a VL and CH1 domain of the third Fab fragment capable of specific binding to the second antigen.

In one aspect, provided is the bispecific antigen binding molecule as defined herein before, wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain and wherein the Fc domain comprises one or more amino acid substitution that reduces the binding affinity of the antibody to an Fc receptor and/or effector function. In one particular aspect, the Fc domain is of human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (EU numbering according to Kabat).
In another aspect, provided is the bispecific antigen binding molecule as defined herein before, wherein the first subunit of the Fc domain comprises knobs and the second subunit of the Fc domain comprises holes according to the knobs into holes method. In one particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering according to Kabat) and the second subunit of the Fc region comprises the amino acid substitutions Y349C, T366S and Y407V (EU numbering according to Kabat).
According to another aspect of the invention, there is provided isolated nucleic acid encoding a bispecific antigen binding molecule as described herein before. The invention further provides a vector, particularly an expression vector, comprising the isolated nucleic acid of the invention, and a host cell comprising the isolated nucleic acid or the expression vector of the invention. In some aspects, the host cell is a eukaryotic cell, particularly a mammalian cell. In another aspect, provided is a method of producing a bispecific antigen binding molecule as described herein before, comprising culturing the host cell as described above under conditions suitable for the expression of the bispecific antigen binding molecule, and isolating the bispecific antigen binding molecule. The invention also encompasses the bispecific antigen binding molecule comprising a modified C-terminal crossfab fragment as produced by the method of the invention.
The invention further provides a pharmaceutical composition comprising a bispecific antigen binding molecule as described herein before and a pharmaceutically acceptable carrier. In one aspect, the pharmaceutical composition comprises an additional therapeutic agent. Also encompassed by the invention is the bispecific antigen binding molecule as described herein before, or the pharmaceutical composition comprising the bispecific antigen binding molecule as defined herein before, for use as a medicament.
In a specific aspect, provided is the bispecific antigen binding molecule as described herein before or the pharmaceutical composition as defined herein before, for use in the treatment of cancer. In another specific aspect, the invention provides the bispecific antigen binding molecule as described herein before for use in the treatment of cancer, wherein the bispecific antigen binding molecule is for administration in combination with a chemotherapeutic agent, radiation and/ or other agents for use in cancer immunotherapy. In another aspect, provided is the bispecific antigen binding molecule as described herein before or the pharmaceutical composition of the invention, for use in up-regulating or prolonging cytotoxic T cell activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1E show schematic representations of bispecific antigen binding molecules which specifically bind to a first antigen and to a second antigen. FIG. 1A shows a schematic representation of a bispecific antigen binding molecule in a 4+1 format consisting of four Fab fragments capable of specific binding to a first antigen combined with one Fab fragment capable of specific binding to a second antigen as crossFab fragment, wherein the VH-Ckappa chain is fused at the C-terminus of the Fc knob chain (tetravalent for the first antigen and monovalent for the second antigen). FIG. 1B shows a schematic representation of a bispecific antigen binding molecule in a 3+1 format consisting of three Fab fragments capable of specific binding to a first antigen combined with one Fab fragment capable of specific binding to a second antigen as crossFab fragment, wherein the VH-Ckappa chain is fused at the C-terminus of the Fc knob chain (trivalent for the first antigen and monovalent for the second antigen). The arm comprising two Fab fragments capable of specific binding to a first antigen fused to each other is on the Fc knob chain. FIG. 1C shows a schematic representation of a bispecific antigen binding molecule in a 3+1 format consisting of three Fab fragments capable of specific binding to a first antigen combined with one Fab fragment capable of specific binding to a second antigen as crossFab fragment, wherein the VH-Ckappa chain is fused at the C-terminus of the Fc knob chain (trivalent for the first antigen and monovalent for the seond antigen). The arm comprising two Fab fragments capable of specific binding to a first antigen fused to each other is on Fc hole chain. FIG. 1D shows a schematic representation of a bispecific antigen binding molecule in a 2+1 format consisting of two Fab fragments capable of specific binding to a first antigen combined with one Fab fragment capable of specific binding to a seond antigen as crossFab fragment, wherein the VH-Ckappa chain is fused at the C-terminus of the Fc knob chain (bivalent for the first antigen and monovalent for the second antigen). FIG. 1E shows a schematic representation of a bispecific FAP-OX40 antibody P1AD4524 in a 4+1 format consisting of four OX40 binding Fab fragments combined with one FAP (4B9) binding moiety as VH and VL domain, wherein the VL domain is fused at the C-terminus of the Fc knob chain and the VH domain is fused at the C-terminus of the Fc hole chain (tetravalent for OX40 and monovalent for FAP). The black point symbolizes knob-into-hole mutations.

FIGS. 2A to 2F show the cellular binding of bispecific antigen binding molecules comprising OX40 clone 49B4 in different formats. The FAP antigen binding domain H212 is the humanized version of FAP clone 212 that is called FAP (1G1a) herein. Human FAP negative tumor cells (A549- NLR)(FIG. 2F), FAP positive fibroblasts (NIH/3T3-huFAP-clone 19) (FIG. 2E), OX40 positive activated PBMC (activated CD4 and CD8 T cells, FIGS. 2A and 2C, respectively) as well as OX40 negative resting PBMC (resting CD4 and CD8 T cells, FIGS. 2B and 2D, respectively) were incubated with indicated serial dilutions of test antibody detected then by fluorescently labeled 2nd antibody against human Fcy. Living cells were gated and the mean fluorescence intensity of the secondary antibody, baseline corrected by the media-only sample, was plotted from duplicates. Error bars indicate the SEM.

FIGS. 3A to 3F show the cellular binding of bispecific antigen binding molecules comprising OX40 clone 8H9 in different formats and in comparison with a bispecific antigen binding molecule comprising OX40 clone 49B4 in 4+1 format (P1AE6838). Human FAP negative tumor cells (A549- NLR)(FIG. 3F), FAP positive fibroblasts (NIH/3T3-huFAP-clone 19) (FIG. 3E), OX40 positive activated PBMC (activated CD4 and CD8 T cells, FIGS. 3A and 3C, respectively) as well as OX40 negative resting PBMC (resting CD4 and CD8 T cells, FIGS. 3B and 3D, respectively) were incubated with indicated serial dilutions of test antibody detected then by fluorescently labeled 2nd antibody against human Fcy. Living cells were gated and the mean fluorescence intensity of the secondary antibody, baseline corrected by the media-only sample, was plotted from duplicates. Error bars indicate the SEM. The clone 8H9 bound with subnanomolar affinity to OX40 positive cells and with comparable strength as tri- and bivalent antibody.

FIGS. 4A to 4F show the cellular binding of bispecific antigen binding molecules comprising OX40 clone MOXR0916 in different formats and in comparison with a bispecific antigen binding molecule comprising OX40 clone 49B4 in 4+1 format (P1AE6838). Human FAP negative tumor cells (A549- NLR)(FIG. 4F), FAP positive fibroblasts (NIH/3T3-huFAP-clone 19) (FIG. 4E), OX40 positive activated PBMC (activated CD4 and CD8 T cells, FIGS. 4A and 4C, respectively) as well as OX40 negative resting PBMC (resting CD4 and CD8 T cells, FIGS. 4B and 4D, respectively) were incubated with indicated serial dilutions of test antibody detected then by fluorescently labeled 2nd antibody against human Fcy. Living cells were gated and the mean fluorescence intensity of the secondary antibody, baseline corrected by the media-only sample, was plotted from duplicates. Error bars indicate the SEM. The clone MOXR0916 bound with nanomolar affinity to OX40 positive cells, with comparable strength as tri- and bivalent antibody.

FIGS. 5A to 5F show the cellular binding of bispecific antigen binding molecules comprising OX40 clone CLC563 in different formats and in comparison with a bispecific antigen binding molecule comprising OX40 clone 49B4 in 4+1 format (P1AE6838). Human FAP negative tumor cells (A549- NLR)(FIG. 5F), FAP positive fibroblasts (NIH/3T3-huFAP-clone 19) (FIG. 5F), OX40 positive activated PBMC (activated CD4 and CD8 T cells, FIGS. 5A and 5C, respectively) as well as OX40 negative resting PBMC (resting CD4 and CD8 T cells, FIGS. 5B and 5D, respectively) were incubated with indicated serial dilutions of test antibody detected then by fluorescently labeled 2nd antibody against human Fcy. Living cells were gated and the mean fluorescence intensity of the secondary antibody, baseline corrected by the media-only sample, was plotted from duplicates. Error bars indicate the SEM. The clone CLC-563 bound with nanomolar affinity to OX40 positive cells, with comparable strength as tri- and bivalent antibody.

FIGS. 6A to 6F show the cellular binding of bispecific antigen binding molecules comprising different variants of OX40 clone 49B4 with amino acid mutations in the VH domain in different formats and in comparison with a bispecific antigen binding molecule comprising OX40 clone 49B4 in 4+1 format (P1AE6838). Human FAP negative tumor cells (A549- NLR)(FIG. 6F), FAP positive fibroblasts (NIH/3T3-huFAP-clone 19) (FIG. 6E), OX40 positive activated PBMC (activated CD4 and CD8 T cells, FIGS. 6A and 6C, respectively) as well as OX40 negative resting PBMC (resting CD4 and CD8 T cells, FIGS. 6B and 6D, respectively) were incubated with indicated serial dilutions of test antibody detected then by fluorescently labeled 2nd antibody against human Fcy. Living cells were gated and the mean fluorescence intensity of the secondary antibody, baseline corrected by the media-only sample, was plotted from duplicates. Error bars indicate the SEM. All antigen binding molecules comprising OX40 (49B4) variants with amino acid mutations showed slightly improved binding to OX40 positive cells compared to antigen binding molecule including clone 49B4.

FIGS. 7A to 7F show the cellular binding of bispecific antigen-binding molecules comprising OX40 clones OX40(49B4_K23E_K73E) or OX40(CLC563) in 3+1 and 4+1 formats as D- and S-variant as indicated. OX40 positive activated PBMC gated on activated CD4 cells (FIGS. 7A, 7C, 7E) and activated CD8 T cells (FIGS. 7C, 7D, 7F), respectively, were incubated with indicated serial dilutions of test antibody detected then by fluorescently labeled 2nd antibody against human Fcy. Living cells were gated and the mean fluorescence intensity of the secondary antibody, baseline corrected by the media-only sample, was plotted from duplicates. FIG. 7A shows the binding of the OX40(CLC563) 3+1 constructs as D- and S-variants to activated CD4 cells and the binding to activated CD8 T cells is shown in FIG. 7B. In FIG. 7C and FIG. 7D is shown the binding of the OX40(CLC563) 4+1 constructs as D- and S-variants on activated CD4 cells and on activated CD8 cells, respectively. The binding of OX40(49B4_K23E_K73E) 4+1 constructs as D- and S-variant to activated CD4 cells and to activated CD8 T cells is shown in FIG. 7E and FIG. 7F, respectively. As control molecules, the untargeted tetravalent OX40(49B4) 4+0 construct (P1AD3690), the tetravalent OX40(49B4)-FAP(4B9) 4+1 construct (P1AD4524) and isotype control were used.

FIGS. 8A to 8F show the NFKB-mediated luciferase expression activity in OX40 expressing reporter cell line HeLa_hOx40_NFκB_Luc1. The concentration of bispecific antigen binding molecules comprising OX40 clones OX40(49B4_K23E_K73E) or OX40(CLC563) in 3+1 and 4+1 formats as D and S variant are blotted against the units of released light (URL) measured after incubation and addition of Luciferase detection solution. Shown is the NFKB induction, either crosslinked with human FAP expressing NIH/3T3 fibroblasts (FIG. 8A for the OX40(CLC563) 3+1 constructs as D- and S-variants, FIG. 8C for the OX40(CLC563) 4+1 constructs as D- and S-variants and FIG. 8E for the OX40(49B4_K23E_K73E) 4+1 constructs as D- and S-variant), or without further crosslinking (FIG. 8B for the OX40(CLC563) 3+1 constructs as D- and S-variants, FIG. 8D for the OX40(CLC563) 4+1 constructs as D- and S-variants and FIG. 8F for the OX40(49B4_K23E_K73E) 4+1 constructs as D- and S-variant). The isotype control antibody did not induce any NFKB activation. All OX40 containing constructs induced dose dependent NFKB activation. The tetravalent format comprising four OX40 Fab fragments induced a certain NFKB activation due to the assembly of the trimeric core OX40 receptor-signaling unit already in the absence of crosslinking. The S and D variant performed similar. Shown is the mean of duplicates. Error bars represent the SEM.

FIGS. 9A to 9F show the primary T cell bioactivity of bispecific antigen-binding molecules comprising OX40 clones OX40(49B4_K23E_K73E) or OX40(CLC563) in 3+1 and 4+1 formats as D and S variant as indicated. The evaluated bioactivity marker was here the CD25 activation marker expression on CD4⁺ T cells (FIG. 9A for OX40(CLC563) 3+1 constructs, FIG. 9C for OX40(CLC563) 4+1 constructs and FIG. 9E for OX40(49B4_K23E_K73E) 4+1 constructs) and CD8⁺ T cells (FIG. 9B for OX40(CLC563) 3+1 constructs, FIG. 9D for OX40(CLC563) 4+1 constructs and FIG. 9F for OX40(49B4_K23E_K73E) 4+1 constructs) cells at endpoint. Increased proliferation and CD25 activation marker expression were observed with FAP-targeted OX40 antigen binding molecules in a dose-dependent manner. The untargeted OX40 molecule showed activity only at the highest tested concentrations whereas isotype control showed no activation after baseline-correction. No statistically significant difference could be detected between the S- and D- variants. Shown is the mean of duplicates. Error bars represent the SEM.

FIGS. 10A to 10F show that co-stimulation with FAP targeted OX40 agonists enhances the cytokine secretion of PBMC induced by CEACAM5 TCB mediated lysis of tumor cells. PBMC were cocultured with MKN45 NLR target cells, FAP+ NIH/3T3-huFAP clone 19, CECAM5 TCB [2 nM] and bispecific antigen binding molecules comprising OX40 clones OX40(49B4_K23E_K73E) or OX40(CLC563) in 3+1 and 4+1 formats as D and S variant as indicated for 48 hrs. The evaluated bioactivity marker was here the fold increase of GM-CSF (FIG. 10A for OX40(CLC563) 3+1 constructs, FIG. 10C for OX40(CLC563) 4+1 constructs and FIG. 10E for OX40(49B4_K23E_K73E) 4+1 constructs) and TNF-α (FIG. 10B for OX40(CLC563) 3+1 constructs, FIG. 10D for OX40(CLC563) 4+1 constructs and FIG. 10F for OX40(49B4_K23E_K73E) 4+1 constructs), over TCB only treated samples in the assay supernatant. Cytokine induction was only seen for FAP-crosslinked OX40 agonists in a dose-dependent manner. The untargeted OX40 control molecule (P1AD3690) and isotype control showed no activity here. The S- variants show a trend to reduced bioactivity compared to the D- variants. Shown is the mean of triplicates.

FIGS. 11A to 11F also show that co-stimulation with FAP-targeted OX40 agonists enhances the cytokine secretion of PBMC induced by CEACAM5 TCB mediated lysis of tumor cells. PBMC were cocultured with MKN45 NLR target cells, FAP+ NIH/3T3-huFAP clone 19, CEACAM5 TCB [2 nM] and bispecific antigen binding molecules comprising OX40 clones OX40(49B4_K23E_K73E) or OX40(CLC563) in 3+1 and 4+1 formats as D and S variant as indicated for 48 hrs. The evaluated bioactivity marker was here the fold increase of IFNy (FIG. 11A for OX40(CLC563) 3+1 constructs, FIG. 11C for OX40(CLC563) 4+1 constructs and FIG. 11E for OX40(49B4_K23E_K73E) 4+1 constructs) and IL-2 (FIG. 11B for OX40(CLC563) 3+1 constructs, FIG. 11D for OX40(CLC563) 4+1 constructs and FIG. 11F for OX40(49B4_K23E_K73E) 4+1 constructs), over TCB only treated samples in the assay supernatant. Cytokine induction was only observed for FAP-crosslinked OX40 agonists in a dose-dependent manner. The untargeted OX40 control molecule (P1AD3690) and isotype control showed no activity here. The S- variants show a trend to reduced bioactivity compared to the D- variant. Shown is the mean of triplicates.

FIG. 12 summarizes the data and shows that co-stimulation with all FAP targeted OX40 agonists enhances the cytokine secretion of PBMC induced by CEACAM5 TCB mediated lysis of tumor cells. The AUC of the dose response curves in FIGS. 37A to 37F and 38A to 38F were calculated and normalized against that of the OX40(CLC563) x FAP(1G1a_EPKSCD) 3+1 antigen binding molecule (P1AF6454, also called 3+1 CLC563/H212-D). Each symbol represents one cytokine in the Box-Whisker Blot.

FIGS. 13A to 13C show that co-stimulation with FAP targeted OX40 agonists suppresses the induction of FoxP3 on Treg cells by TGFβ. Human PBMC preparations containing naive CD4 T cells were cultured in the presence of TGFβ during T cell activation with antibodies against CD28 and CD3. OX40 agonism was provided through serial dilution rows of bispecific antigen binding molecules comprising OX40 clones OX40(CLC563) or OX40(49B4_K23E_K73E) in 3+1 and 4+1 formats as D and S variant. Crosslinking was provided by FAP antigen coated to beads. OX40 agonism interfered with Treg induction visible by reduced FoxP3 expression. Alive CD4⁺CD25⁺Treg singlet cells were gated and the MFI of the αFoxP3 antibody reported. The FoxP3 MFI of each concentration was corrected by the MFI of the sample without OX40 antibody, thus only TGBβ, present. FIG. 13A shows the effect of OX40(CLC563) 3+1 constructs, FIG. 13B for OX40(CLC563) 4+1 constructs and FIG. 13C for OX40(49B4_K23E_K73E) 4+1 constructs. The D and S variant of each FAP targeted OX40 bispecific antigen binding molecule suppressed FoxP3 to a similar range. Shown is the mean of triplicates, error bars represent the SEM.

FIG. 14 shows the results of the NFκB activation assay using engineered Jurkat cells expressing GITR. The concentration of bispecific antigen binding molecules comprising GITR x FAP(4B9) bispecific antibodies in 2+1 formats with C-terminal end EPKSC (SEQ ID NO: 6) as well as S and G variants are blotted against the units of released light (RLU) measured after incubation and addition of Luciferase detection solution. Shown is the NFKB induction, crosslinked with human FAP expressing NIH/3T3 fibroblasts. All three c-terminal variants performed similar. FIG. 14 discloses SEQ ID NOS 6, and 1-2, respectively, in order of appearance.

FIGS. 15A to 15C show the preexisting Anti-Drug Antibody (ADA) reactivity in a panel of human individual plasma samples as measured with the assay described in Example 7.1. High incidence with high signals was observed for the bispecific antigen binding molecule OX40 (49B4) x FAP (4B9) (4+1) as described in WO 2017/060144 A1 wherein a VH and VL domain are c-terminally linked to each of the heavy chains (FIG. 15A). Less incidence was detected for bispecific antigen binding molecules wherein the VH and VL domain fused to the C-termini of the Fc domain were replaced by a Fab fragment. However, there still seem to be preexisting anti-drug antibodies against the Fab fragment as can be seen for OX40 (49B4) x FAP (4B9) (4+1) in FIG. 15B and for OX40 (49B4) x FAP (1G1a) (4+1) in FIG. 15C. 1G1a is a humanized variant of FAP clone 212 (H212).

FIG. 16 compares the preexisting Anti-Drug Antibody (ADA) reactivity of bispecific antigen binding molecules in 2+1 format comprising different anti-OX40 clones (49B4, 8H9, MOX0916 and CLC-563) in a panel of human individual plasma samples.

FIG. 17A shows that the control molecules, i.e. an untargeted tetravalent OX40 (49B4) antigen binding molecule (P1AD3690), the FAP (1G1a) antibody (P1AE1689) or a Germline control antibody (DP47) did not cause preexisting Anti-Drug Antibody (ADA) reactivity, whereas the bispecific antigen molecules comprising a Fab fragment fused at the C-terminus of the Fc domain all caused preexisting IgG interference as shown in FIG. 17B. Surprisingly, the smaller 2+1 molecule induced a slightly higher incidence than the molecules in 3+1 and 4+1 format.

FIGS. 18A to 18C relate to the testing of the preexisting Anti-Drug Antibody (ADA) reactivity of the bispecific antigen binding molecule OX40 (49B4) x FAP (1G1a) (3+1). The molecule comprising a CH1 domain with a “free” C-terminus EPKSC (SEQ ID NO: 6) induces preexisting ADA reactivity as can be seen in FIG. 18A. The individual background signal of the buffer as measured by performing the assay without the drug molecule is shown in FIG. 18B and FIG. 18C shows the preexisting ADA reactivity of the molecule with the background signal substracted. FIG. 18 discloses SEQ ID NO: 6.

FIGS. 19A to 19C: The preexisting Anti-Drug Antibody (ADA) reactivity in a panel of human individual plasma samples of the bispecific antigen binding molecule OX40 (49B4) x FAP (1G1a) (3+1) as determined in FIG. 18C is also shown in FIG. 19A and compared with the preexisting IgG reactivity induced by the bispecific molecule OX40 (MOXR0916) x FAP (1G1a) (3+1) comprising a EPKSCD (SEQ ID NO: 3) terminus (FIG. 19B) or by the bispecific molecule OX40 (MOXR0916) x FAP (1G1a) (3+1) comprising a EPKSCS (SEQ ID NO: 1) terminus (FIG. 19C). A massive reduction was observed with the EPKSD (SEQ ID NO: 163) variant whereas the EPKSCS (SEQ ID NO: 1) variant led to complete elimination of preexisting ADA reactivity. FIG. 19 discloses

SEQ ID NOS

6, 3, and 1, respectively, in order of appearance.

FIGS. 20A to 20C show a respective molecule set in 2+1 format, and confirm the previous results that a bispecific antigen binding molecule OX40 (MOXR0916) x FAP (1G1a) (2+1) with EPKSCD (SEQ ID NO: 3) terminus, P1AF4852 (FIG. 20B) reduces, while a bispecific antigen binding molecule OX40 (MOXR0916) x FAP (1G1a) (2+1) with EPKSCS (SEQ ID NO: 1) terminus, P1AF4858 (FIG. 20C) eliminates the reactivity with preexisting antibodies in plasma compared to a molecule OX40 (49B4) x FAP (1G1a) (2+1) with a free C-terminus EPKSC (SEQ ID NO: 6) (P1AE6840, FIG. 20A). FIG. 20 discloses

SEQ ID NOS

6, 3, and 1, respectively, in order of appearance.

FIGS. 21A to 21H show a set of OX40 (MOXR0916) x FAP (1G1a) molecules with increasing C-terminal extensions. C-terminal extension of an aspartate (Molecule OX40 (MOXR0916) x FAP (1G1a) (2+1) with EPKSCD (SEQ ID NO: 3) terminus, P1AF4852, FIG. 21B) reduces the reactivity with preexisting antibodies in plasma compared to a molecule OX40 (MOXR619) x FAP (1G1a) (2+1) with a free C-terminus EPKSC (SEQ ID NO: 6) (P1AE8872, FIG. 21A), whereas slightly increased reactivity is observed for the C-terminus EPKSCDK (SEQ ID NO: 4) (molecule (MOXR619) x FAP (1G1a) (3+1), P1AF4846, FIG. 21C). High reactivity has been observed for the molecule with the C-terminus EPKSCDKT (SEQ ID NO: 164) (molecule (MOXR619) x FAP (1G1a) (3+1), P1AF4847, FIG. 21D). The reactivity is again reduced for the molecules with the C-terminus EPKSCDKTH (SEQ ID NO: 165) (molecule (MOXR619) x FAP (1G1a) (2+1), P1AF4855, FIG. 21E) and with the C-terminus EPKSCDKTHT (SEQ ID NO: 7) (molecule (MOXR619) x FAP (1G1a) (2+1), P1AF4856, FIG. 21F). Replacement of the C-terminal T with amino acid L, which is not naturally occurring at this position in the upper hinge region, completely eliminates the reactivity with preexisting antibodies in plasma (molecule (MOXR619) x FAP (1G1a) (2+1), P1AF4857, FIG. 21G), comparable with a C-terminal serine (molecule OX40 (MOXR0916) x FAP (1G1a) (2+1) with EPKSCS (SEQ ID NO: 1) terminus, P1AF4858, FIG. 21H). FIG. 21 discloses SEQ ID NOS 6, 3-4, 164-165, 7, 5, and 1, respectively, in order of appearance. FIG. 22 summarizes the results. FIG. 22 discloses SEQ ID NOS 6, 3-4, 164-165, 7, 5, and 1, respectively, in order of appearance.

FIGS. 23A to 23F confirm that the same effect was observed with three other examples. The preexisting ADA reactivity in a panel of human individual plasma samples is shown for OX40 (CLC563) x FAP (1G1a) (3+1) with EPKSCD (SEQ ID NO: 3) terminus (P1AF6454) in FIG. 23A, for OX40 (CLC563) x FAP (1G1a) (3+1) with EPKSCS (SEQ ID NO: 1) terminus (P1AF6455) in FIG. 23B, for OX40 (CLC563) x FAP (1G1a) (4+1) with EPKSCD (SEQ ID NO: 3) terminus (P1AF7205) in FIG. 23C, for OX40 (CLC563) x FAP (1G1a) (4+1) with EPKSCS (SEQ ID NO: 1) terminus (P1AF7217) in FIG. 23D, for OX40 (49B4_K23E_K73E) x FAP (1G1a) (3+1) with EPKSCD (SEQ ID NO: 3) terminus (P1AF6456) in FIG. 23E and for OX40 (49B4_K23E_K73E) x FAP (1G1a) (3+1) with EPKSCS (SEQ ID NO: 1) terminus (P1AF6457) in FIG. 23F. FIG. 23 discloses

SEQ ID NOS

3, 1, 3, 1, 3, and 1, respectively, in order of appearance.

FIGS. 24A to 24C show a set of GITR bispecific antigen binding molecules GITR x FAP (4B9) (2+1) and its C-terminal variants with EPKSCS (SEQ ID NO: 1) terminus (P1AG1036) and with EPKSCG (SEQ ID NO: 2) terminus (P1AG1039). It confirms the previous results that a C-terminal extension of a serine (P1AG1036, FIG. 24B) eliminates the reactivity with preexisting antibodies in plasma compared to the respective molecule with a free C-terminus (P1AE1116, FIG. 24A). It is also shown that similar to the serine extension variant, a further extension variant with a glycine eliminates the unwanted reactivity (P1AG1039, FIG. 24C). FIG. 24 discloses SEQ ID NOS 6 and 1-2, respectively, in order of appearance.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as generally used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa.
As used herein, the term “antigen binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are antibodies, antibody fragments and scaffold antigen binding proteins.
As used herein, the term “antigen binding domain capable of specific binding to an antigen” or “moiety capable of specific binding to an antigen” refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one aspect, the antigen binding domain is able to activate signaling through its target cell antigen. In a particular aspect, the antigen binding domain is able to direct the entity to which it is attached (e.g. the TNF receptor agonistic antibody) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant. Antigen binding domains capable of specific binding to an antigen include antibodies and fragments thereof as further defined herein. In particular, the antigen binding domain capable of specific binding to a target cell antigen is an antigen binding domain capable of specific binding to Fibroblast Activation Protein (FAP).
In relation to an antibody or fragment thereof, the term “antigen binding domain capable of specific binding to an antigen” refers to the part of the molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen binding domain capable of specific antigen binding may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Particularly, an antigen binding domain capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one particular aspect, the “antigen binding domain capable of specific binding to an antigen” is a Fab fragment or a cross-Fab fragment.
The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g. containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen. The term “bispecific” means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells. A bispecific antigen binding molecule as described herein can also form part of a multispecific antibody.
The term “valent” as used within the current application denotes the presence of a specified number of binding sites specific for one distinct antigenic determinant in an antigen binding molecule that are specific for one distinct antigenic determinant. As such, the terms “bivalent”, “trivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding sites, three binding sites, four binding sites, and six binding sites specific for a certain antigenic determinant, respectively, in an antigen binding molecule. In particular aspects of the invention, the bispecific antigen binding molecules according to the invention can be monovalent for a certain antigenic determinant, meaning that they have only one binding site for said antigenic determinant or they can be bivalent or tetravalent for a certain antigenic determinant, meaning that they have two binding sites or four binding sites, respectively, for said antigenic determinant.
The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or µ (IgM), some of which may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.
An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g. scFv); and single domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g. Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S. Pat. No. 6,248,516 B1). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. As used herein, Thus, the term “Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteins from the antibody hinge region. Fab′-SH are Fab’ fragments wherein the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab′)₂ fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region. According to the present invention, the term “Fab fragment” also includes “cross-Fab fragments” or “crossover Fab fragments” as defined below.
The term “cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). This crossover Fab molecule is also referred to as CrossFab (VLVH). On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab molecule is also referred to as CrossFab (CLCH1).
A “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CH1 domain. In addition, these single chain Fab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g. position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).
A “crossover single chain Fab fragment” or “x-scFab” is a is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL form together an antigen-binding site which binds specifically to an antigen and wherein said linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g. position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).
A “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (V_H) and light chains (V_L) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V_H with the C-terminus of the V_L, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.
An “antigen binding molecule that binds to the same epitope” as a reference molecule refers to an antigen binding molecule that blocks binding of the reference molecule to its antigen in a competition assay by 50% or more, and conversely, the reference molecule blocks binding of the antigen binding molecule to its antigen in a competition assay by 50% or more. An “antigen binding molecule that does not bind to the same epitope” as a reference molecule refers to an antigen binding molecule that does not block binding of the reference molecule to its antigen in a competition assay by 50% or more, and conversely, the reference molecule does not block binding of the antigen binding molecule to its antigen in a competition assay by 50% or more.
The term “antigen binding domain” or “antigen binding site” refers to the part of an antigen binding molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen binding molecule may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope,” and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM). The proteins useful as antigens herein can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated. In a particular embodiment the antigen is a human protein. Where reference is made to a specific protein herein, the term encompasses the “full-length”, unprocessed protein as well as any form of the protein that results from processing in the cell. The term also encompasses naturally occurring variants of the protein, e.g. splice variants or allelic variants.
By “specific binding” is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding molecule to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the binding of the antigen binding molecule to the antigen as measured, e.g. by SPR. In certain embodiments, an molecule that binds to the antigen has a dissociation constant (Kd) of ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, < 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g. 10^-8 M or less, e.g. from 10^-8 M to 10^-13 M, e.g. from 10^-9 M to 10^-13 M).
“Affinity” or “binding affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g. antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).
An “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
A “target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, in particular a target cell in a tumor such as a cancer cell or a cell of the tumor stroma. Thus, the target cell antigen is a tumor-associated antigen. In particular, the tumor target cell antigen is Fibroblast Activation Protein (FAP).
The term “Fibroblast activation protein (FAP)”, also known as Prolyl endopeptidase FAP or Seprase (EC 3.4.21), refers to any native FAP from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed FAP as well as any form of FAP that results from processing in the cell. The term also encompasses naturally occurring variants of FAP, e.g., splice variants or allelic variants. In one embodiment, the antigen binding molecule of the invention is capable of specific binding to human, mouse and/or cynomolgus FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession no. Q12884 (version 149, SEQ ID NO:11), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid sequence of a His-tagged human FAP ECD is shown in SEQ ID NO: 12. The amino acid sequence of mouse FAP is shown in UniProt accession no. P97321 (version 126, SEQ ID NO:13), or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. SEQ ID NO:14 shows the amino acid of a His-tagged mouse FAP ECD. SEQ ID NO:15 shows the amino acid of a His-tagged cynomolgus FAP ECD. Preferably, an anti-FAP binding molecule of the invention binds to the extracellular domain of FAP.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.
The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”). Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and
(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.
“Framework” or “FR” refers to variable domain residues other than complementary determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR-H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4.
The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and µ respectively..
A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of “humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding.
The term “CH1 domain” denotes the part of an antibody heavy chain polypeptide that extends approximately from EU position 118 to EU position 215 (EU numbering system according to Kabat). In one aspect, a CH1 domain has the amino acid sequence of ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 16). Usually, a segment having the amino acid sequence of EPKSC (SEQ ID NO:6) is following to link the CH1 domain to the hinge region. The inventors found that a CH1 domain that is not fused to a complete hinge region may lead to reactivity with pre-existing antibodies (ADAs). A CH1 domain with a free C-terminal end can be found for instance in a crossfab fragment.
The term “hinge region” denotes the part of an antibody heavy chain polypeptide that joins in a wild-type antibody heavy chain the CH1 domain and the CH2 domain, e. g. from about position 216 to about position 238 according to the IgG1 EU number system of Kabat, or from about position 226 to about position 230 according to the EU number system of Kabat. The hinge regions of other IgG subclasses can be determined by aligning with the hinge-region cysteine residues of the IgG1 subclass sequence. The hinge region is normally a dimeric molecule consisting of two polypeptides with identical amino acid sequence. The hinge region generally comprises up to 25 amino acid residues and is flexible allowing the associated target binding sites to move independently. The hinge region can be subdivided into three domains: the upper, the core, and the lower hinge domain (see e.g. Roux, et al., J. Immunol. 161 (1998) 4083). In the case of a human IgG1 the upper hinge region has the amino acid sequence of EPKSCDKTHT (SEQ ID NO:7) and corresponds to position 216 to 225 according to the IgG1 EU number system of Kabat. The core hinge region corresponds to positions 226 to 230 according to the IgG1 EU number system of Kabat and has the amino acid sequence of CPXCP (SEQ ID NO:8), wherein X can be P or S. The lower hinge region corresponds to postions 231 to 238 according to the IgG1 EU number system of Kabat and has the amino acid sequence of APELLGGP (SEQ ID NO:9) in the human IgG1 antibody, or the amino acid sequence of APEFLGGP (SEQ ID NO:10) in the human IgG4 antibody. The upper hinge forms a one-turn helix, with little inherent stability, and is exposed to the solvent. The low hinge, which is similar in different subclasses but appears absent in IgA, has an extended conformation. This segment provides segmental flexibility of immunoglobulin molecules. The rigid core forms two parallel polyproline double helices linked by disulfide bridges, the number of which varied between different immunoglobulin classes and subclasses. The length of the central core (or the middle hinge), is different in immunoglobulin molecules of various classes and subclasses and also is variable between species.
The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. The “CH2 domain” of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. (EU numbering system according to Kabat). In one aspect, a CH2 domain has the amino acid sequence of APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 17). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native Fc-region. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain. The “CH3 domain” comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from an amino acid residue at about position 341 to an amino acid residue at about position 447 according to EU numbering system according to Kabat of an IgG). In one aspect, the CH3 domain has the amino acid sequence of GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG(SEQID NO: 18). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with an introduced “protuberance” (“knob”) in one chain thereof and a corresponding introduced “cavity” (“hole”) in the other chain thereof; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as herein described. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
The term “wild-type Fc domain” denotes an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include a native human IgG1 Fc-region (non-A and A allotypes), native human IgG2 Fc-region, native human IgG3 Fc-region, and native human IgG4 Fc-region as well as naturally occurring variants thereof. Wild-type Fc-regions are denoted in SEQ ID NO: 19 (IgG1, caucasian allotype), SEQ ID NO: 20 (IgG1, afroamerican allotype), SEQ ID NO: 21 (IgG2), SEQ ID NO:22 (IgG3) and SEQ ID NO:23 (IgG4). The term “variant (human) Fc domain” denotes an amino acid sequence which differs from that of a “wild-type” (human) Fc domain amino acid sequence by virtue of at least one “amino acid mutation”. In one aspect, the variant Fc-region has at least one amino acid mutation compared to a native Fc-region, e.g. from about one to about ten amino acid mutations, and in one aspect from about one to about five amino acid mutations in a native Fc-region. In one aspect, the (variant) Fc-region has at least about 95 % homology with a wild-type Fc-region.
The “knob-into-hole” technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain. In a further specific embodiment, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
A “region equivalent to the Fc region of an immunoglobulin” is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).
The term “effector function” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.
Fc receptor binding dependent effector functions can be mediated by the interaction of the Fc-region of an antibody with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily, and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC) (see e.g. Van de Winkel, J.G. and Anderson, C.L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcγR. Fc receptor binding is described e.g. in Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492, Capel, P.J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J.E., et al., Ann. Hematol. 76 (1998) 231-248.
Cross-linking of receptors for the Fc-region of IgG antibodies (FcγR) triggers a wide variety of effector functions including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. In humans, three classes of FcγR have been characterized, which are:

FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. Modification in the Fc-region IgG at least at one of the amino acid residues E233-G236, P238, D265, N297, A327 and P329 (numbering according to EU index of Kabat) reduce binding to FcγRI. IgG2 residues at positions 233-236, substituted into IgG1 and IgG4, reduced binding to FcγRI by 103-fold and eliminated the human monocyte response to antibody-sensitized red blood cells (Armour, K.L., et al., Eur. J. Immunol. 29 (1999) 2613-2624).
FcγRII (CD32) binds complexed IgG with medium to low affinity and is widely expressed. This receptor can be divided into two sub-types, FcγRIIA and FcγRIIB. FcγRIIA is found on many cells involved in killing (e.g. macrophages, monocytes, neutrophils) and seems able to activate the killing process. FcγRIIB seems to play a role in inhibitory processes and is found on B cells, macrophages and on mast cells and eosinophils. On B-cells it seems to function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, FcγRIIB acts to inhibit phagocytosis as mediated through FcγRIIA. On eosinophils and mast cells the B-form may help to suppress activation of these cells through IgE binding to its separate receptor. Reduced binding for FcγRIIA is found e.g. for antibodies comprising an IgG Fc-region with mutations at least at one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to EU index of Kabat).
FcγRIII (CD16) binds IgG with medium to low affinity and exists as two types. FcγRIIIA is found on NK cells, macrophages, eosinophils and some monocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Reduced binding to FcγRIIIA is found e.g. for antibodies comprising an IgG Fc-region with mutation at least at one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 (numbering according to EU index of Kabat).

Mapping of the binding sites on human IgG1 for Fc receptors, the above mentioned mutation sites and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, R.L., et al. J. Biol. Chem. 276 (2001) 6591-6604.
The term “ADCC” or “antibody-dependent cellular cytotoxicity” is a function mediated by Fc receptor binding and refers to lysis of target cells by an antibody as reported herein in the presence of effector cells. The capacity of the antibody to induce the initial steps mediating ADCC is investigated by measuring their binding to Fcy receptors expressing cells, such as cells, recombinantly expressing FcγRI and/or FcγRIIA or NK cells (expressing essentially FcγRIIIA). In particular, binding to FcγR on NK cells is measured.
An “activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt accession no. P08637, version 141).
The “Tumor Necrosis factor receptor superfamily” or “TNF receptor superfamily” currently consists of 27 receptors. It is a group of cytokine receptors characterized by the ability to bind tumor necrosis factors (TNFs) via an extracellular cysteine-rich domain (CRD). These pseudorepeats are defined by intrachain disulphides generated by highly conserved cysteine residues within the receptor chains.With the exception of nerve growth factor (NGF), all TNFs are homologous to the archetypal TNF-alpha. In their active form, the majority of TNF receptors form trimeric complexes in the plasma membrame. Accordingly, most TNF receptors contain transmembrane domains (TMDs). Several of these receptors also contain intracellular death domains (DDs) that recruit caspase-interacting proteins following ligand binding to initiate the extrinsic pathway of caspase activation. Other TNF superfamily receptors that lack death domains bind TNF receptor-associated factors and activate intracellular signaling pathways that can lead to proliferation or differentiation. These receptors can also initiate apoptosis, but they do so via indirect mechanisms. In addition to regulating apoptosis, several TNF superfamily receptors are involved in regulating immune cell functions such as B cell homeostasis and activation, natural killer cell activation, and T cell co-stimulation. Several others regulate cell type-specific responses such as hair follicle development and osteoclast development. Members of the TNF receptor superfamily include the following: Tumor necrosis factor receptor 1 (1A) (TNFRSF1A, CD120a), Tumor necrosis factor receptor 2 (1B) (TNFRSF1B, CD120b), Lymphotoxin beta receptor (LTBR, CD18), OX40 (TNFRSF4, CD134), CD40 (Bp50), Fas receptor (Apo-1, CD95, FAS), Decoy receptor 3 (TR6, M68, TNFRSF6B), CD27 (S152, Tp55), CD30 (Ki-1, TNFRSF8), 4-1BB (CD137, TNFRSF9), DR4 (TRAILR1, Apo-2, CD261, TNFRSF10A), DR5 (TRAILR2, CD262, TNFRSF10B), Decoy Receptor 1 (TRAILR3, CD263, TNFRSF10C), Decoy Receptor 2 (TRAILR4, CD264, TNFRSF10D), RANK (CD265, TNFRSF11A), Osteoprotegerin (OCIF, TR1, TNFRSF11B), TWEAK receptor (Fn14, CD266, TNFRSF12A), TACI (CD267, TNFRSF13B), BAFF receptor (CD268, TNFRSF13C), Herpesvirus entry mediator (HVEM, TR2, CD270, TNFRSF14), Nerve growth factor receptor (p75NTR, CD271, NGFR), B-cell maturation antigen (CD269, TNFRSF17), Glucocorticoid-induced TNFR-related (GITR, AITR, CD357, TNFRSF18), TROY (TNFRSF19), DR6 (CD358, TNFRSF21), DR3 (Apo-3, TRAMP, WS-1, TNFRSF25) and Ectodysplasin A2 receptor (XEDAR, EDA2R).
Several members of the tumor necrosis factor receptor (TNFR) family function after initial T cell activation to sustain T cell responses. The term “costimulatory TNF receptor” or “costimulatory TNF receptor superfamily member” or “costimulatory TNF superfamily receptor” refers to a subgroup of TNF receptor superfamily members, which are able to costimulate proliferation and cytokine production of T-cells. The term refers to any native TNF family receptor from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. In specific embodiments of the invention, costimulatory TNF receptors are selected from the group consisting of OX40 (CD134), 4-1BB (CD137), CD27, HVEM (CD270), CD30, and GITR, all of which can have costimulatory effects on T cells. More particularly, the costimulatory TNF receptor superfamily member is selected from the group consisting of OX40 and 4-1BB.
Further information, in particular sequences, of the TNF receptors may be obtained from publically accessible databases such as Uniprot (www.uniprot.org). For instance, particular human costimulatory TNF receptors have the following amino acid sequences: human OX40 (UniProt accession no. P43489, SEQ ID NO:24), human GITR (UniProt accession no. Q9Y5U5, SEQ ID NO:26) and human 4-1BB (UniProt accession no. Q07011, SEQ ID NO:28.
The term “OX40”, as used herein, refers to any native OX40 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed OX40 as well as any form of OX40 that results from processing in the cell. The term also encompasses naturally occurring variants of OX40, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human OX40 is shown in SEQ ID NO:24 (Uniprot P43489, version 112) and the amino acid sequence of an exemplary murine OX40 is shown in SEQ ID NO: 25 (Uniprot P47741, version 101).
The term “OX40 agonist” as used herein includes any moiety that agonizes the OX40/OX40L interaction. OX40 as used in this context refers preferably to human OX40, thus the OX40 agonist is preferably an agonist of human OX40. Typically, the moiety will be an agonistic OX40 antibody or antibody fragment, in particular a Fab fragment.
The terms “anti-OX40 antibody”, “anti-OX40”, “OX40 antibody” and “an antibody that specifically binds to OX40” refer to an antibody that is capable of binding OX40 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting OX40. In one aspect, the extent of binding of an anti-OX40 antibody to an unrelated, non-OX40 protein is less than about 10% of the binding of the antibody to OX40 as measured, e.g., by flow cytometry (FACS). In certain embodiments, an antibody that binds to OX40 has a dissociation constant (K_D) of ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g. 10^-6 M or less, e.g. from 10^-68 M to 10^-13 M, e.g., from 10^-8 M to 10^-10 M).
The term “GITR”, as used herein, refers to any native GITR from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed GITR as well as any form of GITR that results from processing in the cell. The term also encompasses naturally occurring variants of GITR, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human GITR is shown in SEQ ID NO:26 (Uniprot P43489, version 112) and the amino acid sequence of an exemplary cynomolgus GITR is shown in SEQ ID NO: 27.
The term “GITR agonist” as used herein includes any moiety that agonizes the GITR/GITRL interaction. OX40 as used in this context refers preferably to human GITR, thus the GITR agonist is preferably an agonist of human GITR. Typically, the moiety will be an agonistic GITR antibody or antibody fragment, in particular a Fab fragment.
The terms “anti-GITR antibody”, “anti-GITR”, “GITR antibody and “an antibody that specifically binds to GITR” refer to an antibody that is capable of binding GITR with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GITR. In one aspect, the extent of binding of an anti-GITR antibody to an unrelated, non-GITR protein is less than about 10% of the binding of the antibody to GITR as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry (FACS). In certain embodiments, an antibody that binds to GITR has a dissociation constant ( K_D) of ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g. 10^-6 M or less, e.g. from 10^-68 M to 10^-13 M, e.g., from 10^-8 M to 10^-10 M).
The term “4-1BB”, as used herein, refers to any native 4-1BB from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed 4-1BB as well as any form of 4-1BB that results from processing in the cell. The term also encompasses naturally occurring variants of 4-1BB, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human 4-1BB is shown in SEQ ID NO:28 (Uniprot accession no. Q07011).
The terms “anti-4-1BB antibody”, “anti-4-1BB”, “4-1BB antibody and “an antibody that specifically binds to 4-1BB” refer to an antibody that is capable of binding 4-1BB with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting 4-1BB. In one embodiment, the extent of binding of an anti-4-1BB antibody to an unrelated, non-4-1BB protein is less than about 10% of the binding of the antibody to 4-1BB as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry (FACS). In certain embodiments, an antibody that binds to 4-1BB has a dissociation constant ( K_D) of ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g. 10^-6 M or less, e.g. from 10^-68 M to 10^-13 M, e.g., from 10^-8 M to 10^-10 M).
The term “4-1BB agonist” as used herein includes any moiety that agonizes the 4-1BB/4-1BBL interaction. 4-1BB as used in this context refers preferably to human 4-1BB, thus the 4-1BB agonist is preferably an agonist of human 4-1BB. Typically, the moiety will be an agonistic 4-1BB antibody or antibody fragment, in particular a Fab fragment.
The term “peptide linker” refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic linker peptides are, for example, (G₄S)_n (SEQ ID NO: 168), (SG₄)_n (SEQ ID NO: 166) or G₄(SG₄)_n (SEQ ID NO: 167) peptide linkers, wherein “n” is generally a number between 1 and 10, typically between 2 and 4, in particular 2, i.e. the peptides selected from the group consisting of GGGGS (SEQ ID NO:29), GGGGSGGGGS (SEQ ID NO:30), SGGGGSGGGG (SEQ ID NO:31) and GGGGSGGGGSGGGG (SEQ ID NO:32), but also include the sequences GSPGSSSSGS (SEQ ID NO:33), (G4S)₃ (SEQ ID NO:34), (G4S)₄ (SEQ ID NO:35), GSGSGSGS (SEQ ID NO:36), GSGSGNGS (SEQ ID NO:37), GGSGSGSG (SEQ ID NO:38), GGSGSG (SEQ ID NO:39), GGSG (SEQ ID NO:40), GGSGNGSG (SEQ ID NO:41), GGNGSGSG (SEQ ID NO:42) and GGNGSG (SEQ ID NO:43). Peptide linkers of particular interest are (G4S) (SEQ ID NO:29), (G₄S)₂ or GGGGSGGGGS (SEQ ID NO:30), (G4S)₃ (SEQ ID NO:34) and (G4S)₄ (SEQ ID NO:35).
The term “amino acid” as used within this application denotes the group of naturally occurring carboxy α-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
By “fused” or “connected” is meant that the components (e.g. a heavy chain of an antibody and a Fab fragment) are linked by peptide bonds, either directly or via a peptide peptide linker.
“Percent (%) amino acid sequence identity” with respect to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN. SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

In certain embodiments, amino acid sequence variants of the bispecific antigen binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the TNF ligand trimer-containing antigen binding molecules. Amino acid sequence variants of the TNF ligand trimer-containing antigen binding molecules may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecules, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. Sites of interest for substitutional mutagenesis include the HVRs and Framework (FRs). Conservative substitutions are provided in Table B under the heading “Preferred Substitutions” and further described below in reference to amino acid side chain classes (1) to (6). Amino acid substitutions may be introduced into the molecule of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE A

Original Residue	Exemplary Substitutions	Preferred Substitutions
Ala (A)	Val; Leu; Ile	Val
Arg (R)	Lys; Gln; Asn	Lys
Asn (N)	Gln; His; Asp, Lys; Arg	Gln
Asp (D)	Glu; Asn	Glu
Cys (C)	Ser; Ala	Ser
Gln (Q)	Asn; Glu	Asn
Glu (E)	Asp; Gln	Asp
Gly (G)	Ala	Ala
His (H)	Asn; Gln; Lys; Arg	Arg
Ile (I)	Leu; Val; Met; Ala; Phe; Norleucine	Leu
Leu (L)	Norleucine; Ile; Val; Met; Ala; Phe	Ile
Lys (K)	Arg; Gln; Asn	Arg
Met (M)	Leu; Phe; Ile	Leu
Phe (F)	Trp; Leu; Val; Ile; Ala; Tyr	Tyr
Pro (P)	Ala	Ala
Ser (S)	Thr	Thr
Thr (T)	Val; Ser	Ser
Trp (W)	Tyr; Phe	Tyr
Tyr (Y)	Trp; Phe; Thr; Ser	Phe
Val (V)	Ile; Leu; Met; Phe; Ala; Norleucine	Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
The term “amino acid sequence variants” includes substantial variants wherein there are amino acid substitutions in one or more hypervariable region residues of a parent antigen binding molecule (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antigen binding molecule and/or will have substantially retained certain biological properties of the parent antigen binding molecule. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antigen binding molecules displayed on phage and screened for a particular biological activity (e.g. binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antigen binding molecule to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antigen binding molecule complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include bispecific antigen binding molecules of the invention with an N-terminal methionyl residue. Other insertional variants of the molecule include the fusion to the N- or C-terminus to a polypeptide which increases the serum half-life of the bispecific antigen binding molecules.
In certain embodiments, the bispecific antigen binding molecules provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation variants of the molecules may be conveniently obtained by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where the antigen binding molecule comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in TNF family ligand trimer-containing antigen binding molecule may be made in order to create variants with certain improved properties. In one aspect, variants of bispecific antigen binding molecules or antibodies of the invention are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. Such fucosylation variants may have improved ADCC function, see e.g. U.S. Pat. Publication Nos. US 2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). In another aspect, variants of the bispecific antigen binding molecules or antibodies of the invention are provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and/or improved ADCC function., see for example WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function and are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
In certain aspects, it may be desirable to create cysteine engineered variants of the bispecific antigen binding molecules of the invention, e.g., “thioMAbs,” in which one or more residues of the molecule are substituted with cysteine residues. In particular aspects, the substituted residues occur at accessible sites of the molecule. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate. In certain aspects, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antigen binding molecules may be generated as described, e.g., in U.S. Pat. No. 7,521,541.
The term “nucleic acid” or “polynucleotide” includes any compound and/or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5′ to 3′. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and/or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler ert al, Nature Medicine 2017, published online 12 Jun. 2017, doi:10.1038/nm.4356 or EP 2 101 823 B1).
An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
“Isolated nucleic acid encoding a bispecific antigen binding molecule or antibody” refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the bispecific antigen binding molecule or antibody, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
By a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g. ALIGN-2).
The term “expression cassette” refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the invention comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
The term “vector” or “expression vector” is synonymous with “expression construct” and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.
The terms “host cell”, “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
An “effective amount” of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.
A “therapeutically effective amount” of an agent, e.g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human.
The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the molecules of the invention are used to delay development of a disease or to slow the progression of a disease.
The term “cancer” as used herein refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.
The term “chemotherapeutic agent” as used herein refers to a chemical compound useful in the treatment of cancer. In one aspect, the chemotherapeutic agent is an antimetabolite. In one aspect, the antimetabolite is selected from the group consisting of Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Cladribine, Clofarabine, Fludarabine, Mercaptopurine, Pentostatin, Thioguanine, Capecitabine, Cytarabine, Fluorouracil, Floxuridine, and Gemcitabine. In one particular aspect, the antimetabolite is capecitabine or gemcitabine. In another aspect, the antimetabolite is fluorouracil. In one aspect, the chemotherapeutic agent is an agent that affects microtubule formation. In one aspect, the agent that affects microtubule formation is selected from the group consisting of: paclitaxel, docetaxel, vincristine, vinblastine, vindesine, vinorelbin, taxotere, etoposide, and teniposide. In another aspect, the chemotherapeutic agent is an alkylating agent such as cyclophosphamide. In one aspect, the chemotherapeutic agent is a cytotoxic antibiotic such as a topoisomerase II inhibitor. In one aspect, the topoisomerase II inhibitor is doxorubicin.

Bispecific Antigen Binding Molecules of the Invention

The invention relates to bispecific antigen binding molecules comprising a modified C-terminal crossfab fragment. These bispecific antigen binding molecules have improved properties. In particular, these molecules are devoid of neoepitopes that react with anti-drug antibodies (ADA), they have reduced or no reactivity against preexisting anti-drug antibodies (ADA), inparticular anti-hinge autoantibodies (AHA). The bispecific antigen binding molecules comprising this new anti-FAP antibody possess particularly advantageous properties such as improved safety window.By using these molecules an extended dosage range can be given to a patient and thereby a possibly enhanced efficacy can be obtained.
A bispecific antigen binding molecule as described herein comprises a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits. Thus, the bispecific antigen binding molecule as described herein comprises a light chain VL-CH1 that terminates with a part of the hinge region.
In certain aspects, the bispecific antigen binding molecule as described herein comprises a cross-fab fragment with a light chain VL-CH1 comprising a native hinge region or a modified hinge region. A native hinge region is a hinge region normally associated with the CH1 domain of an antibody molecule. In certain aspects, the native hinge region of a presently disclosed bispecific antigen binding molecule can be of the IgG1, IgG2, IgG3 or IgG4 isotype. In particular, the bispecific antigen binding molecule is be of the IgG1 isotype. In certain aspects, the cross-Fab fragment is of the IgG1 isotype comprising a native hinge region. In certain aspects, the cross-Fab fragment is of the IgG4 isotype comprising a native hinge region. A modified hinge region is any hinge that differs in length and/or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions can comprise a complete hinge region derived from an antibody of a different class or subclass from that of the CH1 domain. Thus, for instance, a CH1 domain of class γ1 can be attached to a hinge region of class y4. Alternatively, the modified hinge region can comprise part of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region.
In certain aspects, the native hinge region is altered by substituting, deleting and/or adding one or more amino acid residues to generate a modified hinge region. In certain aspects, the cross-Fab fragment is of the IgG1 isotype comprising a modified hinge region. In certain aspects, the cross-Fab fragment is of the IgG2 isotype comprising a modified hinge region. In certain aspects, the cross-Fab fragment is of the IgG4 isotype comprising a modified hinge region. In certain aspects, a modified hinge region comprises the substitution, deletion and/or addition of one or more amino acids within the upper hinge region. In one aspect, a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions and/or additions at amino acid positions 216 to 225 according to Kabat EU numbering.
In one aspect, the amino acid at position 221, i.e. aspartic acid, can be changed to a serine (S), e.g., D221S, according to EU numbering. In certain aspects, a bispecific antigen binding molecule of the present disclosure comprises a cross-Fab fragment comprising the substitution D221S.
In another aspect, the amino acid at position 221, i.e. aspartic acid, can be changed to a glycine (G), e.g., D221G, according to EU numbering. In certain aspects, a bispecific antigen binding molecule of the present disclosure comprises a cross-Fab fragment comprising the substitution D221G.
In another aspect, the amino acid at position 225, e.g., threonine (T), can be changed to a leucine (L), e.g., T225L, according to EU numbering.. In certain aspects, a bispecific antigen binding molecule of the present disclosure comprises a cross-Fab fragment comprising the substitution T225L.
In one aspect, provided are bispecific antigen binding molecules, comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association, wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit,
(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit, and
(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the light chain VL-CH1 of the cross-fab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).

In one particular aspect, the bispecific antigen binding molecule comprises a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the light chain VL-CH1 of the cross-fab fragment terminates with an amino acid sequence of EPKSCS (SEQ ID NO:1). In one aspect, provided is a bispecific antigen binding molecule, wherein the cross-fab fragment capable of specific binding to a second antigen comprises the amino acid sequence of SEQ ID NO:158.
In another aspect, the bispecific antigen binding molecule comprises a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the light chain VL-CH1 of the cross-fab fragment terminates with an amino acid sequence of EPKSCG (SEQ ID NO:2). In one aspect, provided is a bispecific antigen binding molecule, wherein the cross-fab fragment capable of specific binding to a second antigen comprises the amino acid sequence of SEQ ID NO:159.
In a further aspect, the bispecific antigen binding molecule comprises a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the light chain VL-CH1 of the cross-fab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5). In one aspect, provided is a bispecific antigen binding molecule, wherein the cross-fab fragment capable of specific binding to a second antigen comprises the amino acid sequence of SEQ ID NO:160, SEQ ID NO:161 or SEQ ID NO:162.
In one particular aspect, the cross-fab fragment terminates with an amino acid sequence of EPKSCD (SEQ ID NO:3). In one aspect, provided is a bispecific antigen binding molecule, wherein the cross-fab fragment capable of specific binding to a second antigen comprises the amino acid sequence of SEQ ID NO: 160. In another aspect, the cross-fab fragment terminates with an amino acid of EPKSCDKTHL (SEQ ID NO:5). In one aspect, provided is a bispecific antigen binding molecule, wherein the cross-fab fragment capable of specific binding to a second antigen comprises the amino acid sequence of SEQ ID NO:162.
In one aspect, provided is a bispecific antigen binding molecule, comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association, wherein the bispecific antigen binding molecule comprises

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain of a Fab fragment capable of specific binding to the first antigen and a Fc region subunit,
(b) two light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and
(c) a cross-fab fragment capable of specific binding to the second antigen comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain is connected to the C-terminus of one of the two heavy chains of (a), and wherein the light chain VL-CH1 of the cross-fab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).

In one aspect, the bispecific antigen binding molecule binds bivalently to the first antigen and monovalently to the second antigen (2+1 format). Thus, in one aspect, a bispecific antigen binding molecule is provided that comprises two Fab fragments capable of specific binding to the first antigen and one cross-fab fragment capable of specific binding to the second antigen comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain is connected to the C-terminus of one of the two heavy chains of (a), and wherein the light chain VL-CH1 of the cross-fab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).
In another aspect, provided is a bispecific antigen binding molecule, comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association, wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit,
(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit, and
(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the light chain VL-CH1 of the cross-fab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5), and

Thus, in one aspect, the bispecific antigen binding molecule binds trivalently to the first antigen and monovalently to the second antigen (3+1 format). In one aspect, provided is a bispecific antigen binding molecule, wherein said molecule comprises

(a) a heavy chain comprising a VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc region subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,
(b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen and a Fc region subunit,
(c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and
(d) a light chain comprising a VL and CH1 domain of the third Fab fragment capable of specific binding to the second antigen which terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).

In one further aspect, provided is a bispecific antigen binding molecule, wherein said molecule comprises

(a) a heavy chain comprising a VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit,
(b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen and a Fc region subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,
(c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and
(d) a light chain comprising a VL and CH1 domain of a Fab fragment capable of specific binding to the second antigen which terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).

In another aspect, provided is a bispecific antigen binding molecule, comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association, wherein

Thus, in one aspect, the bispecific antigen binding molecule binds tetravalently to the first antigen and monovalently to the second antigen (4+1 format). In one aspect, provided is a bispecific antigen binding molecule, wherein said molecule comprises

(a) a heavy chain comprising a VH-CH1 chain of a first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc region subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,
(b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen fused at its N-terminus to the VH-CH1 chain of the fifth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc region subunit,
(c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and
(d) a light chain comprising a VL and CH1 domain of a Fab fragment capable of specific binding to the second antigen which terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).

In all of these aspects, the Fc domain is preferably an IgG Fc domain. In particular, the Fc domain is an IgG1 Fc domain or an IgG4 Fc domain. In one particular aspect, the Fc region is an IgG1 Fc domain. In one particular aspect, the Fc domain comprises one or more amino acid substitution that reduces the binding affinity of the antibody to an Fc receptor and/or effector function. More partricularly, the Fc domain is of human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (EU numbering according to Kabat).
In all of these aspects, the Fc domain is composed of a first and a second subunit capable of stable association, In particular, the Fc domain is composed of a first and a second subunit capable of stable association wherein the first subunit of the Fc region comprises knobs and the second subunit of the Fc region comprises holes according to the knobs into holes method. In one particular aspect, wherein the first subunit of the Fc region comprises the amino acid substitutions S354C and T366W (EU numbering according to Kabat) and the second subunit of the Fc region comprises the amino acid substitutions Y349C, T366S and Y407V (EU numbering according to Kabat).

Exemplary Bispecific Antigen Binding Molecules

In one aspect, the provided are bispecific antigen binding molecules comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association; wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit;
(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit,
(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the crossfab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5), wherein the third Fab fragment capable of specific binding to a second antigen is a cross-Fab fragment capable of specific binding to a target cell antigen.

In one aspect, the target cell antigen is a tumor-associated antigen. A tumor-associated antigen or TAA refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell, a cell of the tumor stroma, a malignant B lymphocyte or a melanoma cell. In certain aspects, the target cell antigen is an antigen on the surface of a tumor cell. In one aspect, the TAA is for example selected from the group consisting of Fibroblast Activation Protein (FAP), Carcinoembryonic Antigen (CEA), Folate receptor alpha (FolR1), Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), HER2/neu, p95HER2, EpCAM, HER3, c-Met, CD30, tenascin C, TPBG (5T4), CD19, CD79b, CD20, CD22, CD33, CD37, CD38, BCMA and GPRC5D.
In one particular aspect, the target cell antigen is Fibroblast Activation Protein (FAP).
Thus, in one aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the third Fab fragment is cross-Fab fragment capable of specific binding to FAP, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the crossfab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5),
In one aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the cross-Fab fragment capable of specific binding to FAP comprises (a) a heavy chain variable region (V_HFAP) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:45, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:46, and a light chain variable region (V_LFAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:47, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:48, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:49, or (b) a heavy chain variable region (V_HFAP) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:54, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:55, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:56, and a light chain variable region (V_LFAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:57, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:58, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:59, or (c) a heavy chain variable region (V_HFAP) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:62, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:63, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:64, and a light chain variable region (V_LFAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:65, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:66, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:67.
In one particular aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the cross-Fab fragment capable of specific binding to FAP comprises a heavy chain variable region (V_HFAP) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:45, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:46, and a light chain variable region (V_LFAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:47, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:48, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:49.
In another aspect, the cross-Fab fragment capable of specific binding to FAP comprises a heavy chain variable region (V_HFAP) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:54, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:55, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:56, and a light chain variable region (V_LFAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:57, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:58, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:59.
In one further aspect, provided is a bispecific antigen binding molecule, wherein the cross-Fab fragment capable of specific binding to FAP comprises

(i) a heavy chain variable region (V_HFAP) comprising the amino acid sequence of SEQ ID NO:50 and a light chain variable region (V_LFAP) comprising the amino acid sequence of SEQ ID NO:51, or
(ii) a heavy chain variable region (V_HFAP) comprising the amino acid sequence of SEQ ID NO:52 and a light chain variable region (V_LFAP) comprising the amino acid sequence of SEQ ID NO:53, or
(iii) a heavy chain variable region (V_HFAP) comprising the amino acid sequence of SEQ ID NO:60 and a light chain variable region (V_LFAP) comprising the amino acid sequence of SEQ ID NO:61, or
(iv) a heavy chain variable region (V_HFAP) comprising the amino acid sequence of SEQ ID NO:68 and a light chain variable region (V_LFAP) comprising the amino acid sequence of SEQ ID NO:69.

In one aspect, the cross-Fab fragment capable of specific binding to FAP comprises a heavy chain variable region (V_HFAP) comprising the amino acid sequence of SEQ ID NO:52, and a light chain variable region (V_LFAP) comprising the amino acid sequence of SEQ ID NO:53. In another aspect, the the cross-Fab fragment capable of specific binding to FAP comprises a heavy chain variable region (V_HFAP) comprising the amino acid sequence of SEQ ID NO:60, and a light chain variable region (V_LFAP) comprising the amino acid sequence of SEQ ID NO:61.
In one aspect, the provided are bispecific antigen binding molecules comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association; wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit;
(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit,
(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the crossfab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5), wherein the first antigen is a TNF receptor. In particular, the first and the second Fab fragment are capable of agonistic binding to a TNF receptor. In one aspect, provided are bispecific antigen binding molecules that are characterized by targeted agonistic binding to a TNF receptor (TNFR). In particular, the bispecific antigen binding molecule is a TNFR agonist that is targeted against FAP. In another particular aspect, the bispecific antigen binding molecules of the invention comprise a Fc region composed of a first and a second subunit capable of stable association which comprises mutations that reduce effector function. The use of an Fc region comprising mutations that reduce or abolish effector function will prevent unspecific agonism by crosslinking via Fc receptors and will prevent ADCC of TNFR expressing cells. The bispecific antigen binding molecules as described herein possess the advantage over conventional antibodies capable of specific binding to TNFR in that they selectively induce immune response at the target cells, which are typically close to the tumor, i.e. in the tumor stroma.

In one aspect, the TNFR is OX40, meaning the Fab fragments capable of specific binding to a first antigen are capable of agonistic binding to a TNF receptor are capable of specific binding to OX40, in particular human OX40. Thus, they bind to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:24.
In a further aspect, provided is a bispecific antigen binding molecule, wherein the Fab fragments capable of specific binding to OX40 comprise (a) a heavy chain variable region (V_HOX40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:71, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:72, and a light chain variable region (V_LOX40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:73, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:74, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:75, or (b) a heavy chain variable region (V_HOX40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:78, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:79, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:80, and a light chain variable region (V_LOX40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:81, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:82, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:83, or (c) a heavy chain variable region (V_HOX40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:86, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:87, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:88, and a light chain variable region (V_LOX40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:89, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:90, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:91, or (d) a heavy chain variable region (V_HOX40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:94, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:95, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:96, and a light chain variable region (V_LOX40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:97, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:98, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:99.
In one aspect, the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:71, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:72, and a light chain variable region (V_LOX40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:73, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:74, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:75. In a further aspect, the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:78, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:79, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:80, and a light chain variable region (V_LOX40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:81, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:82, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:83. In another aspect, the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:86, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:87, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:88, and a light chain variable region (V_LOX40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:89, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:90, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:91. In yet another aspect, the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:94, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:95, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:96, and a light chain variable region (V_LOX40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:97, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:98, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:99. In one particular aspect, the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:78, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:79, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:80, and a light chain variable region (V_LOX40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:81, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:82, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:83.
In one aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the Fab fragments capable of specific binding to OX40 comprise

(i) a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:76 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:77, or
(ii) a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:84 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:85, or
(iii) a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:92 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:93, or
(iv) a heavy chain variable region (VnOX40) comprising the amino acid sequence of SEQ ID NO:100 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:101, or
(v) a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:102 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:77, or
(vi) a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:103 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:77, or
(vii) a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:104 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:77.

In one aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:76 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:77. In another aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:84 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:85. In a further aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:92 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:93. In yet a further aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:100 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:101. In one particular aspect, the the Fab fragments capable of specific binding to OX40 (each) comprise a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:84 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:85.
In another aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:102 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:77. In one aspect, the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:103 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:77. In a further aspect, the Fab fragments capable of specific binding to OX40 comprise a heavy chain variable region (V_HOX40) comprising the amino acid sequence of SEQ ID NO:104 and a light chain variable region (V_LOX40) comprising the amino acid sequence of SEQ ID NO:77.
In one aspect, the TNFR is GITR, meaning the Fab fragments capable of specific binding to a first antigen are capable of agonistic binding to a TNF receptor are capable of specific binding to GITR, in particular human GITR. Thus, they bind to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:26.
In one aspect, provided is a bispecific antigen binding molecule, wherein the Fab fragments capable of specific binding to GITR comprise a heavy chain variable region (V_HGITR) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:107, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:108, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:109, and a light chain variable region (V_LGITR) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:110, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:111, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:112.
In one aspect, provided is a bispecific antigen binding molecule as defined herein before, wherein the Fab fragments capable of specific binding to GITR comprise a heavy chain variable region (V_HGITR) comprising the amino acid sequence of SEQ ID NO:113 and a light chain variable region (V_LGITR) comprising the amino acid sequence of SEQ ID NO:114.

Bispecific Antigen Binding Molecules Binding to OX40 and FAP

In one aspect, provided are bispecific antigen binding molecules comprising a first Fab fragment and a second Fab fragment capable of specific binding to OX40, and a third Fab fragment capable of specific binding to FAP, and an Fc domain composed of a first and a second subunit capable of stable association; wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit;
(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit,
(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the crossfab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).

In one aspect, these bispecific molecules bind bivalently to OX40 and monovalently to FAP.
In one aspect, provided is a bispecific antigen binding molecule comprising

(a) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:135, one light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 129, a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:134, and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:140, or
(b) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:135, one light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 142, a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:134, and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:140, or
(c) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:135, one light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 131, a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:134, and a second heavy chain an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:140.

In one aspect, the invention provides a bispecific antigen binding molecule comprising (a) two light chains, each comprising the amino acid sequence of SEQ ID NO:135, one light chain comprising the amino acid sequence of SEQ ID NO:129, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 134, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:140, or (b) two light chains, each comprising the amino acid sequence of SEQ ID NO:135, one light chain comprising the amino acid sequence of SEQ ID NO:142, a first heavy chain comprising the amino acid sequence of SEQ ID NO:134, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:140, or (c) two light chains, each comprising the amino acid sequence of SEQ ID NO:135, one light chain comprising the amino acid sequence of SEQ ID NO:131, a first heavy chain comprising the amino acid sequence of SEQ ID NO:134, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:140.
In another aspect, the bispecific antigen binding molecules as described herein before bind trivalently to OX40 and monovalently to FAP.
In one aspect, provided is a bispecific antigen binding molecule comprising

(a) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:132, a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 130, three light chains each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:128 and a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:129, or
(b) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:132, a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 130, three light chains each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:128 and a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:131, or
(c) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:134, a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 136, three light chains each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 135 and a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:129, or
(d) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:134, a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 136, three light chains each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:135 and a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:137, or
(e) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:134, a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 136, three light chains each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:135 and a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:131.

In one aspect, provided is a bispecific antigen binding molecule comprising (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO:132, a second heavy chain comprising the amino acid sequence of SEQ ID NO:130, three light chains each comprising the amino acid sequence of SEQ ID NO:128 and a light chain comprising the amino acid sequence of SEQ ID NO:129, or (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO:132, a second heavy chain comprising the amino acid sequence of SEQ ID NO:130, three light chains each comprising the amino acid sequence of SEQ ID NO:128 and a light chain comprising the amino acid sequence of SEQ ID NO:131, or (c) a first heavy chain comprising the amino acid sequence of SEQ ID NO:134, a second heavy chain comprising the amino acid sequence of SEQ ID NO:136, three light chains each comprising the amino acid sequence of SEQ ID NO: 135 and a light chain comprising the amino acid sequence of SEQ ID NO:129, or (d) a first heavy chain comprising the amino acid sequence of SEQ ID NO:134, a second heavy chain comprising the amino acid sequence of SEQ ID NO:136, three light chains each comprising the amino acid sequence of SEQ ID NO:135 and a light chain comprising the amino acid sequence of SEQ ID NO:137, or (e) a first heavy chain comprising the amino acid sequence of SEQ ID NO:134, a second heavy chain comprising the amino acid sequence of SEQ ID NO:136, three light chains each comprising the amino acid sequence of SEQ ID NO:135 and a light chain comprising the amino acid sequence of SEQ ID NO:131.
In yet another aspect, the bispecific antigen binding molecules as described herein before bind tetravalently to OX40 and monovalently to FAP.
In one aspect, provided is a bispecific antigen binding molecule comprising

(a) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:128, one light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 129, a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:127, and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:130, or
(b) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:128, one light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 131, a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:127, and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:130, or
(c) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:119, one light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 129, a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:151, and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:152, or
(d) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:119, one light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 131, a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:151, and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:152.

In one aspect, provided is a bispecific antigen binding molecule comprising (a) four light chains, each comprising the amino acid sequence of SEQ ID NO: 128, one light chain comprising the amino acid sequence of SEQ ID NO:129, a first heavy chain comprising the amino acid sequence of SEQ ID NO:127, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:130, or comprising (b) four light chains, each comprising the amino acid sequence of SEQ ID NO:128, one light chain comprising the amino acid sequence of SEQ ID NO:131, a first heavy chain comprising the amino acid sequence of SEQ ID NO:127, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:130, or (c) four light chains, each comprising the amino acid sequence of SEQ ID NO:119, one light chain comprising the amino acid sequence of SEQ ID NO:129, a first heavy chain comprising the amino acid sequence of SEQ ID NO:151, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:152, or (d) four light chains, each comprising the amino acid sequence of SEQ ID NO:119, one light chain comprising the amino acid sequence of SEQ ID NO:131, a first heavy chain comprising the amino acid sequence of SEQ ID NO:151, and a second heavy chain comprising the amino acid sequence of SEQ ID N0:152.

Bispecific Antigen Binding Molecules Binding to GITR and FAP

In one aspect, provided are bispecific antigen binding molecules comprising a first Fab fragment and a second Fab fragment capable of specific binding to GITR, and a third Fab fragment capable of specific binding to FAP, and an Fc domain composed of a first and a second subunit capable of stable association; wherein

In one aspect, these bispecific molecules bind bivalently to GITR and monovalently to FAP.
In one aspect, the invention provides a bispecific antigen binding molecule comprising

(a) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:154, one light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 156, a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:153, and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:155, or
(b) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:154, one light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:157, a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:153, and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:155.

In one aspect, the invention provides a bispecific antigen binding molecule comprising (a) two light chains, each comprising the amino acid sequence of SEQ ID NO:154, one light chain comprising the amino acid sequence of SEQ ID NO:156, a first heavy chain comprising the amino acid sequence of SEQ ID NO:153, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 155, or (b) two light chains, each comprising the amino acid sequence of SEQ ID NO: 154, one light chain comprising the amino acid sequence of SEQ ID NO:157, a first heavy chain comprising the amino acid sequence of SEQ ID NO:153, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:115.

Fc Domain Modifications Reducing Fc Receptor Binding and/or Effector Function

The bispecific antigen binding molecules described herein further comprise an Fc domain composed of a first and a second subunit capable of stable association.
In certain aspects, one or more amino acid modifications may be introduced into the Fc domain of an antibody provided herein, thereby generating an Fc domain variant. The Fc domain variant may comprise a human Fc domain sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc domain) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
The Fc domain confers favorable pharmacokinetic properties to the bispecific antibodies of the invention, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the bispecific antibodies of the invention to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Accordingly, in particular embodiments the Fc domain of the bispecific antibodies of the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is an IgG1 Fc domain.
In one such aspect the Fc domain (or the bispecific antigen binding molecule comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgG1 Fc domain (or the bispecific antigen binding molecule of the invention comprising a native IgG1 Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgG1 Fc domain (or the bispecific antigen binding molecule of the invention comprising a native IgG1 Fc domain). In one aspect, the Fc domain (or the bispecific antigen binding molecule of the invention comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function. In a particular aspect the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a specific aspect, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In one aspect the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a particular aspect, the effector function is ADCC. In one aspect, the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the the bispecific antigen binding molecule of the invention comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgG1 Fc domain (or the the bispecific antigen binding molecule of the invention comprising a native IgG1 Fc domain) to FcRn.
In a particular aspect, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In a particular aspect, the Fc domain of the bispecific antigen binding molecule of the invention comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In another aspect, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In one aspect, the bispecific antigen binding molecule of the invention comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to bispecific antibodies of the invention comprising a non-engineered Fc domain. In a particular aspect, the Fc receptor is an Fcγ receptor. In other aspects, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a specific aspect, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In some aspects the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some aspects, binding affinity to a complement component, specifically binding affinity to C1q, is also reduced. In one aspect, binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the bispecific antigen binding molecule of the invention comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the bispecific antigen binding molecule of the invention comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or the the bispecific antigen binding molecule of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain of the bispecific antigen binding molecule of the invention is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced dendritic cell maturation, or reduced T cell priming.
Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581). Certain antibody variants with improved or diminished binding to FcRs are described, (e.g. U.S. Patent No. 6,737,056; WO 2004/056312, and Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604).
In one aspect, the Fc domain comprises an amino acid substitution at a position of E233, L234, L235, N297, P331 and P329. In some aspects, the Fc domain comprises the amino acid substitutions L234A and L235A (“LALA”). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D or P331S. In more particular embodiments the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”). The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fcγ receptor binding of a human IgG1 Fc domain, as described in PCT Patent Application No. WO 2012/130831 A1. Said document also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions. Such antibody is an IgG1 with mutations L234A and L235A or with mutations L234A, L235A and P329G (numbering according to EU index of Kabat et al, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
In one aspect, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising amino acid substitutions L235E and S228P and P329G. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).
Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer, R.L. et al., J. Immunol. 117 (1976) 587-593, and Kim, J.K. et al., J. Immunol. 24 (1994) 2429-2434), are described in US 2005/0014934. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. A suitable such binding assay is described herein. Alternatively, binding affinity of Fc domains or cell activating bispecific antigen binding molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing FcγIIIa receptor. Effector function of an Fc domain, or bispecific antigen binding molecules of the invention comprising an Fc domain, can be measured by methods known in the art. A suitable assay for measuring ADCC is described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).
The following section describes preferred aspects of the bispecific antigen binding molecules of the invention comprising Fc domain modifications reducing Fc receptor binding and/or effector function. In one aspect, the invention relates to a bispecific antigen binding molecule, comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association; wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit;
(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit,
(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the crossfab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5), and wherein the Fc domain comprises one or more amino acid substitution that reduces effector function. In particular aspect, the Fc domain is of human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).

Fc Domain Modifications Promoting Heterodimerization

The bispecific antigen binding molecules described herein comprise different antigen-binding sites, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain may be comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antigen binding molecules of the invention in recombinant production, it will thus be advantageous to introduce in the Fc domain of the bispecific antigen binding molecules of the invention a modification promoting the association of the desired polypeptides.
Accordingly, in particular aspects provided is a bispecific antigen binding molecule, comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association; wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit;
(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit,
(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the crossfab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5), wherein the Fc domain comprises a modification promoting the association of the first and second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect said modification is in the CH3 domain of the Fc domain.

In a specific aspect said modification is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain. Thus, the invention relates to the bispecific antigen binding molecule comprising (a) at least two antigen binding domains capable of specific binding to OX40, (b) an antigen binding domain capable of specific binding to FAP, and (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein the first subunit of the Fc domain comprises knobs and the second subunit of the Fc domain comprises holes according to the knobs into holes method. In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).
The knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
Accordingly, in one aspect, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen binding molecules described herein an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. In a specific aspect, in the CH3 domain of the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one aspect, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).
In yet a further aspect, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).
In an alternative aspect, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
The C-terminus of the heavy chain of the bispecific antigen binding molecule as reported herein can be a complete C-terminus ending with the amino acid residues PGK. The C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C terminal amino acid residues have been removed. In one preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending PG. In one aspect of all aspects as reported herein, a bispecific antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein, comprises the C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index). In one aspect of all aspects as reported herein, a bispecific antibody comprising a heavy chain including a C-terminal CH3 domain, as specified herein, comprises a C-terminal glycine residue (G446, numbering according to Kabat EU index).

Modifications in the Fab Domains

In one aspect, provided is a bispecific antigen binding molecule, comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association; wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit;
(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit,
(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the crossfab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5). Thus, in one aspect, the invention comprises a bispecific antigen binding molecule, comprising (a) two light chains and two heavy chains of an antibody comprising two Fab fragments capable of specific binding to a first antigen and the Fc region, and (b) a cross-Fab fragment capable of specific binding to a second antigen fused to the C-terminus of one of subunits of the Fc region. The bispecific antigen binding molecule is prepared according to the Crossmab technology.

Multispecific antibodies with a domain replacement/exchange in one binding arm (CrossMab VH-VL or CrossMab CH-CL) are described in detail in WO2009/080252 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191. They clearly reduce the byproducts caused by the mismatch of a light chain against a first antigen with the wrong heavy chain against the second antigen (compared to approaches without such domain exchange).
In another aspect, and to further improve correct pairing, the bispecific antigen binding molecule comprising (a) at least two Fab fragments capable of specific binding to a first antigen, (b) a crossFab fragment capable of specific binding to a second antigen, and (c) a Fc domain composed of a first and a second subunit capable of stable association, can contain different charged amino acid substitutions (so-called “charged residues”). These modifications are introduced in the crossed or non-crossed CH1 and CL domains. In a particular aspect, the invention relates to a bispecific antigen binding molecule, wherein in one of CL domains the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and/or wherein the amino acid at position 124 (EU numbering) has been substituted by lysine (K) and wherein in one of the CH1 domains the amino acids at position 147 (EU numbering) and/or at position 213 (EU numbering) have been substituted by glutamic acid (E).

Polynucleotides

The invention further provides isolated nucleic acid encoding a bispecific antigen binding molecule as described herein or a fragment thereof.
The isolated polynucleotides encoding bispecific antigen binding molecules as disclosed herein may be expressed as a single polynucleotide that encodes the entire antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional antigen binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a separate polynucleotide from the heavy chain portion of the immunoglobulin. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the immunoglobulin.
In some aspects, the isolated polynucleotide encodes a polypeptide comprised in the bispecific molecule according to the invention as described herein.
In certain embodiments the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). RNA of the present invention may be single stranded or double stranded.

Recombinant Methods

Bispecific antigen binding molecules of the invention may be obtained, for example, by recombinant production. For recombinant production one or more polynucleotide encoding the bispecific antigen binding molecule or polypeptide fragments thereof are provided. The one or more polynucleotide encoding the bispecific antigen binding molecule are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one aspect of the invention, a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the bispecific antigen binding molecule (fragment) along with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the bispecific antigen binding molecule or polypeptide fragments thereof (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements. As used herein, a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5′ and 3′ untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the bispecific antigen binding molecule of the invention or polypeptide fragments thereof, or variants or derivatives thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit â-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).
Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if secretion of the bispecific antigen binding molecule or polypeptide fragments thereof is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the bispecific antigen binding molecule of the invention or polypeptide fragments thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, the native signal peptide, e.g. an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse P-glucuronidase.
DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the fusion protein may be included within or at the ends of the polynucleotide encoding a bispecific antigen binding molecule of the invention or polypeptide fragments thereof.
In a further aspect of the invention, a host cell comprising one or more polynucleotides of the invention is provided. In certain aspects, a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one aspect, a host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) a bispecific antigen binding molecule of the invention of the invention. As used herein, the term “host cell” refers to any kind of cellular system which can be engineered to generate the fusion proteins of the invention or fragments thereof. Host cells suitable for replicating and for supporting expression of antigen binding molecules are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the antigen binding molecule for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006).
Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). Standard technologies are known in the art to express foreign genes in these systems. Cells expressing a polypeptide comprising either the heavy or the light chain of an immunoglobulin, may be engineered so as to also express the other of the immunoglobulin chains such that the expressed product is an immunoglobulin that has both a heavy and a light chain.
In one aspect, a method of producing a bispecific antigen binding molecule of the invention or polypeptide fragments thereof is provided, wherein the method comprises culturing a host cell comprising polynucleotides encoding the bispecific antigen binding molecule of the invention or polypeptide fragments thereof, as provided herein, under conditions suitable for expression of the bispecific antigen binding molecule of the invention or polypeptide fragments thereof, and recovering the bispecific antigen binding molecule of the invention or polypeptide fragments thereof from the host cell (or host cell culture medium).
Bispecific molecules of the invention prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the bispecific antigen binding molecule binds. For example, for affinity chromatography purification of fusion proteins of the invention, a matrix with protein A or protein G may be used. Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate an antigen binding molecule essentially as described in the examples. The purity of the bispecific antigen binding molecule or fragments thereof can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like. For example, the bispecific antigen binding molecules expressed as described in the Examples were shown to be intact and properly assembled as demonstrated by reducing and non-reducing SDS-PAGE.

Assays

The antigen binding molecules provided herein may be characterized for their binding properties and/or biological activity by various assays known in the art. In particular, they are characterized by the assays described in more detail in the examples.

1. Binding Assay

Binding of the bispecific antigen binding molecule provided herein to the corresponding target expressing cells may be evaluated for example by using a murine fibroblast cell line expressing human Fibroblast Activation Protein (FAP) and flow cytometry (FACS) analysis. Binding of the bispecific antigen binding molecules provided herein to OX40 or GITR may be determined by using activated human PBMCs as described in Example 2.1 or Example 4.2.2.

2. Activity Assays

Bispecific antigen binding molecules of the invention are tested for biological activity. Biological activity may include efficacy and specificity of the bispecific antigen binding molecules. Efficacy and specificity are demonstrated by assays showing agonistic signaling through the OX40 receptor upon binding of the target antigen. Furthermore, the stimulation of OX40 signaling is measured through induced NFκB activation in human OX40 positive NFκB reporter cells as described in Example 3.1. The stimulation of GITR signaling is also measured through a NFκB -Jurkat Reporter cell assay as described in Example 4.3. The assay for evaluating preexisting Anti-Drug Antibody (ADA) Reactivity is described in Example 5.

Pharmaceutical Compositions, Formulations and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositions comprising any of the bispecific antigen binding molecules provided herein, e.g., for use in any of the below therapeutic methods. In one aspect, a pharmaceutical composition comprises any of the bispecific antigen binding molecules provided herein and at least one pharmaceutically acceptable excipient. In another aspect, a pharmaceutical composition comprises any of the bispecific antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.
Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more bispecific antigen binding molecules dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one bispecific antigen binding molecule according to the invention and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. In particular, the compositions are lyophilized formulations or aqueous solutions. As used herein, “pharmaceutically acceptable excipient” includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers and combinations thereof, as would be known to one of ordinary skill in the art.
Parenteral compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intra-lesional, intravenous, intra-arterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the bispecific antigen binding molecules of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks’ solution, Ringer’s solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the bispecific antigen binding molecules may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antigen binding molecules of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng/mg protein. Suitable pharmaceutically acceptable excipients include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.
Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
In addition to the compositions described previously, the antigen binding molecules may also be lyophilized or formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the fusion proteins may be formulated with suitable polymeric or hydrophobic materials (for example as emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
Pharmaceutical compositions comprising the bispecific antigen binding molecules of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
The bispecific antigen binding molecules described herein may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g. those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
The composition may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

Therapeutic Methods and Compositions

Any of the bispecific antigen binding molecules provided herein may be used in therapeutic methods. For use in therapeutic methods, bispecific antigen binding molecules of the invention can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
In one aspect, bispecific antigen binding molecules of the invention for use as a medicament are provided.
In further aspects, bispecific antigen binding molecules described herein for use (i) in inducing immune stimulation, (ii) in stimulating tumor-specific T cell response, (iii) in causing apoptosis of tumor cells, (iv) in the treatment of cancer, (v) in delaying progression of cancer, (vi) in prolonging the survival of a patient suffering from cancer, (vii) in the treatment of infections are provided. In a particular aspect, bispecific antigen binding molecules for use in treating a disease, in particular for use in the treatment of cancer, are provided.
In certain aspects, bispecific antigen binding molecules as defined herein for use in a method of treatment are provided. In one aspect, provided is a bispecific antigen binding molecule as described herein for use in the treatment of a disease in an individual in need thereof. In certain aspects, the invention provides a bispecific antigen binding molecule for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the bispecific antigen binding molecule. In certain aspects the disease to be treated is cancer. The subject, patient, or “individual” in need of treatment is typically a mammal, more specifically a human.
In one aspect, provided is a method for i) inducing immune stimulation, (ii) stimulating tumor-specific T cell response, (iii) causing apoptosis of tumor cells, (iv) treating of cancer, (v) delaying progression of cancer, (vi) prolonging the survival of a patient suffering from cancer, or (vii) treating of infections, wherein the method comprises administering a therapeutically effective amount of the bispecific antigen binding molecule as described herein to an individual in need thereof.
In a further aspect, the bispecific antigen binding molecule as disclosed herein is provided for the use in the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof. In one aspect, the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain aspects, the disease to be treated is a proliferative disorder, particularly cancer. Examples of cancers include, but are not limited to, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other examples of cancer include carcinoma, lymphoma (e.g., Hodgkin’s and non-Hodgkin’s lymphoma), blastoma, sarcoma, and leukemia. Other cell proliferation disorders that can be treated using the bispecific antigen binding molecule or antibody of the invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. A skilled artisan readily recognizes that in many cases the the bispecific antigen binding molecule or antibody of the invention may not provide a cure but may provide a benefit. In some aspects, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some aspects, an amount of the bispecific antigen binding molecule or antibody of the invention that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount”.
For the prevention or treatment of disease, the appropriate dosage of a bispecific antigen binding molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the specific molecule, the severity and course of the disease, whether the bispecific antigen binding molecule of the invention is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient’s clinical history and response to the bispecific antigen binding molecule, and the discretion of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
The bispecific antigen binding molecule as disclosed herein will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the bispecific antigen binding molecule of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC₅₀ as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
Dosage amount and interval may be adjusted individually to provide plasma levels of the bispecific antigen binding molecule of the invention which are sufficient to maintain therapeutic effect. Therapeutically effective plasma levels may be achieved by administering multiple doses. Levels in plasma may be measured, for example, by HPLC. In cases of local administration or selective uptake, the effective local concentration of the bispecific antigen binding molecule or antibody of the invention may not be related to plasma concentration. One skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
A therapeutically effective dose of the bispecific antigen binding molecule described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of a fusion protein can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD₅₀ (the dose lethal to 50% of a population) and the ED₅₀ (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. Bispecific antigen binding molecules that exhibit large therapeutic indices are preferred. In one aspect, the the bispecific antigen binding molecule exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient’s condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).
The attending physician for patients treated with fusion proteins of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle). At least one active agent in the composition is a bispecific antigen binding molecule as described herein.
The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a bispecific antigen binding molecule as described herein; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

TABLE B

Sequences
SEQ ID NO:	Name	Sequence
1	CH1 connector C-terminal end S-variant	EPKSCS
2	CH1 connector C-terminal end G-variant	EPKSCG
3	CH1 connector C-terminal end D-variant	EPKSCD
4	CH1 connector C-terminal end DK-variant	EPKSCDK
5	CH1 connector C-terminal end DKTHL-variant	EPKSCDKTHL
6	CH1 connector C-terminal end	EPKSC
7	upper hinge region	EPKSCDKTHT
8	core hinge region	CPXCP with X being S or P
9	lower hinge region IgG1	APELLGGP
10	lower hinge region IgG4	APEFLGGP
11	hu FAP	UniProt no. Q12884, version 168 MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPWRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD
12	hu FAP ectodomain+poly-lys-tag+his₆-tag	RPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH
13	Murine FAP	UniProt no. P97321
14	Murine FAP ectodomain+poly-lys-tag+his₆-tag	RPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFRCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGILSGRSQNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH
15	Cynomolgus FAP ectodomain+poly-lys-tag+his₆-tag	RPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH
16	CH1 domain	ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
17	CH2 domain	APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQESTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
18	CH3 domain	GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
19	Fc IgG1, caucasian allotype	ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
20	Fc IgG1, afroamerican allotype	ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
21	Fc IgG2	ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
22	Fc IgG3	ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
23	Fc IgG4	ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
24	hu OX40	Uniprot No. P43489, aa 29-214 LH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ NTVCRPCGPGFYNDWSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI
25	Murine OX40	UniProt no. P47741, version 143 MYVWVQQPTALLLLALTLGVTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDTLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPAFAVLLGLGLGLLAPLTVLLALYLLRKAWRLPNTPKPCWGNSFRTPIQEEHTDAHFTLAKI
26	hu GITR	Uniprot no. Q9Y5U5 MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV
27	Cynomolgus GITR	Uniprot no. A0A2K5WP38 VARHGAMCACGTLCCLALLCAASLGQRPTGGPGCGPGRLLLGTGKDARCCRVHPTRCCRDYQSEECCSEWDCVCVQPEFHCGNPCCTTCQHHPCPSGQGVQPQGKFSFGFRCVDCALGTFSRGHDGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPPGWLTIVLLAVAACVLLLTSAQLGLHIWQLGSQPTGPRETQLLLEVPPSTEDASSCQFPEEERGERLAEEKGRLGDLWV
28	hu 4-1BB	Uniprot no. Q07011 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
29	Peptide linker (G4S)	GGGGS
30	Peptide linker (G4S)₂	GGGGSGGGGS
31	Peptide linker (SG4)₂	SGGGGSGGGG
32	Peptide linker G4(SG4)₂	GGGGSGGGGSGGGG
33	peptide linker	GSPGSSSSGS
34	(G4S)₃ peptide linker	GGGGSGGGGSGGGGS
35	(G4S)₄ peptide linker	GGGGSGGGGSGGGGSGGGGS
36	peptide linker	GSGSGSGS
37	peptide linker	GSGSGNGS
38	peptide linker	GGSGSGSG
39	peptide linker	GGSGSG
40	peptide linker	GGSG
41	peptide linker	GGSGNGSG
42	peptide linker	GGNGSGSG
43	peptide linker	GGNGSG
44	FAP (212) CDR-H1	DYNMD
45	FAP (212) CDR-H2	DIYPNTGGTIYNQKFKG
46	FAP (212) CDR-H3	FRGIHYAMDY
47	FAP (212) CDR-L1	RASESVDNYGLSFIN
48	FAP (212) CDR-L2	GTSNRGS
49	FAP (212) CDR-L3	QQSNEVPYT
50	FAP (212) VH	EVLLQQSGPELVKPGASVKIACKASGYTLTDYNMDWVRQSHGKSLEWIGDIYPNTGGTIYNQKFKGKATLTIDKSSSTAYMDLRSLTSEDTAVYYCTRFRGIHYAMDYWGQGTSVTVSS
51	FAP (212) VL	DIVLTQSPVSLAVSLGQRATISCRASESVDNYGLSFINWFQQKPGQPPKLLIYGTSNRGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSNEVPYTFGGGTNLEIK
52	FAP (1G1a) VH	QVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSS
53	FAP (1G1a) VL	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIK
54	FAP(4B9) CDR-H1	SYAMS
55	FAP(4B9) CDR-H2	AIIGSGASTYYADSVKG
56	FAP(4B9) CDR-H3	GWFGGFNY
57	FAP(4B9) CDR-L1	RASQSVTSSYLA
58	FAP(4B9) CDR-L2	VGSRRAT
59	FAP(4B9) CDR-L3	QQGIMLPPT
60	FAP(4B9) VH	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS
61	FAP(4B9) VL	EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIK
62	FAP (28H1) CDR-H1	SHAMS
63	FAP (28H1) CDR-H2	AIWASGEQYYADSVKG
64	FAP (28H1) CDR-H3	GWLGNFDY
65	FAP (28H1) CDR-L1	RASQSVSRSYLA
66	FAP (28H1) CDR-L2	GASTRAT
67	FAP (28H1) CDR-L3	QQGQVIPPT
68	FAP(28H1) VH	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS
69	FAP(28H1) VL	EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIK
70	OX40 (49B4) CDR-H1	SYAIS
71	OX40 (49B4) CDR-H2	GIIPIFGTANYAQKFQG
72	OX40 (49B4) CDR-H3	EYYRGPYDY
73	OX40 (49B4) CDR-L1	RASQSISSWLA
74	OX40 (49B4) CDR-L2	DASSLES
75	OX40 (49B4) CDR-L3	QQYSSQPYT
76	OX40 (49B4) VH	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSS
77	OX40 (49B4) VL	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIK
78	OX40 (CLC563) CDR-H1	SYAMS
79	OX40 (CLC563) CDR-H2	AISGSGGSTYYADSVKG
80	OX40 (CLC563) CDR-H3	DVGAFDY
81	OX40 (CLC563) CDR-L1	RASQSVSSSYLA
82	OX40 (CLC563) CDR-L2	GASSRAT
83	OX40 (CLC563) CDR-L3	QQYGSSPLT
84	OX40 (CLC563) VH	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSS
85	OX40 (CLC563) VL	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK
86	OX40 (MOXR0916) CDR-H1	DSYMS
87	OX40 (MOXR0916) CDR-H2	DMYPDNGDSSYNQKFRE
88	OX40 (MOXR0916) CDR-H3	APRWYFSV
89	OX40 (MOXR0916) CDR-L1	RASQDISNYLN
90	OX40 (MOXR0916) CDR-L2	YTSRLRS
91	OX40 (MOXR0916) CDR-L3	QQGHTLPPT
92	OX40 (MOXR0916) VH	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS
93	OX40 (MOXR0916) VL	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIK
94	OX40 (8H9) CDR-H1	SYAIS
95	OX40 (8H9) CDR-H2	GIIPIFGTANYAQKFQG
96	OX40 (8H9) CDR-H3	EYGWMDY
97	OX40 (8H9) CDR-L1	RASQSISSWLA
98	OX40 (8H9) CDR-L2	DASSLES
99	OX40 (8H9) CDR-L3	QQYLTYSRFT
100	OX40 (8H9) VH	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSS
101	OX40 (8H9) VL	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYLTYSRFTFGQGTKVEIK
102	OX40 (49B4_K73E) VH	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSS
103	OX40 (49B4_K23T_K73E) VH	QVQLVQSGAEVKKPGSSVKVSCTASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSS
104	OX40 (49B4_K23E_K73E) VH	QVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSS
105	Fc knob chain	DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
106	Fc hole chain	DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
107	GITR (852.2) CDR-H1	SYTMK
108	GITR (852.2) CDR-H2	VISADGGTTDHAASVKG
109	GITR (852.2) CDR-H3	DRANDWITGASFDY
110	GITR (852.2) CDR-L1	RASQSINSYLN
111	GITR (852.2) CDR-L2	ASSLQS
112	GITR (852.2) CDR-L3	QQARSFPLT
113	GITR (852.2) VH	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMKWVRQTPGKGLEWVSVISADGGTTDHAASVKGRFTVSRDNSKNMLYLQMNSLRAEDTAIYYCAKDRANDWITGASFDYWGQGALVTVSS
114	GITR (852.2) VL	DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGNAPKLLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDLATYYCQQARSFPLTFGGGTKVEIK
115	OX40(49B4) VHCH1-OX40(49B4) VHCH1- Fc hole_PGLALA - FAP(4B9) VL	See Table 1
116	OX40(49B4) VHCH1-OX40(49B4) VHCH1- Fc knob_PGLALA - FAP(4B9) VH	See Table 1
117	OX40 (49B4) VLCkappa	See Table 1
118	OX40(49B4) VHCH1-OX40(49B4) VHCH1- Fc knob_PGLALA- FAP(4B9) VHCL	See Table 1
119	OX40(49B4) light chain	See Table 1
120	FAP(4B9) VLCH1-light chain	See Table 1
121	OX40(49B4) VHCH1-OX40(49B4) VHCH1-Fc hole_PGLALA	See Table 1
122	OX40(49B4) VHCH1-OX40(49B4) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	See Table 1
123	FAP(1G1a) VLCH1-light chain	See Table 1
124	OX40(49B4) VHCH1-OX40(49B4) VHCH1-Fc hole_PGLALA	See Table 1
125	OX40(49B4) VHCH1- Fc hole_PGLALA	See Table 1
126	OX40(49B4) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	See Table 1
127	OX40(CLC563) VHCH1-OX40(CLC563) VHCH1-Fc knob_PGLALA-FAP(1G1a) VHCL	See Table 1
128	OX40(CLC563) light chain	See Table 1
129	FAP(1G1a) VLCH1-light chain (EPKSCD)	See Table 1
130	OX40(CLC563) VHCH1-OX40(CLC563) VHCH1-Fc hole_PGLALA	See Table 1
131	FAP(1G1a) VLCH1-light chain (EPKSCS)	See Table 1
132	OX40(CLC563) VHCH1-Fc knob_PGLALA-FAP(1G1a) VHCL	See Table 1
133	OX40(CLC563) VHCH1 Fc hole_PGLALA	See Table 1
134	OX40(MOXR0916) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	See Table 1
135	OX40(MOXR0916) light chain	See Table 1
136	OX40(MOXR0916) VHCH1-OX40(MOXR0916) VHCH1- Fc hole_PGLALA-	See Table 1
137	FAP(1G1a) VLCH1-light chain (EPKSCDK)	See Table 1
138	FAP(1G1a) VLCH1-light chain (EPKSCDKT)	See Table 1
139	FAP(1G1a) VLCH1-light chain (EPKSCDKTH)	See Table 1
140	OX40(MOXR0916) VHCH1- Fc hole_PGLALA	See Table 1
141	FAP(1G1a) VLCH1-light chain (EPKSCDKTHT)	See Table 1
142	FAP(1G1a) VLCH1-light chain (EPKSCDKTHL)	See Table 1
143	OX40(8H9) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	See Table 1
144	OX40(8H9) light chain	See Table 1
145	OX40(8H9) VHCH1-OX40(8H9) VHCH1- Fc hole_PGLALA	See Table 1
146	OX40(8H9) VHCH1- Fc hole_PGLALA	See Table 1
147	OX40(49B4_K73E) VHCH1-OX40(49B4_K73E) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	See Table 3
148	OX40(49B4_K73E) VHCH1-OX40(49B4_K73E) VHCH1-Fc hole_PGLALA	See Table 3
149	OX40(49B4_K23T_K73E) VHCH1-OX40(49B4_K23T_K73E) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	See Table 3
150	OX40(49B4_K23T_K73E) VHCH1-OX40(49B4_K23T_K73E) VHCH1-Fc hole_PGLALA	See Table 3
151	OX40(49B4_K23E_K73E) VHCH1-OX40(49B4_K23E_K73E) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	See Table 3
152	OX40(49B4_K23E_K73E) VHCH1-OX40(49B4_K23E_K73E) VHCH1-Fc hole_PGLALA	See Table 3
153	GITR(852.2) VHCH1- Fc knob_PGLALA- FAP(4B9) VHCL	See Table 14
154	GITR(852.2) light chain	See Table 14
155	GITR(852.2) VHCH1- Fc hole_PGLALA	See Table 14
156	FAP(4B9) VLCH1-light chain (EPKSCS)	See Table 14
157	FAP(4B9) VLCH1-light chain (EPKSCG)	See Table 14
158	CH1-EPKSCS	SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCS
159	CH1-EPKSCG	SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCG
160	CH1-EPKSCD	SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD
161	CH1-EPKSCDK	SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
162	CH1-EPKSCDKTHL	SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL

EXAMPLES

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer’s instructions. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments were either generated by PCR using appropriate templates or were synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. In cases where no exact gene sequence was available, oligonucleotide primers were designed based on sequences from closest homologues and the genes were isolated by RT-PCR from RNA originating from the appropriate tissue. The gene segments flanked by singular restriction endonuclease cleavage sites were cloned into standard cloning / sequencing vectors. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments were designed with suitable restriction sites to allow sub-cloning into the respective expression vectors. All constructs were designed with a 5′-end DNA sequence coding for a leader peptide which targets proteins for secretion in eukaryotic cells.

Protein Purification

Proteins were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a Protein A Sepharose column (GE healthcare) and washed with PBS. Elution of antibodies was achieved at pH 3.0 followed by immediate neutralization of the sample. Aggregated protein was separated from monomeric antibodies by ion exchange chromatography (Poros XS) with equilibration buffer 20 mM His, pH 5.5, 1.47 mS/cm and elution buffer 20 mM His, 500 mM NaCl, pH 5.5, 49.1 mS/cm, (gradient: to 100% elution buffer in 60 CV). In some cases a size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine, 140 mM NaCl pH 6.0 was subsequently performed. Monomeric antibody fractions were pooled, concentrated (if required) using e.g., a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at -20° C. or -80° C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-Page

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer’s instruction. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels) running buffer was used.

CE-SDS

Purity, antibody integrity and molecular weight of bispecific and control antibodies were analyzed by CE-SDS using microfluidic Labchip technology (Caliper Life Science, USA). 5 µl of protein solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according manufacturer’s instructions and analysed on LabChip GXII system using a HT Protein Express Chip. Data were analyzed using LabChip GX Software version 3.0.618.0.

Analytical Size Exclusion Chromatography

The aggregate content of the molecule was analyzed using a BioSuite 250 5 µm. 7.8×300 analytical size-exclusion column (Tosoh) in 200 mM K-Phosophat 250 mM KCl pH 6.2 running buffer at 25° C.

Mass Spectrometry

This section describes the characterization of the multispecific antibodies with VH/VL or CH/CL exchange (CrossMabs) with emphasis on their correct assembly. The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact CrossMabs and deglycosylated/FabALACTICA or alternatively deglycosylated/GingisKHAN digested CrossMabs.
The CrossMabs were deglycosylated with N-Glycosidase F in a phosphate or Tris buffer at 37° C. for up to 17 h at a protein concentration of 1 mg/ml. The FabALACTICA or GingisKHAN (Genovis AB; Sweden) digestions were performed in the buffers supplied by the vendor with 100 µg deglycosylated CrossMabs. Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

Example 1

Generation and Production of Bispecific Antigen Binding Molecules Targeting OX40 and Fibroblast Activation Protein (FAP)

1.1 Generation of Bispecific Antigen Binding Molecules Targeting OX40 and Fibroblast activation protein (FAP)

The cDNAs encoding variable heavy and light chain domains of anti OX40 antibodies (clones 49B4, 8H9 and CLC563 as described in WO 2017/055398 A2, or MOXR0916 as described in WO 2015/153513 A1) as well as anti-FAP antibodies 1G1a (clone 212 as described in WO 2020/070041 A1) or 4B9 (as described in WO 2012/020006 A1) were cloned in frame with the corresponding constant heavy or light chains of human IgG1 in suitable expression plasmids. Expression of heavy and light chain is driven by a chimeric MPSV promoter consisting of the MPSV core promoter and a CMV enhancer element. The expression cassette also contains a synthetic polyA signal at the 3′ end of the cDNAs. In addition the plasmid vectors harbor an origin of replication (EBV OriP) for episomal maintenance of the plasmids.
Bispecific OX40-FAP antibodies were prepared in 2+1, 3+1 and 4+1 format consisting of two, three or four OX40 binding moieties combined with one FAP binding arm at the C-terminus of an Fc (FIGS. 1A to 1 c ). To generate the 2+1, 3+1 and the 4+1 antigen binding molecules the knob-into-hole technology was used to achieve heterodimerization. The S354C/T366W mutations were introduced in the first heavy chain HC1 (Fc knob heavy chain) and the Y349C/T366S/L368A/Y407V mutations were introduced in the second heavy chain HC2 (Fc hole heavy chain). In the 2+1, 3+1 and 4+1 antigen binding molecules the CrossMab technology as described in WO 2010/145792 A1 ensured correct light chain pairing. Independent of the bispecific format, in all cases an effector silent Fc domain (P329G; L234A, L235A) was used to abrogate binding to Fcγ receptors according to the method described in WO 2012/130831 A1. Sequences of the bispecific molecules are shown in Table 1 below.
All genes were transiently expressed under control of a chimeric MPSV promoter consisting of the MPSV core promoter combined with the CMV promoter enhancer fragment. The expression vector also contained the oriP region for episomal replication in EBNA (Epstein Barr Virus Nuclear Antigen) containing host cells.

TABLE 1

Amino acid sequences of the bispecific antigen binding molecules
Molecule	Sequence	Seq ID No
P1AD4524 OX40 (49B4) x FAP (4B9) (4+1) C-terminal VH/VL fusion
OX40(49B4) VHCH1-OX40(49B4) VHCH1- Fc hole_PGLALA -FAP(4B9) VL	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIK	115
OX40(49B4) VHCH1-OX40(49B4) VHCH1- Fc knob_PGLALA - FAP(4B9) VH	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS	116
OX40 (49B4) VLCkappa	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	117

P1AE6836 OX40 (49B4) x FAP (4B9) (4+1) C-terminal crossfab fusion
OX40(49B4) VHCH1-OX40(49B4) VHCH1- Fc knob_PGLALA-FAP(4B9) VHCL	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	118
OX40(49B4) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	119
FAP(4B9) VLCH1-light chain	EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	120
OX40(49B4) VHCH1-OX40(49B4) VHCH1-Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	121

P1AE6838 OX40 (49B4) x FAP (1G1a) (4+1) C-terminal crossfab fusion
OX40(49B4) VHCH1-OX40(49B4) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	122
OX40(49B4) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	119
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40 (49B4) VHCH1-OX40 (49B4) VHCH1-Fc hole PGLALA	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	124

P1AE8786 OX40(49B4) x FAP (1G1a) (3+1) C-terminal crossfab fusion
OX40(49B4) VHCH1-OX40(49B4) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	122
OX40(49B4) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	119
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(49B4) VHCH1- Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	125

P1AE6840 OX40(49B4) x FAP (1G1a) (2+1) C-terminal crossfab fusion
OX40(49B4) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	126
OX40(49B4) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	119
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(49B4) VHCH1- Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	125

P1AF7205 OX40(CLC563) x FAP (1G1a_EPKSCD) (4+1) C-terminal crossfab fusion
OX40(CLC563) VHCH1-OX40(CLC563) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	127
OX40(CLC563) light chain	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	128
FAP(1G1a) VLCH1-light chain (EPKSCD)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD	129
OX40(CLC563) VHCH1-OX40(CLC563) VHCH1-Fc hole_PGLALA	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	130

P1AF7217 OX40 (CLC563) x FAP (1G1a_EPKSCS) (4+1) C-terminal crossfab fusion
OX40(CLC563) VHCH1-OX40(CLC563) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	127
OX40(CLC563) light chain	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	128
FAP(1G1a) VLCH1-light chain (EPKSCS)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCS	131
OX40(CLC563) VHCH1-OX40(CLC563) VHCH1-Fc hole_PGLALA	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	130

P1AE8874 OX40 (CLC563) x FAP (1G1a) (3+1) C-terminal crossfab fusion
OX40(CLC563) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	132
OX40(CLC563) light chain	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	128
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(CLC563) VHCH1-OX40(CLC563) VHCH1-Fc hole_PGLALA	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	130

P1AF6454 OX40 (CLC563) x FAP (1G1a_EPKSCD) (3+1) C-terminal crossfab fusion
OX40(CLC563) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	132
OX40(CLC563) light chain	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	128
FAP(1G1a) VLCH1-light chain (EPKSCD)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD	129
OX40(CLC563) VHCH1- OX40(CLC563) VHCH1-Fc hole_PGLALA	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNAKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	130

P1AF6454 OX40 (CLC563) x FAP (1G1a_EPKSCD) (3+1) C-terminal crossfab fusion
OX40(CLC563) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	132
OX40(CLC563) light chain	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	128
FAP(1G1a) VLCH1-light chain (EPKSCS)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCS	131
OX40(CLC563) VHCH1-OX40(CLC563) VHCH1-Fc hole_PGLALA	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	130

P1AE8871 OX40 (CLC563) x FAP (1G1a) (2+1) C-terminal crossfab fusion
OX40(CLC563) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGOSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGAKTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	132
OX40(CLC563) light chain	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	128
FAP(1G1a) VLCH1-light chain)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(CLC563) VHCH1 Fc hole_PGLALA	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEQVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDOAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	133

P1AE8871 OX40 (CLC563) x FAP (1G1a) (2+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(MOXR09 16) VHCH1-OX40(MOXR09 16) VHCH1- Fc hole_PGLALA-	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVL1QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	136

P1AF4845 OX40 (MOXR0916) x FAP (1G1a_EPKSCD) (3+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCD)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD	129
OX40(MOXR09 16) VHCH1-OX40(MOXR09 16) VHCH1- Fc hole_PGLALA-	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	136

P1AF4846 OX40 (MOXR0916) x FAP (1G1a_EPKSCDK) (3+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCDK)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK	137
OX40(MOXR09 16) VHCH1-OX40(MOXR09 16) VHCH1- Fc hole_PGLALA-	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	136

P1AF4847 OX40 (MOXR0916) x FAP (1G1a_EPKSCDKT) (3+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCDKT)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT	138
OX40(MOXR09 16) VHCH1-OX40(MOXR09 16) VHCH1- Fc hole_PGLALA-	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	136

P1AF4848 OX40 (MOXR0916) x FAP (1G1a_EPKSCDKTH) (3+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCDKTH)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH	139
OX40(MOXR09 16) VHCH1-OX40(MOXR09 16) VHCH1- Fc hole_PGLALA-	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	136

P1AF4851 OX40 (MOXR0916) x FAP (1G1a_EPKSCS) (3+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCS)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCS	131
OX40(MOXR09 16) VHCH1-OX40(MOXR09 16) VHCH1- Fc hole_PGLALA-	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGEVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	136

P1AE8872 OX40 (MOXR0916) x FAP (1G1a) (2+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(MOXR09 16) VHCH1- Fc hole_PGLALA	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	140

P1AF4852 OX40 (MOXR0916) x FAP (1G1a_EPKSCD) (2+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCD)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD	129
OX40(MOXR09 16) VHCH1- Fc hole_PGLALA	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	140

P1AF4855 OX40 (MOXR0916) x FAP (1G1a_EPKSCDKTH) (2+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCDKTH)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH	139
OX40(MOXR09 16) VHCH1- Fc hole_PGLALA	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	140

P1AF4856 OX40 (MOXR0916) x FAP (1G1a_EPKSCDKTHT) (2+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCDKTH T)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT	141
OX40(MOXR09 16) VHCH1- Fc hole_PGLALA	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	140

P1AF4857 OX40 (MOXR0916) x FAP (1G1a_EPKSCDKTHL) (2+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCDKTH L)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL	142
OX40(MOXR09 16) VHCH1- Fc hole_PGLALA	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	140

P1AF4858 OX40 (MOXR0916) x FAP (1G1a_EPKSCS) (2+1) C-terminal crossfab fusion
OX40(MOXR09 16) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	134
OX40(MOXR09 16) light chain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	135
FAP(1G1a) VLCH1-light chain (EPKSCS)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCS	131
OX40(MOXR09 16) VHCH1- Fc hole_PGLALA	EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	140

P1AE8873 OX40 (8H9) x FAP (1G1a) (3+1) C-terminal crossfab fusion
OX40(8H9) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	143
OX40(8H9) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYLTYSRFTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	144
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(8H9) VHCH1-OX40(8H9) VHCH1- Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	145

P1AE8870 OX40 (8H9) x FAP (1G1a) (2+1) C-terminal crossfab fusion
OX40(8H9) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	143
OX40(8H9) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYLTYSRFTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	144
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(8H9) VHCH1- Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	146

1.2 Production of Bispecific Antigen Binding Molecules Targeting FAP and OX40

The molecules were produced by co-transfecting either HEK293-EBNA cells growing in suspension with the mammalian expression vectors using polyethylenimine (PEI) or co-transfecting CHO K1 cells growing in suspension with the mammalian expression using eviFECT (Evitria AG, Switzerland). The cells were transfected with the corresponding expression vectors.
Antibody constructs were expressed by transient transfection of HEK cells grown in suspension with expression vectors encoding the 4 different peptide chains. Transfection of Expi293F™ cells (Gibco™) was performed according to the cell supplier’s instructions using Maxiprep (Macherey-Nagel) preparations of the antibody vectors, Expi293F™ Expression Medium (Gibco™), ExpiFectamine™ 293 Reagent, (Gibco™) and an initial cell density of 2-3 million viable cells/ml in Opti-MEM® 1x Reduced Serum Medium (Gibco®). On the day after transfection (Day 1, 18-22 hours post-transfection), ExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293 Transfection Enhancer 2 was added to the transfected culture. Transfected cultures were incubated at 37° C. in a humidified atmosphere of 8% CO₂ with shaking. Cell culture supernatants were harvested after 7 days of cultivation in shake flasks or stirred fermenters by centrifugation at 3000-5000 g for 20-30 minutes and filtered through a 0.22 µm filter.
For production in CHO K1 cells, CHO K1 cells were grown in eviGrow medium (Evitria AG, Switzerland), a chemically defined, animal-component free, serum-free medium and transfected with eviFect (Evitria AG, Switzerland). After transfection the cells were kept in eviMake (Evitria AG, Switzerland), a chemically defined, animal-component free, serum-free medium, at 37° C. and 5% CO₂ for 7 days. After 7 days the cultivation supernatant was collected for purification by centrifugation for 45 min at maximum speed in a Rotanta 460 RC. The solution was sterile filtered (0.22 µm filter) and kept at 4° C. The concentration of the molecules in the culture medium was either determined by Protein A-HPLC or Protein A-Bio-Layer Interferometry (BLI).
Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were captured on a MabSelect SuRe resin equilibrated with PBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 100 mM sodium acetate, pH 3.0. After neutralization with 1 M Tris pH 9.0, aggregated protein was separated from monomeric antibody species by ion exchange Chromatography (Poros XS) with equilibration buffer 20 mM His, pH 5.5, 1.47 mS/cm and elution buffer 20 mM His, 500 mM NaCl, pH 5.5, 49.1 mS/cm, (gradient: to 100% elution buffer in 60 CV). In some cases a size exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM histidine, 140 mM NaCl, pH 6.0, was subsequently performed. Monomeric protein fractions were pooled, and if required concentrated using e.g. a MILLIPORE Amicon Ultra (30KD MWCO) centrifugal concentrator. Purified proteins were stored at -80° C. Protein quantification was performed using a Nanodrop spectrophotometer and analyzed by CE-SDS under denaturing and reducing conditions and analytical SEC. Sample aliquots were used for subsequent analytical characterization e.g. by CE-SDS, size exclusion chromatography, mass spectrometry and endotoxin determination..
Purity and molecular weight of the bispecific antigen binding molecule after the final purification step were analyzed by CE-SDS analyses in the presence and absence of a reducing agent. The Caliper LabChip GXII system (Caliper Lifescience) was used according to the manufacturer’s instruction.
The aggregate content of the bispecific antigen binding molecule was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM potassium phosphate, 125 mM sodium chloride, 200 mM L-arginine monohydrocloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C.

TABLE 2

Production yield and Quality of bispecific OX40 antigen binding molecules
Molecule	Monomer [%]	CE-SDS (non- reduced) [%]	Yield [mg/l]
P1AE6838 OX40(49B4) x FAP(1G1a) 4+1	99	94	44
P1AE8786 OX40(49B4) x FAP(1G1a) 3+1	99	93	21
P1AE6840 OX40(49B4) x FAP(1G1a) 2+1	98	98	3.7
P1AF7205 OX40(CLC563) x FAP(1G1a_EPKSCD) 4+1	99	96	25
P1AF7217 OX40(CLC563) x FAP(1G1a_EPKSCS) 4+1	99	98	32
P1AE8874 OX40(CLC563) x FAP(1G1a) 3+1	99	100	35
P1AF6454 OX40(CLC563) x FAP(1G1a_EPKSCD) 3+1	98	98	122
P1AF6455 OX40(CLC563) x FAP(1G1a_EPKSCS) 3+1	100	100	19
P1AE8871 OX40(CLC563) x FAP(1G1a) 2+1	96	98	90
P1AE8875 OX40(MOXR0916) x FAP(1G1a) 3+1	98	97	9
P1AF4845 OX40(MOXR0916) x FAP(1G1a_EPKSCD) 3+1	86	100	0.2
P1AF4851 OX40(MOXR0916) x FAP(1G1a_EPKSCS) 3+1	99	100	0.5
P1AE8872 OX40(MOXR0916) x FAP(1G1a) 2+1	95	98	2.4
P1AF4852 OX40(MOXR0916) x FAP(1G1a_EPKSCD) 2+1	95	100	2.7
P1AF4858 OX40(MOXR0916) x FAP(1G1a_EPKSCS) 2+1	98	99	4.6
P1AE8873 OX40(8H9) x FAP(1G1a) 3+1	100	100	13
P1AE8870 OX40(8H9) x FAP(1G1a) 2+1	98	98	13

1.3 Generation of Further Bispecific Antigen Binding Molecules Targeting OX40 and Fibroblast Activation Protein (FAP) - Charge Patch Variants

In analogy to Example 1.1, different variants of a 4+1 bispecific format consisting of four OX40 binding moieties combined with one FAP binding crossfab at the C-terminus of the Fc domain have been prepared. In all these constructs, the variable heavy and light chain domains of the anti-OX40 antibody correspond to the OX40 clone 49B4 as described in WO 2017/055398 A2. The generation and preparation of the FAP antibody 1G1a is described in Example 1. To generate the 4+1 antigen binding molecules, the knob-into-hole technology was used to achieve heterodimerization. The S354C/T366W mutations were introduced in the first heavy chain HC1 (Fc knob heavy chain) and the Y349C/T366S/L368A/Y407V mutations were introduced in the second heavy chain HC2 (Fc hole heavy chain). Furthermore, the CrossMab technology as described in WO 2010/145792 A1 ensures correct light chain pairing. Independent of the bispecific format, in all cases an effector silent Fc (P329G; L234A, L235A) has been used to abrogate binding to Fcγ receptors according to the method described in WO 2012/130831 Al. Amino acid sequences of the bispecific antigen binding molecules are shown in Table 3.
All genes are transiently expressed under control of a chimeric MPSV promoter consisting of the MPSV core promoter combined with the CMV promoter enhancer fragment. The expression vector also contains the oriP region for episomal replication in EBNA (Epstein Barr Virus Nuclear Antigen) containing host cells.

TABLE 3

Amino acid sequences of the bispecific antigen binding molecules
Molecule	Sequence	Seq ID No
P1AE9167 OX40 (49B4_K73E) x FAP (1G1a) (4+1)
OX40(49B4_K7 3E) VHCH1-OX40(49B4_K7 3E) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	147
OX40(49B4) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	119
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(49B4_K7 3E) VHCH1-OX40(49B4_K7 3E) VHCH1-Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	148

P1AE9169 OX40 (49B4_K73E) x FAP (1G1a) (4+1)
OX40(49B4_K2 3T_K73E) VHCH1-OX40(49B4_K2 3T_K73E) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCTASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCTASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	149
OX40(49B4) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	119
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(49B4_K2 3T_K73E) VHCH1-OX40(49B4_K2 3T_K73E) VHCH1-Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCTASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCTASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	150

P1AE9176 OX40 (49B4_K23E_K73E) x FAP (1G1a) (4+1)
OX40(49B4_K2 3E_K73E) VHCH1-OX40(49B4_K2 3E_K73E) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	151
OX40(49B4_K2 3E_K73E) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	119
FAP(1G1a) VLCH1-light chain	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	123
OX40(49B4_K2 3E_K73E) VHCH1-OX40(49B4_K2 3E_K73E) VHCH1-Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	152

P1AF6456 OX40 (49B4_K23E_K73E) x FAP (1G1a_EPKSCD) (4+1)
OX40(49B4_K2 3E_K73E) VHCH1-OX40(49B4_K2 3E_K73E) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	151
OX40(49B4) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	119
FAP(1G1a) VLCH1-light chain (EPKSCD)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD	129
OX40(49B4_K2 3E_K73E) VHCH1-OX40(49B4_K2 3E_K73E) VHCH1-Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	152

P1AF6457 OX40 (49B4_K23E_K73E) x FAP (1G1a_EPKSCS) (4+1)
OX40(49B4_K2 3E_K73E) VHCH1-OX40(49B4_K2 3E_K73E) VHCH1- Fc knob_PGLALA-FAP(1G1a) VHCL	QVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTLTDYNMDWVRQAPGQGLEWIGDIYPNTGGTIYNQKFKGRVTMTIDTSTSTVYMELSSLRSEDTAVYYCTRFRGIHYAMDYWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	151
OX40(49B4) light chain	DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	119
FAP(1G1a) VLCH1-light chain (EPKSCS)	EIVLTQSPATLSLSPGERATLSCRASESVDNYGLSFINWFQQKPGQAPRLLIYGTSNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNEVPYTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCS	131
OX40(49B4_K2 3E_K73E) VHCH1-OX40(49B4_K2 3E_K73E) VHCH1-Fc hole_PGLALA	QVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCGGGGSGGSGGQVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	152

1.4 Production of Bispecific Antigen Binding Molecules Targeting FAP and OX40 (Charge Patch Variants)

Antibodies were expressed by transient transfection of HEK cells grown in suspension with expression vectors encoding the 4 different peptide chains. Transfection into HEK293-F cells (Invitrogen) was performed according to the cell supplier’s instructions using MaxiPREP (Qiagen) preparations of the antibody vectors, Freestyle™ F17 medium (Invitrogen, USA), PEIpro® transfection reagent (Polyscience Europe GmbH) and an initial cell density of 1-2 million viable cells/ml in serum free FreeStyle 293 expression medium (Invitrogen). Cell culture supernatants were harvested after 7 days of cultivation in shake flasks or stirred fermenters by centrifugation at 14000xg for 30 minutes and filtered through a 0.22 µm filter.
Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were captured on a MabSelectSure resin equilibrated with PBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25 mM citrate, pH 3.0. After neutralization with 1 M Tris buffer pH 9.0, aggregated protein was separated from monomeric antibody species by size exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM histidine, 140 mM NaCl, pH 6.0. Monomeric protein fractions were pooled, concentrated if required using e.g. a MILLIPORE Amicon Ultra (30KD MWCO) centrifugal concentrator and stored at -80° C. Sample aliquots were used for subsequent analytical characterization e.g. by CE-SDS, size exclusion chromatography, mass spectrometry and endotoxin determination.

TABLE 4

Production yield and Quality of bispecific OX40 antigen binding molecules
Molecule	Monomer [%]	CE-SDS (non-reduced) [%]	Yield [mg/l]
P1AE9167 OX40(49B4_K73E) x FAP(1G1a) 4+1	99	92	2.7
P1AE9169 OX40(49B4_K23T_K73E) x FAP(1G1a) 4+1	95	98	6.7
P1AE9176 OX40(49B4_K23E_K73E) x FAP(1G1a) 4+1	99	99	32
P1AF6456 OX40(49B4_K23E_K73E) x FAP(1G1a_EPKSCD) 4+1	93	94	2.6
P1AF6457 OX40(49B4_K23E_K73E) x FAP(1G1a_EPKSCS) 4+1	98	96	0.9

Example 2

Characterization of Bispecific Antigen Binding Molecules Targeting OX40 and FAP

2.1 Binding to Naïve Versus Activated Human PBMCs of FAP-Targeted Anti-OX40 Bispecific Antigen Binding Molecules

Human PBMCs were isolated by ficoll density gradient centrifugation. Buffy coats were obtained from the Zürich blood donation center. To isolate fresh peripheral blood mononuclear cells (PBMCs), the buffy coat was diluted with the same volume of DPBS (Gibco by Life Technologies, Cat. No. 14190 326). 50 mL polypropylene centrifuge tubes (TPP, Cat.-No. 91050) were supplied with 15 mL Histopaque 1077 (SIGMA Life Science, Cat.-No. 10771, polysucrose and sodium diatrizoate, adjusted to a density of 1.077 g/mL) and the buffy coat solution was layered above the Histopaque 1077. The tubes were centrifuged for 30 min at 400 x g, room temperature and with low acceleration and no break. Afterwards the PBMCs were collected from the interface, washed three times with DPBS and frozen for later use. PBMC were thawed, washed and resuspended in AIM-V medium (ThermoFischer, Cat. No. 12055091). PBMCs were used unstimulated (binding on resting human PBMCs) or they were stimulated to receive a strong human Ox40 expression on the cell surface of T cells (binding on activated human PBMCs). Therefore thawed PBMCs were cultured for three days at 37° C./5% CO₂ in Aim-V media in 6-well tissue culture plate pre-coated for 2 hours with [2 µg/mL] anti-human CD3 (clone OKT3) and [2 µg/mL] anti-human CD28 (clone CD28.2).
For detection, OX40 naive human PBMCs and activated human PBMCs were mixed. To enable distinction of naive from activated human PBMCs, naïve cells were labeled prior to the binding assay using the eFluor670 cell proliferation dye (eBioscience, Cat.-No.65-0840-85). A 1 to 1 mixture of 1 × 10⁵ naïve, eFluor670 labeled human PBMC and unlabeled activated human PBMCs were then added to each well of a round-bottom 96-well plate (TPP, Cat. No. 92097) and the binding assay was performed.
Cells were first stained 10 min with Zombie Aqua Fixable viability dye (Biolegend, Cat.No. 423102) in DPBS, followed by a 90 minutes incubation at 4° C. in the dark in 50 µL/well FACS buffer containing titrated anti-OX40 bispecific antibody constructs. After three times washing with excess FACS buffer, cells were stained for 30 minutes at 4° C. in the dark in 25 µL/well FACS buffer containing a mixture of fluorescently labeled anti-human CD4 (clone OKT-4, mouse IgG2b, BioLegend, Cat.-No. 317434), anti-human CD8 (clone RPA-T8, mouse IgG1k, BioLegend, Cat.-No. 301042) and Fluorescein isothiocyanate (FITC)-conjugated AffiniPure anti-human IgG Fcy-fragment-specific goat IgG F(ab′)2 fragment (Jackson ImmunoResearch, Cat.-No. 109-096-098). Samples were finally resuspended in 20 µL/well FACS-buffer and acquired the same day using iQue Cell Screener and ForeCyt software (Sartorius).
As can be seen in FIGS. 2A and 2C, the bispecific OX40 (49B4) x FAP antigen binding molecule in tetravalent format (4+1) bound better to OX40 than as a trivalent (3+1) or bivalent (2+1) format (avidity of the 49B4 clone). Along the natural prevalence of OX40 on T cells, the bispecific formats bound stronger to activated CD4 than to CD8 T cells, and had no binding to target negative cells (resting CD4 and CD8 T cells, FIGS. 2B and 2D). As shown in FIGS. 3A and 3C, the bispecific antigen binding molecules comprising clone 8H9 bound with subnanomolar affinity to OX40 positive cells and with comparable strength as tri- and bivalent antibody. Along the natural prevalence of OX40 on T cells, the constructs bound stronger to activated CD4 than CD8 T cells, and had no binding to target negative cells (resting CD4 and CD8 T cells, FIGS. 3B and 3D). In FIGS. 4A and 4C it is shown that bispecific antibodies comprising clone MOX0916 bound with subnanomolar affinity to OX40 positive cells, with comparable strength as trivalent or bivalent antibody. Along the natural prevalence of OX40 on T cells, the constructs bound stronger to activated CD4 than to CD8 T cells, and had no binding to target negative cells (resting CD4 and CD8 T cells, see FIGS. 4B and 4D). Bispecific antibodies comprising clone CLC-563 bound with nanomolar affinity to OX40 positive cells, with comparable strength as trivalent and bivalent antibody as is shown in FIGS. 5A and 5C. Along the natural prevalence of OX40 on T cells, the constructs bound stronger to activated CD4 than to CD8 T cells, and had no binding to target negative cells (resting CD4 and CD8 T cells, see FIGS. 5B and 5D).
In FIGS. 6A and 6C it is shown that all bispecific antigen binding molecules comprising 49B4 amino acid variant based clones showed slightly improved binding to OX40 positive cells compared to the parental antibody 49B4. Along the natural prevalence of OX40 on T cells, the constructs bound stronger to activated CD4 than CD8 T cells, and had no binding to target negative cells (resting CD4 and CD8 T cells, see FIGS. 6B and 6D).
In another experiment, the binding of bispecific antigen binding molecules targeting OX40 and FAP with different C-terminal variants was tested. PBMC were used that were thawed, washed and resuspended in RPMI medium (Gibco, Cat. No. 72400-021) with (10% (v/v) Fetal calf serum (FCS, SIGMA, Cat.-No. F4135). PBMCs were stimulated to receive a strong human Ox40 expression on the cell surface of T cells (binding on activated human PBMCs). Therefore thawed PBMCs were cultured for three days at 37° C./5% CO₂ in in RPMI medium with 10% FCS in 6-well tissue culture plate pre-coated for 2 hours with [2 µg/mL] anti-human CD3 (clone OKT3) and [2 µg/mL] anti-human CD28 (clone CD28.2). For detection, 2 × 10⁵ activated human PBMCs were added to each well of a round-bottom 96-well plate (TPP, Cat. No. 92097) and the binding assay was performed. Cells were first stained 20 min with LIVE/DEAD™ Fixable Aqua Dead Cell Stain (Molecular probes, Cat.No. L34957) in DPBS, followed after one washing step (200 µL 4° C. FACS buffer) by incubation of 90 minutes at 4° C. in the dark in 50 µL/well FACS buffer containing titrated anti-OX40 bispecific antigen binding molecules. After one washing with excess FACS buffer, cells were stained for 30 minutes at 4° C. in the dark in 50 µL/well FACS buffer containing a mixture of fluorescently labeled anti-human CD4 (clone A161A1, BioLegend, Cat.-No. 357406), anti-human CD8 (clone SK1, BioLegend, Cat.-No. 344742) and Phycoerythrin (PE)-conjugated AffiniPure anti-human IgG Fcy-fragment-specific goat IgG F(ab′)2 fragment (Jackson ImmunoResearch, Cat. No. 109-116-098). Samples were finally resuspended in 100 µL/well FACS-buffer and acquired the same day using with BD Fortessa running FACS Diva software.
As can be seen in FIGS. 7A to 7F, all tested bispecific FAP-OX40 bispecific antibodies bound to activated CD4 T cells. Along the natural prevalence of OX40 on T cells, the bispecific formats bound stronger to activated CD4 than to CD8 T cells (FIGS. 7A, 7C, 7E versus FIGS. 7B, 7D and 7F). For all tested compounds the D and the S variant showed comparable binding properties (compare for each plot open vs closed symbols).

2.2 Binding to Human FAP-Expressing Tumor Cells

The binding to cell surface FAP was tested using human fibroblast activating protein (huFAP) expressing NIH/3T3-huFAP clone 19 cells. This cell line was generated by the transfection of the mouse embryonic fibroblast NIH/3T3 cell line (ATCC CRL-1658) with the expression vector pETR4921 to express huFAP under 1.5 µg/mL Puromycin selection. The lack of binding to OX40 negative FAP negative tumor cells was tested using A549 NucLight™ Red Cells (Essen Bioscience, Cat. No. 4491) expressing the NucLight Red fluorescent protein restricted to the nucleus to allow separation from unlabeled human FAP positive NIH/3T3-huFAP clone 19 cells. Parental A549 (ATCC CCL-185) were transduced with the Essen CellPlayer NucLight Red Lentivirus (Essen Bioscience, Cat. No. 4476; EF1α, puromycin) at an MOI of 3 (TU/cell) in the presence of 8 µg/ml polybrene following the standard Essen protocol. This resulted in ≥70% transduction efficiency. Alternatively, the lack of binding to OX40 negative FAP negative tumor cells was tested using the HeLa cell line (ATCC, CCL2), a human cervix adenocarcinoma cell line. Enzyme free cell dissociation buffer was used for detachment to preserve trypsin sensitive surface proteins.
A mixture of 5 × 10⁴ unlabeled NIH/3T3-huFAP clone 19 cells and A549 NucLight™ Red Cells in FACS buffer were added to each well of a round-bottom 96-well plates (TPP, Cat. No. 92097) and the binding assay was performed. Cells were first stained 10 min with Zombie Aqua Fixable viability dye (Biolegend, Cat.No. 423102) in DPBS, followed by a 75 minutes incubation at 4° C. in the dark in 50 µL/well FACS buffer containing titrated anti-OX40 bispecific antibody constructs. Afterwards the cells were washed three times with 200 µL 4° C. FACS buffer and resuspended by a short vortex. Cells were further stained with 25 µL/well of 4° C. cold secondary antibody solution containing Fluorescein isothiocyanate (FITC)-conjugated AffiniPure anti-human IgG Fcy-fragment-specific goat IgG F(ab′)2 fragment (Jackson ImmunoResearch, Cat. No. 109-096-098) and incubated for 30 minutes at 4° C. in the dark. Samples were finally resuspended in 20 µL/well FACS-buffer and acquired the same day using iQue Cell Screener and ForeCyt software (Sartorius).
As can be seen in FIGS. 2E, 3E, 4E, 5E and 6E, all bispecific antigen binding molecules, sharing the FAP (1G1a) antigen binding domain, had comparable binding to human FAP positive fibroblasts (NIH/3T3-huFAP-clone 19). Only in FIG. 3E it is shown, that there was a slightly enhanced binding to human FAP positive fibroblasts (NIH/3T3-huFAP-clone 19) when clone 8H9 was incorporated in the bispecific antigen binding molecules, despite of sharing the same FAP (1G1a) antigen binding domain. No binding was observed to target negative cells (A549-NLR cells, FIGS. 2F, 3F, 4F, 5F and 6F).
In another experiment, a mixture of 2 × 10⁵ NIH/3T3-huFAP clone 19 cells and HeLa cells in FACS buffer were added to each well of a round-bottom 96-well plates (TPP, Cat. No. 92097) and the binding assay was performed. Cells were first stained 20 min with LIVE/DEAD™ Fixable Aqua Dead Cell Stain (Molecular probes, Cat.No. L34957) in DPBS, followed after one washing step with 200 µL 4° C. FACS buffer by a 75 minutes incubation at 4° C. in the dark in 50 µL/well FACS buffer containing titrated anti-OX40 bispecific antibody constructs. Afterwards the cells were washed once with 200 µL FACS buffer at 4° C. and resuspended by a short vortex. Cells were further stained with 50 µL/well of 4° C. cold secondary antibody solution containing Phycoerythrin (PE)-conjugated AffiniPure anti-human IgG Fcy-fragment-specific goat IgG F(ab′)2 fragment (Jackson ImmunoResearch, Cat. No. 109-116-098) and incubated for 30 minutes at 4° C. in the dark. Samples were finally resuspended in 100 µL/well FACS-buffer and acquired the same day using with BD Fortessa running FACS Diva software.
All bispecific antigen binding molecules sharing the FAP (1G1a) antigen binding domain, but comprising a C-terminal S or D variant, had comparable binding to human FAP positive fibroblasts (NIH/3T3-huFAP-clone 19), meaning that the variants had no impact on the binding to FAP (see data in Table 5 below). No binding was observed when targeting negative cells (HeLa cells).

2.3 Summary of Cellular Binding Properties of the Bispecific Antigen Binding Molecules

For evaluation of the binding properties of the FAP-targeted OX40 antibodies human FAP negative tumor cells (A549- NLR or HeLa), FAP positive fibroblasts (NIH/3T3-huFAP-clone 19), OX40 positive activated PBMC (activated CD4 and CD8 T cells) as well as OX40 negative resting PBMC (resting CD4 and CD8 T cells) were incubated with indicated serial dilutions of test antibody detected then by fluorescently labeled 2nd antibody against human Fcy.
All FAP-targeted OX40 antigen binding molecules bound efficiently to human FAP-expressing target cells and had no binding to target negative cells. This is expected to translate in patients to direct tumor-targeting and enrichment of the molecules. Along the natural prevalence of OX40 on T cells, all constructs bound stronger to activated CD4 than to CD8 T cells. The bispecific antigen binding molecules comprising clone 49B4 bound better to OX40 in a tetravalent format (4+1) than in a trivalent (3+1) or bivalent (2+1) format (avidity of the clone; see FIGS. 2A and 2C). All 49B4 amino acid variant based bispecific antigen binding molecules showed slightly improved binding to OX40 positive cells compared to the parental antibody in a tetravalent format (see FIGS. 6A and 6C). The clones 8H9 (FIGS. 3A and 3C) and MOX0916 (FIGS. 4A and 4C) bound with subnanomolar affinity, whereas clone CLC-563 (FIGS. 5A and 5C) bound with nanomolar affinity to OX40 positive cells, with comparable strength as tri- and bivalent formats. For all tested C-terminal variants, the D and the S variant showed comparable binding properties. FAP binding was for all bispecific antigen binding molecules in a comparable nanomolar range.
EC₅₀ values of binding to activated human CD4 T cells and FAP positive tumor cells are summarized in Table 5.

TABLE 5

EC₅₀ values for binding of FAP-targeted OX40 antigen binding molecules to cell surface human FAP and human Ox40 (on CD4⁺ T-cells)
Molecule ID	anti-Ox40 clone	Format	Ox40	FAP
Molecule ID	anti-Ox40 clone	Format	EC₅₀ [nM]
P1AE6838	49B4		4+1	0.03	2.63
P1AE9167	49B4 AA variant K73E	4+1	0.11	2.51
P1AE9169	49B4 AA variant K23T_K73E	4+1	0.12	2.47
P1AE9176	49B4 AA variant K23E_ K73E	4+1	0.20	2.97
P1AE8786	49B4		3+1	17.08	2.91
P1AE6840	49B4		2+1	73.57	2.26
P1AE8873	8H9		3+1	0.11	1.01
P1AE8870	8H9		2+1	0.17	0.95
P1AE8875	MOX0916		3+1	0.69	2.70
P1AE8872	MOX0916		2+1	0.90	1.78
P1AE8874	CLC-563	3+1	3.62	2.52
P1AE8871	CLC-563	2+1	3.19	2.51
P1AF6455	CLC-563, S-variant	3+1	1.47	1.71
P1AF6454	CLC-563, D-variant	3+1	1.64	1.22
P1AF7217	CLC-563, S-variant	4+1	1.46	1.33
P1AF7205	CLC-563, D-variant	4+1	2.11	1.04
P1AF6457	49B4 AA variant K23E_ K73E, S-variant	4+1	0.05	0.69
P1AF6456	49B4 AA variant K23E_ K73E, D variant	4+1	0.07	0.36

2.4 Biophysical and Biochemical Characterization of Bispecific Antigen Binding Molecules Targeting OX40 and FAP

2.4.1 Determination of Thermal Stability

Thermal stability of the FAP-targeted OX40 antigen binding molecules prepared in Example 1 was monitored by Dynamic Light Scattering (DLS) and by monitoring of temperature dependent intrinsic protein fluorescence by applying a temperature ramp using an Optim 2 instrument (Avacta Analytical, UK). 10 µg of filtered protein sample with a protein concentration of 1 mg/ml was applied in duplicate to the Optim 2 instrument. The temperature was ramped from 25° C. to 85° C. at 0.1° C./min, with the ratio of fluorescence intensity at 350 nm/330 nm and scattering intensity at 266 nm being collected. The results are shown in Table 6. The aggregation temperature (T_agg) of all the tested FAP-Ox40 molecules produced in Example 2 is favorable than for the previously described OX40 (49B4) x FAP (4B9) (4+1) bispecific molecule (molecule P1AD4524) as described in WO 2017/060144 A1.

2.4.2 Hydrophobic Interaction Chromatography (HIC)

Apparent hydrophobicity was determined by injecting 20 µg of sample onto a HIC-Ether-5PW (Tosoh) column equilibrated with 25 mM Na-phosphate, 1.5 M ammonium sulfate, pH 7.0. Elution was performed with a linear gradient from 0 to 100% buffer B (25 mM Na-phosphate, pH 7.0) within 60 minutes. Retention times were compared to protein standards with known hydrophobicity. High HIC retention times were obtained for FAP x OX40 bispecific antigen binding molecules containing the clone 8H9 and charged patch variants of clone 49B4. Increased nonspecific interactions have been shown to correlate with high HIC retention time.

2.4.3 FcRn Affinity Chromatography

FcRn was expressed, purified and biotinylated as described (Schlothauer et al., MAbs 2013, 5(4), 576-86). For coupling, the prepared receptor was added to streptavidin-sepharose (GE Healthcare). The resulting FcRn-sepharose matrix was packed in a column housing. The column was equilibrated with 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES) and 140 mM NaCl, pH 5.5 (eluent A) at a 0.5 ml/min flow rate. 30 µg of antibody samples were diluted at a volume ratio of 1: 1 with eluent A and applied to the FcRn column. The column was washed with 5 column volumes of eluent A followed by elution with a linear gradient from 20 to 100% 20 mM Tris/HCl and 140 mM NaCl, pH 8.8 (eluent B) in 35 column volumes. The analysis was performed with a column oven at 25° C. The elution profile was monitored by continuous measurement of the absorbance at 280 nm. Retention times were compared to protein standards with known affinities.

2.4.4 Heparin Affinity Chromatography

Heparin affinity was determined by injecting 30-50 µg of sample onto a TSKgel Heparin-5PW (Tosoh) column equilibrated with 50 mM Tris, pH 7.4. Elution was performed with a linear gradient from 0 to 100% buffer B (50 mM Tris, 1 M NaCl, pH 7.4 mM) within 37 minutes. Retention times were compared to protein standards with known affinities.

TABLE 6

Biophysical and biochemical properties of tested FAP x OX40 bispecific antibodies
Sample		Thermal stability DLS T_agg	Apparent hydrophobicity	FcRn affinity	Heparin affinity
P1AE8870	OX40 (8H9) x FAP (1G1a) 2+1	57.6	0.83	1.81	0.62
P1AE8872	OX40 (MOXR0916) x FAP (1G1a) 2+1	67.5	0.31	0.15	0.58
P1AE8873	OX40 (8H9) x FAP (1G1a) 3+1	56.9	0.89	2.02	0.65
P1AE8874	OX40 (CLC563) x FAP (1G1a) 3+1	66.2	0.15	-0.14	0.51
P1AE8875	OX40 (MOXR0916) x FAP (1G1a) 3+1	67.4	0.32	-0.22	0.6
P1AE9176	OX40 (49B4_K26E_K73E) x FAP (1G1a) 4+1	62	0.61	-0.48	0.5
P1AF7217	OX40 (CLC563) x FAP (1G1a_EPKSCS) 4+1	66	0.14	-0.3	0.5
P1AF7205	OX40 (CLC563) x FAP (1G1a_EPKSCD) 4+1	67	0.14	-0.3	0.5
P1AF6455	OX40 (CLC563) x FAP (1G1a_EPKSCS) 3+1	67	0.14	-0.1	0.5
P1AF6454	OX40 (CLC563) x FAP (1G1a_EPKSCD) 3+1	66	0.14	-0.1	0.5
P1AF6457	OX40 (49B4_K23E_K73E) x FAP (1G1a_EPKSCS) 4+1	62	0.61	-0.5	0.5
P1AF6456	OX40 (49B4_K23E_K73E) x FAP (1G1a_EPKSCD) 4+1	62	0.61	-0.5	0.5
P1AD4524	OX40 (49B4) x FAP (4B9) (4+1)	48	0.56	0	0.68

2.5 Characterization of Binding Potency by Surface Plasmon Resonance (SPR) After Stress

The reduction in binding potency caused by incubation of the molecules for 14 days at 37° C., pH 7.4 and at 40° C., pH 6 was quantified by surface plasmon resonance using a Biacore T200 instrument (GE Healthcare). Samples stored at -80° C. and pH 6 were used as reference. The reference samples and the samples stressed at 40° C. were in 20 mM Histidine buffer, 140 mM NaCl, pH 6.0, and the samples stressed at 37° C. in PBS buffer, pH 7.4, all at a concentration of 1.0 mg/ml. After the stress period (14 days) samples in PBS buffer were dialyzed back to 20 mM Histidine buffer, 140 mM NaCl, pH 6.0 for further analysis.
All SPR experiments were performed at 25° C. with HBS-P+ buffer (10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% Surfactant P20) as running and dilution buffer. Biotinylated human OX40 and FAP, as well as biotinylated anti-hu IgG (Capture Select, Thermo Scientific, #7103262100) were immobilized on a Series S Sensor Chip SA (GE Healthcare, #29104992), resulting in surface densities of at least 1000 resonance units (RU). FAP-OX40 bispecific antibodies with a concentration of 2 µg/ml were injected for 30 s at a flow rate of 5 µl/min, and dissociation was monitored for 120 s. The surface was regenerated by injecting 10 mM glycine buffer, pH 1.5, for 60 s. Bulk refractive index differences were corrected by subtracting blank injections and by subtracting the response obtained from a blank control flow cell. For evaluation, the binding response 5 seconds after injection end was taken.
To normalize the binding signal, the OX40 and FAP binding was divided by the anti-hu IgG response (the signal (RU) obtained upon capture of the FAP x OX40 bispecific antibody on the immobilized anti-hu IgG antibody). The relative binding activity was calculated by referencing each temperature stressed sample to the corresponding, non-stressed sample. As shown in Table 7, all FAP x OX40 bispecific antibodies prepared in Example 1 show an improved binding upon stress to OX40 and FAP, as compared to a previously described FAP-OX40 bispecific antibody as described in WO 2017/060144 A1.

TABLE 7

Binding activity of FAP-OX40 bispecific antibodies to human to Ox40 and FAP after incubation at pH 6/40° C. or pH 7.4/37° C. for 2 weeks
Sample		binding activity [%]
		2 weeks at pH 6.0/40° C.		2 weeks at pH 7.4/37° C.
		FAP	Ox40	FAP	Ox40
P1AE8870	OX40 (8H9) x FAP (1G1a) 2+1	> 90	> 90	> 90	> 90
P1AE8872	OX40 (MOXR0916) x FAP (1G1a) 2+1	> 90	> 90	> 90	> 90
P1AE8873	OX40 (8H9) x FAP (1G1a) 3+1	> 90	> 90	> 90	> 90
P1AE8874	OX40 (CLC563) x FAP (1G1a) 3+1	> 90	> 90	> 90	> 90
P1AE8875	OX40 (MOXR0916) x FAP (1G1a) 3+1	> 90	> 90	> 90	> 90
P1AE9176	OX40 (49B4_K26E_K73E) x FAP (1G1a) 4+1	> 90	> 90	> 90	> 90
P1AF7217	OX40 (CLC563) x FAP (1G1a_EPKSCS) 4+1	98	100	94	100
P1AF7205	OX40 (CLC563) x FAP (1G1a_EPKSCD) 4+1	98	100	94	100
P1AF6455	OX40 (CLC563) x FAP (1G1a_EPKSCS) 3+1	98	100	96	100
P1AF6454	OX40 (CLC563) x FAP (1G1a_EPKSCD) 3+1	99	100	95	99
P1AF6457	OX40 (49B4_K23E_K73E) x FAP (1G1a_EPKSCS) 4+1	99	99	99	99
P1AF6456	OX40 (49B4_K23E_K73E) x FAP (1G1a_EPKSCD) 4+1	99	100	99	100
P1AD4524	OX40 (49B4) x FAP (4B9) (4+1)	~90	> 90	~90	> 90

Example 3

Functional Properties of FAP-targeted Anti-human OX40 Antigen Binding Molecules

3.1 HeLa Cells Expressing Human OX40 and Reporter Gene NFκB-luciferase

Agonistic binding of OX40 to its ligand induces downstream signaling via activation of nuclear factor kappa B (NFκB) (A. D. Weinberg et al., J. Leukoc. Biol. 2004, 75(6), 962-972). The recombinant reporter cell line HeLa_hOx40_NFkB_Luc1 was generated to express human OX40 on its surface. Additionally, it harbors a reporter plasmid containing the luciferase gene under the control of an NFκB-sensitive enhancer segment. OX40 triggering induces dose-dependent activation of NFκB, which translocates to the nucleus, where it binds on the NFκB sensitive enhancer of the reporter plasmid to increase expression of the luciferase protein. Luciferase catalyzes luciferin-oxidation resulting in oxyluciferin which emits light. This can be quantified by a luminometer.
Thus, the capacity of the various FAP-targeted OX40 antigen binding molecules to induce NFκB activation in HeLa_hOx40_NFkB_Luc1 reporter cells was analyzed as a measure for bioactivity.
We tested the NFκB activating capacity of selected FAP-targeted OX40 antigen binding molecules in a bivalent, trivalent and tetravalent FAP-targeted cross-Fab format alone and with hyper-crosslinking of the constructs by either a secondary antibody or a FAP⁺ fibroblast cell line. The crosslinking of FAP-binding antibodies by cell surface FAP was tested using human fibroblast activating protein (huFAP) expressing NIH/3T3-huFAP clone 19. This cell line was generated by the transfection of the mouse embryonic fibroblast NIH/3T3 cell line (ATCC CRL-1658) with the expression vector pETR4921 to express huFAP under 1.5 µg/mL Puromycin selection.
Adherent HeLa_hOX40_NFkB_Luc1 cells were cultured over night at a cell density of 0.2 × 10⁵ cells per well and were stimulated for 6 hours with assay medium containing titrated anti-OX40 antigen binding molecules. For testing the effect of hyper-crosslinking by secondary antibodies, 25 µL/well of medium containing secondary antibody anti-human IgG Fcy-fragment-specific goat IgG F(ab′)2 fragment (Jackson ImmunoResearch, 109-006-098) was added in a 1:2 ratio (primary to secondary antibodies). To test the effect of hyper-crosslinking by cell surface FAP binding, 25 µL/well of medium containing FAP⁺ tumor cells (NIH/3T3-huFAP clone 19) were co-cultured in a 3 to 1 ratio (three times more FAP⁺ tumor cells than reporter cells per well).
After incubation, assay supernatant was aspirated and plates washed two times with DPBS. Quantification of light emission was done using the luciferase 1000 assay system and the reporter lysis buffer (both Promega, Cat.-No. E4550 and Cat-No: E3971) according to manufacturer instructions. Briefly, cells were lysed for 30 minutes on dry ice by addition of 30 µL per well 1x lysis buffer. Cells were thawed for 20 minutes at 37° C. before 100 µL per well provided luciferase assay reagent was added. Light emission was quantified immediately with a Spark10M Tecan microplate reader using 500 ms integration time, without any filter to collect all wavelengths. Emitted relative light units (URL) were corrected by basal luminescence of HeLa_hOx40_NFkB_Luc1 cells and were plotted against the logarithmic primary antibody concentration using Prism7 (GraphPad Software, USA). Curves were fitted using the inbuilt sigmoidal dose response.
Thus, we tested the NFκB activating capacity of selected bispecific OX40 antigen binding molecules in a bivalent, trivalent and tetravalent FAP-targeted cross-Fab format alone and with hyper-crosslinking of the molecules by either a secondary antibody or a FAP⁺ fibroblast cell line. The crosslinking of FAP-binding antibodies by cell surface FAP was tested using human fibroblast activating protein (huFAP) expressing NIH/3T3-huFAP clone 19. All OX40 antigen binding molecules induced dose dependent NKκB activation. The tetravalent and trivalent use of OX40 antibodies induced a certain NFκB activation due to the assembly of the trimeric core OX40 receptor-signaling unit already in the absence of further external crosslinking of the constructs. OX40 antigen binding molecules with bivalent format (2+1) showed accordingly less bioactivity. Additional crosslinking by human FAP expressing fibroblasts via the FAP binding moiety, or by a secondary crosslinking antibody via the Fc region of the OX40 antigen binding molecule further increased the NFκB activation of all antigen binding molecules. For low-nanomolar bivalent OX40 antibodies (49B4, CLC-563), a higher valency of the OX40 resulted also with respect to bioactivity in an avidity gain (4+1 > 3+1 > 2+1), whereas no benefit of a 3+1 over 2+1 format was observed for sub-nanomolar bivalent OX40 binders (8H9, MOXR0916). All amino acid variants of 49B4 in a 4+1 format induced dose dependent NKκB activation to a similar extent than the parental antibody in the 4+1 format.
EC₅₀ values of NFκB induction dose response curves w/o further crosslinking and w/ crosslinking by cell surface human FAP (NIH/3T3 huFAP clone 19) are summarized in Table 8.

TABLE 8

EC₅₀ values for dose responses of NFκB activation by OX40 antigen binding molecules in a FAP targeted format in the presence or absence of cell surface human FAP⁺ fibroblasts
Molecule ID	anti-Ox40 clone	Format	w/ hu FAP	w/o	AUC compared	w/ hu FAP	w/o
Molecule ID	anti-Ox40 clone	Format	EC50 [nM]		to	% of AUC
P1AD3690	49B4	4+0	0.18	0.23	49B4 4+0	-	-
P1AE6838	49B4	4+1	0.04	0.38		225	89
P1AE9167	49B4 AA variant K73E	4+1	0.04	0.57		214	75
P1AE9169	49B4 AA variant K23T_K73E	4+1	0.04	0.39		221	84
P1AE9176	49B4 AA variant K23E_ K73E	4+1	0.04	0.45		206	78
P1AE8786	49B4	3+1	0.20	0.83	49B4 4+1	100	29
P1AE6840	49B4	2+1	0.12	-		77	17
P1AE8873	8H9	3+1	0.14	0.44	8H9 3+1	-	-
P1AE8870	8H9	2+1	0.21	-		75	76
P1AE8875	MOXR0916	3+1	0.31	3.94	MOXR09 16 3+1	-	-
P1AE8872	MOXR0916	2+1	0.19	-		73	no curve
P1AE8874	CLC563	3+1	0.24	0.79	CLC563 3+1	-	-
P1AE8871	CLC563	2+1	0.21	-		63	27

In another experiment, adherent HeLa_hOX40_NFkB_Luc1 cells were cultured over night at a cell density of 0.3 × 10⁵ cells per well and were stimulated for 6 hours with assay medium containing titrated anti-OX40 antigen binding molecules. To test the effect of hyper-crosslinking by cell surface FAP binding, 25 µL/well of medium containing FAP+ tumor cells (NIH/3T3-huFAP clone 19) were co-cultured in a 3 to 1 ratio (three times more FAP+ tumor cells than reporter cells per well).
After incubation, assay supernatant was aspirated and plates washed two times with DPBS. Quantification of light emission was done using the luciferase 1000 assay system and the reporter lysis buffer (both Promega, Cat.-No. E4550 and Cat-No: E3971) according to manufacturer instructions. Briefly, cells were lysed for 30 minutes on dry ice by addition of 30 µL per well 1x lysis buffer. Cells were thawed for 20 minutes at 37° C. before 100 µL per well provided luciferase assay reagent was added. Light emission was quantified immediately with a Spark10M Tecan microplate reader using 500 ms integration time, without any filter to collect all wavelengths. Emitted relative light units (URL) were corrected by basal luminescence of HeLa _hOx40_NFkB_Luc1 cells and were plotted against the logarithmic primary antibody concentration using Prism7 (GraphPad Software, USA). Curves were fitted using the inbuilt sigmoidal dose response.
Thus, we tested the NFκB activating capacity of selected bispecific OX40 antigen binding molecules comprising clones OX40(49B4_K23E_K73E) or OX40(CLC563) in 3+1 and 4+1 formats as D and S variant alone and with hyper-crosslinking of the molecules by FAP+fibroblast cell line. The results are shown in FIGS. 8A to 8F. All OX40 antigen binding molecules induced dose dependent NKκB activation. The tetravalent and trivalent use of OX40 antibodies induced a certain NFκB activation due to the assembly of the trimeric core OX40 receptor-signaling unit already in the absence of further external crosslinking of the constructs (FIGS. 8B, 8D and 8F). Additional crosslinking by human FAP expressing fibroblasts via the FAP binding moiety further increased the NFκB activation of all antigen binding molecules (FIGS. 8A, 8C and 8E). The bioactivity of the D and S variant was of comparable strength.
EC₅₀ values of NFκB induction dose response curves without further crosslinking and with crosslinking by cell surface human FAP (NIH/3T3 huFAP clone 19) are summarized in Table 9.

TABLE 9

EC₅₀ values for dose responses of NFκB activation by OX40 antigen binding molecules (D and S variants) in a FAP targeted format in the presence or absence of cell surface human FAP⁺ fibroblasts
Molecule ID	anti-Ox40 clone	Variant	Format	w/ hu FAP	w/o
Molecule ID	anti-Ox40 clone	Variant	Format	EC₅₀ [nM]
P1AF6455	CLC-563	S	3+1	0.2	0.8
P1AF6454	CLC-563	D	3+1	0.2	1.9
P1AF7217	CLC-563	S	4+1	0.18	0.7
P1AF7205	CLC-563	D	4+1	0.2	0.5
P1AF6457	49B4 AA variant K23E_ K73E	S	4+1	0.02	0.06
P1AF6456	49B4 AA variant K23E_ K73E	D	4+1	0.03	0.08

3.2 OX40 Mediated Co-Stimulation of Sub-Optimally TCR Triggered Resting human PBMCs and Hyper-Crosslinking by Cell Surface FAP

It was shown in Example 4.1 that addition of FAP⁺ tumor cells can strongly increase the NFκB activity induced by FAP targeted OX40 antigen binding molecules in human OX40 positive reporter cell lines by providing strong oligomerization of OX40 receptors. Likewise, we tested all constructs in a primary T cell assay for their ability to rescue suboptimal TCR stimulation of resting human PBMC cells in the presence of NIH/3T3-huFAP clone 19 cells.
Human PBMC preparations contain (1) resting OX40 negative CD4⁺ and CD8⁺ T cells and (2) antigen presenting cells with various Fc- γ receptor molecules on their cell surface e.g. B cells and monocytes. Anti-human CD3 antibody of human IgG1 isotype can bind with its Fc part to the present Fc-y receptor molecules and mediate a prolonged TCR activation on resting OX40 negative CD4 and CD8 T cells. These cells then start to express OX40 within several hours. Functional agonistic compounds against OX40 can signal via the OX40 receptor present on activated CD8⁺ and CD4⁺ T cells and support TCR-mediated stimulation.
Resting human PBMC were stimulated for four days with a suboptimal concentration of anti-CD3 antibody in the presence of irradiated FAP⁺ NIH/3T3-huFAP clone 19 cells and titrated bispecific OX40 antigen binding molecules. Effects on T-cell survival and proliferation were analyzed through monitoring of total cell counts (CD4⁺ or CD8⁺ T cells) and co-staining with fluorescently-labeled antibodies against T-cell activation marker (CD25 expression on CD4⁺ T cells) by flow cytometry. Mouse embryonic fibroblast NIH/3T3-huFAP clone 19 cells were harvested using cell dissociation buffer (Invitrogen, Cat.-No. 13151-014) for 10 minutes at 37° C. Cells were washed once with DPBS. NIH/3T3-huFAP clone 19 cells were irradiated in an xRay irradiator using a dose of 4500 RAD to prevent later overgrowth of human PBMCs by the tumor cell line. Irradiated cells were cultured at a density of 0.2 × 10⁵ cells per well in T cell media in a sterile 96-well round bottom adhesion tissue culture plate (TPP, Cat. No 92097) over night at 37° C./5% CO₂.
Human PBMCs were isolated from fresh blood by ficoll density centrifugation. Cells were added to each well at a density of 0.6 × 10⁵ cells per well. Anti-human CD3 antibody (clone V9, human IgG1) at a final concentration of [10 nM] and FAP-targeted OX40 antigen binding molecules were added at the indicated concentrations. Cells were incubated for four days at 37° C./5% CO₂ prior to analysis.
Cells were first stained 10 min with Zombie Aqua Fixable viability dye (Biolegend, Cat.No. 423102) in DPBS, followed by surface staining with fluorescent dye-conjugated antibodies anti-human CD4 (clone RPA-T4, BioLegend, Cat.-No. 300532), CD8 (clone RPa-T8, BioLegend, Cat.-No. 3010441) and CD25 (clone M-A251, BioLegend, Cat.-No. 356112) for 20 min at 4° C. Cell pellets were washed twice with FACS buffer. Samples were finally resuspended in 20 µL/well FACS-buffer and acquired the same day using iQue Cell Screener and ForeCyt software (Sartorius). Table 10 summarizes the EC₅₀ values for dose responses of CD25 upregulation on CD4⁺ T cells of bispecific OX40 antigen binding molecules in a FAP targeted format following suboptimal TCR stimulation of primary human PBMCs.

TABLE 10

EC₅₀ values for dose responses of CD25 upregulation on CD4⁺ T cells following suboptimal TCR stimulation of primary human PBMCs
MoleculeID	anti-Ox40 clone	Format	EC₅₀ [nM]		AUC compared to reference compound
MoleculeID	anti-Ox40 clone	Format	EC₅₀ [nM]		[%]	+/- SEM
P1AD3690	49B4
	4+0	no curve fit		13	5
P1AE6838	49B4		4+1	0.02/0.003		107	31
P1AE9167	49B4 AA variant K73E	4+1	0.001		137	9
P1AE9169	49B4 AA variant K23T_K73E	4+1	0.001		128	10
P1AE9176	49B4 AA variant K23E_ K73E	4+1	0.001		128	10
P1AE8786	49B4		3+1	0.04		97	16
P1AE6840	49B4		2+1	0.09		59	22
P1AE8873	8H9		3+1	0.01		134	31
P1AE8870	8H9		2+1	0.01		113	32
P1AE8875	MOX0916	3+1	0.02		100	18
P1AE8872	MOX0916		2+1	0.01		106	22
P1AE8874	CLC-563	3+1	0.04		100	0
P1AE8871	CLC-563	2+1	0.06		99	15

Co-stimulation with non-targeted anti-OX40 (49B4) 4+0 format barely rescued sub-optimally TCR stimulated CD4 and CD8 T cells. Hyper-crosslinking of the FAP targeted tetravalent, trivalent and bivalent OX40 antigen binding molecules in the presence of NIH/3T3-huFAP clone 19 cells strongly promoted survival and induced an enhanced activated phenotype in human CD4 T cells for all OX40 clones tested. All amino acid variants of 49B4 in a 4+1 format supported T cell activation to an at least similar, if not even slightly improved extent than the parental antibody in the 4+1 format.
In a further experiment testing the C-terminal S and D variants, human PBMCs were labeled with the CFSE proliferation kit (Thermo Fisher, Cat-No. C34554) for 10 minutes at room temperature according to manufacturer’s instruction at a final concentration of 0.2 [µM] CFSE. Cells were added to each well at a density of 0.6 × 105 cells per well in T cell media. Anti-human CD3 antibody (clone V9, human IgG1) at a final concentration of [10 nM] and the FAP-targeted OX40 antigen binding molecules, both prepared in T cell media, were added at the indicated concentrations. Cells were incubated for four days at 37° C./5% CO₂ prior to analysis.
Cells were first stained 20 min with LIVE/DEAD™ Fixable Aqua Dead Cell Stain (Molecular probes, Cat.No. L34957) in DPBS, followed by one washing step (200 µL 4° C. FACS buffer). Thereafter, surface staining with fluorescent dye-conjugated antibodies anti-human CD4 (clone OKT4, BioLegend, Cat.-No. 317440), CD8 (clone SK-1, BioLegend, Cat.-No. 344714) and CD25 (clone BC96, BioLegend, Cat.-No. 302626) for 30 min at 4° C. Cell pellets were washed twice with dPBS. Samples were finally resuspended in 100 µL/well FACS-buffer and acquired the same day using with BD Fortessa running FACS Diva software. Table 11 summarizes the EC₅₀ values for dose responses of CD25 upregulation on CD4+ T cells of bispecific OX40 antigen binding molecules in a FAP targeted format following suboptimal TCR stimulation of primary human PBMCs.

TABLE 11

EC₅₀ values for dose responses of CD25 upregulation on CD4⁺ T cells following suboptimal TCR stimulation of primary human PBMCs
Molecule ID	anti-Ox40 clone	Variant	Format	EC₅₀ [nM]
P1AF6455	CLC-563	S	3+1	0.003
P1AF6454	CLC-563	D	3+1	0.001
P1AF7217	CLC-563	S	4+1	0.002
P1AF7205	CLC-563	D	4+1	0.001
P1AF6457	49B4 AA variant K23E_ K73E	S		4+1	0.001
P1AF6456	49B4 AA variant K23E_ K73E	D		4+1	0.001

As shown in FIGS. 9A to 9F, co-stimulation with a non-targeted tetravalent anti-OX40 (49B4) (4+0 format) rescued sub-optimally TCR stimulated CD4 and CD8 T cells only at concentrations higher than [1nM]. Hyper-crosslinking of the FAP targeted tetravalent and trivalent OX40 antigen binding molecules in the presence of NIH/3T3-huFAP clone 19 cells strongly promoted proliferation and induced an enhanced activated phenotype in human CD4 (FIGS. 9A, 9C and 9E) and CD8 (FIGS. 9B, 9D and 9F). The S variant performed slightly, but not statistically significant, worse than the D variant for the CLC563 constructs (FIGS. 9A to 9D). All amino acid variants of 49B4 in a 4+1 format supported T cell activation to an at least similar, if not even slightly improved extent than the parental antibody in the 4+1 format. Here, no difference was seen between the S and D variant (FIGS. 9E and 9F).

3.3 OX40 Mediated Co-Stimulation Increases the Secretion of Cytokines of CECAM5 TCB Redirected PBMC That Lyse CEA+ Tumor Cells

One clinically exploited way to recruit the patient’s own immune system to fight cancer are T cell bispecific antibodies (TCB). These molecules are comprised of an agonistic anti-CD3 unit, specific for the T cell receptor (TCR) on T cells, and a targeting moiety specific for a unique cancer antigen. TCBs redirect polyclonal T cells to lyse cancer cells expressing the respective target antigen on their cell surface. No T cell activation occurs in the absence of such target antigen. The TCB used in this example is the CEACAM5 TCB targeting the carcinoembryonic antigen (CEA) and is described in detail in WO 2016/079076 A1. Triggering of the TCR increases, depending on the strength and duration of this primary stimulus, the expression of costimulatory molecules, e.g. OX40, a member of the Tumor necrosis factor receptor (TNFR) superfamily. Concomitant agonistic ligation of this receptor by its respective ligand promotes in turn hallmark T cell effector functions like proliferation, survival and secretion of certain proinflammatory cytokines (GM-CSF, IFN-y, IL-2, TNF-α) (M. Croft et al., Immunol. Rev. 2009, 229(1), 173-191, I. Gramaglia et al., J. Immunol. 1998, 161(12), 6510-6517; S. M. Jensen et al., Seminars in Oncology 2010, 37(5), 524-532). This co-stimulation is needed to raise the full potential of T cells against tumor cells, especially in the context of weak tumor antigen priming, and to sustain the anti-tumor response beyond the first attack allowing for protective memory formation. In certain patients with a strong immune suppressed or exhausted phenotype, only the combination of polyclonal, yet tumor specific T cell recruitment (signal 1) and the restoration of tumor-restricted positive co-stimulation (signal 2) might facilitate sufficient anti-tumor efficacy and prolonged adaptive immune protection. This can persistently drive the tumor microenvironment towards a more immune activating and less immunosuppressive state. FAP-dependent costimulation of OX40 may also facilitate TCB mediated killing of tumor cells at lower intratumoral concentrations which would allow reduction of systemic exposure and correlated side effects. Additionally, the treatment intervals might be prolonged as lower TCB concentration could still be active.
MKN45 NucLight Red (NLR) cells, which naturally express the CEA antigen were used as target cells. MKN-45 (DSMZ; ACC409) were transduced with the Essen CellPlayer NucLight Red Lentivirus Reagent (Essenbioscience, Cat. No. 4476; EF1α, puromycin) at an MOI of 5 (TU/cell) in the presence of 8 µg/mL polybrene following the manufacturer’s instructions to stable express a nuclear-restricted NucLight Red fluorescent protein. This enabled easy separation from non-fluorescent effector T cells or fibroblasts and monitoring of the tumor cell growth by high throughput life fluorescence microscopy.
The crosslinking of FAP-binding antibodies by cell surface FAP was provided by human fibroblast activating protein (huFAP) expressing NIH/3T3-huFAP clone 19 (see Example 3.2). Human PBMCs were isolated from fresh blood by ficoll density centrifugation (see Example 3.1).
MKN45 NucLight Red (NLR) cells were added in each well at a density of 0.1 × 10⁵ cells per well in T cell media. NIH/3T3-huFAP clone 19 were pre-irradiated at 4600 RAD and were then added in each well at a density of 0.1 × 10⁵ cells per well in T cell media. Human PBMC were added to each well at a density of 0.5 × 10⁵ cells per well in T cell media. The CEACAM5 TCB at a final concentration of 2 nM and titrated dilutions of FAP-targeted OX40 antigen binding molecules, both prepared in T cell media, were added at the indicated concentrations. Cells were incubated for three days at 37° C./5% CO₂ prior to analysis. Samples were performed as triplicates.
After 72 hours, the supernatant was collected for subsequent analysis of selected cytokine using the Bio-Plex Pro Human Cytokine 8-Plex Assay Catalogue No. BIO-RAD M50000007A according to manufacturer’s instructions. Triplicate samples were united to equal parts for each tested compound at the respective tested concentration and the mix was analyzed for the cytokine content. The fold increase of a respective cytokine (GM-CSF, IL-2, TNF-α, IFN-y) compared to the concentration in the TCB-only control sample was calculated and plotted against the FAP-OX40 antibody concentration. Dose-response curves were calculated using GraphPAD Prism, AUC and the EC₅₀ values calculated and are reported in Table 24. Dose response curves for GM-CSF and TNF-α are depicted in FIGS. 10A to 10F, and that for IFN-y and IL-2 in FIGS. 11A to 11F. The AUC was normalized for each cytokine to the AUC of the FAP-OX40 antigen binding molecule OX40(CLC563) x FAP(1G1a_EPKSCD) 3+1 (called 3+1 CLC563/H212 -D in the Figure) and plotted for each compound as Box-Whisker plot in FIG. 12 .

TABLE 12

EC₅₀ values for dose responses of increased TCB mediated cytokine secretion following FAP-OX40 costimulation of primary human PBMCs
Molecule ID	anti-Ox40 clone	Variant	Format	EC₅₀ [nM]
P1AF6455	CLC-563	S	3+1	0.058
P1AF6454	CLC-563	D	3+1	0.144
P1AF7217	CLC-563	S	4+1	0.058
P1AF7205	CLC-563	D	4+1	0.034
P1AF6457	49B4 AA variant K23E_ K73E	S		4+1	0.022
P1AF6456	49B4 AA variant K23E_ K73E	D		4+1	0.017

As shown in FIGS. 10A to 10F and FIGS. 11A to 11F, co-stimulation with non-targeted anti-OX40 (49B4) 4+0 format did not enhance the cytokine secretion of PBMC induced by CEACAM5 TCB mediated lysis of tumor cells. Hyper-crosslinking of the FAP targeted tetravalent and trivalent OX40 antigen binding molecules in the presence of NIH/3T3-huFAP clone 19 cells strongly promoted the secretion of GM-CSF (FIGS. 10A, 10C and 10E) and of TNF-α (FIGS. 10B, 10D and 10F), as well as that of IFN-y (FIGS. 11A, 11C and 11E) and of IL-2 (FIGS. 11B, 11D and 11F). The S-variant performed slightly, but not statistically significant, worse than the D-variant for all tested constructs (FIG. 12 ). The amino acid variants of 49B4 in a 4+1 format supported T cell activation stronger than the parental clone in the 4+1 format. Here, the CLC563 showed stronger agonistic potential as tetra than as trivalent FAP targeted OX40 agonist (FIG. 12 ).

3.4 OX40 Mediated Co-Stimulation Reduces TGFβ Induced FoxP3 Expression

CD4+Foxp3+ T regulatory cells (Tregs) play a critical role in immune homeostasis and peripheral tolerance (Sakaguchi S, Yamaguchi T, Nomura T, Ono M, Cell 2008, 133(5), 775-87). Their development, lineage stability, and suppressor functions are dependent on the expression of the transcription factor FoxP3, which is a “master” regulator of Treg identity (Hori S, Nomura T, Sakaguchi S, Science 2003, 299(5609), 1057-61). In addition to their thymic origin, CD4+FoxP3+ Treg cells can also be induced in the periphery from naive CD4+ T cells following activation, which are often called inducible Tregs (iTregs) or peripheral Tregs (pTregs) (Curotto de Lafaille MA, Lafaille J, Immunity 2009, 30(5), 626-35). The best characterized conditions for the induction of iTregs in vitro is the combination of transforming growth factor β (TGF-β) and CD28 costimulation. This cytokine potently induces de novo FoxP3 expression, which programs the conversion of activated conventional T cells to iTregs (Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM, J Exp. Med. 2003, 198(12), 1875-86). OX40 signaling has been described to inhibit FoxP3 expression and Treg induction (Zhang X, Xiao X, Lan P, et al., Cell Rep. 2018, 24(3), 607-618). It was shown in examples 4.1 to 4.3 that all FAP-OX40 bispecific antigen binding molecules were able to induce NFκB, and promote TCR stimulation resulting in enhanced activation phenotype and increased cytokine secretion. Likewise, we tested all constructs in a primary T cell assay for their ability to suppress TGF-β mediated FoxP3 induction.
Human PBMC preparations containing naive CD4 T cells were cultured in the presence of TGFβ during T cell activation with antibodies against CD28 and CD3. FoxP3 induction as well as OX40 expression occurs within several hours. Functional agonistic compounds against OX40, e.g. bispecific antigen binding molecules comprising OX40 clones OX40(49B4_K23E_K73E) or OX40(CLC563) n 3+1 and 4+1 formats as D- and S-variant, can signal via the OX40 receptor present on the activated CD4+ T cells, when crosslinking by FAP is provided (here FAP antigen coated to beads). This interferes with Treg induction visible by reduced FoxP3 expression.
Sterile 96-well round bottom adhesion tissue culture plates (TPP, Cat. No 92097) were pre-coated with anti-human CD3 antibodies (eBioscience, Cat.No. 16-0037-85) in dPBS at a concentration of 3 µg/mL for two hours 37° C./5% CO₂. Dynabeads® M-280 Streptavidin (ThermoFisher, Cat. No. 11205D) were coated with biotinylated human FAP antigen (Roche, P1AD8986; 0.01 µg protein for 1 µg beads) in dPBS for 30 minutes at room temperature according to manufacturer’s instructions before storage at 4° C. for long-term in dPBS containing 0.1% (w/v) BSA. Human PBMCs were isolated from fresh blood by ficoll density centrifugation. Cells were labeled with the CFSE proliferation kit (Thermo Fisher, Cat-No. C34554) for 10 minutes at room temperature according to manufacturer’s instruction at a final concentration of 0.4 µM CFSE. Cells were then added to each well of the pre-coated plates at a density of 1 × 10⁵ cells per well in serum-free X-Vivo15 medium (Lonza, Cat. No. BE02-054Q). Recombinant human TGF-β (2 ng/mL, R&D Systems, Cat.No. 240-B-010), monoclonal anti-human CD28 (1 µg/mL, eBioscience, Cat.No. 16-0289-85) and FAP coated beads (2 × 10⁵ cells per well) were added. Titrated dilutions of FAP-targeted OX40 antigen binding molecules were added at the indicated concentrations. Cells were incubated for three days at 37° C./5% CO₂ prior to analysis.
Cells were first stained 10 min with LIVE/DEAD™ Fixable Aqua Dead Cell Stain (Molecular probes, Cat.No. L34957) in DPBS, followed by one washing step (200 µL 4° C. FACS buffer). Thereafter, surface staining with fluorescent dye-conjugated antibodies anti-human CD4 (clone RPA-T4, BioLegend, Cat.-No. 300532), CD8 (clone RPA-T8, BioLegend, Cat.-No. 301042) and CD25 (clone BC96, BioLegend, Cat.-No. 302610) for 30 min at 4° C. Cell pellets were washed twice with FACS buffer, before cells were fixed and permeabilized in FoxP3 Fixation/Permeabilization working solution (FoxP3 staining Kit, eBioscience, Cat.No. 00-5521) for 60 minutes at room temperature in the dark according to manufacturer’s instructions. After washing twice with 1x Perm buffer solution (FoxP3 staining Kit, eBioscience, Cat.No. 00-5521), cells were stained with fluorescent dye-conjugated antibody against FoxP3 (clone 29D6, BioLegend, Cat.-No. 20108) in 1x Perm buffer for 40 minutes at room temperature in the dark. Cell pellets were washed twice with 1x Perm buffer and finally resuspended in 100 µL/well FACS-buffer and acquired the same day using with BD Fortessa running FACS Diva software. Alive CD4+ CD25+Treg singlet cells were gated and the MFI of the αFoxP3 antibody reported. The FoxP3 MFI of each concentration was corrected by the MFI of the sample without OX40 bispecific antibody, thus only TGBβ, present.
FIGS. 13A to 13C show that FAP-OX40 bispecific antigen bispecific antigen binding molecules suppressed FoxP3 induction on resting CD4 T cells activated in the presence of TGFβ in a dose dependent manner. The D- and S-variant of each FAP-OX40 bispecific antibody showed similar bioactivity properties. In Table 13 the EC₅₀ values were summarized for the FAP-OX40 agonists dose-responses of FoxP3 suppression (FoxP3 MFI) on CD4+ CD25+Treg cells.

TABLE 13

EC₅₀ values of FAP-OX40 suppressed FoxP3 induction on TGFβ-exposed resting CD4 T cells
Molecule ID	anti-Ox40 clone	Variant	Format	EC₅₀ [nM]
P1AF6455	CLC-563	S	3+1	0.25
P1AF6454	CLC-563	D	3+1	0.11
P1AF7217	CLC-563	S	4+1	0.01
P1AF7205	CLC-563	D	4+1	0.02
P1AF6457	49B4 AA variant K23E_ K73E	S		4+1	0.01
P1AF6456	49B4 AA variant K23E_ K73E	D		4+1	0.02

Example 4

Generation and Production of Bispecific Antigen Binding Molecules Targeting GITR and Fibroblast Activation Protein (FAP)

4.1 Generation and Production of Bispecific Antigen Binding Molecules targeting GITR and Fibroblast Activation Protein (FAP)

The cDNAs encoding variable heavy and light chain domains of anti GITR antibody 852.2 (generated by rabbit immunization with human GITR, SEQ ID NO:26 and cynomolgus GITR, SEQ ID NO:27) as well as anti-FAP antibody 4B9 (as described in WO 2012/020006 A1) were cloned in frame with the corresponding constant heavy or light chains of human IgG1 in suitable expression plasmids. Expression of heavy and light chain is driven by a CMV promoter. The expression cassette also contains a synthetic polyA signal at the 3′ end of the cDNAs.
Bispecific GITR-FAP antibodies were prepared in 2+1 format consisting of two GITR binding moieties combined with one FAP binding arm at the C-terminus of an Fc (in accordance with FIG. 1A). To generate the 2+1 antigen binding molecules the knob-into-hole technology was used to achieve heterodimerization. The S354C/T366W mutations were introduced in the first heavy chain HC1 (Fc knob heavy chain) and the Y349C/T366S/L368A/Y407V mutations were introduced in the second heavy chain HC2 (Fc hole heavy chain). In the 2+1 antigen binding molecules the CrossMab technology as described in WO 2010/145792 A1 ensured correct light chain pairing. Independent of the bispecific format, in all cases an effector silent Fc domain (P329G; L234A, L235A) was used to abrogate binding to Fcy receptors according to the method described in WO 2012/130831 A1. Sequences of the bispecific molecules are shown in Table 14 below.

TABLE 14

Amino acid sequences of the bispecific antigen binding molecules
Molecule	Sequence	Seq ID No
P1AE1116 GITR(852.2) x FAP (4B9) (2+1) C-terminal crossfab fusion
GITR(852.2) VHCH1- Fc knob_PGLALA-FAP(4B9) VHCL	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMKWVRQTPGKGLEWVSVISADGGTTDHAASVKGRFTVSRDNSKNMLYLQMNSLRAEDTAIYYCAKDRANDWITGASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	153
GITR(852.2) light chain	DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGNAPKLLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDLATYYCQQARSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	154
FAP(4B9) VLCH1-light chain	EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC	120
GITR(852.2) VHCH1- Fc hole_PGLALA	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMKWVRQTPGKGLEWVSVISADGGTTDHAASVKGRFTVSRDNSKNMLYLQMNSLRAEDTAIYYCAKDRANDWITGASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	155

P1AG1036 GITR(852.2) x FAP (4B9_ERPKSCS) (2+1) C-terminal crossfab fusion
GITR(852.2) VHCH1- Fc knob_PGLALA-FAP(4B9) VHCL	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMKWVRQTPGKGLEWVSVISADGGTTDHAASVKGRFTVSRDNSKNMLYLQMNSLRAEDTAIYYCAKDRANDWITGASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	153
GITR(852.2) light chain	DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGNAPKLLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDLATYYCQQARSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	154
FAP(4B9) VLCH1-light chain (EPKSCS)	EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCS	156
GITR(852.2) VHCH1- Fc hole_PGLALA	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMKWVRQTPGKGLEWVSVISADGGTTDHAASVKGRFTVSRDNSKNMLYLQMNSLRAEDTAIYYCAKDRANDWITGASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	155

P1AG1039 GITR(852.2) x FAP (4B9_ERPKSCG) (2+1) C-terminal crossfab fusion
GITR(852.2) VHCH1- Fc knob_PGLALA-FAP(4B9) VHCL	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMKWVRQTPGKGLEWVSVISADGGTTDHAASVKGRFTVSRDNSKNMLYLQMNSLRAEDTAIYYCAKDRANDWITGASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	153
GITR(852.2) light chain	DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGNAPKLLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDLATYYCQQARSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	154
FAP(4B9) VLCH1-light chain (EPKSCG)	EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCG	157
GITR(852.2) VHCH1- Fc hole_PGLALA	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMKWVRQTPGKGLEWVSVISADGGTTDHAASVKGRFTVSRDNSKNMLYLQMNSLRAEDTAIYYCAKDRANDWITGASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP	155

Antibody constructs were expressed by transient transfection of HEK cells grown in suspension with expression vectors encoding the 4 different peptide chains. Transfection of Expi293F™ cells (Gibco™) was performed according to the cell supplier’s instructions using Maxiprep (Macherey-Nagel) preparations of the antibody vectors, Expi293F™ Expression Medium (Gibco™), ExpiFectamine™ 293 Reagent, (Gibco™) and an initial cell density of 2-3 million viable cells/ml in Opti-MEM® 1x Reduced Serum Medium (Gibco®). On the day after transfection (Day 1, 18-22 hours post-transfection), ExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293 Transfection Enhancer 2 was added to the transfected culture. Transfected cultures were incubated at 37° C. in a humidified atmosphere of 8% CO₂ with shaking. Cell culture supernatants were harvested after 7 days of cultivation in shake flasks or stirred fermenters by centrifugation at 3000-5000 g for 20-30 minutes and filtered through a 0.22 µm filter.
Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were captured on a MabSelect SuRe resin equilibrated with PBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 100 mM sodium acetate, pH 3.0. After neutralization with 1 M Tris pH 9.0, aggregated protein was separated from monomeric antibody species by ion exchange Chromatography (Poros XS) with equilibration buffer 20 mM His, pH 5.5, 1.47 mS/cm and elution buffer 20 mM His, 500 mM NaCl, pH 5.5, 49.1 mS/cm, (gradient: to 100% elution buffer in 60 CV). In some cases a size exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM histidine, 140 mM NaCl, pH 6.0, was subsequently performed. Monomeric protein fractions were pooled, and if required concentrated using e.g. a MILLIPORE Amicon Ultra (30KD MWCO) centrifugal concentrator. Purified proteins were stored at -80° C. Protein quantification was performed using a Nanodrop spectrophotometer and analyzed by CE-SDS under denaturing and reducing conditions and analytical SEC. Sample aliquots were used for subsequent analytical characterization e.g. by CE-SDS, size exclusion chromatography, mass spectrometry and endotoxin determination..
Purity and molecular weight of the bispecific antigen binding molecule after the final purification step were analyzed by CE-SDS analyses in the presence and absence of a reducing agent. The Caliper LabChip GXII system (Caliper Lifescience) was used according to the manufacturer’s instruction.
The aggregate content of the bispecific antigen binding molecule was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM potassium phosphate, 125 mM sodium chloride, 200 mM L-arginine monohydrocloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C.

TABLE 15

Production yield and Quality of bispecific GITR antigen binding molecules
Molecule	Monomer [%]	CE-SDS (non-reduced) [%]	Yield [mg/l]
P1AE1116 GITR(852.2) × FAP (4B9) (2+1) C-terminal crossfab fusion	82	77	18.0
P1AG1036 GITR(852.2) × FAP (4B9_EPKSCS) (2+1) C-terminal crossfab fusion	99	92	10.7
P1AG1039 GITR(852.2) × FAP (4B9_EPKSCG) (2+1) C-terminal crossfab fusion	99	94	6.0

4.2 Characterization of Bispecific Antigen Binding Molecules Targeting GITR and FAP

4.2.1 Simultaneous Binding of Bispecific GITR Antigen Binding Molecules to GITR and FAP

The capacity of binding simultaneously human GITR Fc(kih) and human FAP can be assessed by surface plasmon resonance (SPR). All SPR experiments are performed on a Biacore T200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany). Biotinylated human GITR Fc (kih) is directly coupled to a flow cell of a streptavidin (SA) sensor chip. Immobilization levels up to 1000 resonance units (RU) are used. The bispecific antibodies targeting GITR and FAP are passed at a concentration range of 200 nM with a flow of 30 µL/minute through the flow cells over 90 seconds and dissociation was set to zero sec. Human FAP is injected as second analyte with a flow of 30 µL/minute through the flow cells over 90 seconds at a concentration of 500 nM. The dissociation is monitored for 120 sec. Bulk refractive index differences are corrected for by subtracting the response obtained in a reference flow cell, where no protein is immobilized. All bispecific constructs can bind simultaneously human GITR and human FAP.

4.2.2 Binding to GITR-Expressing RPMI-8226 Cells, HEK Cells, Activated Human T Cells or FAP-Expressing 3T3 Cells

The binding to cell surface GITR was tested by using various cells. RPMI-8226 (ATCC® CCL-155™) were isolated from human myeloma patient and these cells endogenously express high levels of GITR at the cell surface (Liu et al., Novel Tumor Suppressor Function of Glucocorticoid-Induced TNF Receptor GITR in Multiple Myeloma, 2013, PLoS ONE 8(6): e66982). Cell lines were engineered by gene knock-out using a CRISPR/CAS9 kit (CAS9 and gRNA expression vectors) provided by GenScript to obtain a control cell line RPMI-8226 GITR^-/- without GITR expression according to the manufacturer’s instructions. In brief, lentiviral based vectors containing CAS9 and GITR specific gRNAs were used to transduce RPMI-8226 cells using polybrene. After selection cell pools were sorted for GFP and GITR expression and GFP+/GITR- cells were selected. Single clones were generated from these pools and analyzed by FACS for GITR expression. From clones that were negatively tested for GITR expression genomic DNA was isolated, the GITR locus was amplified and sequenced. RPMI-8226 GITR^+/+ and GITR^-/- cell lines were grown in RPMI1640 Medium GlutaMAX Supplement HEPES (Thermo Fischer, Cat.No. 72400-054) supplemented with 10 % FBS (Thermo Fischer, Cat.No. 16140-071), 1 mM sodium-pyruvate (Thermo Fischer, Cat.No. 11360-088) and 50 U/mL penicillin-streptomycin (Thermo Fischer, Cat.No. 15070-063).
HEK293T (ATCC CRL11268) cell lines overexpressing human or cyno GITR/TNFRSF18 were generated by transduction with virus-like particles (VLP). Lentivirus-based virus-like particles were produced by co-transfection of HEK293T cells with ViraSafe™ Lentiviral Packaging plasmids (Cell Biolabs) and lentiviral expression vectors coding for either human or cyno GITR/TNFRSF18. Plasmid transfections into HEK293T cells were performed with Lipofectamine LTX (Invitrogen, #15338100) according the manufacturer’s instructions. Transfections were done in 6-well plates seeded with 6 × 10⁵ cells/well the day before transfection and 2.5 µg of plasmid DNA. Each transfection contained 0.4 µg of pRSV-Rev packaging vector, 0.4 µg of pCgpV packaging vector, 0.4 µg of pCMV-VSV-G envelop vector, and 1.3 µg of either human or cyno GITR/TNFRSF18 expression vector. The VLP-containing supernatant was collected after 48 h and filtered through 0.45 µm pore-sized polyethersulfone membrane. To generate stable GITR/TNFRSF18 expressing cell lines, HEK293T cells were seeded at 1.0 × 10⁶ cells/well in 6-well plates and overlaid with 1 mL of VLP-containing supernatant. Transductions were carried out by spinoculation at 800 × g and at 32° C. for 30 min in an Eppendorf centrifuge 5810 table-top centrifuge (Eppendorf). Viral supernatant was exchanged for fresh media 12 h after spinoculation. Three days after transduction, puromycin was added to 1 µg/mL and the cells were cultured for several passages. After initial selection, the cells with the highest cell surface expression of GITR/TNFRSF18 were sorted by BD FACSAria III cell sorter (BD Biosciences) and cultured to establish stable cell clones. The expression level and stability was confirmed by FACS analysis using a human/cyno cross-reactive APC-conjugated anti-GITR/TNFRSF18 antibody (eBioscience, #17-5875-42) over a period of 4 weeks. GITR-expressing HEK cells or parental GITR negative HEK cells (HEK WT) as control were grown in DMEM medium (Thermo Fischer, Cat.No. 42430-082) supplemented with 10% FBS (Thermo Fischer, Cat.No. 16140-071), 50 U/mL penicillin-streptomycin (Thermo Fischer, Cat.No. 15070-063) and GlutaMAX (Thermo Fischer, Cat.No. 35050-061) with 1 µg/mL puromycin (Thermo Fischer, Cat.No. A11138-03) for GITR-expressing cells only.
Binding to GITR on activated human T cells was performed by isolating peripheral blood mononuclear cells (PBMCs) from buffy coats (Blutspende Zurich) using standard density gradient procedure (Stemcell, Cat.No. 07851). PBMCs were resuspended in T cell medium consisting of RPMI1640 Medium GlutaMAX Supplement HEPES (Thermo Fischer, Cat.No. 72400-054) supplemented with 10 % FBS (Thermo Fischer, Cat.No. 16140-071), 1 mM sodium-pyruvate (Thermo Fischer, Cat.No. 11360-088), 1X MEM Non-Essential Amino Acids Solution (Thermo Fischer, Cat.No. 11140035), 50 µM 2-Mercaptoethanol (Thermo Fischer, Cat.No. 31350-010) and 50 U/mL penicillin-streptomycin (Thermo Fischer, Cat.No. 15070-063). PBMCs were activated 48 hours at 37° C., 5% CO₂ with 300 U/ml Proleukin (Novartis, Aldesleukin) and 2.5 ug/ml Leucoagglutinin PHA-L (Sigma, Cat.No. L4144) in T cell medium.
The binding to cell surface FAP was tested using 3T3 cells (ATCC CRL-1658) that were transfected to express human fibroblast activating protein (huFAP). 3T3 cells were grown in DMEM medium (Thermo Fischer, Cat.No. 31966047) supplemented with 10% CS (Sigma Cat.No. C8056) while 3T3-huFAP cells were grown in the same medium supplemented with 1.5 µg/mL puromycin (Thermo Fischer, Cat.No. A11138-03).
For binding experiments, adherent cells were harvested with trypsin (Thermo Fischer, Cat.No. 25300096) and washed with PBS (Thermo Fischer, Cat.No. 20012-019). Control cells (HEK WT, parental 3T3 or RPMI-8226 GITR^-/-) were labeled with 0.25 µM of CellTrace™ CFSE Cell Proliferation Kit (Thermo Fischer, Cat.No. C34554) in PBS for 7 minutes at 37° C. Cells were washed twice with respective media and once with PBS + 2% FBS before final resuspension in PBS + 2% FBS. 10⁵ control cells were mixed with 10⁵ unlabeled target cells expressing GITR or FAP in round-bottom 96-well plates (TPP, Cat.No. 92097). Plates were centrifuged at 4° C., 3 minutes at 600 × g and supernatant flicked off. Cells were resuspended in 50 µL/well of 4° C. cold PBS + 2% FBS containing various titrated antibody constructs and incubated for 60 minutes at 4° C. Plates were washed twice with cold PBS + 2% FBS to remove unbound construct. Cells were stained with Zombie Aqua Fixable Viability Kit (Biolegend Cat.No. 423102) and species-specific secondary antibody (Jackson ImmunoResearch Cat.No. 109-116-098, 115-116-071 or 111-116-144) in PBS for 30 minutes at 4° C. Plates were washed twice with PBS + 2% FBS. Cells were resuspended in 100 µL/well FACS buffer (eBioscience Cat.No. 00-4222-26) for acquisition using a 4-laser LSR II (BD Bioscience with DIVA software).

4.2.3 Functional Properties of of Bispecific GITR Antigen Binding Molecules to GITR and FAP

One approach to measure the potency of GITR agonists evaluates NFκB activation using engineered Jurkat cells expressing GITR on the cell surface which have the luciferase gene under NFκB control (Promega GITR bioassay, Cat.No. CS184003). For this, NIH/3T3-huFAP clone 19 cells and engineered Jurkat cells were seeded in 96-well flat-bottom plates (TPP Cat.No. 92096) following manufacturer’s instructions in presence of titrations of the molecules to enable FAP-mediated binding of the bispecific molecules on NIH/3T3-huFAP clone 19 cell surface and thereby to potentiate the oligomerization of human GITR on the cell surface of the Jurkat cells. This oligomerization of GITR resulted in expression of luciferase by the Jurkat cells and measurable luminescence after six hours incubation at 37° C., 5% CO₂. Luminescence was measured by adding an equal volume of Bio-Glo or One-Glo reagents (Promega, Cat.No. G7941 or E6120) to the wells and acquired on a multimode plate reader (Tecan Spark or Perkin Elmer Envision) according to manufacturer’s instructions.
Thus, we tested the NFκB activating capacity of the bispecific GITR antigen binding molecules in 2+1 formats with the different C-terminal variants. The results are shown in FIG. 14 . All GITR antigen binding molecules induced dose dependent NKκB activation. The bioactivity of the EPKSCS (SEQ ID NO: 1) and EPKSCG (SEQ ID NO: 2) variant was of comparable strength to the molecule P1AE1116 with the C-terminal end EPKSC (SEQ ID NO:6).

Example 5

Evaluation and Improvement of Preexisting Anti-Drug Antibody Reactivity

5.1 Evaluation of Preexisting Anti-Drug Antibody (ADA) Reactivity

For the evaluation of the root cause of preexisting IgG interference, an exploratory ELISA in human individual plasma samples to detect anti-drug antibodies (ADA) was performed. It uses a biotinylated anti-PGLALA antibody against the amino acid mutations L234A, L235A and P329G (“PGLALA modification”) in the Fc domain of the drug as a capture reagent and a digoxigenin labeled FcγReceptor I (CD64) as detection reagent, which binds to a human IgG with no PGLALA modification that is part of the ADA-drug complex. To compare preexisting IgG interference to different drug molecules, this assay was performed on the same panel of naive human individual plasma samples.
2 µg/mL biotinylated anti-PGLALA antibody is coated to the streptavidin coated microtiterplate in the first step. In parallel, naïve human individual plasma samples (BioIVT) were pre-incubated with a buffer containing 6.7×10^-9 mol/L of the respective drug molecule for 30 minutes at room temperature to allow the formation of the Drug-anti-drug antibody complex. After incubation of the samples and a washing step, human IgG with no PGLALA modification that is part of the immune complexes and bound to the surface can be detected with 0.5 µg/mL digoxigenin labeled FcγReceptor I (CD64). After washing, a polyclonal anti-digoxigenin-horseradish peroxidase (HRP) conjugate (50 mU/mL) was added and incubated. After another washing step and the addition of the ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) substrate solution to the microtiterplate, the HRP of the antibody enzyme conjugate catalyzes the color reaction. An ELISA reader measures the absorption at 405 nm wavelength. [(Wessels et al, Bioanalysis 2017, 9(11), 849-859).

5.2 Improvement of Preexisting Anti-Drug Antibody (ADA) Reactivity

The bispecific antigen binding molecule OX40 (49B4) x FAP (4B9) (4+1) VH/VL as described in WO 2017/060144 A1 was explored in naive human plasma samples for preexisting IgG interference. High incidence with high signals was observed as shown in FIG. 15A. Holland et al., J. Clin. Immunol. 2013, 33, 1192-1203 published that a similar VH/VL fragment as included in OX40 (49B4) x FAP (4B9) (4+1) VH/VL in another type of molecule has shown reactivity to human anti-VH autoantibodies. Therefore, it was questioned if a replacement of the VH/VL fragment by a Fab fragment carrying the FAP binder can lead to less ADA reactivity in the antigen binding molecules of the present invention.
An antigen binding molecule P1AE6836, similar to OX40 (49B4) x FAP (4B9) (4+1) VH/VL, but with a cross-Fab fragment carrying the FAP binder, was produced and investigated for preexisting IgG reactivity in the same panel of naive human plasma samples, as shown in FIGS. 15A to 15C. P1AE6836 still showed preexisting IgG Interference, with reduced signals compared to OX40 (49B4) x FAP (4B9) (4+1) VH/VL, and in different individual human samples, indicating a different type of preexisting anti-drug antibodies against the cross-Fab fragment carrying the FAP binder (FIG. 15B). A similar molecule, but comprising the humanized FAP clone 1G1a (P1AE6838, FIG. 15C), confirmed the results obtained with the molecule comprising the FAP 4B9 clone (P1AE6836), indicating that the FAP clone is not the root cause of the preexisting IgG reactivity.
As can be seen in FIG. 16 , all bispecific antigen binding molecules, sharing the FAP (1G1a) antigen binding domain, but different anti-OX40 Clones (49B4, 8H9, MOX0916 and CLC-563) showed the same preexisting IgG interference, indicating that the OX40 Clone is not the root cause of preexisting IgG reactivity.
In FIG. 17A a subset of human individual plasma samples was tested with control molecules comprising the OX40(49B4) clone (P1AD3690) (4+0), an untargeted molecule comprising four OX40(49B4) Fab fragments, a FAP(1G1a) molecule (P1AE1689) comprising the humanized FAP(1G1a) Fab fragment), and the Germline control antibody (P1AD5108, DP47). These molecules do not cause preexisting IgG reactivity and therefore show low background signals. FIG. 17B shows that all valences for OX40 (2+1, 3+1 and 4+1) result in preexisting IgG interference with a slight increasing trend of signal height 2+1>3+1>4+1, presumably due to sterical hindrance.
Literature (Kim et al, MABS 2016, 8, 1536-1547) suggests that several proteases associated with invasive diseases are able to cleave antibodies in the hinge-region, thus generating neoepitopes for anti-hinge antibodies. We thus produced molecules with different C-terminal amino acids in the CH1 domain of the Fab fragment that is fused to the C-terminus of the Fc domain and thus has a “free” hinge-like region. Whereas the original bispecific antibody has a C-terminal amino acid sequence of EPKSC (SEQ ID NO:6), variants with C-terminal amino acid sequences of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5) were produced.
To evaluate the C-terminal extension variants of antigen binding molecules OX40 (49B4) x FAP (1G1a) (3+1), the same panel of human individual serum samples was tested (FIG. 18A) and its individual background signal substracted (FIG. 18C). The individual background signal was measured by performing the assay without the drug molecule (FIG. 18B).
An extension of the C-terminus by naturally occurring aspartate at this position of the upper hinge region was generated in the molecule OX40 (MOXR0916) x FAP (1G1a) (3+1) (P1AF4845) (FIG. 19B). This modification resulted in a reduction of preexisting IgG reactivity compared to the molecule P1AE8786 with a free C-terminus (FIG. 19A). To eliminate the preexisting IgG reactivity completely, a variant with a C-terminal serine was generated. This serine is not naturally located at this position of the upper hinge region. The extension of the C-terminus by a serine of molecule P1AF4851 led to a complete elimination of the preexisting IgG reactivity, as shown in FIG. 19C.
FIGS. 20A to 20C show a respective molecule set in 2+1 format, and confirm the previous results that a C-terminal extension of an aspartate (Molecule OX40 (MOXR0916) x FAP (1G1a) (2+1) with EPKSCD (SEQ ID NO: 3) terminus, P1AF4852, FIG. 20B) reduces, while a C-terminal serine (molecule OX40 (MOXR0916) x FAP (1G1a) (2+1) with EPKSCS (SEQ ID NO: 1) terminus, P1AF4858, FIG. 20C) eliminates the reactivity with preexisting antibodies in plasma compared to a molecule OX40 (49B4) x FAP (1G1a) (2+1) with a free C-terminus EPKSC (SEQ ID NO: 6) (P1AE6840, FIG. 20A).
FIGS. 21A to 21H show a set of OX40 (MOXR0916) x FAP (1G1a) molecules with increasing C-terminal extensions. C-terminal extension of an aspartate (Molecule OX40 (MOXR0916) x FAP (1G1a) (2+1) with EPKSCD (SEQ ID NO: 3) terminus, P1AF4852, FIG. 21B) reduces the reactivity with preexisting antibodies in plasma compared to a molecule OX40 (MOXR619) x FAP (1G1a) (2+1) with a free C-terminus EPKSC (SEQ ID NO: 6) (P1AE8872, FIG. 21A), whereas slightly increased reactivity is observed for the C-terminus EPKSCDK (SEQ ID NO: 4) (molecule (MOXR619) x FAP (1G1a) (3+1), P1AF4846, FIG. 21C). High reactivity has been observed for the molecule with the C-terminus EPKSCDKT (SEQ ID NO: 164) (molecule (MOXR619) x FAP (1G1a) (3+1), P1AF4847, FIG. 21D). The reactivity is again reduced for the molecules with the C-terminus EPKSCDKTH (SEQ ID NO: 165) (molecule (MOXR619) x FAP (1G1a) (2+1), P1AF4855, FIG. 21E) and with the C-terminus EPKSCDKTHT (SEQ ID NO: 7) (molecule (MOXR619) x FAP (1G1a) (2+1), P1AF4856, FIG. 21F). Replacement of the C-terminal T with amino acid L, which is not naturally occurring at this position in the upper hinge region, completely eliminates the reactivity with preexisting antibodies in plasma (molecule (MOXR619) x FAP (1G1a) (2+1), P1AF4857, FIG. 21G), comparable with a C-terminal serine (molecule OX40 (MOXR0916) x FAP (1G1a) (2+1) with EPKSCS (SEQ ID NO: 1) terminus, P1AF4858, FIG. 21H). FIG. 22 summarizes the results.
As can be seen in FIGS. 23A to 23F, three further molecule examples strengthen the finding that the additional C-terminal serine abolishes the preexisting ADA reactivity completely (FIGS. 23B, 23D and 23F), whereas the C-terminal aspartate reduces the unwanted interference (FIGS. 23A, 23C and 23E).
Furthermore, a set of GITR bispecific antigen binding molecules was also tested, i.e. the bispecific antigen binding molecule GITR x FAP (4B9) (2+1) and its C-terminal variants with EPKSCS (SEQ ID NO: 1) terminus (P1AG1036) and with EPKSCG (SEQ ID NO: 2) terminus (P1AG1039). The results are shown in FIGS. 24A to 24C. It confirms the previous results that a C-terminal extension of a serine (P1AG1036, FIG. 24B) eliminates the reactivity with preexisting antibodies in plasma compared to the respective molecule with a free C-terminus (P1AE1116, FIG. 24A). Similar to the serine extension variant, a further extension variant with a glycine, which is also not naturally located at this position of the upper hinge region, was generated. The glycine extension variant also eliminates the unwanted reactivity (P1AG1039, FIG. 24C).

Claims

1. A bispecific antigen binding molecule, comprising a first Fab fragment and a second Fab fragment capable of specific binding to a first antigen, and a third Fab fragment capable of specific binding to a second antigen, and an Fc domain composed of a first and a second subunit capable of stable association, wherein

(a) the first Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the first Fc domain subunit;

(b) the second Fab fragment capable of specific binding to a first antigen is fused at its C-terminus to the N-terminus of the second Fc domain subunit,

(c) the third Fab fragment capable of specific binding to a second antigen is a cross-fab fragment, wherein the constant domains CH1 and CL are replaced by each other and the N-terminus of the VH domain is fused to the C-terminus of one of Fc domain subunits, and wherein the CH1 domain of the cross-fab fragment terminates with an amino acid sequence selected from the group consisting of EPKSCS (SEQ ID NO:1), EPKSCG (SEQ ID NO:2), EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).

2. The bispecific antigen binding molecule of claim 1, wherein the bispecific antigen binding molecule has reduced or no reactivity towards pre-existing anti-drug antibodies.

3. The bispecific antigen binding molecule of claim 1, wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence of EPKSCS (SEQ ID NO:1).

4. The bispecific antigen binding molecule of claim 1, wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence of EPKSCG (SEQ ID NO:2).

5. The bispecific antigen binding molecule of claim 1, wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence selected from the group consisting of EPKSCD (SEQ ID NO:3), EPKSCDK (SEQ ID NO:4) and EPKSCDKTHL (SEQ ID NO:5).

6. The bispecific antigen binding molecule of claim 1, wherein the CH1 domain of the crossfab fragment terminates with the amino acid sequence of EPKSCD (SEQ ID NO:3).

7. The bispecific antigen binding molecule of claim 1 wherein the bispecific antigen binding molecule binds bivalently to the first antigen and monovalently to the second antigen.

8. The bispecific antigen binding molecule of claim 1, comprising

(a) two heavy chains, each heavy chain comprising a VH and CH1 domain of a Fab fragment capable of specific binding to the first antigen and a Fc domain subunit,

(b) two light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and

(c) a cross-fab fragment capable of specific binding to the second antigen comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain is connected to the C-terminus of one of the two heavy chains of (a).

9. The bispecific antigen binding molecule of claim 1, wherein the antigen binding molecule comprises a fourth Fab fragment capable of specific binding to the first antigen, wherein the fourth Fab fragment is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen.

10. The bispecific antigen binding molecule of claim 1 wherein the bispecific antigen binding molecule binds trivalently to the first antigen and monovalently to the second antigen.

11. The bispecific antigen binding molecule of claim 1, comprising

(a) a heavy chain comprising a VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,

(b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen and a Fc domain subunit,

(c) three light chains, each light chain comprising a VL and CL domain of a Fab fragment capable of specific binding to the first antigen, and

(d) a light chain comprising a VL and CH1 domain of the third Fab fragment capable of specific binding to the second antigen.

12. The bispecific antigen binding molecule of claim 1, comprising

(a) a heavy chain comprising a VH-CH1 chain of the first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit,

(b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen and a Fc region subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc domain subunit, optionally via a peptide linker,

(d) a light chain comprising a VL and CH1 domain of a Fab fragment capable of specific binding to the second antigen.

13. The bispecific antigen binding molecule of claim 1, wherein the antigen binding molecule comprises a fifth Fab fragment capable of specific binding to the first antigen, wherein the fifth Fab fragment is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the second Fab fragment capable of specific binding to the first antigen.

14. The bispecific antigen binding molecule of claim 1, wherein the bispecific antigen binding molecule binds tetravalently to the first antigen and monovalently to the second antigen.

15. The bispecific antigen binding molecule of claim 1, comprising

(a) a heavy chain comprising a VH-CH1 chain of a first Fab fragment capable of specific binding to the first antigen fused at its N-terminus to the VH-CH1 chain of the fourth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit, and a VH-CL chain of the third Fab fragment capable of specific binding to the second antigen fused to the C-terminus of the Fc region subunit, optionally via a peptide linker,

(b) a heavy chain comprising a VH-CH1 domain of the second Fab fragment capable of specific binding to first antigen fused at its N-terminus to the VH-CH1 chain of the fifth Fab fragment capable of specific binding to the first antigen, optionally via a peptide linker, a Fc domain subunit,

16. The bispecific antigen binding molecule of claim 1, wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain and/or wherein the Fc domain comprises one or more amino acid substitution that reduces the binding affinity of the antibody to an Fc receptor and/or effector function.

17. The bispecific antigen binding molecule of claim 1, wherein the Fc domain is of human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (EU numbering according to Kabat).

18. The bispecific antigen binding molecule of claim 1, wherein the first subunit of the Fc domain comprises knobs and the second subunit of the Fc domain comprises holes according to the knobs into holes method.

19. The bispecific antigen binding molecule of claim 1, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering according to Kabat) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (EU numbering according to Kabat).

20. An isolated nucleic acid encoding the bispecific antigen binding molecule of claim 1.

21. An expression vector comprising the isolated nucleic acid of claim 20.

22. A host cell comprising the isolated nucleic acid of claim 20.

23. A method of producing a bispecific antigen binding molecule, comprising culturing the host cell of claim 22 under conditions suitable for the expression of the bispecific antigen binding molecule, and isolating the bispecific antigen binding molecule.

24. A pharmaceutical composition comprising the bispecific antigen binding molecule of claim 1 and a pharmaceutically acceptable carrier.

25. (canceled)