WO2023165516A1 - Anti-pd-l1 and vegf bispecific antibody and use thereof - Google Patents
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- WO2023165516A1 WO2023165516A1 PCT/CN2023/079017 CN2023079017W WO2023165516A1 WO 2023165516 A1 WO2023165516 A1 WO 2023165516A1 CN 2023079017 W CN2023079017 W CN 2023079017W WO 2023165516 A1 WO2023165516 A1 WO 2023165516A1
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Definitions
- the invention belongs to the field of biomedicine, and in particular relates to a bispecific antibody targeting PD-L1 and VEGF and its preparation method and application.
- PD-L1 belongs to a type of transmembrane protein on the cell membrane, and is expressed on T cells, B cells and other immune cells and tumor cells.
- Tasuku Honjo et al. discovered and proved that when PD-L1 on the tumor cell membrane combines with PD-1 on immune cells such as T cells, the tumor cells send out inhibitory signals, and then T cells cannot recognize tumor cells and have a killing effect on tumor cells , the immune function of the body is suppressed (Chamoto, K., Al-Habsi, M., & Honjo, T. (2017). Current topics in microbiology and immunology, 410, 75-97).
- pathological detection indicators such as PD-L1 expression level and tumor mutation burden (TMB) indicate a low response rate of anti-PD-1/PD-L1 antibodies, which currently only have an effect on less than 40% of solid tumors, and 30% of patients in the A certain amount of drug resistance appears after treatment.
- TMB tumor mutation burden
- VEGF-A also known as VEGF.
- VEGF activates VEGFR2 (the main receptor tyrosine kinase receptor that mediates angiogenesis) to promote mitosis of vascular endothelial cells and increased vascular permeability, thereby promoting new vessel sprouting.
- VEGFR2 the main receptor tyrosine kinase receptor that mediates angiogenesis
- the high expression of VEGF in the tumor microenvironment can amplify the immunosuppressive effect of PD-(L)1. Therefore, targeting VEGF or VEGFR2 can effectively inhibit abnormal vascular proliferation (Ferrara, N. (2010). Mol Biol Cell 21 (5): 687-690.).
- Bevacizumab The anti-VEGF monoclonal antibody Bevacizumab (Bevacizumab) has good efficacy and target safety in tumor treatment and eye diseases related to abnormal blood vessel proliferation (Pfisterer, J., et al. (2020). Lancet Oncol 21(5):699-709. Bhandari, S., et al. (2020). Ophthalmology 127(5):608-615.).
- anti-VEGF monoclonal antibodies can improve blood vessels, promote T cell infiltration into tumors, inhibit DC maturation, promote T cell activation and activation, improve tumor microenvironment, and increase anti-PD-1/PD-L1 Drug response rate, further through T cell-mediated tumor killing, restore tumor immune function (Socinski, M.A., et al.(2018).N Engl J Med 378(24):2288-2301.N Engl J Med.2018Jun 14 ; 378(24):2288-2301. N Engl J Med 2020; 382:1894-1905).
- bispecific antibodies have more potential in reducing toxic side effects and dosage, and have better safety; at the same time, anti-PD-L1 has better safety than anti-PD-1 It can better target into the tumor microenvironment, and can improve the toxic and side effects on normal tissues.
- bispecific antibody drugs the biological activity of candidate antibodies is an important factor that needs to be taken into consideration. Although many bispecific antibodies have been proposed, due to the diversity of therapeutic functional and therapeutic behavior requirements of different therapeutic products, there is no theoretical prediction method that can be generally applied to most different desired molecular combinations. Therefore, it is often necessary to develop bispecific antibodies with good biological activity for specific targets and antibody compositions.
- Anti-PD-L1 and VEGF bispecific antibodies in the prior art are mainly anti-PD-L1 antibody fused to VEGFR1 protein (US20200172623A1, WO2020200210A1) or Mab-VHH anti-PD-L1 and VEGF bispecific antibody (WO2021147829A1), these Bispecific antibodies all contain full-length IgG1 antibodies, which have problems of large molecular weight and druggability.
- the inventors developed a bispecific nanobody targeting VEGF and PD-L1 at the same time, with a molecular weight of only 107KDa, which has potential With the advantages of strong tumor tissue infiltration and small dosage, it is superior to major competitors in terms of clinical dosage and safety. Further, the in vitro VEGF neutralizing activity of the anti-VEGF nanobody part of the bispecific antibody of the present invention is better than that of the marketed Bevacizumab, and in the animal model of angiogenesis, in the case of equal dose administration, the blood vessels in vivo The production inhibitory activity is also superior to that of Bevacizumab.
- the anti-PD-L1 nanobody part of the bispecific antibody of the present invention is similar to the marketed Atezolizumab in terms of in vitro blocking activity and cell binding level. Furthermore, compared with monoclonal antibody combination and other bispecific antibodies of the prior art, the bispecific antibody of the present invention can simultaneously block PD-1/PD-L1 and VEGF/VEGFR signaling pathways, and improve blood vessel and At the same time, by restoring immunity, it has a more significant effect of inhibiting tumors, and has a very broad application prospect.
- the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, said antibody comprising one or two VHH domains specifically binding to PD-L1 (VHH PD-L1 ) and one or two VHH domains (VHH VEGF ) specifically binding to VEGF, wherein the VHH VEGF comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO: 2, and preferably, wherein the VHH PD -L1 comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:1.
- the VHH PD-L1 and VHH VEGF domains may be included in a tandem form on the same polypeptide chain (for example, by means of a linker and/or an Fc region in tandem on a polypeptide chain); or Linked to the immunoglobulin Fc region in a Fab-like structure (for example, by means of the immunoglobulin CH1 and CL constant domains to form a Fab-like structure).
- the bispecific antibody comprises a polypeptide chain comprising a first VHH domain, an Fc region, a linker and a second VHH domain from the N-terminus to the C-terminus, wherein the first VHH domain and the second VHH domain are The two VHH domains specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 And the second antigen is VEGF.
- the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus.
- the Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region.
- the Fc region polypeptide is linked at the N-terminus to the first VHH domain through an immunoglobulin hinge region sequence.
- the Fc region polypeptide is connected to the second VHH domain at the C-terminus via a flexible linker peptide, such as (G 4 S) 3 (SEQ ID NO: 19).
- the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 7 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity amino acid sequence.
- the bispecific antibody comprises a polypeptide chain comprising a first VHH domain, a linker, a second VHH domain and an Fc region from the N-terminus to the C-terminus, wherein the first VHH domain and The second VHH domain specifically binds the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD- L1 and the second antigen is VEGF.
- said first and second VHH domains are connected in tandem via a linker, eg ( G4S ) 3 (SEQ ID NO: 19).
- the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus.
- the Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region.
- the Fc region polypeptide is at the N-terminus Linked to the second VHH domain by an immunoglobulin hinge region sequence.
- the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 8 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity amino acid sequence.
- said bispecific antibody comprises a first and a second polypeptide chain, wherein,
- the first polypeptide chain comprises a first VHH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus;
- the second polypeptide chain comprises a second VHH domain and a CL domain from the N-terminus to the C-terminus;
- first VHH domain and the CH1 domain in the first polypeptide chain are paired with the second VHH domain and the CL domain in the second polypeptide chain to form a Fab-like structure
- the first VHH structural domain and the second VHH structural domain specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the The first antigen is PD-L1 and the second antigen is VEGF.
- the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus.
- the Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region.
- the Fc region polypeptide is connected to the CH1 domain at the N-terminus through an immunoglobulin hinge region sequence.
- the first polypeptide chain comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 9 or 11 Amino acid sequences with % or 99% identity.
- the second polypeptide chain comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 10 or 12 Amino acid sequences with % or 99% identity.
- the present invention also provides nucleic acids encoding the polypeptide chains of the bispecific antibodies of the present invention, vectors and host cells comprising them.
- the present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention, and its in vitro and in vivo uses, especially in the treatment or diagnosis of diseases.
- FIG. 1A-1D show schematic structures of candidate bispecific antibodies.
- BsAb10 has the format of FIG. 1A;
- BsAb11 has the format of FIG. 1B;
- BsAb12 has the format of FIG. 1C;
- BsAb13 has the format of FIG. 1D.
- Figures 2A-2B show gel electrophoresis profiles of candidate bispecific antibodies.
- the samples in Fig. 2A are BsAb10 and the reference IPI respectively; the samples in Fig. 2B are BsAb11, BsAb12, BsAb13 and the reference IPI respectively.
- Figures 3A-3D show the SEC-HPLC monomer detection profiles of candidate bispecific antibodies.
- Figure 3A is the SEC-HPLC monomer detection spectrum of BsAb10;
- Figure 3B is the SEC-HPLC monomer detection spectrum of BsAb11;
- Figure 3C is the SEC-HPLC monomer detection spectrum of BsAb12;
- Figure 3D is the SEC-HPLC monomer detection spectrum of BsAb13 Atlas.
- Figure 4 shows the binding activity of candidate bispecific antibodies to recombinant protein VEGF-His.
- Figure 5 shows the binding activity of candidate bispecific antibodies to recombinant protein PD-L1-His.
- Figure 6 shows the binding activity of candidate bispecific antibodies to huPD-L1-CHO-K cells.
- Figures 7A-7B show the binding activity of candidate bispecific antibodies to both VEGF and PD-L1.
- Figure 7A shows the activity of candidate bispecific antibodies that first bind to recombinant protein VEGF-Fc and then bind to recombinant protein PD-L1-Fc;
- Figure 7B shows that candidate bispecific antibodies first bind to recombinant protein PD-L1-Fc and then bind to recombinant protein PD-L1-Fc Binding activity to recombinant protein VEGF-Fc.
- Figure 8 shows the detection of the blocking activity of candidate bispecific antibodies against PD-1/PD-L1 by luciferase reporter gene method.
- Figure 9 shows the detection of the blocking activity of candidate bispecific antibodies against VEGF/VEGFR2 by luciferase reporter gene method.
- Figure 10 shows the inhibitory effect of candidate bispecific antibodies on tumor growth in the COLO205 mouse transplantation model.
- Figure 11 shows the inhibitory effect of candidate bispecific antibodies on tumor growth in the A431 mouse transplantation model.
- the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps. Where “comprising” is mentioned, the expression also covers “consisting of” unless the context clearly dictates otherwise. For example, when referring to a polypeptide chain comprising a specific amino acid sequence, it is also contemplated that the polypeptide chain consists of the specific amino acid sequence.
- VEGF refers to vascular endothelial growth factor A (VEGF-A) protein.
- VEGF-A produces multiple isoforms through alternative splicing of exons during transcription, isoforms such as VEGF121, VEGF165, VEGF189, and VEGF206.
- VEGF refers in particular to the isoform VEGF165, for example the human VEGF165 protein described under UniProtKB-P15692 (in particular, the amino acid sequence without amino acids 27-191 of the signal peptide).
- VEGF165 is active in angiogenesis, vessel formation, and endothelial cell growth; can induce endothelial cell proliferation, promote cell migration, inhibit apoptosis, and induce vascular permeability.
- VEGF165 binds to the receptors FLT1/VEGFR1 and KDR/VEGFR2; and to the protein Neuropilin-1 (NRP1).
- NRP1 Neuropilin-1
- mice with NRP1 gene deletion on killer T cells produced better protection against secondary tumors, including relatively "cold” tumors such as melanoma in B16.F10 mice, against PD1
- the response to immunotherapy was also more positive (Chang Liu et al, Neuropilin-1 is a T cell memory checkpoint limiting long-term antitumor immunity, Nature Immunology (2020). DOI: 10.1038/s41590-020-0733-2).
- antigen-binding specificity for VEGF refers to a binding site or a binding domain in a molecule that specifically binds to VEGF.
- the term "PD-L1" refers to programmed cell death 1 ligand 1 (Programmed cell death 1 ligand 1). As a ligand for the inhibitory receptor PD-1, this protein regulates T cell activation thresholds and limits T cell effector responses.
- the PD1/PD-L1-mediated immunosuppressive pathway can be used by tumors to attenuate anti-tumor immunity and evade clearance by the immune system, thereby promoting tumor survival. Blocking this pathway reverses the exhausted T cell phenotype and normalizes antitumor responses, facilitating cancer immunotherapy.
- the term PD-L1 herein refers especially to human PD-L1 protein, eg human PD-L1 protein under accession number UniProtKB-Q9NZQ7.
- antigen-binding specificity for PD-L1 refers to a binding site or binding domain in a molecule that specifically binds to PD-L1.
- the terms "antigen-binding site” and “antigen-binding specificity” are used interchangeably to refer to the region of the antibody that actually binds to the antigen.
- the PD-L1 antigen-binding site is provided by the VHH domain from anti-PD-L1; the VEGF antigen-binding site is provided by the VHH domain from anti-VEGF.
- the term "bind” or “specifically bind” means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
- the ability of an antigen binding site to bind a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, for example, by detecting antibodies by ELISA assays described in Examples 4.1, 4.2 or 4.4 The ability to bind to PD-L1 or VEGF protein or both, or to detect the ability of the antibody to bind to PD-L1 expressed on the cell surface by the FACS assay described in Example 4.3.
- ELISA enzyme-linked immunosorbent assay
- an antibody having "blocking activity" on PD-L1 means that the antibody blocks the binding of PD-L1 to the receptor PD-1, and/or reduces the function of PD-L1/PD-1 signal transmission.
- Assays that can be used to determine this blocking activity can be FACS assays known in the art; or reporter gene-based signaling pathway blockade assays, such as the reporter gene-based assay described in Example 5.
- the PD-L1 blocking activity of the antibody to be tested can be determined with reference to the PD-L1/PD-1 binding or PD-L1/PD-1 signaling level in the absence of the antibody and/or in the presence of a positive antibody.
- the neutralizing activity of an antibody to VEGF refers to the function of the antibody to block the signaling pathway between VEGF and the receptor VEGFR2.
- An assay that can be used to measure this activity can be, for example, a reporter gene-based signaling pathway blockade assay, such as the reporter gene-based assay described in Example 6.
- the neutralizing activity of the antibody to be tested against VEGF can be determined with reference to the VEGF/VEGFR2 signaling level in the absence of the antibody and/or in the presence of a positive antibody.
- variable region or “variable domain” of an antibody refers to the domains of the heavy or light chain of an antibody that participate in the binding of the antibody to an antigen.
- the variable regions of antibodies can be further subdivided into hypervariable regions (ie, complementarity determining regions (CDRs)) and more conserved regions interspersed between hypervariable regions (ie, framework regions (FRs)).
- CDRs complementarity determining regions
- FRs framework regions
- the antigen binding site is composed of a single heavy chain variable domain (also referred to herein as a VHH domain). supply.
- this single heavy chain variable domain VHH comprises four FR regions and three CDR regions, and is arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
- CDR region or “CDR” or “hypervariable region”
- VHH antibody variable domain
- the CDRs are primarily responsible for binding to antigenic epitopes. Numbering sequentially from the N-terminus of the antibody chain, the CDRs located in the VHH variable region domain are called CDR1, CDR2, and CDR3.
- CDR sequence Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat ( Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT ) (International Immunogenetics Information System, World Wide Web imgt.cines.fr/), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).
- CDR or “CDR sequence” covers a CDR sequence determined in any of the above ways and combinations thereof.
- VHH domain the antigen-binding site that specifically binds to the antigen is provided by the VHH domain.
- VHH or "VHH domain” is used herein to refer to a heavy chain variable domain derived from a heavy chain antibody (herein sometimes referred to as a Nanobody) lacking a light chain, also referred to as a single Variable domain (sVD). Unlike the conventional VH of four-chain immunoglobulins, the VHH domain does not need to be paired with a light chain variable domain to form an antigen-binding site.
- VHH molecules may be derived from antibodies produced in Camelidae species such as llamas, alpacas, dromedaries, llamas and guanacos. In some instances, for therapeutic use of a VHH, it is desirable to reduce its immunogenicity. Thus, preferably, in one embodiment, the invention provides antibodies comprising a humanized VHH domain.
- the VHH domain from anti-PD-L1 and the VHH domain from anti-VEGF preferably via an immunoglobulin hinge region or a linker , separately connected to the opposite sides of the Fc region polypeptide of the antibody; or in the form of both in series (for example, from N-terminal to C-terminal, VHH PD-L1 -linker-VHH VEGF ; or VHH VEGF -linked Sub-VHH PD-L1 ), linked to the N-terminal side of the Fc region polypeptide of the antibody.
- Fab-like structure is used herein to refer to, as shown in Figure 1C and Figure 1D, similar to that in a conventional four-chain IgG antibody consisting of a heavy chain variable region and a heavy chain constant region CH1 with complementary light chain variable regions and A structure formed by CL pairing of the light chain constant region, but in which the heavy chain variable region is replaced by a VHH domain and the light chain variable region is replaced by a different VHH domain.
- the Fab-like structure comprises the first chain consisting of VHH PD-L1 and the CH1 domain of the constant region of the immunoglobulin heavy chain and the CL domain of the constant region of the immunoglobulin light chain consisting of VHH VEGF and composed of the second chain.
- the Fab-like structure comprises a first chain consisting of VHH VEGF and the CH1 domain of the constant region of the immunoglobulin heavy chain and a CL structure composed of VHH PD-L1 and the constant region of the immunoglobulin light chain The domain consists of the second chain.
- the VHH domain from anti-PD-L1 and the VHH domain from anti-VEGF are in a Fab-like structure, preferably by immunoglobulin
- the hinge region is connected to the N-terminal side of the immunoglobulin Fc region polypeptide.
- the Fab-like structure is linked to the Fc region polypeptide through its CH1 domain.
- immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
- IgG class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two light chains and two heavy chains that are disulfide bonded. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2 and CH3 ). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL).
- VH heavy chain variable region
- CL light chain constant domain
- an IgG immunoglobulin In an IgG molecule, usually the VH-CH1 of the heavy chain is paired with the VL-CL of the light chain to form a Fab fragment that specifically binds the antigen.
- an IgG immunoglobulin essentially consists of two Fab molecules and two dimerized Fc regions linked by the immunoglobulin hinge region.
- the heavy chains of immunoglobulins can be assigned to one of five classes, called ⁇ (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG), or ⁇ (IgM), based on the type of their constant region, one of which is These classes can be further divided into subclasses such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), ⁇ 4 (IgG4), ⁇ 1 (IgA1 ) and ⁇ 2 (IgA2).
- the light chains of immunoglobulins can also be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains.
- Immunoglobulin heavy chain constant region domain means a constant region domain from or obtained from or derived from an immunoglobulin heavy chain, including the heavy chain constant regions CH1, CH2 covalently linked sequentially from the N-terminus to the C-terminus , CH3 and optionally heavy chain constant region CH4. In most cases, heavy The chain constant regions CH1 and CH2 are connected by the hinge region of the heavy chain, but they can also be connected by a flexible linking peptide when appropriate.
- the antibody of the present invention comprises a polypeptide chain consisting of immunoglobulin heavy chain constant region CH1-hinge region-CH2-CH3.
- the antibody of the present invention comprises an Fc region polypeptide chain composed of immunoglobulin heavy chain constant regions CH2-CH3.
- immunoglobulin constant domains can be selected based on the intended function of the antibody.
- the constant domain may be an IgA, IgD, IgE, IgG or IgM domain, especially an immunoglobulin constant domain of human IgG, for example, a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably of human IgG1 constant domain.
- the CH1 and Fc fragments of an antibody can both be from IgG1.
- Immunoglobulin light chain constant region domain refers to a constant region domain CL from or obtained from or derived from an immunoglobulin light chain.
- the light chain CL constant region of an immunoglobulin based on its amino acid sequence, may be a kappa light chain CL domain and a lambda light chain CL domain.
- Fc domain or “Fc region” or “Fc region polypeptide” are used interchangeably to refer to a C-terminal region polypeptide containing at least a part of the constant region derived from an immunoglobulin heavy chain.
- the term includes native sequence Fc region polypeptides and variant Fc region polypeptides.
- a native immunoglobulin "Fc domain” comprises two or three constant domains, a CH2 domain, a CH3 domain and optionally a CH4 domain.
- the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of the heavy chain derived from antibodies of the IgG, IgA and IgD classes; and the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of the heavy chain of antibodies of the IgE class.
- amino acid residue numbering in the Fc region or heavy chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system described in 1991 (also known as the EU Index) is used for numbering.
- Fc domain or "Fc region” or “Fc polypeptide” does not include the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region of an immunoglobulin cl.
- “Native sequence Fc region” polypeptides encompass naturally occurring Fc region sequences of various immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions , 20 October 2014, doi: 10.3389/fimmu.2014.00520.).
- the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain.
- the C-terminal terminal lysine (Lys447) of the Fc region may or may not be present.
- the human IgG heavy chain Fc region bears at the N-terminus a hinge sequence or a partial hinge sequence of a native immunoglobulin, for example a sequence from E216 to T225 or a sequence from D221 to T225 according to EU numbering.
- variant Fc region or “Fc region variant” polypeptide herein, used interchangeably herein, refers to an Fc region polypeptide comprising a modification relative to a native sequence Fc region polypeptide.
- Fc region variant polypeptides of the invention are defined by the amino acid modifications that make them up.
- L234A is an Fc region variant with alanine substituted for leucine at position 234 relative to the parental polypeptide, where numbering is according to the EU index.
- Modifications can be additions, deletions or substitutions. Substitutions can include naturally occurring amino acids and non-naturally occurring amino acids. Variants may contain unnatural amino acids.
- the purpose of the modification may be to alter the binding of the Fc region to its receptor and the effector functions elicited thereby.
- effector functions refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype.
- immunoglobulin 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, downregulation of cell surface receptors (eg, B-cell receptors), and B-cell activation.
- the antibody of the invention may have altered effector functions relative to an antibody with a wild-type Fc region, such as reduced or eliminated ADCC activity and the like.
- the term "flexible linker” or “linker” or “linker” is used interchangeably to refer to a short amino acid sequence consisting of amino acids such as glycine (G) and/or serine ( S) and/or threonine residues (T), or from the hinge region of an immunoglobulin.
- the connecting peptide is 5-50 amino acids in length, eg, 10, 15, 20, 25, 30 amino acids in length.
- the connecting peptide comprises the amino acid sequence (G 4 S) n (SEQ ID NO: 20), wherein n is an integer equal to or greater than 1, for example, n is 2, 3, 4, 5, 6 or 7 an integer of .
- the connecting peptide comprises the amino acid sequence TS(G 4 S) n (SEQ ID NO: 21), wherein n is an integer equal to or greater than 1, for example, n is 2, 3, 4, 5, 6 or Integer of 7.
- the connecting peptide is a hinge region from an immunoglobulin, for example comprising a hinge region amino acid sequence of "CPPC", for example, the amino acid sequence "EPKSCDKTHTCPPCP” (SEQ ID NO: 22) or "EPKSSDKTHTCPPCP” (SEQ ID NO:23).
- the connecting peptide that can be used to link the various domains in the antibody of the present invention can also be, for example but not limited to, the following amino acid sequence: GGG (SEQ ID NO: 24); DGGGS (SEQ ID NO: 25); TGEKP (SEQ ID NO :26); GGRR (SEQ ID NO:27); EGKSSGSGSESKVD (SEQ ID NO:28); KESGSVSSEQLAQFRSLD (SEQ ID NO:29); GGRRGGGS (SEQ ID NO:30); LRQRDGERP (SEQ ID NO:31); (SEQ ID NO:32) and GSTSGSGKPGSGEGSTKG (SEQ ID NO:33).
- suitable flexible linker peptides can be rationally designed using computer programs to model the three-dimensional structures of proteins and peptides, or by phage display methods.
- chimeric antibody is an antibody in which (a) the constant region, or part thereof, has been altered, substituted or exchanged such that the antigen binding site corresponds to a constant region of a different or altered class, effector function and/or species or linking entirely different molecules (e.g., enzymes, toxins, hormones, growth factors, drugs) etc. that confer new properties on the chimeric antibody; Variable regions are altered, substituted or swapped.
- a “humanized antibody” is an antibody that retains the antigen-specific reactivity of a non-human antibody (eg, an alpaca heavy chain antibody), while being less immunogenic when administered to humans as a therapeutic. This can be achieved, for example, by retaining the non-human antigen binding sites and replacing the remainder of the antibodies with their human counterparts (ie, the constant regions and the parts of the variable regions that do not participate in binding are those of human antibodies).
- a non-human antibody eg, an alpaca heavy chain antibody
- Percent identity (%) of an amino acid sequence refers to after aligning a candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without taking into account any When conservative substitutions are taken as part of sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular amino acid sequence shown in this specification.
- the invention contemplates variants of the antibodies of the invention that have a substantial degree of identity, for example at least 80%, 85% identity, relative to the antibodies and sequences thereof specifically disclosed herein. %, 90%, 95%, 97%, 98%, or 99% or higher. Such variants may contain conservative modifications.
- conservative modifications include substitutions, deletions or additions to a polypeptide sequence that result in the substitution of an amino acid for a chemically similar amino acid.
- Conservative substitution tables providing functionally similar amino acids are well known in the art.
- conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues and alleles of the invention.
- the following 8 groups contain amino acids that are mutually conservative substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N) , glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine Acid (C), Methionine (M) (See eg, Creighton, Proteins (1984)).
- the term "conservative sequence modification” is used to refer to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence.
- host cell refers to a cell into which an exogenous polynucleotide 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.
- a host cell is any type of cellular system that can be used to produce antibodies of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
- Host cells include cultured cells as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.
- expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed.
- Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system.
- Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses) that incorporate recombinant polynucleotides. virus and adeno-associated virus).
- 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 rodents). mouse).
- domesticated animals e.g., cows, sheep, cats, dogs, and horses
- primates e.g., humans and non-human primates such as monkeys
- rabbits e.g., mice and rodents.
- rodents e.g., mice and rodents.
- an individual is a human being.
- treatment refers to clinical intervention intended to alter the natural course of disease in the individual being treated. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
- the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.
- treatment encompasses anti-tumor biological effects that can be induced by human intervention (e.g., administration of drugs such as antibodies of the invention), including, but not limited to, for example, reduction in tumor volume, tumor Decreased cell number, decreased tumor cell proliferation, or decreased tumor cell survival.
- human intervention e.g., administration of drugs such as antibodies of the invention
- cancer and “tumor” are used interchangeably to refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth.
- examples of cancer include, but are not limited to, carcinoma, solid tumors, and liquid tumors.
- the present invention provides a VHH domain-based bispecific antibody specifically binding to PD-L1 and VEGF, said antibody comprising a VHH domain specifically binding to PD-L1 (also abbreviated herein as VHH PD-L1 ) and a VHH domain specifically binding to VEGF (also abbreviated herein as VHH VEGF ).
- the VHH PD-L1 and VHH VEGF domains may be in a tandem form contained on the same polypeptide chain (for example, by means of a linker and/or or Fc regions in series on one polypeptide chain); or can form a Fab-like structure (for example, by means of immunoglobulin CH1 and CL constant domains to form a Fab-like structure).
- the bispecific antibody according to the invention further comprises an Fc region.
- said VHH PD-L1 and VHH VEGF domains may be in a tandem form comprising both VHH PD-L1 and VHH VEGF domains (for example, as shown in Figure 1A and Figure 1B ), or in a Fab-like structure comprising both VHH PD-L1 and VHH VEGF domains (eg, as shown in Figure 1C and Figure 1D ), linked to the Fc region.
- the bispecific antibody according to the present invention can be a tetravalent antibody that specifically binds PD-L1 and VEGF.
- bispecific antibody of the present invention and the bispecific antibody of the present invention composed of them are described in detail below. Those skilled in the art can understand that unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of the present invention. And, those skilled in the art will understand that unless the context clearly indicates otherwise, the antibodies of the invention (including antibodies in any form, such as diabodies and tetrabodies) may comprise any such combination of features.
- VHH VEGF domain and VHH PD-L1 domain according to the invention
- the antigen-binding site that specifically binds to PD-L1 is provided by the VHH PD-L1 according to the present invention; and
- the antigen binding site that specifically binds VEGF is provided by the VHH VEGF according to the invention.
- a VHH PD-L1 domain according to the invention comprises a VHH domain from an anti-PD-L1 Nanobody.
- the VHH comprises CDR1, CDR2 and CDR3 sequences having the variable region shown in SEQ ID NO:1.
- the CDR sequence range of the variable region SEQ ID NO: 1 amino acid sequence can be defined according to the Kabat, AbM, Chothia, Contact or IMGT schemes, or can be defined according to any two or more or all of these definition schemes. Those skilled in the art can easily know the CDR sequences defined by these definitions through http://www.abysis.org/abysis/.
- the VHH PD-L1 domain according to the invention comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence defined according to AbM in the variable region having SEQ ID NO:1.
- the VHH PD-L1 domain according to the invention comprises
- the VHH PD-L1 domain comprises the amino acid sequence shown in SEQ ID NO:1. In yet another embodiment, the VHH PD-L1 domain comprises at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 1 and retains the ability to specifically bind PD-L1 amino acid sequence. In yet another preferred embodiment, the VHH PD-L1 domain comprises one or more (preferably 1-10, more preferably 1-5) amino acid additions compared to SEQ ID NO: 1, Amino acid sequences that are deleted and/or substituted (eg, conservatively substituted) and retain the ability to specifically bind PD-L1. Preferably, said amino acid additions, deletions and/or substitutions do not occur in the CDR regions. Most preferably, in the bispecific antibody according to the present invention, the VHH PD-L1 domain according to the present invention comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.
- a VHH VEGF domain according to the invention comprises a VHH domain from an anti-VEGF Nanobody.
- the VHH comprises CDR1, CDR2 and CDR3 sequences of the variable region shown in SEQ ID NO:2.
- the CDR sequence range of the variable region SEQ ID NO: 2 amino acid sequence can be defined according to the Kabat, AbM, Chothia, Contact or IMGT schemes, or can be defined according to any two or more or all of these definition schemes. Those skilled in the art can easily know the CDR sequences defined by these definitions through http://www.abysis.org/abysis/.
- the VHH VEGF domain according to the invention comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence defined according to AbM in the variable region having SEQ ID NO:2.
- the VHH VEGF domain according to the invention comprises
- the VHH VEGF domain comprises the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the VHH VEGF domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2 and retains the ability to specifically bind VEGF. In yet another preferred embodiment, the VHH VEGF domain comprises one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and and/or amino acid sequences that are substituted (eg, conservatively substituted) and retain the ability to specifically bind VEGF. Preferably, said amino acid additions, deletions and/or substitutions do not occur in the CDR regions. Most preferably, in the bispecific antibody according to the present invention, the VHH VEGF domain according to the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO:2.
- the bispecific antibody of the present invention in addition to the aforementioned VHH VEGF domain and VHH PD-L1 domain, in some embodiments, it may also include an immunoglobulin constant domain, for example, the immunoglobulin constant regions CH2 and CH3 Constituent Fc region, and/or CH1 and CL constant domains in a Fab-like structure.
- an immunoglobulin constant domain for example, the immunoglobulin constant regions CH2 and CH3 Constituent Fc region, and/or CH1 and CL constant domains in a Fab-like structure.
- the invention provides a bispecific antibody comprising an Fc region polypeptide chain consisting of immunoglobulin heavy chain constant regions CH2-CH3.
- the present invention provides a bispecific antibody comprising an Fc region and further comprising an immunoglobulin CH1 domain and a CL domain, preferably, wherein the CH1 domain passes through an immunoglobulin hinge region connects the Fc region polypeptide, and more preferably, the antibody comprises a polypeptide chain consisting of an immunoglobulin heavy chain constant region CH1-hinge region-CH2-CH3.
- the immunoglobulin constant domains comprised in the bispecific antibody according to the invention including the CH2 and CH3 constant domains in the Fc region and the CH1 and CL constant domains in the Fab-like structure (if present), May be, independently of each other, an immunoglobulin constant domain from human IgG, eg, a constant domain of human IgGl, IgG2, IgG3 or IgG4, preferably a constant domain of human IgGl.
- immunoglobulin constant domains comprised in the bispecific antibodies according to the invention may independently of each other be native sequence constant domains (for example, Human native sequence constant domain) or an amino acid sequence variant thereof, preferably a native sequence constant domain.
- the Fc region comprised in the bispecific antibody according to the invention is an Fc region sequence from human IgG1.
- the Fc region has the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence at least 90%, 95% or 99% identical thereto, or no more than Amino acid sequence with 1-10 (preferably 1-5) amino acid residues altered.
- the bispecific antibody according to the present invention may comprise two polypeptide chains having an Fc region, for example, the di-chain antibody and the tetra-chain antibody of the present invention as shown in FIG. 1 , wherein the two polypeptide chains The Fc region of is capable of pairing and dimerization.
- both the first Fc region polypeptide and the second Fc region polypeptide constituting the dimerized Fc region of the antibody may be Fc domains from IgG, such as human IgG1, IgG2, IgG3 or IgG4, preferably human IgG1 and may independently of each other be a native sequence Fc region and a variant Fc region as defined herein, provided that said first Fc region polypeptide and said second Fc region polypeptide can pair and dimerize.
- the first Fc region and the second Fc region are native sequence Fc regions, especially human IgG1 native sequence Fc regions.
- the first Fc region and the second Fc region have the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence at least 90%, 95% or 99% identical thereto, or identical thereto An amino acid sequence that has no more than 1-10 (preferably 1-5) amino acid residue changes.
- the antibody according to the present invention may comprise a Fab-like structure having a heavy chain constant region CH1 domain and a light chain constant region CL domain, wherein the CH1 structure The domain is preferably linked C-terminally to the first VHH domain and the light chain constant region CL domain is linked C-terminally to the second VHH domain.
- the heavy chain constant regions CH1 and CL constituting the antibody may be constant domains from IgG, such as human IgG1, IgG2, IgG3 or IgG4, preferably human IgG1.
- the CL domain of the antibody may be a Kappa light chain CL domain or a Lamda light chain CL domain, preferably a Kappa light chain CL domain.
- the CH1 and CL domains may independently of each other be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof, provided that the CH1 structure is included Domain and the antibody polypeptide chain of the first VHH domain connected at its N-terminus, and the antibody polypeptide chain comprising said CL domain and the second VHH domain connected at its N-terminus can be paired with each other to form a Fab according to the invention like structure.
- the CH1 and CL domains are native sequence constant domains, especially human IgG1 native sequence CH1 domains and CL domains. More preferably, the CH1 domain has the amino acid sequence shown in SEQ ID NO:4. More preferably, the CL domain has the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6.
- linkers can be used to connect antibody components.
- a VHH domain according to the present invention may be linked to the N-terminal or C-terminal of the Fc region via a linker, or may be linked to another VHH domain according to the present invention via a linker.
- Linkers that can be used in the antibodies of the present invention are not particularly limited. Those skilled in the art can easily determine the available linker sequences according to the components to be linked and the position of the link.
- the VHH domains according to the invention are in tandem form (eg, as shown in Figures 1A and 1B ) or in a Fab-like structure (eg, as shown in Figure 1C and 1D), connected to the Fc region polypeptide chain through a linker.
- linker e.g., as shown in Figures 1A and 1B
- linker e.g., as shown in Figure 1C and 1D
- the polypeptide chains of the Fc region are connected at the N-terminus to a single VHH domain or two VHH domains in tandem, or a Fab-like structure via a linker, wherein the linker preferably comprises (one or two) cysteine Amino acid residues, which are capable of forming (one or two) disulfide bonds when the two antibody polypeptide chains comprising the Fc region pair to stabilize the structure of the bispecific antibody thus formed.
- the linker preferably comprises (one or two) cysteine Amino acid residues, which are capable of forming (one or two) disulfide bonds when the two antibody polypeptide chains comprising the Fc region pair to stabilize the structure of the bispecific antibody thus formed.
- the two-chain antibody structure and the four-chain antibody structure shown in FIG. 1 shown in FIG. 1 . Therefore, in a preferred embodiment, the Fc region polypeptide chain contained in the bispecific antibody of the present invention has a linker sequence comprising "CPPC" at the N-terminus.
- the Fc region polypeptide chain contained in the bispecific antibody of the present invention has a hinge region derived from an immunoglobulin at the N-terminus, for example, the amino acid sequence of the hinge region comprising "CPPC", for example, amino acid The sequence "EPKSCDKTHTCPPCP” (SEQ ID NO:22) or "EPKSSDKTHTCPPCP” (SEQ ID NO:23); more preferably, the amino acid sequence "EPKSCDKTHTCPPCP" (SEQ ID NO:22).
- the Fc region polypeptide chain is connected to the VHH domain at the C-terminus by a linker, wherein the linker is preferably a flexible connecting peptide of 5-50 amino acids, preferably comprising glycine (G) and/or serine (S) and/or a linking peptide of a threonine residue (T).
- the linker is 5-50 amino acids in length, eg, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers.
- the linker comprises the amino acid sequence (G 4 S) n , wherein n is an integer equal to or greater than 1, eg, n is an integer of 2, 3, 4, 5, 6 or 7.
- the linker consists of the amino acid sequence (G 4 S) 3 .
- the VHH VEGF domain and the VHH PD-L1 domain according to the invention form a tandem VHH domain via a linker.
- the first and second domains are connected in series through a linker, wherein the linker is a flexible connecting peptide of 5-50 amino acids.
- the linker is 5-50 amino acids in length, eg, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers.
- the linker is a linker peptide comprising glycine (G) and/or serine (S) and/or threonine residues (T).
- the linker comprises the amino acid sequence (G 4 S) n (SEQ ID NO: 20), wherein n is an integer equal to or greater than 1, for example, n is 2, 3, 4, 5, 6 or Integer of 7.
- the linker consists of the amino acid sequence (G 4 S) 3 (SEQ ID NO: 19).
- the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, wherein the antibody comprises a VHH domain specifically binding to a first antigen and a VHH domain specifically binding to a second antigen , wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF.
- the VHH domain specifically binding to VEGF comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:2.
- the VHH domain specifically binding to PD-L1 comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:1.
- the VHH domain specifically binding to VEGF comprises the amino acid sequence shown in SEQ ID NO: 2, or has at least 80%, 85%, 90%, 95%, or An amino acid sequence with 99% identity, or an amino acid sequence with one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and/or substitutions compared to SEQ ID NO:2 , most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence shown in SEQ ID NO: 2.
- the VHH domain specifically binding to PD-L1 comprises the amino acid sequence shown in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 1 Amino acid sequence with % or 99% identity, or with one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and/or substitutions compared to SEQ ID NO: 1 Amino acid sequence, most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.
- the bispecific antibody according to the present invention further comprises a linker comprising 10-20 amino acids in length, preferably comprising the amino acid sequence (G 4 S) 3 (SEQ ID NO: 19) .
- the bispecific antibody according to the present invention further comprises an Fc region of an immunoglobulin, wherein the Fc region is an IgG Fc region, for example, the Fc domain of human IgG1, IgG2, IgG3 or IgG4, preferably human The Fc domain of IgG1, more preferably the Fc region carries an immunoglobulin hinge region sequence at the N-terminus.
- the bispecific antibody according to the invention comprises a polypeptide chain comprising VHH PD-L1 and VHH VEGF domains connected in series by means of an Fc region and a linker.
- the bispecific antibody comprises a polypeptide chain comprising a first VHH domain, an Fc region, a linker and a second VHH domain from the N-terminus to the C-terminus, wherein the first VHH domain and the second VHH domain
- the two VHH domains specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and The second antigen is VEGF.
- the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 7 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or amino acid sequences with 99% identity.
- the bispecific antibody according to the invention comprises a polypeptide chain comprising VHH PD-L1 and VHH VEGF domains connected in series by means of a linker.
- the bispecific antibody comprises a polypeptide chain comprising from the N-terminus to the C-terminus Containing a first VHH domain, a linker, a second VHH domain and an Fc region, wherein the first VHH domain and the second VHH domain specifically bind to a first antigen and a second antigen, respectively, wherein the first antigen and The second antigens are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF.
- the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 8 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or amino acid sequences with 99% identity.
- the bispecific antibody according to the invention comprises a first and a second polypeptide chain forming a Fab-like structure.
- the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first VHH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus; the The second polypeptide chain comprises a second VHH domain and a CL domain from the N-terminus to the C-terminus; wherein the first VHH domain and the CH1 domain in the first polypeptide chain are the same as the second VHH in the second polypeptide chain domain and CL domain pair to form a Fab-like structure; wherein the first VHH domain and the second VHH domain specifically bind a first antigen and a second antigen, respectively, wherein the first and second antigens are different and independent of each other is selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the
- the bispecific antibody comprises first and second polypeptide chains, wherein the first antigen is PD-L1 and the second antigen is VEGF, wherein the first polypeptide chain comprises SEQ The amino acid sequence shown in ID NO: 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and
- the two polypeptide chains comprise an amino acid sequence shown in SEQ ID NO: 10 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith amino acid sequence.
- the antibody may comprise two polypeptide chains comprising an Fc region, wherein the two polypeptide chains Dimers can be formed by dimerizing association of the Fc region.
- the two polypeptide chains that associate to form a dimer need not be identical.
- asymmetric modifications can be introduced in one or both polypeptide chains (eg, in the Fc region of the polypeptide chains) without affecting the desired target activity.
- the two polypeptide chains are identical. For example, as shown in Figures 1A and 1B.
- the present invention provides a bispecific antibody that specifically binds PD-L1 and VEGF, wherein said antibody comprises two polypeptide chains, said two polypeptide chains may be the same or different, wherein:
- Each polypeptide chain comprises a first VHH domain, an Fc region, a linker, and a second VHH domain from N-terminus to C-terminus (as shown in Figure 1A); or each polypeptide chain comprises from N-terminus to C-terminus A first VHH domain, a linker, a second VHH domain and an Fc region (as shown in Figure 1B);
- the Fc region polypeptides of the two polypeptide chains are paired to form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen respectively, wherein the The first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF.
- the first antigen is PD-L1 and the second antigen is VEGF; in another embodiment, the first antigen is VEGF and the second antigen is PD-L1.
- the present invention provides a bispecific antibody that specifically binds PD-L1 and VEGF, wherein the antibody comprises two polypeptide chains, which may be the same or different, wherein:
- Each polypeptide chain comprises a first VHH domain, an Fc region, a linker and a second VHH domain from the N-terminus to the C-terminus,
- the Fc region polypeptides of the two polypeptide chains are paired and form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen
- the two antigens are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 and the second antigen is VEGF.
- the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus.
- the Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region.
- the Fc region polypeptide is linked at the N-terminus to the first VHH domain through an immunoglobulin hinge region sequence. Still preferably, the Fc region polypeptide is connected to the second VHH domain at the C-terminus through a flexible linking peptide with a length of 10-20 amino acids, such as (G 4 S) 3 .
- the first VHH domain is a VHH PD-L1 domain according to the present invention, preferably, comprising the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprising SEQ ID NOs: 13- 15 CDR1-3 sequence; most preferably comprising the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH VEGF domain according to the present invention, preferably comprising SEQ ID NO: 2
- the CDR1-3 sequence of the variable region more preferably comprising the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprising the variable region sequence shown in SEQ ID NO:2.
- the two polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 7 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences of 98% or 99% identity.
- the two polypeptide chains are identical and each comprise the amino acid sequence of SEQ ID NO:7.
- the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, wherein the antibody comprises two polypeptide chains, and the two polypeptide chains may be the same or different, wherein :
- Each polypeptide chain comprises a first VHH domain, a linker, a second VHH domain and an Fc region from the N-terminus to the C-terminus,
- the Fc region polypeptides of the two polypeptides pair and form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen respectively, wherein the first antigen and The second antigens are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 and the second antigen is VEGF.
- the linker is a flexible connecting peptide with a length of 10-20 amino acids, such as (G 4 S) 3 (SEQ ID NO: 19) .
- the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus.
- the Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region.
- the Fc region polypeptide is linked at the N-terminus to a tandem VHH domain through an immunoglobulin hinge region sequence.
- the first VHH domain is a VHH PD-L1 domain according to the present invention, preferably, comprising the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprising SEQ ID NOs: 13- 15 CDR1-3 sequence; most preferably comprising the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH VEGF domain according to the present invention, preferably comprising SEQ ID NO: 2
- the CDR1-3 sequence of the variable region more preferably comprising the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprising the variable region sequence shown in SEQ ID NO:2.
- the two polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 8 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences of 98% or 99% identity.
- the two polypeptide chains are identical and each comprise the amino acid sequence of SEQ ID NO:8.
- said antibody may comprise two first polypeptide chains comprising an Fc region and two The second polypeptide chain, wherein the two first polypeptide chains can form a dimer through dimerization association of the Fc region.
- the two first polypeptide chains comprised in the bispecific antibody need not be identical; and similarly, the two second polypeptide chains need not be identical either.
- the two first polypeptide chains are identical and the two second polypeptide chains are also identical.
- the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, wherein the antibody comprises two first polypeptide chains and two second polypeptide chains, wherein the two The first polypeptide chains may be the same or different, and the two second polypeptide chains may be the same or different, wherein:
- the two first polypeptide chains each comprise a first VHH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus;
- the two second polypeptide chains each comprise a second VHH domain and a CL domain from the N-terminus to the C-terminus;
- first VHH structural domain and the second VHH structural domain specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF.
- first antigen is PD-L1 and the second antigen is VEGF; or in another embodiment, the first antigen is VEGF and the second antigen is PD-L1.
- the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus.
- the Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region.
- the Fc region polypeptide is connected to the CH1 domain at the N-terminus through an immunoglobulin hinge region sequence.
- the first VHH domain is a VHH PD-L1 domain according to the present invention, preferably, comprising the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprising SEQ ID NOs: 13- 15 CDR1-3 sequence; most preferably comprising the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH VEGF domain according to the present invention, preferably comprising SEQ ID NO: 2
- the CDR1-3 sequence of the variable region more preferably comprising the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprising the variable region sequence shown in SEQ ID NO:2.
- the bispecific antibody of the invention comprises two first polypeptide chains and two second polypeptide chains, wherein the first antigen is PD-L1 and the second antigen is VEGF, wherein:
- the two first polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 9 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
- the two second polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 10 and have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% % identity amino acid sequence.
- the two first polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO:9; and the two second polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO:10.
- the bispecific antibody of the invention comprises two first polypeptide chains and two second polypeptide chains, wherein the first antigen is VEGF and the second antigen is PD-L1, wherein:
- the two first polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 11 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences;
- the two second polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 12 and have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% % identity amino acid sequence.
- the two first polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO: 11; and the two second polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO: 12.
- the invention also contemplates an embodiment in which the CH1 and CL domains in the first and second polypeptide chains are exchanged.
- the two Fc regions are linked by the CL domain of the Fab-like structure through an immunoglobulin hinge region.
- the invention provides methods for producing antibodies of the invention.
- the polypeptide chains of the antibodies of the invention can be obtained, eg, by solid-state peptide synthesis (eg, Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions.
- polynucleotides encoding any polypeptide chain and/or multiple polypeptide chains of the antibody can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
- the polynucleotides can be readily isolated and sequenced using conventional methods.
- polynucleotides encoding one or more polypeptide chains of an antibody of the invention are provided.
- the invention provides a vector, preferably an expression vector, comprising one or more polynucleotides of the invention.
- the invention provides a method for producing an antibody of the invention, said method comprising: culturing a host cell comprising a polypeptide chain encoding said antibody under conditions suitable for expressing said polypeptide chain; and The antibody is produced by assembling the polypeptide chains under conditions suitable for assembly of the polypeptide chains into the antibody.
- Expression vectors can be constructed using methods well known to those skilled in the art.
- Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
- the expression vector can be transfected or introduced into a suitable host cell.
- a suitable host cell for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.
- the invention also provides host cells comprising one or more polynucleotides of the invention.
- host cells comprising an expression vector of the invention are provided.
- the term "host cell” refers to any kind of cellular system that can be engineered to produce the antibodies of the invention.
- Host cells suitable for replicating and supporting expression of the antibodies of the invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors as needed, and large numbers of cells containing the vector can be grown for inoculation of large-scale fermenters to obtain sufficient quantities of the antibodies of the invention for clinical use.
- Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells and the like. Mammalian cell lines suitable for suspension culture can be used.
- Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2) , CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2/0, etc.
- the host cell is a CHO, HEK293 or NSO cell.
- Antibodies prepared by the methods described herein can be purified by known art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. After purification, the antibodies of the invention can be determined by any of a variety of well known assays.
- the well-known analytical methods include size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.
- the physical/chemical properties and/or biological activities of the antibodies provided herein can be identified, screened or characterized by a variety of assays known in the art.
- the bispecific antibody of the present invention exhibits good production properties in recombinant production in mammalian host cells such as CHO cells, in particular, good expression yield and good by-product profile.
- the transient expression level of the bispecific antibody of the present invention reaches above 50 ⁇ g/mL, preferably above 150 ⁇ g/mL; After purification, the purity of the product is determined by SEC-HPLC, showing a purity of more than 95%, preferably more than 96%, 97%, 98% or 99%.
- the determination of the transient expression level is determined according to the method described in Example 3; the determination of the purity of the product after one-step protein A purification is performed according to the SEC-HPLC monomer purity identification method described in Example 3.4.
- the invention provides compositions, eg, pharmaceutical compositions, comprising an antibody described herein formulated together with a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
- the pharmaceutical compositions of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion).
- an antibody of the invention is the sole active ingredient in a pharmaceutical composition.
- a pharmaceutical composition may comprise an antibody described herein and more than one therapeutic agent.
- the invention also provides pharmaceutical combinations comprising an antibody described herein and more than one therapeutic agent.
- Therapeutic agents suitable for use in the pharmaceutical compositions and drug combinations of the invention may be therapeutic agents selected from any of the following categories (i)-(iv): (i) agents that enhance antigen presentation (e.g., tumor antigen presentation) Drugs; (ii) drugs that enhance effector cell responses (eg, B cell and/or T cell activation and/or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor suppressive effects.
- agents that enhance antigen presentation e.g., tumor antigen presentation
- Drugs e.g., tumor antigen presentation
- drugs that enhance effector cell responses eg, B cell and/or T cell activation and/or mobilization
- drugs that reduce immunosuppression e.g., B cell and/or T cell activation and/or mobilization
- drugs that reduce immunosuppression e.g., B cell and/or T cell activation and/or mobilization
- drugs that reduce immunosuppression e.g., B cell and/or T cell activ
- compositions of the invention can be in a variety of forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories.
- liquid solutions eg, injectable solutions and infusible solutions
- dispersions or suspensions e.g., liposomes, and suppositories.
- the preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions.
- the preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection.
- the antibody is administered by intravenous infusion or injection.
- the antibody is administered by intramuscular, intraperitoneal or subcutaneous injection.
- parenteral administration and “parenteral administration” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.
- compositions should generally be sterile and stable under the conditions of manufacture and storage.
- Compositions can be formulated as solutions, microemulsions, dispersions, liposomes or in lyophilized form.
- Sterile injectable solutions can be prepared by incorporating the active compound (ie, antibody) in the required amount in an appropriate solvent, followed by filtered sterilization.
- dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and other ingredients. Coating agents such as lecithin and the like can be used.
- proper fluidity of the solution can be maintained by using a surfactant.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition substances which delay absorption, for example, monostearate salts and gelatin.
- the pharmaceutical composition of the present invention may contain "therapeutically effective amount” or “prophylactically effective amount” of the antibody of the present invention.
- a “therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result. can be based on a variety of factors such as disease status, individual age, A therapeutically effective amount for such changes as sex and weight. A therapeutically effective amount is one in which any noxious or detrimental effects are outweighed by the therapeutically beneficial effects.
- a “therapeutically effective amount” preferably inhibits a measurable parameter (e.g. tumor growth rate) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60% and still more relative to an untreated subject. Preferably at least about 80%.
- the ability of the antibodies of the invention to inhibit a measurable parameter eg, tumor volume
- prophylactically effective amount refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, the prophylactically effective amount is less than the therapeutically effective amount because the prophylactic dose is used in the subject before or at an earlier stage of the disease.
- Kits comprising the antibodies described herein are also within the scope of the invention.
- a kit may comprise one or more other elements including, for example: instructions for use; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.
- the present invention provides in vivo and in vitro uses and methods of application of the antibodies of the present invention.
- the uses and methods of the invention involve the in vivo and/or in vitro application of the antibodies of the invention to:
- the antibody of the present invention or a pharmaceutical composition comprising the antibody of the present invention is used as a drug for treating and/or preventing a disease or as a diagnostic tool for a disease in an individual, preferably, the individual is a mammal, more preferably Preferably a human.
- the present invention provides methods and uses of using the antibodies of the present invention, especially bispecific antibodies, to treat cancer, wherein the cancer can be PD-L1 positive solid tumors or blood cancers, for example selected from head and neck squamous cell carcinoma, Melanoma, renal cell carcinoma, non-small cell lung cancer, bladder cancer, urothelial carcinoma, gastric cancer, colon cancer, colorectal cancer, ovarian cancer, breast cancer, lung cancer, cervical cancer, glioblastoma, pancreatic cancer, prostate cancer Carcinoma, esophageal cancer, lymphoma, liver cancer, microsatellite unstable solid tumors.
- said cancer is colorectal cancer, liver cancer, ovarian cancer, breast cancer, lung cancer.
- the invention provides in vitro or in vivo diagnostic methods for detecting the presence of relevant antigens in biological samples such as serum, semen or urine or tissue biopsy samples (eg, from hyperproliferative or cancerous lesions).
- the diagnostic method comprises: (i) contacting a sample (and optionally, a control sample) with an antibody as described herein or administering the antibody to a subject under conditions that allow the interaction to occur and (ii) detecting the detected Complex formation between the antibody and the sample (and optionally, a control sample) is performed. Formation of the complex indicates the presence of the relevant antigen and may indicate suitability or need for treatment and/or prophylaxis as described herein.
- the invention provides a diagnostic kit comprising an antibody described herein and instructions for use.
- Embodiment 1 raw material preparation
- the extracellular region of human PD-L1 (UniProtKB-Q9NZQ7), the extracellular region of human PD-1 (UniProtKB-Q15116) and human VEGF (UniProtKB-P15692) were synthesized by General Biosystems (Anhui) Co., Ltd. for the target fragment gene.
- Each target fragment was amplified by PCR, and a His tag or hIgG1Fc (shown in the amino acid sequence SEQ ID NO: 3) was introduced at the C-terminus by primers, and then respectively constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination .
- the constructed recombinant protein expression vectors were respectively transformed into Escherichia coli DH5 ⁇ , cultured overnight at 37°C, and then plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids and For eukaryotic expression.
- OEGA endotoxin-free plasmid extraction kit
- Recombinant protein VEGF-His, recombinant protein VEGF-Fc, recombinant protein PD-L1-His, recombinant protein PD-L1-Fc, recombinant protein PD-1-His and recombinant protein PD-1-Fc were all passed through the Expi293 transient expression system (ThermoFisher, A14635) expression, see Expi293 TM Expression System Kit Manual for the transient method. After 5-7 days of transfection, the cell expression supernatant was centrifuged at a high speed of 15000 g for 10 min.
- the resulting His-tagged recombinant protein expression supernatant was affinity-purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with gradient concentrations of imidazole.
- the filter concentration tube (Millipore, UFC901096) was replaced with PBS buffer; the resulting Fc-tagged recombinant protein expression supernatant was filtered through a 0.22 ⁇ m filter membrane, and then purified by Protein A/G affinity chromatography column affinity method.
- the target protein was eluted with 100mM glycine salt (pH3.0), then concentrated and replaced, and then frozen at -80°C after being identified by SDS-PAGE and qualified for activity identification.
- anti-human VEGF antibody P30-10-26 (VHH VEGF -hIgG1Fc, VHH VEGF amino acid sequence shown in SEQ ID NO: 2) is derived from patent application CN202110995278.7; anti-human PD-L1 antibody D21-4 (VHH PD-L1 -hIgG1Fc, VHH PD-L1 amino acid sequence shown in SEQ ID NO: 1) is derived from the patent application PCT/CN2020/125301; the anti-VEGF control antibody Bevacizumab (Bevacizumab, abbreviated as Beva) sequence is derived from Drug Bank (Drug Bank No: DB00112); anti-PD-L1 control antibody Atezolizumab (Ate) sequence is derived from Drug Bank (Drug Bank No: DB11595); anti-PD-L1 and VEGF control bispecific
- Bevacizumab Bevacizumab, abbreviated as Beva
- Beva anti-PD-L1 control
- the antibodies were all expressed using the transient system (ExpiCHO).
- ExpiCHO transient system
- the expressed cell suspension was subjected to high-speed centrifugation and the supernatant was taken, and the obtained supernatant was filtered through a 0.22 ⁇ m filter membrane, and then purified by using a Protein A/G affinity chromatography column affinity method. After purification, the target protein was eluted with 100 mM glycine salt (pH 3.0), then concentrated and replaced, and then frozen at -80°C after being identified by SDS-PAGE and qualified for activity identification.
- the CHO-K cells (Thermo, A1461801) were passaged to 5 ⁇ 10 6 cells/mL, and the next day, the constructed cells containing full-length human PD-L1 Plasmid introduction of (UniProtKB-Q9NZQ7) gene sequence in CHO-K cells.
- the cells after electroporation were transferred to CD-CHO medium (Gibco, 10743029), and placed in a cell culture incubator at 37°C for 48 hours. Then, 2000 cells/well were plated into a 96-well plate, and MSX (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C) were added to a final concentration of 30 ⁇ M for pressurized screening.
- MSX Micropore, GSS-1015-F
- GS supplement Sigma, 58672C
- the pGL4.30 plasmid (promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into HEK293 cells ( CRL-1573 TM ) to obtain HEK293-NFAT cell line.
- the HEK293-NFAT cells were passaged to 2 ⁇ 105 cells/mL, and the next day, the electroporation kit (Invitrogen, MPK10096) and electroporation instrument (Invitrogen, MP922947) were used to transform the constructed human VEGFR2 gene sequence (NCBI Gene ID: 3791) plasmid into HEK293-NFAT cells.
- the electroporated cells were transferred to DMEM medium (Gibco, 12634010), and placed in a 37°C cell culture incubator for 48 hours. Then, 1000 cells/well were plated in a 96-well plate, and puromycin at a final concentration of 2 ⁇ g/mL was added for pressure selection. The single-cell clones grown in the 96-well plate were picked, and after expanded culture, the HEK293-VEGFR2-NFAT cell line was obtained by adding a luciferase catalytic substrate to detect the fluorescent signal and identify it.
- the pGL4.30 plasmid (promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into Jurkat cells ( TIB-152) to obtain Jurkat-NFAT cell line.
- stably transform the full-length expression gene sequence of PD-1 NCBI Gene ID: 5133
- Monoclonal cell lines were screened, and Jurkat-PD-1-NFAT cell lines were obtained by FACS identification after expansion.
- the scFv sequence of OKT-3 (Drug Bank No: DB00075) was electrotransfected into CHO cells to obtain CHO-CD3L stably transfected cell line, on this basis, the full-length expression gene sequence of PD-L1 was stably transfected (NCBI Gene ID: 29126), The method was consistent with 1.3.2. The final concentration of 30 mM methionine iminosulfone and 8 ⁇ g/mL puromycin were added to screen monoclonal cell lines, and the CHO-PD-L1-CD3L cell line was obtained by FACS identification after expansion.
- This example describes the construction of exemplary anti-PD-L1 and VEGF bispecific antibody (BsAb) structures and expression vectors.
- BsAb bispecific antibody
- Four constructs were designed and constructed: the amino acid sequence of VHH PD-L1 is from D21-4, and its variable region amino acid sequence is shown in SEQ ID NO:1; the VHH VEGF amino acid sequence is from P30-10-26, and its variable region The amino acid sequence is shown in SEQ ID NO: 2; the amino acid sequence of the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 19).
- Exemplary BsAb constructs are shown in Table 1 and the corresponding amino acid sequences are provided in Table 2.
- BsAb10 Contains two identical polypeptide chains, including anti-PD-L1 VHH domain, IgG1 heavy chain hinge region, IgG1 heavy chain Fc, linker and anti-VEGF VHH domain from N-terminal to C-terminal.
- BsAb10 has the format of Figure 1A.
- BsAb11 Contains two identical polypeptide chains, including anti-PD-L1 VHH domain, linker, anti-VEGF VHH domain, IgG1 heavy chain hinge region and IgG1 heavy chain Fc from N-terminal to C-terminal.
- BsAb11 has the format of Figure 1B.
- Construct BsAb12 Contains two identical first polypeptide chains, including anti-PD-L1 VHH domain from N-terminal to C-terminal, IgG1 heavy Chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, IgG1 heavy chain Fc); contains two identical second polypeptide chains, from N-terminal to C-terminal contains anti-VEGF VHH domain, antibody Kappa light chain CL domain.
- BsAb12 has the format of Figure 1C.
- BsAb13 Contains two identical first polypeptide chains, including anti-VEGF VHH domain from N-terminal to C-terminal, IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, IgG1 Heavy chain Fc); Contains two identical second polypeptide chains, including VHH domain of anti-PD-L1 and CL domain of antibody kappa light chain from N-terminus to C-terminus.
- BsAb13 has the format of Figure 1D.
- fragments of the variable region and constant region of each antibody are amplified by PCR method, and the fragments are connected by overlap extension PCR method, and then constructed into modified eukaryotic expression vectors by homologous recombination method
- the full-length gene of the polypeptide chain constituting the complete construct.
- the constructed vectors containing the full-length gene of the polypeptide chain of the construct were respectively transformed into Escherichia coli DH5 ⁇ , and cultured overnight at 37°C.
- the endotoxin-free plasmid extraction kit (OMEGA, D6950-01) was used for plasmid extraction to obtain an endotoxin-free construct polypeptide chain plasmid for eukaryotic expression.
- Example 3 Expression, purification, and analysis of physicochemical properties of anti-PD-L1 and VEGF bispecific antibody
- Example 2 The construct in Example 2 was expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: on the day of transfection, confirm that the cell density was about 7 ⁇ 10 6 to 1 ⁇ 10 7 cells/mL, and the cell survival At this point, adjust the cells to a final concentration of 6 ⁇ 10 6 cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C.
- OptiPRO TM SFM Dilute the target plasmid with 4°C pre-cooled OptiPRO TM SFM (add 1 ⁇ g of plasmid to 1 mL of the medium), and at the same time, dilute ExpiFectamine TM CHO with OptiPRO TM SFM, then mix the two in equal volumes and gently blow and mix to prepare Form ExpiFectamine TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5min, slowly add to the prepared cell suspension and shake gently at the same time, and finally place in a cell culture shaker at 37°C, 8% CO 2 conditions under cultivation.
- the cell culture supernatant expressing the target protein was centrifuged at a high speed of 15000g for 10min, and the resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then purified with 100mM sodium acetate (pH3.0 ) to elute the target protein, then neutralize it with 1M Tris-HCl, and finally replace the obtained protein into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096).
- the concentration of the purified bispecific antibody in Example 3.1 was measured with a verified ultra-micro spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300), and the measured A280 value was divided by the theoretical extinction coefficient of the antibody. The value is used as the antibody concentration value for follow-up research. After passing the quality inspection, it is aliquoted and stored at -80°C.
- Non-reducing solution preparation 1 ⁇ g of candidate bispecific antibody and reference product IPI (the IPI is the abbreviation of Ipilimumab (Ipilimumab), prepared by the method in Example 3.1) was added to 5 ⁇ SDS loading buffer and 40mM Iodoacetamide was heated in a dry bath at 75°C for 10 minutes, cooled to room temperature, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was taken.
- IPI is the abbreviation of Ipilimumab (Ipilimumab)
- Preparation of reducing solution Add 2 ⁇ g of candidate bispecific antibody and reference IPI to 5 ⁇ SDS loading buffer and 5 mM DTT, heat in a dry bath at 100°C for 10 min, cool to room temperature, centrifuge at 12,000 rpm for 5 min, and take the supernatant.
- SEC-HPLC results of candidate bispecific antibodies are as follows: the percentages of high molecular polymers, antibody monomers and low molecular substances in the sample were calculated according to the area normalization method, and the results are shown in Figures 3A-3D and Table 3.
- huPD-L1-CHO-K cells Collect the huPD-L1-CHO-K cells in the exponential growth phase, centrifuge at 300g to remove the supernatant, resuspend the cells in FACS buffer (PBS containing 1% BSA), count and adjust the density of the cell suspension to 2 ⁇ 10 6 individual/mL. Subsequently, huPD-L1-CHO-K cells were added to a 96-well round bottom plate at 100 ⁇ L per well, and centrifuged at 300 g to remove the supernatant. Different concentrations of candidate bispecific antibody and control antibody D21-4 dilutions were added to the corresponding wells, the cells were resuspended and incubated at 4°C for 30 min.
- a PE-labeled anti-human-IgG-Fc flow antibody (Abcam, 98596) was added, resuspended and incubated at 4°C for 30 min. After the incubated cell mixture was washed three times, 200 ⁇ L of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). Data were analyzed using PRISM TM (GraphPad Software, San Diego, CA), and EC50 values were calculated.
- PRISM TM GraphPad Software, San Diego, CA
- bispecific antibodies BsAb10 and BsAb12 showed better binding ability to PD-L1 expressed on cells than control antibody D21-4.
- the recombinant protein PD-L1-Fc was coated on a 96-well ELISA plate overnight at 4°C. On the next day, the well plate was washed 3 times with PBST and then blocked with 5% skim milk for 2 hours. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibodies were added and incubated for 1 hour.
- the luciferase reporter gene system was used to detect the activity of candidate anti-PD-L1 and VEGF bispecific antibodies in blocking the PD-1/PD-L1 signaling pathway, and the combination of PD-1 and PD-L1 can block the downstream signal of CD3 Transduced to inhibit luciferase expression system, when adding anti-PD-L1 and VEGF bispecific antibody, the blocking effect is reversed, luciferase expression, stimulated with antibodies with different concentration gradients, will have antibody concentration-dependent A specific fluorescence readout curve allows the evaluation of the blocking activity of the antibody.
- the luciferase reporter gene system was used to detect the activity of candidate anti-PD-L1 and VEGF bispecific antibodies in blocking the PD-1/PD-L1 signaling pathway. The specific method is as follows:
- the HEK293-VEGFR2-NFAT cell line was used (the VEGF recombinant protein was added to the cell line culture system to activate the cell line through the VEGF-VEGFR2 signaling axis). Transcription and expression of the internal NFAT luciferase reporter gene, adding the catalytic substrate of luciferase to generate a fluorescent signal) as a material to detect the binding of the candidate bispecific antibody to VEGF-VEGFR2 to block the expression of the downstream NFAT luciferase reporter gene ability.
- the specific implementation is as follows:
- Adjust the HEK293-VEGFR2-NFAT cell line to 4 ⁇ 10 5 cells/mL add 100 ⁇ L per well to a new 96-well cell culture plate, and place it in a 37°C cell culture incubator.
- the candidate bispecific antibody and control antibody P30-10-26 were serially diluted in DMEM medium, 60 ng/mL VEGF-Fc was added, mixed and incubated at room temperature for 30 min. Subsequently, the co-incubated serially diluted antibody and VEGF-Fc mixture was added to a 96-well cell culture plate, and cultured in a 37° C. incubator for 18 hours. After the incubation, 30 ⁇ L of luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and the fluorescence value of the 96-well plate was detected after shaking for 2 minutes.
- Example 7 In vivo tumor suppression experiment of anti-PD-L1 and VEGF bispecific antibody (COLO205 mouse tumor-bearing model)
- NOG mice purchased from Shanghai Weitong Lihua, strain: NOD.Cg-Prkdc scid Il2rg tm1Sug /JicCrl
- the experimental mice were kept in an independent ventilated box with constant temperature and humidity, and the temperature of the feeding room was 21 -24°C, humidity 30-53%.
- the huPD-L1-COLO205 cells (a stably transfected cell line overexpressing human PD-L1 obtained by stably transfecting PD-L1 (Gene ID: 29126) into COLO205 cells (purchased from Beina Biotech, catalog number: BNCC338682)) were 3 ⁇ 10 6 mice/mouse were subcutaneously injected into the right back (day 0), and then randomly divided into groups (6 mice per group): PBS treatment group, Atezolizumab administration group, Bevacizumab administration group , Atezolizumab+Bevacizumab combined administration group, W3256 administration group, AK112 administration group and bispecific antibody BsAb10 administration group, each administration group has a high-dose group, in which the bispecific antibody BsAb10 administration group and AK112 The administration group also set up a middle and low dose group.
- PBMC peripheral blood mononuclear cell
- ip intraperitoneal injection
- Example 8 In vivo tumor suppression experiment of anti-PD-L1 and VEGF bispecific antibody (A431 mouse tumor-bearing model)
- mice 6-8 weeks old female NSG mice (purchased from Shanghai Nsweeping Model Organisms, strain: M-NSG) were used, and the experimental mice were kept in an independent ventilation box with constant temperature and humidity. %.
- A431 cells human epidermal cancer cells
- PBS treatment group respectively , D21-4 low-dose administration group (0.37mpk (mg/kg)), P30-10-26 low-dose administration group (0.37mpk), bispecific antibody BsAb10 high-dose administration group (2mpk), bispecific Antibody BsAb10 low-dose administration group (0.5mpk), D21-4 and P30-10-26 high-dose combination treatment group (1.48+1.48mpk), D21-4 and P30-10-26 low-dose combination treatment group (0.37 +0.37mpk).
- PBMC C2106025
- ip intraperitoneal injection
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Abstract
The present invention relates to a bispecific antibody targeting PD-L1 and VEGF and a method for preparing same, and also relates to a pharmaceutical composition comprising the bispecific antibody and use thereof in treatment and diagnosis.
Description
本发明属于生物医药领域,具体涉及一种靶向PD-L1和VEGF的双特异性抗体及其制备方法和应用。The invention belongs to the field of biomedicine, and in particular relates to a bispecific antibody targeting PD-L1 and VEGF and its preparation method and application.
PD-L1属于细胞膜上的一型跨膜蛋白,表达在T细胞、B细胞等免疫细胞以及肿瘤细胞上。Tasuku Honjo等发现并证明,当肿瘤细胞膜上的PD-L1与T细胞等免疫细胞上的PD-1结合后,肿瘤细胞发出抑制性信号,进而T细胞不能识别肿瘤细胞和对肿瘤细胞产生杀伤作用,机体的免疫功能受到抑制(Chamoto,K.,Al-Habsi,M.,&Honjo,T.(2017).Current topics in microbiology and immunology,410,75-97)。陈列平等首次通过PD-L1阻断性抗体联合T细胞治愈约60%头颈癌小鼠(Chen,L et al.(2003).Cancer research,63(19),6501-6505.)。目前已有10个抗PD-1或PD-L1单抗在中国获批上市,适应症针对PD-L1阳性的NSCLC、头颈鳞癌和黑色素瘤等多种实体瘤(Radvanyi,et al,P.(2013).letter.Clinical cancer research,19(19),5541)。然而,PD-L1表达水平、肿瘤突变负荷(TMB)等病理检测指标预示抗PD-1/PD-L1抗体响应率低,目前只对少于40%的实体瘤有作用,且30%患者在用药后出现一定的耐药性。PD-L1 belongs to a type of transmembrane protein on the cell membrane, and is expressed on T cells, B cells and other immune cells and tumor cells. Tasuku Honjo et al. discovered and proved that when PD-L1 on the tumor cell membrane combines with PD-1 on immune cells such as T cells, the tumor cells send out inhibitory signals, and then T cells cannot recognize tumor cells and have a killing effect on tumor cells , the immune function of the body is suppressed (Chamoto, K., Al-Habsi, M., & Honjo, T. (2017). Current topics in microbiology and immunology, 410, 75-97). For the first time, Chen Lieping cured about 60% of head and neck cancer mice by combining PD-L1 blocking antibody with T cells (Chen, L et al. (2003). Cancer research, 63 (19), 6501-6505.). At present, 10 anti-PD-1 or PD-L1 monoclonal antibodies have been approved for marketing in China, and the indications are for PD-L1 positive NSCLC, head and neck squamous cell carcinoma and melanoma and other solid tumors (Radvanyi, et al, P. (2013). letter. Clinical cancer research, 19(19), 5541). However, pathological detection indicators such as PD-L1 expression level and tumor mutation burden (TMB) indicate a low response rate of anti-PD-1/PD-L1 antibodies, which currently only have an effect on less than 40% of solid tumors, and 30% of patients in the A certain amount of drug resistance appears after treatment.
促进血管生成最关键的细胞因子为VEGF-A(也称VEGF)。VEGF激活VEGFR2(介导血管生成主要的受体酪氨酸激酶受体)促进血管内皮细胞有丝分裂和血管渗透性增加,从而促进新血管出芽。同时,VEGF在肿瘤微环境中的高表达能够放大PD-(L)1的免疫抑制效应。因此,靶向VEGF或者VEGFR2能够有效抑制异常的血管增生(Ferrara,N.(2010).Mol Biol Cell 21(5):687-690.)。抗VEGF单抗贝伐珠单抗(Bevacizumab)在肿瘤治疗和在血管异常增生相关的眼病中,都具有较好的疗效和靶点安全性(Pfisterer,J.,et al.(2020).Lancet Oncol 21(5):699-709.Bhandari,S.,et al.(2020).Ophthalmology 127(5):608-615.)。The most critical cytokine to promote angiogenesis is VEGF-A (also known as VEGF). VEGF activates VEGFR2 (the main receptor tyrosine kinase receptor that mediates angiogenesis) to promote mitosis of vascular endothelial cells and increased vascular permeability, thereby promoting new vessel sprouting. At the same time, the high expression of VEGF in the tumor microenvironment can amplify the immunosuppressive effect of PD-(L)1. Therefore, targeting VEGF or VEGFR2 can effectively inhibit abnormal vascular proliferation (Ferrara, N. (2010). Mol Biol Cell 21 (5): 687-690.). The anti-VEGF monoclonal antibody Bevacizumab (Bevacizumab) has good efficacy and target safety in tumor treatment and eye diseases related to abnormal blood vessel proliferation (Pfisterer, J., et al. (2020). Lancet Oncol 21(5):699-709. Bhandari, S., et al. (2020). Ophthalmology 127(5):608-615.).
抗VEGF单抗除了具有抗血管生成作用外,能够改善血管,促进T细胞浸润进入肿瘤,并抑制DC成熟,促进T细胞的启动和活化,改善肿瘤微环境,提高抗PD-1/PD-L1药物响应率,进一步通过T细胞介导的肿瘤杀伤,恢复肿瘤免疫功能(Socinski,M.A.,et al.(2018).N Engl J Med 378(24):2288-2301.N Engl J Med.2018Jun 14;378(24):2288-2301.N Engl J Med 2020;382:1894-1905)。最新的临床试验结果证实,在肝癌、肺癌、子宫内膜癌和肾癌等肿瘤治疗中抗VEGF单抗联合抗PD-L1单抗,表现出了比化药或者抗VEGF单抗或抗PD-L1单抗更优越的药效,取得非常重大的临床突破。目前抗PD-L1/VEGF单抗联用和抗PD-1/VEGF双特异性抗体均进展至临床三期,治疗相关不良反应分别为91%和63.4%,三级以上治疗相关不良反应分别为40%和19.5%,说明双特异性抗体相较于单抗联用在减少毒副作用和给药剂量上更具有潜力,具有更好的安全性;同时,抗PD-L1相对于抗PD-1能够更好地靶向进入到肿瘤微环境中,能改善对正常组织的毒副作用。In addition to its anti-angiogenic effect, anti-VEGF monoclonal antibodies can improve blood vessels, promote T cell infiltration into tumors, inhibit DC maturation, promote T cell activation and activation, improve tumor microenvironment, and increase anti-PD-1/PD-L1 Drug response rate, further through T cell-mediated tumor killing, restore tumor immune function (Socinski, M.A., et al.(2018).N Engl J Med 378(24):2288-2301.N Engl J Med.2018Jun 14 ; 378(24):2288-2301. N Engl J Med 2020; 382:1894-1905). The latest clinical trial results confirm that anti-VEGF monoclonal antibody combined with anti-PD-L1 monoclonal antibody is more effective than chemical drugs or anti-VEGF monoclonal antibody or anti-PD-L1 monoclonal antibody in the treatment of liver cancer, lung cancer, endometrial cancer and kidney cancer. The superior efficacy of L1 monoclonal antibody has achieved a very significant clinical breakthrough. At present, both anti-PD-L1/VEGF monoclonal antibody combination and anti-PD-1/VEGF bispecific antibody have progressed to clinical phase III, and treatment-related adverse reactions were 91% and 63.4%, respectively. 40% and 19.5%, indicating that compared with monoclonal antibodies, bispecific antibodies have more potential in reducing toxic side effects and dosage, and have better safety; at the same time, anti-PD-L1 has better safety than anti-PD-1 It can better target into the tumor microenvironment, and can improve the toxic and side effects on normal tissues.
在双特异性抗体药物的开发中,候选抗体的生物学活性是需要纳入考量的重要因素。尽管目前有许多双特异性抗体被提出,但由于不同治疗产品在治疗功能性和治疗行为上的要求具有多样性,目前尚不存在可以普遍适用于大多数不同期望分子组合的理论预测方法。因此,往往需要就具体靶点和抗体组成有针对性开发在生物活性上表现良好的双特异性抗体。
In the development of bispecific antibody drugs, the biological activity of candidate antibodies is an important factor that needs to be taken into consideration. Although many bispecific antibodies have been proposed, due to the diversity of therapeutic functional and therapeutic behavior requirements of different therapeutic products, there is no theoretical prediction method that can be generally applied to most different desired molecular combinations. Therefore, it is often necessary to develop bispecific antibodies with good biological activity for specific targets and antibody compositions.
发明内容Contents of the invention
现有技术中的抗PD-L1和VEGF双特异性抗体主要为抗PD-L1抗体融合VEGFR1蛋白(US20200172623A1、WO2020200210A1)或Mab-VHH型抗PD-L1和VEGF双特异性抗体(WO2021147829A1),这些双特异性抗体都含有IgG1型全长抗体,存在分子量较大和成药性问题。通过深入研究,本发明人在开发的抗PD-L1纳米抗体和抗VEGF纳米抗体的基础上,开发了同时靶向VEGF和PD-L1的双特异性纳米抗体,分子量仅为107KDa,具有潜在的肿瘤组织浸润强和给药剂量小的优势,在临床剂量和安全性上优于主要竞品。进一步地,本发明双特异性抗体中的抗VEGF纳米抗体部分的体外VEGF中和活性效果优于已上市的Bevacizumab,并且在血管生成的动物模型上,在等质量给药的情况下,体内血管生成抑制活性也优于Bevacizumab。同时,本发明双特异性抗体中的抗PD-L1纳米抗体部分在体外阻断活性和细胞结合水平上,与已上市Atezolizumab类似。更进一步地,本发明双特异性抗体相较于单抗联用和其它现有技术的双特异性抗体,能够同时阻断PD-1/PD-L1以及VEGF/VEGFR信号通路,在改善血管和肿瘤微环境的同时,通过恢复免疫,具有更加显著的抑制肿瘤效果,具有非常广阔的应用前景。Anti-PD-L1 and VEGF bispecific antibodies in the prior art are mainly anti-PD-L1 antibody fused to VEGFR1 protein (US20200172623A1, WO2020200210A1) or Mab-VHH anti-PD-L1 and VEGF bispecific antibody (WO2021147829A1), these Bispecific antibodies all contain full-length IgG1 antibodies, which have problems of large molecular weight and druggability. Through in-depth research, on the basis of the developed anti-PD-L1 nanobody and anti-VEGF nanobody, the inventors developed a bispecific nanobody targeting VEGF and PD-L1 at the same time, with a molecular weight of only 107KDa, which has potential With the advantages of strong tumor tissue infiltration and small dosage, it is superior to major competitors in terms of clinical dosage and safety. Further, the in vitro VEGF neutralizing activity of the anti-VEGF nanobody part of the bispecific antibody of the present invention is better than that of the marketed Bevacizumab, and in the animal model of angiogenesis, in the case of equal dose administration, the blood vessels in vivo The production inhibitory activity is also superior to that of Bevacizumab. At the same time, the anti-PD-L1 nanobody part of the bispecific antibody of the present invention is similar to the marketed Atezolizumab in terms of in vitro blocking activity and cell binding level. Furthermore, compared with monoclonal antibody combination and other bispecific antibodies of the prior art, the bispecific antibody of the present invention can simultaneously block PD-1/PD-L1 and VEGF/VEGFR signaling pathways, and improve blood vessel and At the same time, by restoring immunity, it has a more significant effect of inhibiting tumors, and has a very broad application prospect.
因此,在一方面,本发明提供了一种特异性结合PD-L1和VEGF的双特异性抗体,所述抗体包括特异性结合PD-L1的一个或两个VHH结构域(VHHPD-L1)和特异性结合VEGF的一个或两个VHH结构域(VHH VEGF),其中所述VHHVEGF包含SEQ ID NO:2所示的VHH结构域的CDR1-3序列,且优选地,其中所述VHHPD-L1包含SEQ ID NO:1所示的VHH结构域的CDR1-3序列。Therefore, in one aspect, the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, said antibody comprising one or two VHH domains specifically binding to PD-L1 (VHH PD-L1 ) and one or two VHH domains (VHH VEGF ) specifically binding to VEGF, wherein the VHH VEGF comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO: 2, and preferably, wherein the VHH PD -L1 comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:1.
在一个实施方案中,所述的VHHPD-L1和VHHVEGF结构域可以以包含在同一条多肽链上的串联形式(例如借助于连接子和/或Fc区串联在一条多肽链上);或以Fab样结构(例如,借助于免疫球蛋白CH1和CL恒定结构域形成Fab样结构),连接在免疫球蛋白Fc区上。In one embodiment, the VHH PD-L1 and VHH VEGF domains may be included in a tandem form on the same polypeptide chain (for example, by means of a linker and/or an Fc region in tandem on a polypeptide chain); or Linked to the immunoglobulin Fc region in a Fab-like structure (for example, by means of the immunoglobulin CH1 and CL constant domains to form a Fab-like structure).
在一个优选实施方案中,所述双特异性抗体包含从N端至C端包含第一VHH结构域、Fc区、连接子和第二VHH结构域的多肽链,其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同且独立地选自PD-L1和VEGF,优选地所述第一抗原为PD-L1且第二抗原为VEGF。优选地,所述Fc区为来自IgG,尤其是IgG1的Fc区,从N端到C端包含CH2和CH3结构域序列。所述Fc区可以为天然序列Fc区或变体Fc区,优选天然序列Fc区。优选地,Fc区多肽在N端通过免疫球蛋白铰链区序列与第一VHH结构域连接。再优选地,Fc区多肽在C端通过柔性连接肽,例如(G4S)3(SEQ ID NO:19),与第二VHH结构域连接。优选地,所述多肽链包含SEQ ID NO:7所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。In a preferred embodiment, the bispecific antibody comprises a polypeptide chain comprising a first VHH domain, an Fc region, a linker and a second VHH domain from the N-terminus to the C-terminus, wherein the first VHH domain and the second VHH domain are The two VHH domains specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 And the second antigen is VEGF. Preferably, the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is linked at the N-terminus to the first VHH domain through an immunoglobulin hinge region sequence. Still preferably, the Fc region polypeptide is connected to the second VHH domain at the C-terminus via a flexible linker peptide, such as (G 4 S) 3 (SEQ ID NO: 19). Preferably, the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 7 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity amino acid sequence.
在另一优选实施方案中,所述双特异性抗体包含从N端至C端包含第一VHH结构域、连接子、第二VHH结构域和Fc区的多肽链,其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同且独立地选自PD-L1和VEGF,优选地所述第一抗原为PD-L1且第二抗原为VEGF。优选地,所述第一和第二VHH结构域通过连接子,例如(G4S)3(SEQ ID NO:19),串联。优选地,所述Fc区为来自IgG,尤其是IgG1的Fc区,从N端到C端包含CH2和CH3结构域序列。所述Fc区可以为天然序列Fc区或变体Fc区,优选天然序列Fc区。优选地,所述Fc区多肽在N端
通过免疫球蛋白铰链区序列与第二VHH结构域连接。优选地,所述多肽链包含SEQ ID NO:8所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。In another preferred embodiment, the bispecific antibody comprises a polypeptide chain comprising a first VHH domain, a linker, a second VHH domain and an Fc region from the N-terminus to the C-terminus, wherein the first VHH domain and The second VHH domain specifically binds the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD- L1 and the second antigen is VEGF. Preferably, said first and second VHH domains are connected in tandem via a linker, eg ( G4S ) 3 (SEQ ID NO: 19). Preferably, the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is at the N-terminus Linked to the second VHH domain by an immunoglobulin hinge region sequence. Preferably, the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 8 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity amino acid sequence.
在再一优选实施方案中,所述双特异性抗体包含第一和第二多肽链,其中,In yet another preferred embodiment, said bispecific antibody comprises a first and a second polypeptide chain, wherein,
所述第一多肽链从N端至C端包含第一VHH结构域、CH1结构域和Fc区;The first polypeptide chain comprises a first VHH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus;
所述第二多肽链从N端至C端包含第二VHH结构域和CL结构域;The second polypeptide chain comprises a second VHH domain and a CL domain from the N-terminus to the C-terminus;
其中第一多肽链中的第一VHH结构域和CH1结构域与第二多肽链中的第二VHH结构域和CL结构域配对形成Fab样结构;wherein the first VHH domain and the CH1 domain in the first polypeptide chain are paired with the second VHH domain and the CL domain in the second polypeptide chain to form a Fab-like structure;
其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同且独立地选自PD-L1和VEGF,优选地所述第一抗原为PD-L1且第二抗原为VEGF。优选地,所述Fc区为来自IgG,尤其是IgG1的Fc区,从N端到C端包含CH2和CH3结构域序列。所述Fc区可以为天然序列Fc区或变体Fc区,优选天然序列Fc区。优选地,Fc区多肽在N端通过免疫球蛋白铰链区序列与CH1结构域连接。优选地,第一多肽链包含SEQ ID NO:9或11所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。优选地,第二多肽链包含SEQ ID NO:10或12所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。Wherein the first VHH structural domain and the second VHH structural domain specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the The first antigen is PD-L1 and the second antigen is VEGF. Preferably, the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is connected to the CH1 domain at the N-terminus through an immunoglobulin hinge region sequence. Preferably, the first polypeptide chain comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 9 or 11 Amino acid sequences with % or 99% identity. Preferably, the second polypeptide chain comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 10 or 12 Amino acid sequences with % or 99% identity.
再一方面,本发明也提供编码本发明双特异性抗体的多肽链的核酸、包含其的载体和宿主细胞。In another aspect, the present invention also provides nucleic acids encoding the polypeptide chains of the bispecific antibodies of the present invention, vectors and host cells comprising them.
再一方面,本发明也提供包含本发明双特异性抗体的药物组合物、及其体外和体内用途,尤其是在疾病的治疗或诊断中的用途。In another aspect, the present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention, and its in vitro and in vivo uses, especially in the treatment or diagnosis of diseases.
图1A-1D显示了候选双特异性抗体的示意性结构。BsAb10具有图1A的形式;BsAb11具有图1B的形式;BsAb12具有图1C的形式;BsAb13具有图1D的形式。Figures 1A-1D show schematic structures of candidate bispecific antibodies. BsAb10 has the format of FIG. 1A; BsAb11 has the format of FIG. 1B; BsAb12 has the format of FIG. 1C; BsAb13 has the format of FIG. 1D.
图2A-2B显示了候选双特异性抗体的凝胶电泳图。图2A样品分别为BsAb10和参考品IPI;图2B样品分别为BsAb11、BsAb12、BsAb13和参考品IPI。Figures 2A-2B show gel electrophoresis profiles of candidate bispecific antibodies. The samples in Fig. 2A are BsAb10 and the reference IPI respectively; the samples in Fig. 2B are BsAb11, BsAb12, BsAb13 and the reference IPI respectively.
图3A-3D显示了候选双特异性抗体的SEC-HPLC单体检测图谱。图3A为BsAb10的SEC-HPLC单体检测图谱;图3B为BsAb11的SEC-HPLC单体检测图谱;图3C为BsAb12的SEC-HPLC单体检测图谱;图3D为BsAb13的SEC-HPLC单体检测图谱。Figures 3A-3D show the SEC-HPLC monomer detection profiles of candidate bispecific antibodies. Figure 3A is the SEC-HPLC monomer detection spectrum of BsAb10; Figure 3B is the SEC-HPLC monomer detection spectrum of BsAb11; Figure 3C is the SEC-HPLC monomer detection spectrum of BsAb12; Figure 3D is the SEC-HPLC monomer detection spectrum of BsAb13 Atlas.
图4显示了候选双特异性抗体与重组蛋白VEGF-His的结合活性。Figure 4 shows the binding activity of candidate bispecific antibodies to recombinant protein VEGF-His.
图5显示了候选双特异性抗体与重组蛋白PD-L1-His的结合活性。Figure 5 shows the binding activity of candidate bispecific antibodies to recombinant protein PD-L1-His.
图6显示了候选双特异性抗体与huPD-L1-CHO-K细胞的结合活性。Figure 6 shows the binding activity of candidate bispecific antibodies to huPD-L1-CHO-K cells.
图7A-7B显示了候选双特异性抗体同时与VEGF和PD-L1的结合活性。图7A显示了候选双特异性抗体先与重组蛋白VEGF-Fc结合再与重组蛋白PD-L1-Fc结合的活性;图7B显示了候选双特异性抗体先与重组蛋白PD-L1-Fc结合再与重组蛋白VEGF-Fc结合的活性。
Figures 7A-7B show the binding activity of candidate bispecific antibodies to both VEGF and PD-L1. Figure 7A shows the activity of candidate bispecific antibodies that first bind to recombinant protein VEGF-Fc and then bind to recombinant protein PD-L1-Fc; Figure 7B shows that candidate bispecific antibodies first bind to recombinant protein PD-L1-Fc and then bind to recombinant protein PD-L1-Fc Binding activity to recombinant protein VEGF-Fc.
图8显示了通过荧光素酶报告基因方法检测候选双特异性抗体对PD-1/PD-L1的阻断活性。Figure 8 shows the detection of the blocking activity of candidate bispecific antibodies against PD-1/PD-L1 by luciferase reporter gene method.
图9显示了通过荧光素酶报告基因方法检测候选双特异性抗体对VEGF/VEGFR2的阻断活性。Figure 9 shows the detection of the blocking activity of candidate bispecific antibodies against VEGF/VEGFR2 by luciferase reporter gene method.
图10显示了候选双特异性抗体在COLO205小鼠移植模型中对肿瘤生长的抑制作用。Figure 10 shows the inhibitory effect of candidate bispecific antibodies on tumor growth in the COLO205 mouse transplantation model.
图11显示了候选双特异性抗体在A431小鼠移植模型中对肿瘤生长的抑制作用。Figure 11 shows the inhibitory effect of candidate bispecific antibodies on tumor growth in the A431 mouse transplantation model.
发明详述Detailed description of the invention
除非另外限定,否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外,本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the appended claims.
I.定义I. Definition
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5%的下限和比指定数字数值大5%的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit of 5% less and an upper limit of 5% greater than the stated numerical value.
如本文中所用,术语“包含”或“包括”意指包括所述的要素、整数或步骤,但是不排除任意其他要素、整数或步骤。在述及“包含”时,除非上下文有明确相反说明,否则该表述也涵盖“由……组成”。例如,当述及一个多肽链包含一个特定氨基酸序列时,也涵盖该多肽链由该特定氨基酸序列组成的情形。As used herein, the term "comprising" or "comprising" means including stated elements, integers or steps, but not excluding any other elements, integers or steps. Where "comprising" is mentioned, the expression also covers "consisting of" unless the context clearly dictates otherwise. For example, when referring to a polypeptide chain comprising a specific amino acid sequence, it is also contemplated that the polypeptide chain consists of the specific amino acid sequence.
在本文中,术语“VEGF”指血管内皮生长因子A(Vascular endothelial growth factor A,VEGF-A)蛋白。VEGF-A在转录过程中通过外显子可变拼接产生多种同种型,VEGF121、VEGF165、VEGF189和VEGF206等同种型。在本文中,VEGF尤其指同种型VEGF165,例如UniProtKB-P15692下记载的人VEGF165蛋白(尤其是,无信号肽的氨基酸27-191的氨基酸序列)。As used herein, the term "VEGF" refers to vascular endothelial growth factor A (VEGF-A) protein. VEGF-A produces multiple isoforms through alternative splicing of exons during transcription, isoforms such as VEGF121, VEGF165, VEGF189, and VEGF206. In this context, VEGF refers in particular to the isoform VEGF165, for example the human VEGF165 protein described under UniProtKB-P15692 (in particular, the amino acid sequence without amino acids 27-191 of the signal peptide).
VEGF165在血管发生、脉管形成和内皮细胞生长方面具有活性;可以诱导内皮细胞增殖、促进细胞迁移、抑制凋亡和诱导血管通透性。VEGF165与受体FLT1/VEGFR1和KDR/VEGFR2结合;并与神经纤毛蛋白-1(NRP1)的蛋白质结合。研究证实,阻断VEGF与受体VEGFR的结合在肿瘤治疗和在血管异常增生相关的眼病中,具有较好的疗效和靶点安全性。近来的研究也发现,NRP1蛋白在抑制对癌症的免疫应答中起着重要作用。与正常小鼠相比,杀伤性T细胞上NRP1基因缺失的小鼠对继发性肿瘤(包括B16.F10小鼠黑色素瘤等相对“冷”的肿瘤)产生了更好的预防作用,对抗PD1免疫治疗的反应也更积极(Chang Liu et al,Neuropilin-1is a T cell memory checkpoint limiting long-term antitumor immunity,Nature Immunology(2020).DOI:10.1038/s41590-020-0733-2)。VEGF165 is active in angiogenesis, vessel formation, and endothelial cell growth; can induce endothelial cell proliferation, promote cell migration, inhibit apoptosis, and induce vascular permeability. VEGF165 binds to the receptors FLT1/VEGFR1 and KDR/VEGFR2; and to the protein Neuropilin-1 (NRP1). Studies have confirmed that blocking the combination of VEGF and the receptor VEGFR has good curative effect and target safety in tumor treatment and eye diseases related to abnormal blood vessel proliferation. Recent studies have also found that the NRP1 protein plays an important role in suppressing the immune response to cancer. Compared with normal mice, mice with NRP1 gene deletion on killer T cells produced better protection against secondary tumors, including relatively "cold" tumors such as melanoma in B16.F10 mice, against PD1 The response to immunotherapy was also more positive (Chang Liu et al, Neuropilin-1 is a T cell memory checkpoint limiting long-term antitumor immunity, Nature Immunology (2020). DOI: 10.1038/s41590-020-0733-2).
在本文中,“针对VEGF的抗原结合特异性”是指分子中特异性结合VEGF的结合位点或结合结构域。Herein, "antigen-binding specificity for VEGF" refers to a binding site or a binding domain in a molecule that specifically binds to VEGF.
在本文中,术语“PD-L1”是指程序性细胞死亡1配体1(Programmed cell death 1ligand 1)。作为抑制性受体PD-1的配体,该蛋白调节T细胞的激活阈值并限制T细胞的效应子反应。PD1/PD-L1介导的免疫抑制途径可以被肿瘤用于减弱抗肿瘤免疫以及逃逸免疫系统的清除作用,从而促进肿瘤存活。阻断该途径可以逆转耗竭的T细胞表型并使抗肿瘤反应正常化,促进癌症免疫治疗。该术语PD-L1在本文中尤其是指人PD-L1蛋白,例如登录号UniProtKB-Q9NZQ7下人PD-L1蛋白。
Herein, the term "PD-L1" refers to programmed cell death 1 ligand 1 (Programmed cell death 1 ligand 1). As a ligand for the inhibitory receptor PD-1, this protein regulates T cell activation thresholds and limits T cell effector responses. The PD1/PD-L1-mediated immunosuppressive pathway can be used by tumors to attenuate anti-tumor immunity and evade clearance by the immune system, thereby promoting tumor survival. Blocking this pathway reverses the exhausted T cell phenotype and normalizes antitumor responses, facilitating cancer immunotherapy. The term PD-L1 herein refers especially to human PD-L1 protein, eg human PD-L1 protein under accession number UniProtKB-Q9NZQ7.
在本文中,“针对PD-L1的抗原结合特异性”是指分子中特异性结合PD-L1的结合位点或结合结构域。Herein, "antigen-binding specificity for PD-L1" refers to a binding site or binding domain in a molecule that specifically binds to PD-L1.
如本文所用的术语“抗原结合位点”与“抗原结合特异性”可以互换使用,表示抗体与抗原实际结合的区域。优选地,在本发明双特异性抗体中,PD-L1抗原结合位点由来自抗PD-L1的VHH结构域提供;VEGF抗原结合位点由来自抗VEGF的VHH结构域提供。As used herein, the terms "antigen-binding site" and "antigen-binding specificity" are used interchangeably to refer to the region of the antibody that actually binds to the antigen. Preferably, in the bispecific antibody of the present invention, the PD-L1 antigen-binding site is provided by the VHH domain from anti-PD-L1; the VEGF antigen-binding site is provided by the VHH domain from anti-VEGF.
如本文所用,术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗原结合位点与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)或本领域已知的常规结合测定法测定,例如,通过实施例4.1、4.2或4.4描述的ELISA测定法检测抗体与PD-L1或VEGF蛋白或两者的结合能力,或通过实施例4.3描述的FACS测定法检测抗体与细胞表面表达的PD-L1的结合能力。As used herein, the term "bind" or "specifically bind" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, for example, by detecting antibodies by ELISA assays described in Examples 4.1, 4.2 or 4.4 The ability to bind to PD-L1 or VEGF protein or both, or to detect the ability of the antibody to bind to PD-L1 expressed on the cell surface by the FACS assay described in Example 4.3.
在本文中,抗体对PD-L1具有“阻断活性”是指抗体阻断PD-L1与受体PD-1结合,和/或减少PD-L1/PD-1的信号传递的功能。可以用于测定此阻断活性的试验可以是本领域已知的FACS测定法;或基于报告基因的信号途径阻断测定试验,例如实施例5中描述的基于报告基因的检测法。可以参照在不存在抗体时和/或存在阳性抗体时的PD-L1/PD-1结合或PD-L1/PD-1信号传递水平,确定待测抗体对PD-L1的阻断活性。Herein, an antibody having "blocking activity" on PD-L1 means that the antibody blocks the binding of PD-L1 to the receptor PD-1, and/or reduces the function of PD-L1/PD-1 signal transmission. Assays that can be used to determine this blocking activity can be FACS assays known in the art; or reporter gene-based signaling pathway blockade assays, such as the reporter gene-based assay described in Example 5. The PD-L1 blocking activity of the antibody to be tested can be determined with reference to the PD-L1/PD-1 binding or PD-L1/PD-1 signaling level in the absence of the antibody and/or in the presence of a positive antibody.
在本文中,抗体对VEGF的“中和活性”是指抗体阻断VEGF与受体VEGFR2的信号通路的功能。可以用于测定该活性的试验可以是例如基于报告基因的信号通路阻断测定试验,例如实施例6中描述的基于报告基因的检测法。可以参照在不存在抗体时和/或存在阳性抗体时的VEGF/VEGFR2的信号传递水平,确定待测抗体对VEGF的中和活性。Herein, the "neutralizing activity" of an antibody to VEGF refers to the function of the antibody to block the signaling pathway between VEGF and the receptor VEGFR2. An assay that can be used to measure this activity can be, for example, a reporter gene-based signaling pathway blockade assay, such as the reporter gene-based assay described in Example 6. The neutralizing activity of the antibody to be tested against VEGF can be determined with reference to the VEGF/VEGFR2 signaling level in the absence of the antibody and/or in the presence of a positive antibody.
术语抗体的“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链或轻链的结构域。抗体的可变区可以进一步再划分为超变区(即,互补决定区(CDR))和间插在超变区之间的较为保守的区域(即,构架区(FR))。在重链抗体(在本文中也称作纳米抗体),例如来自骆驼科的重链抗体的情况下,抗原结合位点由单个重链可变结构域(在本文中也称作VHH结构域)提供。与IgG抗体的重链可变区VH一样,该单个重链可变结构域VHH包含四个FR区和三个CDR区,并从氨基端到羧基端以如下顺序排列:FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。The term "variable region" or "variable domain" of an antibody refers to the domains of the heavy or light chain of an antibody that participate in the binding of the antibody to an antigen. The variable regions of antibodies can be further subdivided into hypervariable regions (ie, complementarity determining regions (CDRs)) and more conserved regions interspersed between hypervariable regions (ie, framework regions (FRs)). In the case of heavy chain antibodies (also referred to herein as Nanobodies), such as those from Camelidae, the antigen binding site is composed of a single heavy chain variable domain (also referred to herein as a VHH domain). supply. Like the heavy chain variable region VH of an IgG antibody, this single heavy chain variable domain VHH comprises four FR regions and three CDR regions, and is arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
抗体的“互补决定区”,在本文中与“CDR区”或“CDR”或“高变区”可互换使用,是抗体可变结构域(例如VHH)中在序列上高度可变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。从抗体链的N-端开始顺序编号,位于VHH可变区结构域中的CDR称作CDR1、CDR2和CDR3。在一个给定的可变区氨基酸序列中,可以采用本领域公知的多种方案确定其CDR序列,例如:基于抗体的三维结构和CDR环的拓扑学的Chothia,基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath),Contact(University College London),国际ImMunoGeneTics database(IMGT)(国际免疫遗传学信息系统,万维网imgt.cines.fr/),以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义(North等,“A New Clustering of Antibody CDR Loop Con格式ions”,Journal of Molecular Biology,406,228-256(2011))。本领域技术人员可以在http://www.abysis.org/abysis/上,容易地确定任一给定的抗体可变区氨基酸序列的CDR
序列范围,包括Kabat、AbM、Chothia、Contact和IMGT方案定义的CDR的区域范围及其组合范围。除非另有说明,否则在本发明中,术语“CDR”或“CDR序列”涵盖以上述任一种方式及其组合确定的CDR序列。The "complementarity determining region" of an antibody, used interchangeably herein with "CDR region" or "CDR" or "hypervariable region", is a region within an antibody variable domain (e.g., VHH) that is highly variable in sequence and forms Structurally defined loops ("hypervariable loops") and/or regions containing antigen contact residues ("antigen contact points"). The CDRs are primarily responsible for binding to antigenic epitopes. Numbering sequentially from the N-terminus of the antibody chain, the CDRs located in the VHH variable region domain are called CDR1, CDR2, and CDR3. In a given variable region amino acid sequence, various schemes known in the art can be used to determine its CDR sequence, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat ( Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT ) (International Immunogenetics Information System, World Wide Web imgt.cines.fr/), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)). Those skilled in the art can readily determine the CDRs of any given antibody variable region amino acid sequence at http://www.abysis.org/abysis/ Sequence range, including the range of CDR regions defined by Kabat, AbM, Chothia, Contact and IMGT schemes and their combined ranges. Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the above ways and combinations thereof.
在本发明的双特异性抗体中,特异性结合抗原的抗原结合位点由VHH结构域提供。术语“VHH”或“VHH结构域”在本文中用于指,从缺乏轻链的重链抗体(在本文中,有时也称作纳米抗体)衍生的重链可变结构域,也称作单可变结构域(sVD)。与四链免疫球蛋白的常规VH不同,VHH结构域无需与轻链可变结构域配对来形成抗原结合位点。这种VHH分子可以衍生自骆驼科物种(例如骆驼、羊驼、单峰驼、驼羊和原驼)中产生的抗体。在一些情况下,对于VHH的治疗应用,期望的是降低其免疫原性。因此,优选地,在一个实施方案中,本发明提供包含人源化VHH结构域的抗体。在根据本发明的一些实施方案中,在根据本发明的双特异性抗体中,来自抗PD-L1的VHH结构域和来自抗VEGF的VHH结构域,优选地通过免疫球蛋白铰链区或连接子,分别单独地连接在抗体的Fc区多肽上的相对两侧;或以两者串联的形式(例如,从N端到C端,VHHPD-L1-连接子-VHHVEGF;或VHHVEGF-连接子-VHHPD-L1),连接在抗体的Fc区多肽的N端侧。In the bispecific antibody of the present invention, the antigen-binding site that specifically binds to the antigen is provided by the VHH domain. The term "VHH" or "VHH domain" is used herein to refer to a heavy chain variable domain derived from a heavy chain antibody (herein sometimes referred to as a Nanobody) lacking a light chain, also referred to as a single Variable domain (sVD). Unlike the conventional VH of four-chain immunoglobulins, the VHH domain does not need to be paired with a light chain variable domain to form an antigen-binding site. Such VHH molecules may be derived from antibodies produced in Camelidae species such as llamas, alpacas, dromedaries, llamas and guanacos. In some instances, for therapeutic use of a VHH, it is desirable to reduce its immunogenicity. Thus, preferably, in one embodiment, the invention provides antibodies comprising a humanized VHH domain. In some embodiments according to the invention, in the bispecific antibody according to the invention, the VHH domain from anti-PD-L1 and the VHH domain from anti-VEGF, preferably via an immunoglobulin hinge region or a linker , separately connected to the opposite sides of the Fc region polypeptide of the antibody; or in the form of both in series (for example, from N-terminal to C-terminal, VHH PD-L1 -linker-VHH VEGF ; or VHH VEGF -linked Sub-VHH PD-L1 ), linked to the N-terminal side of the Fc region polypeptide of the antibody.
“Fab样结构”在本文中用于指,如图1C和图1D中所示,类似于常规四链IgG抗体中由重链可变区和重链恒定区CH1与互补轻链可变区和轻链恒定区CL配对形成的结构,但是在该结构中重链可变区被一个VHH结构域替代,轻链可变区被另一不同的VHH结构域替代。在根据本发明的一个实施方案中,Fab样结构包含由VHHPD-L1与免疫球蛋白重链恒定区CH1结构域组成的第一链和由VHHVEGF与免疫球蛋白轻链恒定区CL结构域组成的第二链。在根据本发明的再一实施方案中,Fab样结构包含由VHHVEGF与免疫球蛋白重链恒定区CH1结构域组成的第一链和由VHHPD-L1与免疫球蛋白轻链恒定区CL结构域组成的第二链。在根据本发明的又一些实施方案中,在根据本发明的双特异性抗体中,来自抗PD-L1的VHH结构域和来自抗VEGF的VHH结构域以Fab样结构,优选地通过免疫球蛋白铰链区,连接在免疫球蛋白Fc区多肽的N端侧。优选地,所述Fab样结构通过其CH1结构域连接所述Fc区多肽。"Fab-like structure" is used herein to refer to, as shown in Figure 1C and Figure 1D, similar to that in a conventional four-chain IgG antibody consisting of a heavy chain variable region and a heavy chain constant region CH1 with complementary light chain variable regions and A structure formed by CL pairing of the light chain constant region, but in which the heavy chain variable region is replaced by a VHH domain and the light chain variable region is replaced by a different VHH domain. In one embodiment according to the present invention, the Fab-like structure comprises the first chain consisting of VHH PD-L1 and the CH1 domain of the constant region of the immunoglobulin heavy chain and the CL domain of the constant region of the immunoglobulin light chain consisting of VHH VEGF and composed of the second chain. In yet another embodiment according to the present invention, the Fab-like structure comprises a first chain consisting of VHH VEGF and the CH1 domain of the constant region of the immunoglobulin heavy chain and a CL structure composed of VHH PD-L1 and the constant region of the immunoglobulin light chain The domain consists of the second chain. In still other embodiments according to the present invention, in the bispecific antibody according to the present invention, the VHH domain from anti-PD-L1 and the VHH domain from anti-VEGF are in a Fab-like structure, preferably by immunoglobulin The hinge region is connected to the N-terminal side of the immunoglobulin Fc region polypeptide. Preferably, the Fab-like structure is linked to the Fc region polypeptide through its CH1 domain.
术语“免疫球蛋白分子”指具有天然存在抗体的结构的蛋白质。例如,IgG类免疫球蛋白是由二硫键键合的两条轻链和两条重链组成的约150,000道尔顿的异四聚体糖蛋白。从N端至C端,每条免疫球蛋白重链具有一个重链可变区(VH),也称作重链可变结构域,随后是三个重链恒定结构域(CH1、CH2和CH3)。类似地,从N端至C端,每条免疫球蛋白轻链具有一个轻链可变区(VL),也称作轻链可变结构域,随后是一个轻链恒定结构域(CL)。在IgG分子中,通常重链的VH-CH1与轻链的VL-CL配对形成特异性结合抗原的Fab片段。因此,一个IgG免疫球蛋白基本上由借助免疫球蛋白铰链区连接的两个Fab分子和两个二聚化的Fc区组成。免疫球蛋白的重链可以基于其恒定区的类型,归属5个类别之一,称作α(IgA)、δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM),其中某些类别可以进一步划分成亚类,例如γ1(IgG1)、γ2(IgG2)、γ3(IgG3)、γ4(IgG4)、α1(IgA1)和α2(IgA2)。免疫球蛋白的轻链也可以基于其恒定结构域的氨基酸序列而划分成两种类型之一,称作κ和λ。The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two light chains and two heavy chains that are disulfide bonded. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2 and CH3 ). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). In an IgG molecule, usually the VH-CH1 of the heavy chain is paired with the VL-CL of the light chain to form a Fab fragment that specifically binds the antigen. Thus, an IgG immunoglobulin essentially consists of two Fab molecules and two dimerized Fc regions linked by the immunoglobulin hinge region. The heavy chains of immunoglobulins can be assigned to one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), based on the type of their constant region, one of which is These classes can be further divided into subclasses such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1 ) and α2 (IgA2). The light chains of immunoglobulins can also be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains.
“免疫球蛋白重链恒定区结构域”是指来自或获自或衍生自免疫球蛋白重链的恒定区结构域,包括从N端至C端顺序共价连接的重链恒定区CH1、CH2、CH3和任选地重链恒定区CH4。在大多数情况下,重
链恒定区CH1和CH2之间通过重链铰链区连接,但在适宜时,也可以通过柔性连接肽连接。在本发明的一些优选实施方案中,本发明抗体包含由免疫球蛋白重链恒定区CH1-铰链区-CH2-CH3组成的多肽链。在本发明的另一些优选实施方案中,本发明抗体包含由免疫球蛋白重链恒定区CH2-CH3组成的Fc区多肽链。在本文中,免疫球蛋白恒定结构域可以根据抗体的预期功能进行选择。例如,恒定结构域可以是IgA、IgD、IgE、IgG或IgM结构域,尤其是人IgG的免疫球蛋白恒定结构域,例如,人IgG1,IgG2,IgG3或IgG4的恒定结构域,优选人IgG1的恒定结构域。作为一个例子,抗体的CH1和Fc片段可以均来自IgG1。"Immunoglobulin heavy chain constant region domain" means a constant region domain from or obtained from or derived from an immunoglobulin heavy chain, including the heavy chain constant regions CH1, CH2 covalently linked sequentially from the N-terminus to the C-terminus , CH3 and optionally heavy chain constant region CH4. In most cases, heavy The chain constant regions CH1 and CH2 are connected by the hinge region of the heavy chain, but they can also be connected by a flexible linking peptide when appropriate. In some preferred embodiments of the present invention, the antibody of the present invention comprises a polypeptide chain consisting of immunoglobulin heavy chain constant region CH1-hinge region-CH2-CH3. In other preferred embodiments of the present invention, the antibody of the present invention comprises an Fc region polypeptide chain composed of immunoglobulin heavy chain constant regions CH2-CH3. Herein, immunoglobulin constant domains can be selected based on the intended function of the antibody. For example, the constant domain may be an IgA, IgD, IgE, IgG or IgM domain, especially an immunoglobulin constant domain of human IgG, for example, a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably of human IgG1 constant domain. As an example, the CH1 and Fc fragments of an antibody can both be from IgG1.
“免疫球蛋白轻链恒定区结构域”是指来自或获自或衍生自免疫球蛋白轻链的恒定区结构域CL。免疫球蛋白的轻链CL恒定区,基于其氨基酸序列,可以是κ轻链CL结构域和λ轻链CL结构域。"Immunoglobulin light chain constant region domain" refers to a constant region domain CL from or obtained from or derived from an immunoglobulin light chain. The light chain CL constant region of an immunoglobulin, based on its amino acid sequence, may be a kappa light chain CL domain and a lambda light chain CL domain.
在本文中,术语“Fc结构域”或“Fc区”或“Fc区多肽”可互换使用,用于指源自免疫球蛋白重链的含有至少一部分恒定区的C端区域多肽。该术语包括天然序列Fc区多肽和变体Fc区多肽。天然的免疫球蛋白“Fc结构域”包含两个或三个恒定结构域,即CH2结构域、CH3结构域和可选的CH4结构域。例如,在天然抗体中,免疫球蛋白Fc结构域包含源自IgG、IgA和IgD类抗体的重链的第二和第三恒定结构域(CH2结构域和CH3结构域);或者包含源自IgM和IgE类抗体的重链的第二、第三和第四恒定结构域(CH2结构域、CH3结构域和CH4结构域)。除非本文中另外说明,否则Fc区或重链恒定区中的氨基酸残基编号根据如Kabat等人,Sequences of Proteins of Immunological Interes,第5版,Public Health Service,National Institutes of Health,Bethesda,MD,1991中所述的EU编号体系(也称作EU索引)进行编号。在本文中,术语“Fc结构域”或“Fc区”或“Fc多肽”不包括免疫球蛋白的重链可变区VH和轻链可变区VL以及重链恒定区CH1和轻链恒定区CL。Herein, the terms "Fc domain" or "Fc region" or "Fc region polypeptide" are used interchangeably to refer to a C-terminal region polypeptide containing at least a part of the constant region derived from an immunoglobulin heavy chain. The term includes native sequence Fc region polypeptides and variant Fc region polypeptides. A native immunoglobulin "Fc domain" comprises two or three constant domains, a CH2 domain, a CH3 domain and optionally a CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of the heavy chain derived from antibodies of the IgG, IgA and IgD classes; and the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of the heavy chain of antibodies of the IgE class. Unless otherwise stated herein, amino acid residue numbering in the Fc region or heavy chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system described in 1991 (also known as the EU Index) is used for numbering. As used herein, the term "Fc domain" or "Fc region" or "Fc polypeptide" does not include the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region of an immunoglobulin cl.
“天然序列Fc区”多肽涵盖天然存在的各种免疫球蛋白Fc区序列,例如各种Ig亚型以及其同种异型的Fc区序列(Gestur Vidarsson等,IgG subclasses and allotypes:from structure to effector functions,20October2014,doi:10.3389/fimmu.2014.00520.)。在一些实施方案中,人IgG重链Fc区具有自Cys226或自Pro230延伸至重链羧基端的氨基酸序列。然而,Fc区的C端末端赖氨酸(Lys447)可以存在或不存在。在再一些实施方案中,人IgG重链Fc区在N端带有天然免疫球蛋白的铰链序列或部分铰链序列,例如根据EU编号,E216到T225的序列或D221到T225的序列。"Native sequence Fc region" polypeptides encompass naturally occurring Fc region sequences of various immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions , 20 October 2014, doi: 10.3389/fimmu.2014.00520.). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. However, the C-terminal terminal lysine (Lys447) of the Fc region may or may not be present. In still other embodiments, the human IgG heavy chain Fc region bears at the N-terminus a hinge sequence or a partial hinge sequence of a native immunoglobulin, for example a sequence from E216 to T225 or a sequence from D221 to T225 according to EU numbering.
本文中的术语“变体Fc区”或“Fc区变体“多肽,在本文中可互换使用,指相对于天然序列Fc区多肽包含修饰的Fc区多肽。本发明的Fc区变体多肽按照组成它们的氨基酸修饰来定义。因此,例如,L234A是相对于亲本多肽在234位用丙氨酸取代亮氨酸的Fc区变体,其中编号按照EU索引。修饰可以是添加,缺失或取代。取代可以包括天然存在的氨基酸和非天然存在的氨基酸。变体可以包含非天然氨基酸。修饰的目的可以是旨在改变由Fc区与其受体的结合及其由此引发的效应子功能。The term "variant Fc region" or "Fc region variant" polypeptide herein, used interchangeably herein, refers to an Fc region polypeptide comprising a modification relative to a native sequence Fc region polypeptide. Fc region variant polypeptides of the invention are defined by the amino acid modifications that make them up. Thus, for example, L234A is an Fc region variant with alanine substituted for leucine at position 234 relative to the parental polypeptide, where numbering is according to the EU index. Modifications can be additions, deletions or substitutions. Substitutions can include naturally occurring amino acids and non-naturally occurring amino acids. Variants may contain unnatural amino acids. The purpose of the modification may be to alter the binding of the Fc region to its receptor and the effector functions elicited thereby.
术语“效应子功能”指随免疫球蛋白同种型变动的归因于免疫球蛋白Fc区的那些生物学活性。免疫球蛋白效应子功能的例子包括:C1q结合和补体依赖的细胞毒性(CDC)、Fc受体结合作用、抗体依赖的细胞介导的细胞毒性(ADCC)、抗体依赖的细胞吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞摄取抗原、下调细胞表面受体(例如B细胞受体)和B细胞活化。根据抗体的预期用途,本发明的抗体可以相对于具有野生型Fc区的抗体具有改变的效应子功能,例如降低或消除的ADCC活性等。
The term "effector functions" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin 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, downregulation of cell surface receptors (eg, B-cell receptors), and B-cell activation. Depending on the intended use of the antibody, the antibody of the invention may have altered effector functions relative to an antibody with a wild-type Fc region, such as reduced or eliminated ADCC activity and the like.
在本文中,术语“柔性连接肽”或“连接子”或“连接肽”可互换使用,是指由氨基酸组成的短氨基酸序列,例如单独或组合使用的甘氨酸(G)和/或丝氨酸(S)和/或苏氨酸残基(T),或来自免疫球蛋白的铰链区。在一个实施方案中,连接肽具有5-50个氨基酸长度,例如,10、15、20、25、30个氨基酸长度。在一个实施方案中,连接肽包含氨基酸序列(G4S)n(SEQ ID NO:20),其中n是等于或大于1的整数,例如,n是2、3、4、5、6或7的整数。在一个实施方案中,连接肽包含氨基酸序列TS(G4S)n(SEQ ID NO:21),其中n是等于或大于1的整数,例如,n是2、3、4、5、6或7的整数。在再一实施方案中,连接肽为来自免疫球蛋白的铰链区,例如包含“CPPC”的铰链区氨基酸序列,例如,氨基酸序列“EPKSCDKTHTCPPCP”(SEQ ID NO:22)或“EPKSSDKTHTCPPCP”(SEQ ID NO:23)。可以用于本发明抗体中连接各结构域的连接肽还可以是,例如但不限于,如下氨基酸序列:GGG(SEQ ID NO:24);DGGGS(SEQ ID NO:25);TGEKP(SEQ ID NO:26);GGRR(SEQ ID NO:27);EGKSSGSGSESKVD(SEQ ID NO:28);KESGSVSSEQLAQFRSLD(SEQ ID NO:29);GGRRGGGS(SEQ ID NO:30);LRQRDGERP(SEQ ID NO:31);LRQKDGGGSERP(SEQ ID NO:32)和GSTSGSGKPGSGEGSTKG(SEQ ID NO:33)。或者,可以使用计算机程序模拟蛋白和肽的三维结构,或通过噬菌体展示方法,来合理地设计合适的柔性连接肽。As used herein, the term "flexible linker" or "linker" or "linker" is used interchangeably to refer to a short amino acid sequence consisting of amino acids such as glycine (G) and/or serine ( S) and/or threonine residues (T), or from the hinge region of an immunoglobulin. In one embodiment, the connecting peptide is 5-50 amino acids in length, eg, 10, 15, 20, 25, 30 amino acids in length. In one embodiment, the connecting peptide comprises the amino acid sequence (G 4 S) n (SEQ ID NO: 20), wherein n is an integer equal to or greater than 1, for example, n is 2, 3, 4, 5, 6 or 7 an integer of . In one embodiment, the connecting peptide comprises the amino acid sequence TS(G 4 S) n (SEQ ID NO: 21), wherein n is an integer equal to or greater than 1, for example, n is 2, 3, 4, 5, 6 or Integer of 7. In yet another embodiment, the connecting peptide is a hinge region from an immunoglobulin, for example comprising a hinge region amino acid sequence of "CPPC", for example, the amino acid sequence "EPKSCDKTHTCPPCP" (SEQ ID NO: 22) or "EPKSSDKTHTCPPCP" (SEQ ID NO:23). The connecting peptide that can be used to link the various domains in the antibody of the present invention can also be, for example but not limited to, the following amino acid sequence: GGG (SEQ ID NO: 24); DGGGS (SEQ ID NO: 25); TGEKP (SEQ ID NO :26); GGRR (SEQ ID NO:27); EGKSSGSGSESKVD (SEQ ID NO:28); KESGSVSSEQLAQFRSLD (SEQ ID NO:29); GGRRGGGS (SEQ ID NO:30); LRQRDGERP (SEQ ID NO:31); (SEQ ID NO:32) and GSTSGSGKPGSGEGSTKG (SEQ ID NO:33). Alternatively, suitable flexible linker peptides can be rationally designed using computer programs to model the three-dimensional structures of proteins and peptides, or by phage display methods.
术语“嵌合抗体”是这样的抗体,其中(a)将恒定区或其部分改变、替换或交换,从而抗原结合位点与不同的或改变的类别、效应子功能和/或物种的恒定区或赋予嵌合抗体新性能的完全不同的分子(例如,酶、毒素、激素、生长因子、药物)等连接;或(b)将可变区或其部分用具有不同或改变的抗原特异性的可变区改变、替换或交换。The term "chimeric antibody" is an antibody in which (a) the constant region, or part thereof, has been altered, substituted or exchanged such that the antigen binding site corresponds to a constant region of a different or altered class, effector function and/or species or linking entirely different molecules (e.g., enzymes, toxins, hormones, growth factors, drugs) etc. that confer new properties on the chimeric antibody; Variable regions are altered, substituted or swapped.
“人源化抗体”是一种保留非人类抗体(例如羊驼重链抗体)的抗原特异性反应性,同时作为治疗药对人施用时免疫原性较低的抗体。这可以例如通过保留非人类抗原结合位点并且将抗体的剩余部分替换成它们的人类相应部分(即,恒定区以及可变区中不参与结合的部分为人类抗体的相应部分)来实现。A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (eg, an alpaca heavy chain antibody), while being less immunogenic when administered to humans as a therapeutic. This can be achieved, for example, by retaining the non-human antigen binding sites and replacing the remainder of the antibodies with their human counterparts (ie, the constant regions and the parts of the variable regions that do not participate in binding are those of human antibodies).
氨基酸序列的“同一性百分数(%)”是指将候选序列与本说明书中所示的具体氨基酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后,并且不考虑任何保守置换作为序列同一性的一部分时,候选序列中与本说明书中所示的具体氨基酸序列的氨基酸残基相同的氨基酸残基百分数。在一些实施方案中,本发明考虑本发明抗体的变体,所述变体相对于在本文中具体公开的抗体及其序列而言具有相当程度的同一性,例如同一性为至少80%、85%、90%、95%、97%、98%或99%或更高。所述变体可以包含保守性修饰。"Percent identity (%)" of an amino acid sequence refers to after aligning a candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without taking into account any When conservative substitutions are taken as part of sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular amino acid sequence shown in this specification. In some embodiments, the invention contemplates variants of the antibodies of the invention that have a substantial degree of identity, for example at least 80%, 85% identity, relative to the antibodies and sequences thereof specifically disclosed herein. %, 90%, 95%, 97%, 98%, or 99% or higher. Such variants may contain conservative modifications.
对于多肽序列,“保守性修饰”包括对多肽序列的置换、缺失或添加,它们导致某个氨基酸置换为化学上相似的氨基酸。提供功能上相似氨基酸的保守性置换表是本领域熟知的。这类保守性修饰的变体相对于本发明的多态性变体、物种间同源物和等位基因而言是附加的并且不排斥它们。以下8组含有互为保守替换的氨基酸:1)丙氨酸(A)、甘氨酸(G);2)天冬氨酸(D)、谷氨酸(E);3)天冬酰胺(N)、谷氨酰胺(Q);4)精氨酸(R)、赖氨酸(K);5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V);6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W);7)丝氨酸(S)、苏氨酸(T);和8)半胱氨酸(C)、甲硫氨酸(M)(参阅例如,Creighton,Proteins(1984))。在一些实施方案中,术语“保守序列修饰”用于指不显著影响或改变含有氨基酸序列的抗体的结合特征的氨基酸修饰。
With respect to polypeptide sequences, "conservative modifications" include substitutions, deletions or additions to a polypeptide sequence that result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues and alleles of the invention. The following 8 groups contain amino acids that are mutually conservative substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N) , glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine Acid (C), Methionine (M) (See eg, Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence.
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞,包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”,这包括原代转化的细胞和从其衍生的子代。宿主细胞是可以用来产生本发明抗体的任何类型的细胞系统,包括真核细胞,例如,哺乳动物细胞、昆虫细胞、酵母细胞;和原核细胞,例如,大肠杆菌细胞。宿主细胞包括培养的细胞,也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。The term "host cell" refers to a cell into which an exogenous polynucleotide 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. A host cell is any type of cellular system that can be used to produce antibodies of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells. Host cells include cultured cells as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.
术语“表达载体”是指包含重组多核苷酸的载体,其包含有效连接要表达的核苷酸序列的表达控制序列。表达载体包含足够的用于表达的顺式作用元件;用于表达的其它元件可以由宿主细胞提供或在体外表达系统中。表达载体包括本领域已知的所有那些,包括被掺入重组多核苷酸的粘粒、质粒(例如,裸的或包含在脂质体中)和病毒(例如,慢病毒、逆转录病毒、腺病毒和腺伴随病毒)。The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses) that incorporate recombinant polynucleotides. virus and adeno-associated virus).
术语“个体”或“受试者”可互换地使用,是指哺乳动物。哺乳动物包括但不限于驯化动物(例如,奶牛、绵羊、猫、犬和马)、灵长类(例如,人和非人灵长类如猴)、兔和啮齿类(例如,小鼠和大鼠)。特别地,个体是人。The terms "individual" or "subject" are used interchangeably to refer to 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 rodents). mouse). In particular, an individual is a human being.
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态,以及缓解或改善预后。在一些实施方案中,本发明的抗体用来延缓疾病发展或用来减慢疾病的进展。在涉及肿瘤或癌症治疗的情形下,“治疗”涵盖可以通过人为干预手段(例如,药物如本发明抗体的施用)引起的抗肿瘤生物学效果,包括但不限于,例如,肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。The term "treatment" refers to clinical intervention intended to alter the natural course of disease in the individual being treated. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease. In the context of tumor or cancer treatment, "treatment" encompasses anti-tumor biological effects that can be induced by human intervention (e.g., administration of drugs such as antibodies of the invention), including, but not limited to, for example, reduction in tumor volume, tumor Decreased cell number, decreased tumor cell proliferation, or decreased tumor cell survival.
术语“癌症”和“肿瘤”可互换使用,指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。癌症的例子包括但不限于癌,实体瘤和液体瘤。The terms "cancer" and "tumor" are used interchangeably to refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, solid tumors, and liquid tumors.
以下就本发明的各方面进行详细描述。Various aspects of the present invention are described in detail below.
I.本发明的双特异性抗体I. Bispecific antibodies of the invention
在一方面,本发明提供了一种基于VHH结构域的特异性结合PD-L1和VEGF的双特异性抗体,所述抗体包含特异性结合PD-L1的VHH结构域(在本文中也缩写为VHHPD-L1)和特异性结合VEGF的VHH结构域(在本文中也缩写为VHHVEGF)。In one aspect, the present invention provides a VHH domain-based bispecific antibody specifically binding to PD-L1 and VEGF, said antibody comprising a VHH domain specifically binding to PD-L1 (also abbreviated herein as VHH PD-L1 ) and a VHH domain specifically binding to VEGF (also abbreviated herein as VHH VEGF ).
在一些特定的实施方案中,在本发明的双特异性抗体中,所述的VHHPD-L1和VHHVEGF结构域可以为包含在同一条多肽链上的串联形式(例如借助于连接子和/或Fc区串联在一条多肽链上);或可以形成Fab样结构(例如,借助于免疫球蛋白CH1和CL恒定结构域形成Fab样结构)。In some specific embodiments, in the bispecific antibody of the present invention, the VHH PD-L1 and VHH VEGF domains may be in a tandem form contained on the same polypeptide chain (for example, by means of a linker and/or or Fc regions in series on one polypeptide chain); or can form a Fab-like structure (for example, by means of immunoglobulin CH1 and CL constant domains to form a Fab-like structure).
在一些特定的实施方案中,根据本发明的双特异性抗体还包含Fc区。在根据本发明的双特异性抗体中,所述的VHHPD-L1和VHHVEGF结构域可以以包含VHHPD-L1和VHHVEGF结构域两者的串联形式(例如,如图1A和图1B所示)、或以包含VHHPD-L1和VHHVEGF结构域两者的Fab样结构(例如,如图1C和图1D所示),连接在Fc区上。由于包含Fc区的抗体多肽链可以通过Fc区的二聚化作用而缔合,因此,在一些实施方案中,根据本发明的双特异性抗体可以为特异性结合PD-L1和VEGF的四价二链抗体,或可以为特
异性结合PD-L1和VEGF的四价四链抗体。In some specific embodiments, the bispecific antibody according to the invention further comprises an Fc region. In the bispecific antibody according to the present invention, said VHH PD-L1 and VHH VEGF domains may be in a tandem form comprising both VHH PD-L1 and VHH VEGF domains (for example, as shown in Figure 1A and Figure 1B ), or in a Fab-like structure comprising both VHH PD-L1 and VHH VEGF domains (eg, as shown in Figure 1C and Figure 1D ), linked to the Fc region. Since antibody polypeptide chains comprising the Fc region can associate through dimerization of the Fc region, in some embodiments, the bispecific antibody according to the present invention can be a tetravalent antibody that specifically binds PD-L1 and VEGF. Two-chain antibody, or can be specific Quadrivalent 4-chain antibody that heterosexually binds PD-L1 and VEGF.
以下就本发明双特异性抗体的组件及由其组成的本发明双特异性抗体分别进行详述描述。本领域技术人员可以理解,除非上下文有明确相反指示,这些组件的任何技术特征的任何组合均在本发明考虑范畴之中。并且,本领域技术人员可以理解,除非上下文有明确相反指示,本发明的抗体(包括任何形式的抗体,例如二链抗体和四链抗体)可以包含任何这样的组合特征。The components of the bispecific antibody of the present invention and the bispecific antibody of the present invention composed of them are described in detail below. Those skilled in the art can understand that unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of the present invention. And, those skilled in the art will understand that unless the context clearly indicates otherwise, the antibodies of the invention (including antibodies in any form, such as diabodies and tetrabodies) may comprise any such combination of features.
根据本发明的VHHVEGF结构域和VHHPD-L1结构域VHH VEGF domain and VHH PD-L1 domain according to the invention
在根据本发明的双特异性抗体(包括本发明的二链抗体和本发明的四链抗体)中,特异性结合PD-L1的抗原结合位点由根据本发明的VHHPD-L1提供;且特异性结合VEGF的抗原结合位点由根据本发明的VHHVEGF提供。In the bispecific antibody according to the present invention (including the diabody of the present invention and the tetrabody of the present invention), the antigen-binding site that specifically binds to PD-L1 is provided by the VHH PD-L1 according to the present invention; and The antigen binding site that specifically binds VEGF is provided by the VHH VEGF according to the invention.
根据本发明的VHHPD-L1结构域包含来自抗PD-L1纳米抗体的VHH结构域。优选地所述VHH包含具有SEQ ID NO:1所示的可变区的CDR1、CDR2和CDR3序列。可变区SEQ ID NO:1氨基酸序列的CDR序列范围可以根据Kabat、AbM、Chothia、Contact或IMGT方案定义,或可以根据这些定义方案中的任何两者或多者或全部进行定义。本领域技术人员可以容易地通过http://www.abysis.org/abysis/获知由这些定义方式界定的CDR序列。在一个优选的实施方案中,根据本发明的VHHPD-L1结构域包含在具有SEQ ID NO:1的可变区中根据AbM定义的CDR1序列、CDR2序列和CDR3序列。A VHH PD-L1 domain according to the invention comprises a VHH domain from an anti-PD-L1 Nanobody. Preferably, the VHH comprises CDR1, CDR2 and CDR3 sequences having the variable region shown in SEQ ID NO:1. The CDR sequence range of the variable region SEQ ID NO: 1 amino acid sequence can be defined according to the Kabat, AbM, Chothia, Contact or IMGT schemes, or can be defined according to any two or more or all of these definition schemes. Those skilled in the art can easily know the CDR sequences defined by these definitions through http://www.abysis.org/abysis/. In a preferred embodiment, the VHH PD-L1 domain according to the invention comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence defined according to AbM in the variable region having SEQ ID NO:1.
在再一个优选的实施方案中,根据本发明的VHHPD-L1结构域包含In yet another preferred embodiment, the VHH PD-L1 domain according to the invention comprises
(i)包含SEQ ID NO:13或由SEQ ID NO:13组成的CDR1;(i) CDR1 comprising or consisting of SEQ ID NO:13;
(ii)包含SEQ ID NO:14或由SEQ ID NO:14组成的CDR2;和(ii) a CDR2 comprising or consisting of SEQ ID NO: 14; and
(iii)包含SEQ ID NO:15或由SEQ ID NO:15组成的CDR3。(iii) CDR3 comprising or consisting of SEQ ID NO:15.
在一个实施方案中,所述VHHPD-L1结构域包含SEQ ID NO:1所示的氨基酸序列。在再一实施方案中,所述VHHPD-L1结构域包含与SEQ ID NO:1具有至少80%、85%、90%、95%或99%同一性并且保留特异性结合PD-L1的能力的氨基酸序列。在再一优选实施方案中,所述VHHPD-L1结构域包含与SEQ ID NO:1相比具有一个或多个(优选地1-10个,更优选地1-5个)氨基酸的添加、缺失和/或取代(例如,保守性取代)并且保留特异性结合PD-L1的能力的氨基酸序列。优选地,所述氨基酸的添加、缺失和/或取代不发生在CDR区中。最优选地,在根据本发明的双特异性抗体中,根据本发明的VHHPD-L1结构域包含SEQ ID NO:1的氨基酸序列,或由SEQ ID NO:1所示的氨基酸序列组成。In one embodiment, the VHH PD-L1 domain comprises the amino acid sequence shown in SEQ ID NO:1. In yet another embodiment, the VHH PD-L1 domain comprises at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 1 and retains the ability to specifically bind PD-L1 amino acid sequence. In yet another preferred embodiment, the VHH PD-L1 domain comprises one or more (preferably 1-10, more preferably 1-5) amino acid additions compared to SEQ ID NO: 1, Amino acid sequences that are deleted and/or substituted (eg, conservatively substituted) and retain the ability to specifically bind PD-L1. Preferably, said amino acid additions, deletions and/or substitutions do not occur in the CDR regions. Most preferably, in the bispecific antibody according to the present invention, the VHH PD-L1 domain according to the present invention comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.
根据本发明的VHHVEGF结构域包含来自抗VEGF纳米抗体的VHH结构域。优选地,所述VHH包含具有SEQ ID NO:2所示的可变区的CDR1、CDR2和CDR3序列。可变区SEQ ID NO:2氨基酸序列的CDR序列范围可以根据Kabat、AbM、Chothia、Contact或IMGT方案定义,或可以根据这些定义方案中的任何两者或多者或全部进行定义。本领域技术人员可以容易地通过http://www.abysis.org/abysis/获知由这些定义方式界定的CDR序列。在一个优选的实施方案中,根据本发明的VHHVEGF结构域包含在具有SEQ ID NO:2的可变区中根据AbM定义的CDR1序列、CDR2序列和CDR3序列。A VHH VEGF domain according to the invention comprises a VHH domain from an anti-VEGF Nanobody. Preferably, the VHH comprises CDR1, CDR2 and CDR3 sequences of the variable region shown in SEQ ID NO:2. The CDR sequence range of the variable region SEQ ID NO: 2 amino acid sequence can be defined according to the Kabat, AbM, Chothia, Contact or IMGT schemes, or can be defined according to any two or more or all of these definition schemes. Those skilled in the art can easily know the CDR sequences defined by these definitions through http://www.abysis.org/abysis/. In a preferred embodiment, the VHH VEGF domain according to the invention comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence defined according to AbM in the variable region having SEQ ID NO:2.
在再一个优选的实施方案中,根据本发明的VHHVEGF结构域包含In yet another preferred embodiment, the VHH VEGF domain according to the invention comprises
(i)包含SEQ ID NO:16或由SEQ ID NO:16组成的CDR1;
(i) CDR1 comprising or consisting of SEQ ID NO: 16;
(ii)包含SEQ ID NO:17或由SEQ ID NO:17组成的CDR2;和(ii) a CDR2 comprising or consisting of SEQ ID NO: 17; and
(iii)包含SEQ ID NO:18或由SEQ ID NO:18组成的CDR3。(iii) CDR3 comprising or consisting of SEQ ID NO:18.
在一个实施方案中,所述VHHVEGF结构域包含SEQ ID NO:2所示的氨基酸序列。在再一实施方案中,所述VHHVEGF结构域包含与SEQ ID NO:2具有至少80%、85%、90%、95%或99%同一性并且保留特异性结合VEGF的能力的氨基酸序列。在再一优选实施方案中,所述VHHVEGF结构域包含与SEQ ID NO:2相比具有一个或多个(优选地1-10个,更优选地1-5个)氨基酸的添加、缺失和/或取代(例如,保守性取代)并且保留特异性结合VEGF的能力的氨基酸序列。优选地,所述氨基酸的添加、缺失和/或取代不发生在CDR区中。最优选地,在根据本发明的双特异性抗体中,根据本发明的VHHVEGF结构域包含SEQ ID NO:2的氨基酸序列,或由SEQ ID NO:2所示的氨基酸序列组成。In one embodiment, the VHH VEGF domain comprises the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the VHH VEGF domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2 and retains the ability to specifically bind VEGF. In yet another preferred embodiment, the VHH VEGF domain comprises one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and and/or amino acid sequences that are substituted (eg, conservatively substituted) and retain the ability to specifically bind VEGF. Preferably, said amino acid additions, deletions and/or substitutions do not occur in the CDR regions. Most preferably, in the bispecific antibody according to the present invention, the VHH VEGF domain according to the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO:2.
免疫球蛋白恒定结构域immunoglobulin constant domain
根据本发明的双特异性抗体,除前述的VHHVEGF结构域和VHHPD-L1结构域外,在一些实施方案中,还可以包含免疫球蛋白恒定结构域,例如由免疫球蛋白恒定区CH2和CH3组成的Fc区,和/或位于Fab样结构中的CH1和CL恒定结构域。According to the bispecific antibody of the present invention, in addition to the aforementioned VHH VEGF domain and VHH PD-L1 domain, in some embodiments, it may also include an immunoglobulin constant domain, for example, the immunoglobulin constant regions CH2 and CH3 Constituent Fc region, and/or CH1 and CL constant domains in a Fab-like structure.
在本发明的一些实施方案中,因此,本发明提供了包含由免疫球蛋白重链恒定区CH2-CH3组成的Fc区多肽链的双特异性抗体。在本发明的另一些实施方案中,本发明提供了包含Fc区以及还包含免疫球蛋白CH1结构域和CL结构域的双特异性抗体,优选地,其中所述CH1结构域通过免疫球蛋白铰链区连接所述Fc区多肽,且更优选地,所述抗体包含由免疫球蛋白重链恒定区CH1-铰链区-CH2-CH3组成的多肽链。包含在根据本发明的双特异性抗体中的免疫球蛋白恒定结构域,包括在Fc区中的CH2和CH3恒定结构域以及在Fab样结构中的CH1和CL恒定结构域(如果存在的话),可以彼此独立地是来自人IgG的免疫球蛋白恒定结构域,例如,人IgG1、IgG2、IgG3或IgG4的恒定结构域,优选人IgG1的恒定结构域。此外,包含在根据本发明的双特异性抗体中的免疫球蛋白恒定结构域,包括恒定区CH2和CH3以及CH1和CL(如果存在的话),可以彼此独立地是天然序列恒定结构域(例如,人天然序列恒定结构域)或其氨基酸序列变体,优选天然序列恒定结构域。In some embodiments of the invention, therefore, the invention provides a bispecific antibody comprising an Fc region polypeptide chain consisting of immunoglobulin heavy chain constant regions CH2-CH3. In some other embodiments of the present invention, the present invention provides a bispecific antibody comprising an Fc region and further comprising an immunoglobulin CH1 domain and a CL domain, preferably, wherein the CH1 domain passes through an immunoglobulin hinge region connects the Fc region polypeptide, and more preferably, the antibody comprises a polypeptide chain consisting of an immunoglobulin heavy chain constant region CH1-hinge region-CH2-CH3. the immunoglobulin constant domains comprised in the bispecific antibody according to the invention, including the CH2 and CH3 constant domains in the Fc region and the CH1 and CL constant domains in the Fab-like structure (if present), May be, independently of each other, an immunoglobulin constant domain from human IgG, eg, a constant domain of human IgGl, IgG2, IgG3 or IgG4, preferably a constant domain of human IgGl. Furthermore, the immunoglobulin constant domains comprised in the bispecific antibodies according to the invention, including the constant regions CH2 and CH3 and CH1 and CL (if present), may independently of each other be native sequence constant domains (for example, Human native sequence constant domain) or an amino acid sequence variant thereof, preferably a native sequence constant domain.
在一个优选的实施方案中,包含在根据本发明的双特异性抗体中的Fc区为来自人IgG1的Fc区序列。在一个更优选的实施方案中,所述Fc区具有SEQ ID NO:3所示的氨基酸序列,或与其具有至少90%、95%或99%同一性的氨基酸序列,或与其相比具有不超过1-10个(优选地1-5个)氨基酸残基改变的氨基酸序列。In a preferred embodiment, the Fc region comprised in the bispecific antibody according to the invention is an Fc region sequence from human IgG1. In a more preferred embodiment, the Fc region has the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence at least 90%, 95% or 99% identical thereto, or no more than Amino acid sequence with 1-10 (preferably 1-5) amino acid residues altered.
在一些特定的实施方案中,根据本发明的双特异性抗体可以包含两个具有Fc区的多肽链,例如,如图1所示的本发明二链抗体和四链抗体,其中两个多肽链的Fc区能够配对并二聚化。在这样的实施方案中,构成抗体的二聚化Fc区的第一Fc区多肽和第二Fc区多肽均可以为来自IgG,例如人IgG1、IgG2、IgG3或IgG4的Fc结构域,优选人IgG1的Fc结构域;且可以彼此独立地为本文所定义的天然序列Fc区和变体Fc区,条件是所述第一Fc区多肽和所述第二Fc区多肽可以配对并二聚化。优选地,所述第一Fc区和第二Fc区为天然序列Fc区,尤其是人IgG1天然序列Fc区。更优选地,所述第一Fc区和所述第二Fc区具有SEQ ID NO:3所示的氨基酸序列,或与其具有至少90%、95%或99%同一性的氨基酸序列,或与其相比具有不超过1-10个(优选地1-5个)氨基酸残基改变的氨基酸序列。
In some specific embodiments, the bispecific antibody according to the present invention may comprise two polypeptide chains having an Fc region, for example, the di-chain antibody and the tetra-chain antibody of the present invention as shown in FIG. 1 , wherein the two polypeptide chains The Fc region of is capable of pairing and dimerization. In such an embodiment, both the first Fc region polypeptide and the second Fc region polypeptide constituting the dimerized Fc region of the antibody may be Fc domains from IgG, such as human IgG1, IgG2, IgG3 or IgG4, preferably human IgG1 and may independently of each other be a native sequence Fc region and a variant Fc region as defined herein, provided that said first Fc region polypeptide and said second Fc region polypeptide can pair and dimerize. Preferably, the first Fc region and the second Fc region are native sequence Fc regions, especially human IgG1 native sequence Fc regions. More preferably, the first Fc region and the second Fc region have the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence at least 90%, 95% or 99% identical thereto, or identical thereto An amino acid sequence that has no more than 1-10 (preferably 1-5) amino acid residue changes.
在再一个实施方案中,根据本发明的抗体(例如,本发明的四链抗体)可以包含具有重链恒定区CH1结构域和轻链恒定区CL结构域的Fab样结构,其中所述CH1结构域优选连接在第一VHH结构域的C端,轻链恒定区CL结构域连接在第二VHH结构域的C端。在这样的双特异性抗体中,组成抗体的所述重链恒定区CH1和CL可以为来自IgG,例如人IgG1、IgG2、IgG3或IgG4的恒定结构域,优选人IgG1的恒定结构域。抗体的CL结构域可以为Kappa轻链CL结构域或Lamda轻链CL结构域,优选Kappa轻链CL结构域。在这样的双特异性抗体中,所述CH1和CL结构域可以彼此独立地是天然序列恒定结构域(例如,人天然序列恒定结构域)或其氨基酸序列变体,条件是包含所述CH1结构域和在其N端连接的第一VHH结构域的抗体多肽链,与包含所述CL结构域和在其N端连接的第二VHH结构域的抗体多肽链可以相互配对形成根据本发明的Fab样结构。优选地,所述CH1和CL结构域为天然序列恒定结构域,尤其是人IgG1天然序列CH1结构域和CL结构域。再优选地,所述CH1结构域具有SEQ ID NO:4所示的氨基酸序列。再优选地,所述CL结构域具有SEQ ID NO:5或SEQ ID NO:6所示的氨基酸序列。In yet another embodiment, the antibody according to the present invention (for example, the four-chain antibody of the present invention) may comprise a Fab-like structure having a heavy chain constant region CH1 domain and a light chain constant region CL domain, wherein the CH1 structure The domain is preferably linked C-terminally to the first VHH domain and the light chain constant region CL domain is linked C-terminally to the second VHH domain. In such bispecific antibodies, the heavy chain constant regions CH1 and CL constituting the antibody may be constant domains from IgG, such as human IgG1, IgG2, IgG3 or IgG4, preferably human IgG1. The CL domain of the antibody may be a Kappa light chain CL domain or a Lamda light chain CL domain, preferably a Kappa light chain CL domain. In such bispecific antibodies, the CH1 and CL domains may independently of each other be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof, provided that the CH1 structure is included Domain and the antibody polypeptide chain of the first VHH domain connected at its N-terminus, and the antibody polypeptide chain comprising said CL domain and the second VHH domain connected at its N-terminus can be paired with each other to form a Fab according to the invention like structure. Preferably, the CH1 and CL domains are native sequence constant domains, especially human IgG1 native sequence CH1 domains and CL domains. More preferably, the CH1 domain has the amino acid sequence shown in SEQ ID NO:4. More preferably, the CL domain has the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6.
连接子Linker
在根据本发明的双特异抗体中,抗体组件之间可以采用连接子进行连接。例如,根据本发明的VHH结构域可以通过连接子连接在Fc区的N端或C端,或可以通过连接子连接在另一根据本发明的VHH结构域上。可以用于本发明的抗体中的连接子并无特定限制。本领域技术人员可以根据待连接的组件和连接位置容易地确定可用的连接子序列。In the bispecific antibody according to the present invention, linkers can be used to connect antibody components. For example, a VHH domain according to the present invention may be linked to the N-terminal or C-terminal of the Fc region via a linker, or may be linked to another VHH domain according to the present invention via a linker. Linkers that can be used in the antibodies of the present invention are not particularly limited. Those skilled in the art can easily determine the available linker sequences according to the components to be linked and the position of the link.
在一些实施方案中,在根据本发明的双特异性抗体中,根据本发明的VHH结构域以串联形式(例如,如图1A和1B所示)或在Fab样结构中(例如,如图1C和1D所示),通过连接子,连接在Fc区多肽链上。本领域技术人员可以根据VHH的连接位置,即,连接在Fc区N端或C端而选择不同的连接子。In some embodiments, in the bispecific antibody according to the invention, the VHH domains according to the invention are in tandem form (eg, as shown in Figures 1A and 1B ) or in a Fab-like structure (eg, as shown in Figure 1C and 1D), connected to the Fc region polypeptide chain through a linker. Those skilled in the art can select different linkers according to the connection position of the VHH, that is, the connection at the N-terminal or C-terminal of the Fc region.
在一个实施方案中,Fc区多肽链通过连接子在N端连接单个VHH结构域或串联的两个VHH结构域、或Fab样结构,其中所述连接子优选包含(一个或两个)半胱氨酸残基,由此能够在包含Fc区的两条抗体多肽链配对时形成(一对或两对)二硫键,以稳定由此形成的双特异性抗体的结构。例如,图1所示的二链抗体结构和四链抗体结构。因此,在一个优选实施方案中,包含在本发明双特异性抗体中的Fc区多肽链在N端带有包含“CPPC”的连接子序列。在再一优选的实施方案中,包含在本发明双特异性抗体中的Fc区多肽链在N端带有来自免疫球蛋白的铰链区,例如包含“CPPC”的铰链区氨基酸序列,例如,氨基酸序列“EPKSCDKTHTCPPCP”(SEQ ID NO:22)或“EPKSSDKTHTCPPCP”(SEQ ID NO:23);更优选,氨基酸序列“EPKSCDKTHTCPPCP”(SEQ ID NO:22)。In one embodiment, the polypeptide chains of the Fc region are connected at the N-terminus to a single VHH domain or two VHH domains in tandem, or a Fab-like structure via a linker, wherein the linker preferably comprises (one or two) cysteine Amino acid residues, which are capable of forming (one or two) disulfide bonds when the two antibody polypeptide chains comprising the Fc region pair to stabilize the structure of the bispecific antibody thus formed. For example, the two-chain antibody structure and the four-chain antibody structure shown in FIG. 1 . Therefore, in a preferred embodiment, the Fc region polypeptide chain contained in the bispecific antibody of the present invention has a linker sequence comprising "CPPC" at the N-terminus. In yet another preferred embodiment, the Fc region polypeptide chain contained in the bispecific antibody of the present invention has a hinge region derived from an immunoglobulin at the N-terminus, for example, the amino acid sequence of the hinge region comprising "CPPC", for example, amino acid The sequence "EPKSCDKTHTCPPCP" (SEQ ID NO:22) or "EPKSSDKTHTCPPCP" (SEQ ID NO:23); more preferably, the amino acid sequence "EPKSCDKTHTCPPCP" (SEQ ID NO:22).
在另一个实施方案中,Fc区多肽链通过连接子在C端连接VHH结构域,其中所述连接子优选为5-50个氨基酸的柔性连接肽,优选地包含甘氨酸(G)和/或丝氨酸(S)和/或苏氨酸残基(T)的连接肽。在一个实施方案中,所述连接子具有5-50个氨基酸长度,例如,10、15、20、25或30个氨基酸长度,或具有落入任何两个整数之间的氨基酸长度。在一个实施方案中,连接子包含氨基酸序列(G4S)n,其中n是等于或大于1的整数,例如,n是2、3、4、5、6或7的整数。在一个优选实施方案中,连接子由氨基酸序列(G4S)3组成。In another embodiment, the Fc region polypeptide chain is connected to the VHH domain at the C-terminus by a linker, wherein the linker is preferably a flexible connecting peptide of 5-50 amino acids, preferably comprising glycine (G) and/or serine (S) and/or a linking peptide of a threonine residue (T). In one embodiment, the linker is 5-50 amino acids in length, eg, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers. In one embodiment, the linker comprises the amino acid sequence (G 4 S) n , wherein n is an integer equal to or greater than 1, eg, n is an integer of 2, 3, 4, 5, 6 or 7. In a preferred embodiment, the linker consists of the amino acid sequence (G 4 S) 3 .
在再一些实施方案中,在根据本发明的双特异性抗体中,根据本发明的VHHVEGF结构域和VHHPD-L1结构域通过连接子形成串联VHH结构域。在具有这种串联VHH结构域的根据本发明双特异性抗体中,优
选地,所述第一和第二结构域之间通过连接子串联,其中所述连接子为5-50个氨基酸的柔性连接肽。在一个实施方案中,所述连接子具有5-50个氨基酸长度,例如,10、15、20、25或30个氨基酸长度,或具有落入任何两个整数之间的氨基酸长度。在一个实施方案中,连接子为包含甘氨酸(G)和/或丝氨酸(S)和/或苏氨酸残基(T)的连接肽。在再一实施方案中,连接子包含氨基酸序列(G4S)n(SEQ ID NO:20),其中n是等于或大于1的整数,例如,n是2、3、4、5、6或7的整数。在一个优选实施方案中,连接子由氨基酸序列(G4S)3(SEQ ID NO:19)组成。In still other embodiments, in the bispecific antibody according to the invention, the VHH VEGF domain and the VHH PD-L1 domain according to the invention form a tandem VHH domain via a linker. In the bispecific antibody according to the invention having such tandem VHH domains, preferably Optionally, the first and second domains are connected in series through a linker, wherein the linker is a flexible connecting peptide of 5-50 amino acids. In one embodiment, the linker is 5-50 amino acids in length, eg, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers. In one embodiment, the linker is a linker peptide comprising glycine (G) and/or serine (S) and/or threonine residues (T). In yet another embodiment, the linker comprises the amino acid sequence (G 4 S) n (SEQ ID NO: 20), wherein n is an integer equal to or greater than 1, for example, n is 2, 3, 4, 5, 6 or Integer of 7. In a preferred embodiment, the linker consists of the amino acid sequence (G 4 S) 3 (SEQ ID NO: 19).
本发明的双特异性抗体实施方案Bispecific antibody embodiments of the invention
在一个方面,本发明提供了一种特异性结合PD-L1和VEGF的双特异性抗体,其中所述抗体包括特异性结合第一抗原的VHH结构域和特异性结合第二抗原的VHH结构域,其中第一抗原和第二抗原彼此不同并独立地选自PD-L1和VEGF。In one aspect, the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, wherein the antibody comprises a VHH domain specifically binding to a first antigen and a VHH domain specifically binding to a second antigen , wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF.
在一个实施方案中,所述特异性结合VEGF的VHH结构域包含SEQ ID NO:2所示的VHH结构域的CDR1-3序列。在另一实施方案中,所述特异性结合PD-L1的VHH结构域包含SEQ ID NO:1所示的VHH结构域的CDR1-3序列。In one embodiment, the VHH domain specifically binding to VEGF comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:2. In another embodiment, the VHH domain specifically binding to PD-L1 comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:1.
在另一实施方案中,所述特异性结合VEGF的VHH结构域包含SEQ ID NO:2所示的氨基酸序列,或与SEQ ID NO:2具有至少80%、85%、90%、95%或99%同一性的氨基酸序列,或与SEQ ID NO:2相比具有一个或多个(优选地1-10个,更优选地1-5个)氨基酸的添加、缺失和/或取代的氨基酸序列,最优选地,所述VHH结构域包含SEQ ID NO:2的氨基酸序列,或由SEQ ID NO:2所示的氨基酸序列组成。In another embodiment, the VHH domain specifically binding to VEGF comprises the amino acid sequence shown in SEQ ID NO: 2, or has at least 80%, 85%, 90%, 95%, or An amino acid sequence with 99% identity, or an amino acid sequence with one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and/or substitutions compared to SEQ ID NO:2 , most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence shown in SEQ ID NO: 2.
在再一实施方案中,所述特异性结合PD-L1的VHH结构域包含SEQ ID NO:1所示的氨基酸序列,或与SEQ ID NO:1具有至少80%、85%、90%、95%或99%同一性的氨基酸序列,或与SEQ ID NO:1相比具有一个或多个(优选地1-10个,更优选地1-5个)氨基酸的添加、缺失和/或取代的氨基酸序列,最优选地,所述VHH结构域包含SEQ ID NO:1的氨基酸序列,或由SEQ ID NO:1所示的氨基酸序列组成。In yet another embodiment, the VHH domain specifically binding to PD-L1 comprises the amino acid sequence shown in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 1 Amino acid sequence with % or 99% identity, or with one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and/or substitutions compared to SEQ ID NO: 1 Amino acid sequence, most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.
在一些实施方案中,根据本发明的双特异性抗体还包含连接子,所述连接子包含10-20个氨基酸长度,优选地,包含氨基酸序列(G4S)3(SEQ ID NO:19)。在再一些实施方案中,根据本发明的双特异性抗体还包含免疫球蛋白的Fc区,其中,Fc区为IgG Fc区,例如,人IgG1、IgG2、IgG3或IgG4的Fc结构域,优选人IgG1的Fc结构域,更优选所述Fc区在N端带有免疫球蛋白铰链区序列。In some embodiments, the bispecific antibody according to the present invention further comprises a linker comprising 10-20 amino acids in length, preferably comprising the amino acid sequence (G 4 S) 3 (SEQ ID NO: 19) . In some further embodiments, the bispecific antibody according to the present invention further comprises an Fc region of an immunoglobulin, wherein the Fc region is an IgG Fc region, for example, the Fc domain of human IgG1, IgG2, IgG3 or IgG4, preferably human The Fc domain of IgG1, more preferably the Fc region carries an immunoglobulin hinge region sequence at the N-terminus.
在一些优选实施方案中,根据本发明的双特异性抗体包含多肽链,所述多肽链包含借助于Fc区和连接子串联的VHHPD-L1和VHH VEGF结构域。优选地,所述双特异性抗体包含多肽链,所述多肽链从N端到C端包含第一VHH结构域、Fc区、连接子和第二VHH结构域,其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同并独立地选自PD-L1和VEGF,优选地,第一抗原是PD-L1且第二抗原是VEGF。优选地,所述双特异性抗体包含SEQ ID NO:7所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。In some preferred embodiments, the bispecific antibody according to the invention comprises a polypeptide chain comprising VHH PD-L1 and VHH VEGF domains connected in series by means of an Fc region and a linker. Preferably, the bispecific antibody comprises a polypeptide chain comprising a first VHH domain, an Fc region, a linker and a second VHH domain from the N-terminus to the C-terminus, wherein the first VHH domain and the second VHH domain The two VHH domains specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and The second antigen is VEGF. Preferably, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 7 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or amino acid sequences with 99% identity.
在另一些优选实施方案中,根据本发明的双特异性抗体包含多肽链,所述多肽链包含借助于连接子串联的VHHPD-L1和VHHVEGF结构域。优选地,所述双特异性抗体包含多肽链,所述多肽链从N端至C端包
含第一VHH结构域、连接子、第二VHH结构域和Fc区,其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同并独立地选自PD-L1和VEGF,优选地,第一抗原是PD-L1且第二抗原是VEGF。优选地,所述双特异性抗体包含SEQ ID NO:8所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。In other preferred embodiments, the bispecific antibody according to the invention comprises a polypeptide chain comprising VHH PD-L1 and VHH VEGF domains connected in series by means of a linker. Preferably, the bispecific antibody comprises a polypeptide chain comprising from the N-terminus to the C-terminus Containing a first VHH domain, a linker, a second VHH domain and an Fc region, wherein the first VHH domain and the second VHH domain specifically bind to a first antigen and a second antigen, respectively, wherein the first antigen and The second antigens are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 8 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or amino acid sequences with 99% identity.
在再一些优选实施方案中,根据本发明的双特异性抗体包含形成Fab样结构的第一和第二多肽链。优选地,双特异性抗体包含第一多肽链和第二多肽链,其中所述第一多肽链从N端至C端包含第一VHH结构域、CH1结构域和Fc区;所述第二多肽链从N端至C端包含第二VHH结构域和CL结构域;其中第一多肽链中的第一VHH结构域和CH1结构域与第二多肽链中的第二VHH结构域和CL结构域配对形成Fab样结构;其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同且独立地选自PD-L1和VEGF,优选地,第一抗原是PD-L1且第二抗原是VEGF。在这样的实施方案中,优选地,所述双特异性抗体包含第一和第二多肽链,其中第一抗原为PD-L1且第二抗原为VEGF,其中,第一多肽链包含SEQ ID NO:9所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列;且第二多肽链包含SEQ ID NO:10所示的氨基酸序列与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。In further preferred embodiments, the bispecific antibody according to the invention comprises a first and a second polypeptide chain forming a Fab-like structure. Preferably, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first VHH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus; the The second polypeptide chain comprises a second VHH domain and a CL domain from the N-terminus to the C-terminus; wherein the first VHH domain and the CH1 domain in the first polypeptide chain are the same as the second VHH in the second polypeptide chain domain and CL domain pair to form a Fab-like structure; wherein the first VHH domain and the second VHH domain specifically bind a first antigen and a second antigen, respectively, wherein the first and second antigens are different and independent of each other is selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF. In such embodiments, preferably, the bispecific antibody comprises first and second polypeptide chains, wherein the first antigen is PD-L1 and the second antigen is VEGF, wherein the first polypeptide chain comprises SEQ The amino acid sequence shown in ID NO: 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and The two polypeptide chains comprise an amino acid sequence shown in SEQ ID NO: 10 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith amino acid sequence.
本发明的二链双特异性抗体实施方案Two-chain bispecific antibody embodiments of the invention
在具有串联排列形式的VHHPD-L1和VHHVEGF结构域的本发明双特异性抗体的一些实施方案中,所述抗体可以包含两条包含Fc区的多肽链,其中所述的两条多肽链能够通过Fc区的二聚化缔合而形成二聚体。如本领域技术人员理解,缔合形成二聚体的两条多肽链并不需要完全相同。例如,可以在一条或两条多肽链中(例如在多肽链的Fc区),引入不对称修饰,而不影响期望目标活性。但在一些方面,优选地,所述两条多肽链相同。例如,如图1A和1B所示。In some embodiments of the bispecific antibody of the invention having VHH PD-L1 and VHH VEGF domains arranged in tandem, the antibody may comprise two polypeptide chains comprising an Fc region, wherein the two polypeptide chains Dimers can be formed by dimerizing association of the Fc region. As will be appreciated by those skilled in the art, the two polypeptide chains that associate to form a dimer need not be identical. For example, asymmetric modifications can be introduced in one or both polypeptide chains (eg, in the Fc region of the polypeptide chains) without affecting the desired target activity. However, in some aspects, preferably, the two polypeptide chains are identical. For example, as shown in Figures 1A and 1B.
因此,在一个方面,本发明提供了特异性结合PD-L1和VEGF的双特异性抗体,其中所述抗体包括两条多肽链,所述两条多肽链可以相同或不同,其中:Therefore, in one aspect, the present invention provides a bispecific antibody that specifically binds PD-L1 and VEGF, wherein said antibody comprises two polypeptide chains, said two polypeptide chains may be the same or different, wherein:
每条多肽链各自从N端到C端包含第一VHH结构域、Fc区、连接子和第二VHH结构域(如图1A所示);或每条多肽链各自从N端到C端包含第一VHH结构域、连接子、第二VHH结构域和Fc区(如图1B所示);Each polypeptide chain comprises a first VHH domain, an Fc region, a linker, and a second VHH domain from N-terminus to C-terminus (as shown in Figure 1A); or each polypeptide chain comprises from N-terminus to C-terminus A first VHH domain, a linker, a second VHH domain and an Fc region (as shown in Figure 1B);
其中两条所述多肽链的Fc区多肽配对并形成二聚化Fc区,其中所述第一VHH结构域和所述第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同且独立地选自PD-L1和VEGF。在一个实施方案中,所述第一抗原为PD-L1且第二抗原为VEGF;在另一实施方案中,所述第一抗原为VEGF且第二抗原为PD-L1。The Fc region polypeptides of the two polypeptide chains are paired to form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen respectively, wherein the The first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF. In one embodiment, the first antigen is PD-L1 and the second antigen is VEGF; in another embodiment, the first antigen is VEGF and the second antigen is PD-L1.
在该方面的一些实施方案中,本发明提供了特异性结合PD-L1和VEGF的双特异性抗体,其中所述抗体包括两条多肽链,所述两条多肽链可以相同或不同,其中:In some embodiments of this aspect, the present invention provides a bispecific antibody that specifically binds PD-L1 and VEGF, wherein the antibody comprises two polypeptide chains, which may be the same or different, wherein:
每条多肽链各自从N端至C端包含第一VHH结构域、Fc区、连接子和第二VHH结构域,
Each polypeptide chain comprises a first VHH domain, an Fc region, a linker and a second VHH domain from the N-terminus to the C-terminus,
其中两条多肽链的Fc区多肽配对并形成二聚化Fc区,其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同且独立地选自PD-L1和VEGF,优选地所述第一抗原为PD-L1且第二抗原为VEGF。优选地,所述Fc区为来自IgG,尤其是IgG1的Fc区,从N端到C端包含CH2和CH3结构域序列。所述Fc区可以为天然序列Fc区或变体Fc区,优选天然序列Fc区。优选地,Fc区多肽在N端通过免疫球蛋白铰链区序列与第一VHH结构域连接。再优选地,Fc区多肽在C端通过10-20个氨基酸长度的柔性连接肽,例如(G4S)3,与第二VHH结构域连接。优选地,第一VHH结构域为根据本发明的VHHPD-L1结构域,优选地,包含SEQ ID NO:1所示可变区的CDR1-3序列,更优选地包含SEQ ID NOs:13-15的CDR1-3序列;最优选地包含SEQ ID NO:1所示的可变区序列;且第二VHH结构域为根据本发明的VHHVEGF结构域,优选地,包含SEQ ID NO:2所示可变区的CDR1-3序列,更优选地包含SEQ ID NOs:16-18的CDR1-3序列;最优选地包含SEQ ID NO:2所示的可变区序列。优选地,所述的两条多肽链分别包含SEQ ID NO:7所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。最优选地,所述两条多肽链相同,且分别包含SEQ ID NO:7的氨基酸序列。The Fc region polypeptides of the two polypeptide chains are paired and form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen The two antigens are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is linked at the N-terminus to the first VHH domain through an immunoglobulin hinge region sequence. Still preferably, the Fc region polypeptide is connected to the second VHH domain at the C-terminus through a flexible linking peptide with a length of 10-20 amino acids, such as (G 4 S) 3 . Preferably, the first VHH domain is a VHH PD-L1 domain according to the present invention, preferably, comprising the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprising SEQ ID NOs: 13- 15 CDR1-3 sequence; most preferably comprising the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH VEGF domain according to the present invention, preferably comprising SEQ ID NO: 2 The CDR1-3 sequence of the variable region, more preferably comprising the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprising the variable region sequence shown in SEQ ID NO:2. Preferably, the two polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 7 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences of 98% or 99% identity. Most preferably, the two polypeptide chains are identical and each comprise the amino acid sequence of SEQ ID NO:7.
在该方面的另一些实施方案中,本发明提供了特异性结合PD-L1和VEGF的双特异性抗体,其中所述抗体包括两条多肽链,所述两条多肽链可以相同或不同,其中:In other embodiments of this aspect, the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, wherein the antibody comprises two polypeptide chains, and the two polypeptide chains may be the same or different, wherein :
每条多肽链各自从N端至C端包含第一VHH结构域、连接子、第二VHH结构域和Fc区,Each polypeptide chain comprises a first VHH domain, a linker, a second VHH domain and an Fc region from the N-terminus to the C-terminus,
其中所述两条多肽的Fc区多肽配对并形成二聚化Fc区,其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同且独立地选自PD-L1和VEGF,优选地所述第一抗原为PD-L1且第二抗原为VEGF。优选地,所述连接子为10-20个氨基酸长度的柔性连接肽,例如(G4S)3(SEQ ID NO:19)。再优选地,所述Fc区为来自IgG,尤其是IgG1的Fc区,从N端到C端包含CH2和CH3结构域序列。所述Fc区可以为天然序列Fc区或变体Fc区,优选天然序列Fc区。优选地,Fc区多肽在N端通过免疫球蛋白铰链区序列与串联的VHH结构域连接。优选地,第一VHH结构域为根据本发明的VHHPD-L1结构域,优选地,包含SEQ ID NO:1所示可变区的CDR1-3序列,更优选地包含SEQ ID NOs:13-15的CDR1-3序列;最优选地包含SEQ ID NO:1所示的可变区序列;且第二VHH结构域为根据本发明的VHHVEGF结构域,优选地,包含SEQ ID NO:2所示可变区的CDR1-3序列,更优选地包含SEQ ID NOs:16-18的CDR1-3序列;最优选地包含SEQ ID NO:2所示的可变区序列。优选地,所述的两条多肽链分别包含SEQ ID NO:8所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。最优选地,所述两条多肽链相同,且分别包含SEQ ID NO:8的氨基酸序列。Wherein the Fc region polypeptides of the two polypeptides pair and form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen respectively, wherein the first antigen and The second antigens are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the linker is a flexible connecting peptide with a length of 10-20 amino acids, such as (G 4 S) 3 (SEQ ID NO: 19) . Still more preferably, the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is linked at the N-terminus to a tandem VHH domain through an immunoglobulin hinge region sequence. Preferably, the first VHH domain is a VHH PD-L1 domain according to the present invention, preferably, comprising the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprising SEQ ID NOs: 13- 15 CDR1-3 sequence; most preferably comprising the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH VEGF domain according to the present invention, preferably comprising SEQ ID NO: 2 The CDR1-3 sequence of the variable region, more preferably comprising the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprising the variable region sequence shown in SEQ ID NO:2. Preferably, the two polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 8 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences of 98% or 99% identity. Most preferably, the two polypeptide chains are identical and each comprise the amino acid sequence of SEQ ID NO:8.
本发明的四链双特异性抗体实施方案Four-chain bispecific antibody embodiments of the invention
在具有包含在Fab样结构中的VHHPD-L1和VHHVEGF结构域的本发明双特异性抗体的一些实施方案中,所述抗体可以包含两条包含Fc区的第一多肽链和两条第二多肽链,其中所述的两条第一多肽链能够通过Fc区的二聚化缔合而形成二聚体。如本领域技术人员理解,包含在所述双特异性抗体中的两条第一多肽链并不需要完全相同;且类似地,两条第二多肽链也不需要完全相同。例如,可以在一条或两条第一多肽链
和/或第二多肽链中引入不对称修饰,而不影响期望目标活性。但在一些方面,优选地,两条第一多肽链相同,且两条第二多肽链也相同。In some embodiments of the bispecific antibody of the invention having VHH PD-L1 and VHH VEGF domains comprised in a Fab-like structure, said antibody may comprise two first polypeptide chains comprising an Fc region and two The second polypeptide chain, wherein the two first polypeptide chains can form a dimer through dimerization association of the Fc region. As understood by those skilled in the art, the two first polypeptide chains comprised in the bispecific antibody need not be identical; and similarly, the two second polypeptide chains need not be identical either. For example, in one or both first polypeptide chains and/or introduce asymmetric modifications into the second polypeptide chain without affecting the desired target activity. However, in some aspects, preferably, the two first polypeptide chains are identical and the two second polypeptide chains are also identical.
因此,在又一个方面,本发明提供了特异性结合PD-L1和VEGF的双特异性抗体,其中所述抗体包含两条第一多肽链和两条第二多肽链,其中,两条第一多肽链可以相同或不同,两条第二多肽链可以相同或不同,其中:Therefore, in yet another aspect, the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, wherein the antibody comprises two first polypeptide chains and two second polypeptide chains, wherein the two The first polypeptide chains may be the same or different, and the two second polypeptide chains may be the same or different, wherein:
所述的两条第一多肽链各自从N端至C端包含第一VHH结构域、CH1结构域和Fc区;The two first polypeptide chains each comprise a first VHH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus;
所述的两条第二多肽链各自从N端至C端包含第二VHH结构域和CL结构域;The two second polypeptide chains each comprise a second VHH domain and a CL domain from the N-terminus to the C-terminus;
其中两条第一多肽链中的Fc区多肽配对并形成二聚化Fc区,且wherein the Fc region polypeptides in the two first polypeptide chains pair and form a dimerized Fc region, and
其中两条第一多肽链中的第一VHH结构域和CH1结构域分别与两条第二多肽链中的第二VHH结构域和CL结构域配对形成两个Fab样结构;wherein the first VHH domain and the CH1 domain in the two first polypeptide chains are paired with the second VHH domain and the CL domain in the two second polypeptide chains respectively to form two Fab-like structures;
其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,其中所述第一抗原和第二抗原彼此不同且独立地选自PD-L1和VEGF。例如,如图1C和图1D所示。在一个实施方案中,所述第一抗原为PD-L1且第二抗原为VEGF;或在另一实施方案中,所述第一抗原为VEGF且第二抗原为PD-L1。优选地,所述Fc区为来自IgG,尤其是IgG1的Fc区,从N端到C端包含CH2和CH3结构域序列。所述Fc区可以为天然序列Fc区或变体Fc区,优选天然序列Fc区。优选地,Fc区多肽在N端通过免疫球蛋白铰链区序列与CH1结构域连接。优选地,第一VHH结构域为根据本发明的VHHPD-L1结构域,优选地,包含SEQ ID NO:1所示可变区的CDR1-3序列,更优选地包含SEQ ID NOs:13-15的CDR1-3序列;最优选地包含SEQ ID NO:1所示的可变区序列;且第二VHH结构域为根据本发明的VHHVEGF结构域,优选地,包含SEQ ID NO:2所示可变区的CDR1-3序列,更优选地包含SEQ ID NOs:16-18的CDR1-3序列;最优选地包含SEQ ID NO:2所示的可变区序列。Wherein the first VHH structural domain and the second VHH structural domain specifically bind the first antigen and the second antigen respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF. For example, as shown in Figure 1C and Figure 1D. In one embodiment, the first antigen is PD-L1 and the second antigen is VEGF; or in another embodiment, the first antigen is VEGF and the second antigen is PD-L1. Preferably, the Fc region is from IgG, especially IgG1, and includes CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is connected to the CH1 domain at the N-terminus through an immunoglobulin hinge region sequence. Preferably, the first VHH domain is a VHH PD-L1 domain according to the present invention, preferably, comprising the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprising SEQ ID NOs: 13- 15 CDR1-3 sequence; most preferably comprising the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH VEGF domain according to the present invention, preferably comprising SEQ ID NO: 2 The CDR1-3 sequence of the variable region, more preferably comprising the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprising the variable region sequence shown in SEQ ID NO:2.
在该方面的一个实施方案中,本发明的双特异性抗体包含两条第一多肽链和两条第二多肽链,其中第一抗原为PD-L1且第二抗原为VEGF,其中:In one embodiment of this aspect, the bispecific antibody of the invention comprises two first polypeptide chains and two second polypeptide chains, wherein the first antigen is PD-L1 and the second antigen is VEGF, wherein:
两条第一多肽链分别包含SEQ ID NO:9所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列;且The two first polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 9 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences; and
两条第二多肽链分别包含SEQ ID NO:10所示的氨基酸序列与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。The two second polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 10 and have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% % identity amino acid sequence.
最优选地,两条第一多肽链相同,分别包含SEQ ID NO:9的氨基酸序列;且两条第二多肽链相同,分别包含SEQ ID NO:10的氨基酸序列。Most preferably, the two first polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO:9; and the two second polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO:10.
在该方面的一个实施方案中,本发明的双特异性抗体包含两条第一多肽链和两条第二多肽链,其中第一抗原为VEGF且第二抗原为PD-L1,其中:In one embodiment of this aspect, the bispecific antibody of the invention comprises two first polypeptide chains and two second polypeptide chains, wherein the first antigen is VEGF and the second antigen is PD-L1, wherein:
两条第一多肽链分别包含SEQ ID NO:11所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列;且The two first polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 11 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences; and
两条第二多肽链分别包含SEQ ID NO:12所示的氨基酸序列与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。
The two second polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 12 and have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% % identity amino acid sequence.
最优选地,两条第一多肽链相同,分别包含SEQ ID NO:11的氨基酸序列;且两条第二多肽链相同,分别包含SEQ ID NO:12的氨基酸序列。Most preferably, the two first polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO: 11; and the two second polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO: 12.
在根据本发明的四链抗体的上述实施方案中,本发明也考虑第一和第二多肽链中的CH1与CL结构域交换的实施方案。在具有CH1/CL结构域交换的此实施方案中,优选地,两个Fc区通过免疫球蛋白铰链区连接所述Fab样结构的CL结构域。In the above-described embodiments of the four-chain antibody according to the invention, the invention also contemplates an embodiment in which the CH1 and CL domains in the first and second polypeptide chains are exchanged. In this embodiment with CH1/CL domain exchange, preferably the two Fc regions are linked by the CL domain of the Fab-like structure through an immunoglobulin hinge region.
II.本发明的抗体的生产和纯化II. Production and Purification of Antibodies of the Invention
再一方面,本发明提供用于生产本发明抗体的方法。为了产生本发明的抗体,可以例如通过固态肽合成(例如Merrifield固相合成)或重组生产获得本发明的抗体的多肽链,并在适宜条件下使其装配。In yet another aspect, the invention provides methods for producing antibodies of the invention. To produce the antibodies of the invention, the polypeptide chains of the antibodies of the invention can be obtained, eg, by solid-state peptide synthesis (eg, Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions.
为了重组生产,可以将编码所述抗体的任意一条多肽链和/或多条多肽链的多核苷酸分离并插入一个或多个载体中以便进一步在宿主细胞中克隆和/或表达。使用常规方法,可以轻易地分离所述多核苷酸并将其测序。在一个实施方案中,提供了编码本发明抗体的一条或多条多肽链的多核苷酸。在再一实施方案中,本发明提供了包含本发明的一种或多种多核苷酸的载体,优选地表达载体。因此,在一个实施方案中,本发明提供用于生产本发明抗体的方法,所述方法包括:在适于表达所述抗体的多肽链的条件下培养包含编码所述多肽链的宿主细胞;和在适于所述多肽链装配为所述抗体的条件下使多肽链装配产生所述抗体。For recombinant production, polynucleotides encoding any polypeptide chain and/or multiple polypeptide chains of the antibody can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells. The polynucleotides can be readily isolated and sequenced using conventional methods. In one embodiment, polynucleotides encoding one or more polypeptide chains of an antibody of the invention are provided. In yet another embodiment, the invention provides a vector, preferably an expression vector, comprising one or more polynucleotides of the invention. Accordingly, in one embodiment, the invention provides a method for producing an antibody of the invention, said method comprising: culturing a host cell comprising a polypeptide chain encoding said antibody under conditions suitable for expressing said polypeptide chain; and The antibody is produced by assembling the polypeptide chains under conditions suitable for assembly of the polypeptide chains into the antibody.
可以使用本领域技术人员熟知的方法来构建表达载体。表达载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
一旦已经制备了用于表达的包含本发明的一种或多种多核苷酸的表达载体,则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的,例如,原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质体的转染或其他常规技术。Once an expression vector comprising one or more polynucleotides of the invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.
在一个实施方案中,本发明也提供了包含一种或多种本发明多核苷酸的宿主细胞。在一些实施方案中,提供了包含本发明表达载体的宿主细胞。如本文所用,术语“宿主细胞”指可以工程化以产生本发明的抗体的任何种类的细胞系统。适于复制和支持本发明的抗体表达的宿主细胞是本领域熟知的。根据需要,这类细胞可以用特定表达载体转染或转导,并且可以培育大量含有载体的细胞用于接种大规模发酵器以获得足够量的本发明抗体用于临床应用。合适的宿主细胞包括原核微生物,如大肠杆菌,真核微生物如丝状真菌或酵母,或各种真核细胞,如中国仓鼠卵巢细胞(CHO)、昆虫细胞等。可以使用适于悬浮培养的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)、人胚肾系(HEK293或293F细胞)、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、人肝脏细胞(HepG2)、CHO细胞、NSO细胞、骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu,Methods in Molecular Biology,第248卷(B.K.C.Lo编著,Humana Press,Totowa,NJ),第255-268页(2003)。在一个优选的实施方案中,所述宿主细胞是CHO、HEK293或NSO细胞。In one embodiment, the invention also provides host cells comprising one or more polynucleotides of the invention. In some embodiments, host cells comprising an expression vector of the invention are provided. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce the antibodies of the invention. Host cells suitable for replicating and supporting expression of the antibodies of the invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors as needed, and large numbers of cells containing the vector can be grown for inoculation of large-scale fermenters to obtain sufficient quantities of the antibodies of the invention for clinical use. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells and the like. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2) , CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2/0, etc. For a review of mammalian host cell lines suitable for protein production see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. B.K.C. Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell.
通过本文所述方法制备的抗体,可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。在纯化后,可以通过多种熟知分析方法中的任一种方法确定本发明的抗
体的纯度,所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。可以通过本领域已知的多种测定法,鉴定、筛选或表征本文提供的抗体的物理/化学特性和/或生物学活性。Antibodies prepared by the methods described herein can be purified by known art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. After purification, the antibodies of the invention can be determined by any of a variety of well known assays. The well-known analytical methods include size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical/chemical properties and/or biological activities of the antibodies provided herein can be identified, screened or characterized by a variety of assays known in the art.
在一个优选的实施方案中,本发明双特异性抗体在哺乳动物宿主细胞例如CHO细胞中重组生产中表现出良好的生产性质,尤其是,良好的表达产量和良好的副产物谱。在一个实施方案中,本发明双特异性抗体的瞬时表达量达到50μg/mL以上,优选地150μg/mL以上;在再一实施方案中,本发明双特异性抗体在一步蛋白A亲和层析纯化后,应用SEC-HPLC测定产物纯度,展示出95%以上,优选96%、97%、98%或99%以上的纯度。优选地,瞬时表达量的测定按照实施例3所述的方法测定;一步蛋白A纯化后的产物纯度测定按照实施例3.4所述的SEC-HPLC单体纯度鉴定方法进行。In a preferred embodiment, the bispecific antibody of the present invention exhibits good production properties in recombinant production in mammalian host cells such as CHO cells, in particular, good expression yield and good by-product profile. In one embodiment, the transient expression level of the bispecific antibody of the present invention reaches above 50 μg/mL, preferably above 150 μg/mL; After purification, the purity of the product is determined by SEC-HPLC, showing a purity of more than 95%, preferably more than 96%, 97%, 98% or 99%. Preferably, the determination of the transient expression level is determined according to the method described in Example 3; the determination of the purity of the product after one-step protein A purification is performed according to the SEC-HPLC monomer purity identification method described in Example 3.4.
III.药物组合物、药物联合和试剂盒III. Pharmaceutical Compositions, Drug Combinations and Kits
在一个方面,本发明提供了组合物,例如,药物组合物,所述组合物包含与可药用载体配制在一起的本文所述的抗体。如本文所用,“可药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。本发明的药物组合物适于静脉内、肌内、皮下、肠胃外、直肠、脊髓或表皮施用(例如,通过注射或输注)。在一些实施方案中,本发明抗体是药物组合物中的唯一活性成分。在另一些实施方案中,药物组合物可以包含本文所述的抗体与一种以上治疗剂。In one aspect, the invention provides compositions, eg, pharmaceutical compositions, comprising an antibody described herein formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutical compositions of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion). In some embodiments, an antibody of the invention is the sole active ingredient in a pharmaceutical composition. In other embodiments, a pharmaceutical composition may comprise an antibody described herein and more than one therapeutic agent.
在另一方面,本发明也提供包含本文所述的抗体与一种以上治疗剂的药物联合。In another aspect, the invention also provides pharmaceutical combinations comprising an antibody described herein and more than one therapeutic agent.
适用于本发明的药物组合物和药物联合中的治疗剂可以为选自以下类别(i)-(iv)中任一类别的治疗剂:(i)增强抗原呈递(例如,肿瘤抗原呈递)的药物;(ii)增强效应细胞反应(例如,B细胞和/或T细胞活化和/或动员)的药物;(iii)减少免疫抑制的药物;(iv)具有抑制肿瘤作用的药物。Therapeutic agents suitable for use in the pharmaceutical compositions and drug combinations of the invention may be therapeutic agents selected from any of the following categories (i)-(iv): (i) agents that enhance antigen presentation (e.g., tumor antigen presentation) Drugs; (ii) drugs that enhance effector cell responses (eg, B cell and/or T cell activation and/or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor suppressive effects.
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型,如液态溶液剂(例如,可注射用溶液剂和可输注溶液剂)、分散体剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。常见的优选组合物处于可注射用溶液剂或可输注溶液剂形式。优选的施用模式是肠胃外(例如,静脉内、皮下、腹腔(i.p.)、肌内)注射。在一个优选实施方案中,通过静脉内输注或注射施用抗体。在另一个优选实施方案中,通过肌内、腹腔或皮下注射施用抗体。The compositions of the invention can be in a variety of forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection. In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular, intraperitoneal or subcutaneous injection.
如本文所用的短语“肠胃外施用“和“肠胃外方式施用”意指除了肠施用和局部施用之外的施用模式,通常通过注射施用,并且包括但不限于静脉内、肌内、动脉内、皮内、腹腔、经气管、皮下注射和输注。The phrases "parenteral administration" and "parenteral administration" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.
治疗性组合物一般应当是无菌的并且在制造和储存条件下稳定。可以将组合物配制为溶液、微乳液、分散体、脂质体或冻干形式。可以通过将活性化合物(即抗体)以要求的量加入适宜的溶剂中,随后过滤消毒,制备无菌可注射溶液剂。通常,通过将所述活性化合物并入无菌溶媒中来制备分散体,所述无菌溶媒含有基础分散介质和其他成分。可以使用包衣剂如卵磷脂等。在分散体的情况下,可以通过使用表面活性剂来维持溶液剂的适宜流动性。可以通过在组合物中包含延迟吸收的物质例如单硬脂酸盐和明胶而引起可注射组合物的延长吸收。Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. Compositions can be formulated as solutions, microemulsions, dispersions, liposomes or in lyophilized form. Sterile injectable solutions can be prepared by incorporating the active compound (ie, antibody) in the required amount in an appropriate solvent, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and other ingredients. Coating agents such as lecithin and the like can be used. In the case of a dispersion, proper fluidity of the solution can be maintained by using a surfactant. Prolonged absorption of the injectable compositions can be brought about by including in the composition substances which delay absorption, for example, monostearate salts and gelatin.
本发明的药物组合物可以包含“治疗有效量”或“预防有效量”的本发明所述抗体。“治疗有效量”指以需要的剂量并持续需要的时间段,有效实现所需治疗结果的量。可以根据多种因素如疾病状态、个体的年龄、
性别和重量等变动治疗有效量。治疗有效量是任何有毒或有害作用不及治疗有益作用的量。相对于未治疗的受试者,“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20%、更优选地至少约40%、甚至更优选地至少约60%和仍更优选地至少约80%。可以在预示人肿瘤中的功效的动物模型系统中评价本发明的抗体抑制可度量参数(例如,肿瘤体积)的能力。The pharmaceutical composition of the present invention may contain "therapeutically effective amount" or "prophylactically effective amount" of the antibody of the present invention. A "therapeutically effective amount" refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result. can be based on a variety of factors such as disease status, individual age, A therapeutically effective amount for such changes as sex and weight. A therapeutically effective amount is one in which any noxious or detrimental effects are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g. tumor growth rate) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60% and still more relative to an untreated subject. Preferably at least about 80%. The ability of the antibodies of the invention to inhibit a measurable parameter (eg, tumor volume) can be evaluated in animal model systems predictive of efficacy in human tumors.
“预防有效量”指以需要的剂量并持续需要的时间段,有效实现所需预防结果的量。通常,由于预防性剂量在受试者中在疾病较早阶段之前或在疾病较早阶段使用,故预防有效量小于治疗有效量。A "prophylactically effective amount" refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, the prophylactically effective amount is less than the therapeutically effective amount because the prophylactic dose is used in the subject before or at an earlier stage of the disease.
包含本文所述抗体的试剂盒也处于本发明的范围内。试剂盒可以包含一个或多个其他要素,例如包括:使用说明书;其他试剂,例如标记物或用于偶联的试剂;可药用载体;和用于施用至受试者的装置或其他材料。Kits comprising the antibodies described herein are also within the scope of the invention. A kit may comprise one or more other elements including, for example: instructions for use; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.
IV.本发明分子的用途和方法IV. Uses and methods of the molecules of the invention
在一个方面,本发明提供了本发明抗体的体内、体外用途和应用方法。In one aspect, the present invention provides in vivo and in vitro uses and methods of application of the antibodies of the present invention.
在一些实施方案中,本发明的用途和方法涉及将本发明抗体在体内和/或体外应用于:In some embodiments, the uses and methods of the invention involve the in vivo and/or in vitro application of the antibodies of the invention to:
-结合人PD-L1和人VEGF;或- binds human PD-L1 and human VEGF; or
-阻断人PD-L1/PD-1信号途径;或- Block the human PD-L1/PD-1 signaling pathway; or
-中和VEGF活性;或- neutralizes VEGF activity; or
-抑制肿瘤细胞生长,- inhibition of tumor cell growth,
或用于制备用于上述任一项用途的药物。Or for the preparation of medicines for any of the above purposes.
在一些实施方案中,本发明抗体或包含本发明抗体的药物组合物用作在个体中治疗和/或预防疾病的药物或用作疾病的诊断工具,优选地,所述个体是哺乳动物,更优选地是人。In some embodiments, the antibody of the present invention or a pharmaceutical composition comprising the antibody of the present invention is used as a drug for treating and/or preventing a disease or as a diagnostic tool for a disease in an individual, preferably, the individual is a mammal, more preferably Preferably a human.
在一些实施方案中,本发明提供应用本发明的抗体尤其是双特异性抗体治疗癌症的方法和用途,其中所述癌症可以是PD-L1阳性实体瘤或血液癌症,例如选自头颈鳞癌,黑色素瘤,肾细胞癌、非小细胞肺癌、膀胱癌、尿路上皮癌、胃癌、结肠癌、结直肠癌、卵巢癌、乳腺癌、肺癌、宫颈癌、胶质母细胞癌、胰腺癌、前列腺癌、食管癌、淋巴瘤、肝癌、微卫星不稳定型实体瘤。优选地,所述癌症是结直肠癌、肝癌、卵巢癌、乳腺癌、肺癌。In some embodiments, the present invention provides methods and uses of using the antibodies of the present invention, especially bispecific antibodies, to treat cancer, wherein the cancer can be PD-L1 positive solid tumors or blood cancers, for example selected from head and neck squamous cell carcinoma, Melanoma, renal cell carcinoma, non-small cell lung cancer, bladder cancer, urothelial carcinoma, gastric cancer, colon cancer, colorectal cancer, ovarian cancer, breast cancer, lung cancer, cervical cancer, glioblastoma, pancreatic cancer, prostate cancer Carcinoma, esophageal cancer, lymphoma, liver cancer, microsatellite unstable solid tumors. Preferably, said cancer is colorectal cancer, liver cancer, ovarian cancer, breast cancer, lung cancer.
在一个方面,本发明提供了体外或体内检测生物样品,例如血清、精液或尿或组织活检样品(例如,来自过度增生性或癌性病灶)中存在相关抗原的诊断方法。该诊断方法包括:(i)在允许相互作用发生的条件下使样品(和任选地,对照样品)与如本文所述的抗体接触或向受试者施用所述抗体和(ii)检测所述抗体和样品(和任选地,对照样品)之间复合物的形成。复合物的形成表示存在相关抗原,并且可以显示本文所述治疗和/或预防的适用性或需求。In one aspect, the invention provides in vitro or in vivo diagnostic methods for detecting the presence of relevant antigens in biological samples such as serum, semen or urine or tissue biopsy samples (eg, from hyperproliferative or cancerous lesions). The diagnostic method comprises: (i) contacting a sample (and optionally, a control sample) with an antibody as described herein or administering the antibody to a subject under conditions that allow the interaction to occur and (ii) detecting the detected Complex formation between the antibody and the sample (and optionally, a control sample) is performed. Formation of the complex indicates the presence of the relevant antigen and may indicate suitability or need for treatment and/or prophylaxis as described herein.
在一个实施方案中,本发明提供了包含本文所述抗体和使用说明书的诊断试剂盒。In one embodiment, the invention provides a diagnostic kit comprising an antibody described herein and instructions for use.
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
The following examples are described to aid in the understanding of the present invention. The examples are not intended and should not be construed in any way as limiting the scope of the invention.
实施例1原材料制备Embodiment 1 raw material preparation
1.1抗原制备1.1 Antigen preparation
人PD-L1(UniProtKB-Q9NZQ7)的胞外区、人PD-1(UniProtKB-Q15116)的胞外区和人VEGF(UniProtKB-P15692)由通用生物系统(安徽)有限公司进行目的片段基因合成。PCR扩增各目的片段,并通过引物在C端引入His标签或者hIgG1Fc(氨基酸序列SEQ ID NO:3所示),然后通过同源重组的方法分别构建至真核表达载体pcDNA3.4(Invitrogen)。将构建好的各重组蛋白表达载体分别转化到大肠杆菌DH5α中,37℃过夜培养,然后利用无内毒素质粒提取试剂盒(OMEGA,D6950-01)进行质粒提取,得到无内毒素的各质粒以供真核表达使用。The extracellular region of human PD-L1 (UniProtKB-Q9NZQ7), the extracellular region of human PD-1 (UniProtKB-Q15116) and human VEGF (UniProtKB-P15692) were synthesized by General Biosystems (Anhui) Co., Ltd. for the target fragment gene. Each target fragment was amplified by PCR, and a His tag or hIgG1Fc (shown in the amino acid sequence SEQ ID NO: 3) was introduced at the C-terminus by primers, and then respectively constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination . The constructed recombinant protein expression vectors were respectively transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids and For eukaryotic expression.
重组蛋白VEGF-His、重组蛋白VEGF-Fc、重组蛋白PD-L1-His、重组蛋白PD-L1-Fc、重组蛋白PD-1-His和重组蛋白PD-1-Fc均通过Expi293瞬转表达系统(ThermoFisher,A14635)表达,瞬转方法参见Expi293TM表达系统试剂盒说明书。在转染5-7天后,将细胞表达上清于15000g高速离心10min。所得His标签重组蛋白表达上清用Ni Smart Beads 6FF(常州天地人和生物科技有限公司,SA036050)进行亲和纯化,然后用梯度浓度的咪唑洗脱目的蛋白,洗脱下来的各蛋白分别通过超滤浓缩管(Millipore,UFC901096)置换至PBS缓冲液中;所得Fc标签重组蛋白表达上清经0.22μm滤膜过滤后,采用Protein A/G亲和层析柱亲和法进行纯化,纯化后用100mM甘氨酸盐(pH3.0)洗脱目的蛋白,然后浓缩和置换经SDS-PAGE鉴定和活性鉴定合格后于-80℃冻存。Recombinant protein VEGF-His, recombinant protein VEGF-Fc, recombinant protein PD-L1-His, recombinant protein PD-L1-Fc, recombinant protein PD-1-His and recombinant protein PD-1-Fc were all passed through the Expi293 transient expression system (ThermoFisher, A14635) expression, see Expi293 TM Expression System Kit Manual for the transient method. After 5-7 days of transfection, the cell expression supernatant was centrifuged at a high speed of 15000 g for 10 min. The resulting His-tagged recombinant protein expression supernatant was affinity-purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with gradient concentrations of imidazole. The filter concentration tube (Millipore, UFC901096) was replaced with PBS buffer; the resulting Fc-tagged recombinant protein expression supernatant was filtered through a 0.22 μm filter membrane, and then purified by Protein A/G affinity chromatography column affinity method. The target protein was eluted with 100mM glycine salt (pH3.0), then concentrated and replaced, and then frozen at -80°C after being identified by SDS-PAGE and qualified for activity identification.
1.2对照抗体制备1.2 Preparation of control antibody
在本实施例中,抗人VEGF抗体P30-10-26(VHHVEGF-hIgG1Fc,VHHVEGF氨基酸序列SEQ ID NO:2所示)源自专利申请CN202110995278.7;抗人PD-L1抗体D21-4(VHHPD-L1-hIgG1Fc,VHHPD-L1氨基酸序列SEQ ID NO:1所示)源自专利申请PCT/CN2020/125301;抗VEGF对照抗体贝伐珠单抗(Bevacizumab,简写Beva)序列来源于Drug Bank(Drug Bank No:DB00112);抗PD-L1对照抗体阿特珠单抗(Atezolizumab,简写Ate)序列来源于Drug Bank(Drug Bank No:DB11595);抗PD-L1和VEGF的对照双特异性抗体W3256序列来源于WO2021147829A1;抗PD-1和VEGF的对照双特异性抗体AK112序列来源于US20210340239A1中的VP101序列。分别将上述氨基酸序列转化成基因序列后由通用生物科技股份有限公司进行目的片段基因合成。PCR扩增各目的片段,然后通过同源重组的方法构建至真核表达载体pcDNA3.4(Invitrogen)。In this example, anti-human VEGF antibody P30-10-26 (VHH VEGF -hIgG1Fc, VHH VEGF amino acid sequence shown in SEQ ID NO: 2) is derived from patent application CN202110995278.7; anti-human PD-L1 antibody D21-4 (VHH PD-L1 -hIgG1Fc, VHH PD-L1 amino acid sequence shown in SEQ ID NO: 1) is derived from the patent application PCT/CN2020/125301; the anti-VEGF control antibody Bevacizumab (Bevacizumab, abbreviated as Beva) sequence is derived from Drug Bank (Drug Bank No: DB00112); anti-PD-L1 control antibody Atezolizumab (Ate) sequence is derived from Drug Bank (Drug Bank No: DB11595); anti-PD-L1 and VEGF control bispecific The anti-antibody W3256 sequence is derived from WO2021147829A1; the anti-PD-1 and VEGF control bispecific antibody AK112 sequence is derived from the VP101 sequence in US20210340239A1. After the above amino acid sequences were converted into gene sequences, General Biotechnology Co., Ltd. carried out gene synthesis of the target fragment. Each target fragment was amplified by PCR, and then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination.
所述抗体均采用瞬转系统(ExpiCHO)进行表达,瞬转方法参见ExpiCHOTM Expression System Kit User Guide。将表达好的细胞混悬液进行高速离心并取上清,所得上清经0.22μm滤膜过滤后,采用Protein A/G亲和层析柱亲和法进行纯化。纯化后,用100mM甘氨酸盐(pH3.0)洗脱目的蛋白,然后浓缩和置换,经SDS-PAGE鉴定和活性鉴定合格后于-80℃冻存。The antibodies were all expressed using the transient system (ExpiCHO). For the transient method, see ExpiCHO ™ Expression System Kit User Guide. The expressed cell suspension was subjected to high-speed centrifugation and the supernatant was taken, and the obtained supernatant was filtered through a 0.22 μm filter membrane, and then purified by using a Protein A/G affinity chromatography column affinity method. After purification, the target protein was eluted with 100 mM glycine salt (pH 3.0), then concentrated and replaced, and then frozen at -80°C after being identified by SDS-PAGE and qualified for activity identification.
1.3细胞株构建1.3 Cell line construction
1.3.1 huPD-L1-CHO-K细胞株的构建1.3.1 Construction of huPD-L1-CHO-K cell line
将CHO-K细胞(Thermo,A1461801)传代至5×106个/mL,次日使用电转试剂盒(Invitrogen,MPK10096)和电转仪(Invitrogen,MP922947)将构建好的包含全长人PD-L1(UniProtKB-Q9NZQ7)基因序列的质粒导入
CHO-K细胞中。将电转后的细胞移至CD-CHO培养基(Gibco,10743029)中,放置于37℃细胞培养箱中培养48h。然后按2000个/孔铺到96孔板中,加入终浓度30μM MSX(Millipore,GSS-1015-F)和GS supplement(Sigma,58672C)进行加压筛选。挑取96孔板中长出的单细胞克隆,扩大培养后通过FACS鉴定获得huPD-L1-CHO-K细胞株。The CHO-K cells (Thermo, A1461801) were passaged to 5×10 6 cells/mL, and the next day, the constructed cells containing full-length human PD-L1 Plasmid introduction of (UniProtKB-Q9NZQ7) gene sequence in CHO-K cells. The cells after electroporation were transferred to CD-CHO medium (Gibco, 10743029), and placed in a cell culture incubator at 37°C for 48 hours. Then, 2000 cells/well were plated into a 96-well plate, and MSX (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C) were added to a final concentration of 30 μM for pressurized screening. Single-cell clones grown in 96-well plates were picked and expanded to obtain huPD-L1-CHO-K cell lines through FACS identification.
1.3.2 HEK293-VEGFR2-NFAT细胞株的构建1.3.2 Construction of HEK293-VEGFR2-NFAT cell line
将含有NF-AT-re核酸序列的pGL4.30质粒(promega,#E8481)电转至HEK293细胞(CRL-1573TM)获得HEK293-NFAT细胞株。将HEK293-NFAT细胞传代至2×105个/mL,次日使用电转试剂盒(Invitrogen,MPK10096)和电转仪(Invitrogen,MP922947)将构建好的包含全长人VEGFR2基因序列(NCBI Gene ID:3791)的质粒导入HEK293-NFAT细胞中。将电转后的细胞移至DMEM培养基(Gibco,12634010)中,放置于37℃细胞培养箱中培养48h。然后按1000个细胞/孔铺到96孔板中,加入终浓度2μg/mL的嘌呤霉素进行加压筛选。挑取96孔板中长出的单细胞克隆,扩大培养后通过加入荧光素酶的催化底物检测荧光信号鉴定获得HEK293-VEGFR2-NFAT细胞株。The pGL4.30 plasmid (promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into HEK293 cells ( CRL-1573 TM ) to obtain HEK293-NFAT cell line. The HEK293-NFAT cells were passaged to 2× 105 cells/mL, and the next day, the electroporation kit (Invitrogen, MPK10096) and electroporation instrument (Invitrogen, MP922947) were used to transform the constructed human VEGFR2 gene sequence (NCBI Gene ID: 3791) plasmid into HEK293-NFAT cells. The electroporated cells were transferred to DMEM medium (Gibco, 12634010), and placed in a 37°C cell culture incubator for 48 hours. Then, 1000 cells/well were plated in a 96-well plate, and puromycin at a final concentration of 2 μg/mL was added for pressure selection. The single-cell clones grown in the 96-well plate were picked, and after expanded culture, the HEK293-VEGFR2-NFAT cell line was obtained by adding a luciferase catalytic substrate to detect the fluorescent signal and identify it.
1.3.3 Jurkat-PD-1-NFAT细胞株的构建1.3.3 Construction of Jurkat-PD-1-NFAT cell line
将含有NF-AT-re核酸序列的pGL4.30质粒(promega,#E8481)电转至Jurkat细胞(TIB-152)获得Jurkat-NFAT细胞株。在此基础上稳转PD-1的全长表达基因序列(NCBI Gene ID:5133),方法与1.3.2一致,加入终浓度2μg/mL的嘌呤霉素和0.5μg/mL潮霉素B溶液筛选单克隆细胞株,扩大培养后通过FACS鉴定获得Jurkat-PD-1-NFAT细胞株。The pGL4.30 plasmid (promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into Jurkat cells ( TIB-152) to obtain Jurkat-NFAT cell line. On this basis, stably transform the full-length expression gene sequence of PD-1 (NCBI Gene ID: 5133), the method is consistent with 1.3.2, add puromycin and 0.5 μg/mL hygromycin B solution at a final concentration of 2 μg/mL Monoclonal cell lines were screened, and Jurkat-PD-1-NFAT cell lines were obtained by FACS identification after expansion.
1.3.4 CHO-PD-L1-CD3L细胞株的构建1.3.4 Construction of CHO-PD-L1-CD3L cell line
将OKT-3(Drug Bank No:DB00075)的scFv序列电转至CHO细胞获得CHO-CD3L稳转细胞株,在此基础上稳转PD-L1的全长表达基因序列(NCBI Gene ID:29126),方法与1.3.2一致,加入终浓度30mM蛋氨酸亚氨基代砜和8μg/mL嘌呤霉素筛选单克隆细胞株,扩大培养后通过FACS鉴定获得CHO-PD-L1-CD3L细胞株。The scFv sequence of OKT-3 (Drug Bank No: DB00075) was electrotransfected into CHO cells to obtain CHO-CD3L stably transfected cell line, on this basis, the full-length expression gene sequence of PD-L1 was stably transfected (NCBI Gene ID: 29126), The method was consistent with 1.3.2. The final concentration of 30 mM methionine iminosulfone and 8 μg/mL puromycin were added to screen monoclonal cell lines, and the CHO-PD-L1-CD3L cell line was obtained by FACS identification after expansion.
实施例2抗PD-L1和VEGF双特异性抗体的构建Example 2 Construction of anti-PD-L1 and VEGF bispecific antibody
本实施例描述了示例性抗PD-L1和VEGF双特异性抗体(BsAb)的结构和表达载体的构建。设计并构建了4种构建体:VHH PD-L1氨基酸序列来自D21-4,其可变区氨基酸序列如SEQ ID NO:1所示;VHH VEGF氨基酸序列来自P30-10-26,其可变区氨基酸序列如SEQ ID NO:2所示;连接子氨基酸序列为GGGGSGGGGSGGGGS(SEQ ID NO:19)。示例性BsAb构建体如表1所示,对应的氨基酸序列提供于表2中。This example describes the construction of exemplary anti-PD-L1 and VEGF bispecific antibody (BsAb) structures and expression vectors. Four constructs were designed and constructed: the amino acid sequence of VHH PD-L1 is from D21-4, and its variable region amino acid sequence is shown in SEQ ID NO:1; the VHH VEGF amino acid sequence is from P30-10-26, and its variable region The amino acid sequence is shown in SEQ ID NO: 2; the amino acid sequence of the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 19). Exemplary BsAb constructs are shown in Table 1 and the corresponding amino acid sequences are provided in Table 2.
构建体BsAb10:含有两条相同的多肽链,从N端到C端包含抗PD-L1的VHH结构域、IgG1重链铰链区、IgG1重链Fc、连接子和抗VEGF的VHH结构域。BsAb10具有图1A的形式。Construct BsAb10: Contains two identical polypeptide chains, including anti-PD-L1 VHH domain, IgG1 heavy chain hinge region, IgG1 heavy chain Fc, linker and anti-VEGF VHH domain from N-terminal to C-terminal. BsAb10 has the format of Figure 1A.
构建体BsAb11:含有两条相同的多肽链,从N端到C端包含抗PD-L1的VHH结构域、连接子、抗VEGF的VHH结构域、IgG1重链铰链区和IgG1重链Fc。BsAb11具有图1B的形式。Construct BsAb11: Contains two identical polypeptide chains, including anti-PD-L1 VHH domain, linker, anti-VEGF VHH domain, IgG1 heavy chain hinge region and IgG1 heavy chain Fc from N-terminal to C-terminal. BsAb11 has the format of Figure 1B.
构建体BsAb12:含有两条相同的第一多肽链,从N端到C端包含抗PD-L1的VHH结构域、IgG1重
链恒定区结构域(包括IgG1重链CH1、IgG1重链铰链区、IgG1重链Fc);含有两条相同的第二多肽链,从N端到C端包含抗VEGF的VHH结构域、抗体κ轻链CL结构域。BsAb12具有图1C的形式。Construct BsAb12: Contains two identical first polypeptide chains, including anti-PD-L1 VHH domain from N-terminal to C-terminal, IgG1 heavy Chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, IgG1 heavy chain Fc); contains two identical second polypeptide chains, from N-terminal to C-terminal contains anti-VEGF VHH domain, antibody Kappa light chain CL domain. BsAb12 has the format of Figure 1C.
构建体BsAb13:含有两条相同的第一多肽链,从N端到C端包含抗VEGF的VHH结构域、IgG1重链恒定区结构域(包括IgG1重链CH1、IgG1重链铰链区、IgG1重链Fc);含有两条相同的第二多肽链,从N端到C端包含抗PD-L1的VHH结构域、抗体κ轻链CL结构域。BsAb13具有图1D的形式。Construct BsAb13: Contains two identical first polypeptide chains, including anti-VEGF VHH domain from N-terminal to C-terminal, IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, IgG1 Heavy chain Fc); Contains two identical second polypeptide chains, including VHH domain of anti-PD-L1 and CL domain of antibody kappa light chain from N-terminus to C-terminus. BsAb13 has the format of Figure 1D.
根据构建体的结构,通过PCR方法扩增获取各个抗体可变区及恒定区的片段,通过重叠延伸PCR法将各个片段连接起来,再通过同源重组方法分别构建至经过改造的真核表达载体质粒pcDNA3.4(Invitrogen)上,组成完整的构建体多肽链全长基因。将构建好的含构建体多肽链全长基因的载体分别转化到大肠杆菌DH5α中,37℃过夜培养。利用无内毒素质粒提取试剂盒(OMEGA,D6950-01)进行质粒提取,得到无内毒素的构建体多肽链质粒以供真核表达使用。According to the structure of the construct, fragments of the variable region and constant region of each antibody are amplified by PCR method, and the fragments are connected by overlap extension PCR method, and then constructed into modified eukaryotic expression vectors by homologous recombination method On the plasmid pcDNA3.4 (Invitrogen), the full-length gene of the polypeptide chain constituting the complete construct. The constructed vectors containing the full-length gene of the polypeptide chain of the construct were respectively transformed into Escherichia coli DH5α, and cultured overnight at 37°C. The endotoxin-free plasmid extraction kit (OMEGA, D6950-01) was used for plasmid extraction to obtain an endotoxin-free construct polypeptide chain plasmid for eukaryotic expression.
表1抗PD-L1和VEGF双特异性抗体的构建体
Table 1 Constructs of anti-PD-L1 and VEGF bispecific antibodies
Table 1 Constructs of anti-PD-L1 and VEGF bispecific antibodies
表2抗PD-L1和VEGF双特异性抗体的氨基酸序列
Table 2 Amino acid sequences of anti-PD-L1 and VEGF bispecific antibodies
Table 2 Amino acid sequences of anti-PD-L1 and VEGF bispecific antibodies
实施例3抗PD-L1和VEGF双特异性抗体的表达、纯化、理化性质分析Example 3 Expression, purification, and analysis of physicochemical properties of anti-PD-L1 and VEGF bispecific antibody
3.1抗PD-L1和VEGF双特异性抗体的表达、纯化3.1 Expression and purification of anti-PD-L1 and VEGF bispecific antibody
实施例2的构建体是通过ExpiCHO瞬转表达系统(Thermo Fisher,A29133)表达的,具体方法如下:转染当天,确认细胞密度为7×106至1×107个/mL左右,细胞存活率>98%,此时,用37℃预温的新鲜ExpiCHO表达培养基将细胞调整到终浓度为6×106个/mL。用4℃预冷的OptiPROTMSFM稀释目的质粒(向1mL所述培养基中加入1μg质粒),同时,用OptiPROTMSFM稀释ExpiFectamineTMCHO,再将两者等体积混合并轻轻吹打混匀制备成ExpiFectamineTMCHO/质粒DNA混合液,室温孵育1-5min,缓慢加入到准备好的细胞悬液中并同时轻轻摇晃,最后置于细胞培养摇床中,在37℃、8%CO2条件下培养。在转染后18-22h,向培养液中添加ExpiCHOTMEnhancer和ExpiCHOTMFeed,摇瓶放置于32℃摇床和5%CO2条件下继续培养。在转染后的第5天,添加相同体积的ExpiCHOTMFeed,缓慢加入的同时轻轻混匀细胞混悬液。在转染7-15天后,将表达有目的蛋白的细胞培养上清于15000g高速离心10min,所得上清用MabSelect SuRe LX(GE,17547403)进行亲和纯化,然后用100mM乙酸钠(pH3.0)洗脱目的蛋白,接着用1M Tris-HCl中和,最后通过超滤浓缩管(Millipore,UFC901096)将所得蛋白置换至PBS缓冲液中。
The construct in Example 2 was expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: on the day of transfection, confirm that the cell density was about 7×10 6 to 1×10 7 cells/mL, and the cell survival At this point, adjust the cells to a final concentration of 6×10 6 cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C. Dilute the target plasmid with 4°C pre-cooled OptiPRO TM SFM (add 1 μg of plasmid to 1 mL of the medium), and at the same time, dilute ExpiFectamine TM CHO with OptiPRO TM SFM, then mix the two in equal volumes and gently blow and mix to prepare Form ExpiFectamine TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5min, slowly add to the prepared cell suspension and shake gently at the same time, and finally place in a cell culture shaker at 37°C, 8% CO 2 conditions under cultivation. 18-22 hours after transfection, add ExpiCHO ™ Enhancer and ExpiCHO ™ Feed to the culture medium, and place the shaker flask in a shaker at 32°C and 5% CO 2 to continue culturing. On day 5 after transfection, the same volume of ExpiCHO TM Feed was added, and the cell suspension was mixed gently while adding slowly. After 7-15 days of transfection, the cell culture supernatant expressing the target protein was centrifuged at a high speed of 15000g for 10min, and the resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then purified with 100mM sodium acetate (pH3.0 ) to elute the target protein, then neutralize it with 1M Tris-HCl, and finally replace the obtained protein into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096).
3.2抗PD-L1和VEGF双特异性抗体的浓度测定3.2 Concentration determination of anti-PD-L1 and VEGF bispecific antibody
将实施例3.1经纯化的双特异性抗体用经验证过的超微量分光光度计(杭州奥盛仪器有限公司,Nano-300)进行浓度测定,将经测定的A280数值除以抗体理论消光系数后的数值作为后续研究的抗体浓度值,质检合格后分装并保存于-80℃。The concentration of the purified bispecific antibody in Example 3.1 was measured with a verified ultra-micro spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300), and the measured A280 value was divided by the theoretical extinction coefficient of the antibody. The value is used as the antibody concentration value for follow-up research. After passing the quality inspection, it is aliquoted and stored at -80°C.
3.3抗PD-L1和VEGF双特异性抗体的SDS-PAGE鉴定3.3 SDS-PAGE identification of anti-PD-L1 and VEGF bispecific antibody
非还原溶液制备:1μg候选双特异性抗体以及参考品IPI(所述IPI是伊匹木单抗(Ipilimumab)的缩写,通过实施例3.1的方法制备获得)加入5×SDS上样缓冲液和40mM碘代乙酰胺,75℃干浴加热10min,冷却到室温后,12000rpm离心5min,取上清。还原溶液制备:2μg候选双特异性抗体以及参考品IPI加入5×SDS上样缓冲液和5mM DTT,100℃干浴加热10min,冷却到室温后,12000rpm离心5min,取上清。将上清加入Bis-tris 4-15%梯度胶(购于金斯瑞)中,恒压110V电泳,当考马斯亮蓝迁移到凝胶底部,停止运行,取出凝胶片置考马斯亮蓝染色液中1-2h,弃去染色液,加入脱色液,根据需要更换2-3次脱色液,脱色至凝胶背景透明后保存在去离子水中。脱色后用EPSON V550彩色扫描仪扫描,通过ImageJ按照峰面积归一法计算还原和非还原条带纯度。Non-reducing solution preparation: 1 μg of candidate bispecific antibody and reference product IPI (the IPI is the abbreviation of Ipilimumab (Ipilimumab), prepared by the method in Example 3.1) was added to 5×SDS loading buffer and 40mM Iodoacetamide was heated in a dry bath at 75°C for 10 minutes, cooled to room temperature, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was taken. Preparation of reducing solution: Add 2 μg of candidate bispecific antibody and reference IPI to 5×SDS loading buffer and 5 mM DTT, heat in a dry bath at 100°C for 10 min, cool to room temperature, centrifuge at 12,000 rpm for 5 min, and take the supernatant. Add the supernatant to Bis-tris 4-15% gradient gel (purchased from GenScript), electrophoresis at a constant voltage of 110V, stop running when the Coomassie brilliant blue migrates to the bottom of the gel, take out the gel slice and place it in the Coomassie brilliant blue staining solution Incubate for 1-2 hours, discard the staining solution, add destaining solution, replace the destaining solution 2-3 times as needed, decolorize until the gel background is transparent, and store in deionized water. After decolorization, it was scanned with EPSON V550 color scanner, and the purity of reduced and non-reduced bands was calculated by ImageJ according to the peak area normalization method.
结果如图2A-2B所示:候选双特异性抗体和参考品IPI还原胶和非还原胶的条带均符合预期大小,且还原胶纯度都在大于95%。The results are shown in Figures 2A-2B: the bands of the candidate bispecific antibody and the reference product IPI reduced gel and non-reduced gel all conformed to the expected size, and the purity of the reduced gel was greater than 95%.
3.4抗PD-L1和VEGF双特异性抗体的SEC-HPLC单体纯度鉴定3.4 SEC-HPLC monomer purity identification of anti-PD-L1 and VEGF bispecific antibody
材料准备:1、流动相:150mmol/L磷酸缓冲液,pH 7.4;2、样品制备:候选双特异性抗体均用流动相溶液稀释到0.5mg/mL。Agilent HPLC 1100色谱柱(XBridge BEH SEC 3.5μm,7.8mm I.D.×30cm,Waters)流速设为0.8mL/min,进样体积20μL,VWD检测器波长为280nm和214nm。Material preparation: 1. Mobile phase: 150 mmol/L phosphate buffer, pH 7.4; 2. Sample preparation: Candidate bispecific antibodies were diluted to 0.5 mg/mL with mobile phase solution. Agilent HPLC 1100 chromatographic column (XBridge BEH SEC 3.5μm, 7.8mm I.D.×30cm, Waters) flow rate was set to 0.8mL/min, injection volume was 20μL, VWD detector wavelength was 280nm and 214nm.
候选双特异性抗体的SEC-HPLC结果如下:按照面积归一法计算样品中高分子聚合物,抗体单体和低分子物质百分比,结果显示在图3A-3D和表3。The SEC-HPLC results of candidate bispecific antibodies are as follows: the percentages of high molecular polymers, antibody monomers and low molecular substances in the sample were calculated according to the area normalization method, and the results are shown in Figures 3A-3D and Table 3.
表3抗PD-L1和VEGF双特异性抗体的理化数据
Table 3 Physicochemical data of anti-PD-L1 and VEGF bispecific antibodies
Table 3 Physicochemical data of anti-PD-L1 and VEGF bispecific antibodies
实施例4抗PD-L1和VEGF双特异性抗体的亲和活性分析Example 4 Affinity activity analysis of anti-PD-L1 and VEGF bispecific antibody
4.1基于ELISA方法检测候选双特异性抗体对重组蛋白VEGF-His的结合能力4.1 Detection of binding ability of candidate bispecific antibodies to recombinant protein VEGF-His based on ELISA method
在96孔ELISA板上包被重组蛋白VEGF-His,4℃过夜。次日,将孔板用PBST洗3次后用5%脱脂牛奶封闭2h,用PBST洗板3次后,加入不同浓度的候选双特异性抗体、对照抗体贝伐珠单抗和P30-10-26孵育1h。之后,用PBST清洗3次后加入二抗Anti-human-IgG-Fc-HRP(abcam,ab79225)并孵育1h。孵育完成后,PBST洗板六次,加TMB(SurModics,TMBS-1000-01)显色。根据显色结果,加入2M HCl终止反应,通过酶标仪(Molecular Devices,SpecterMax 190)在OD450下读板。利用PRISMTM(GraphPad Software,
San Diego,CA)分析数据,并且计算EC50值。Coat the recombinant protein VEGF-His on a 96-well ELISA plate, overnight at 4°C. On the next day, the well plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 h. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibody, control antibody bevacizumab and P30-10- 26 for 1 h. Afterwards, after washing with PBST for 3 times, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 h. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop the color. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Using PRISM TM (GraphPad Software, San Diego, CA) analyzed data and calculated EC50 values.
ELISA结合测定结果如图4和表4所示,双特异性抗体BsAb11、BsAb12和BsAb13表现出优于对照抗体贝伐珠单抗的与VEGF结合的能力,同时与对照抗体P30-10-26相当。The results of the ELISA binding assay are shown in Figure 4 and Table 4, the bispecific antibodies BsAb11, BsAb12 and BsAb13 exhibited better binding ability to VEGF than the control antibody bevacizumab, and comparable to the control antibody P30-10-26 .
4.2基于ELISA方法检测候选双特异性抗体对重组蛋白PD-L1-His的结合能力4.2 Detection of binding ability of candidate bispecific antibodies to recombinant protein PD-L1-His based on ELISA method
在96孔ELISA板上包被重组蛋白PD-L1-His,4℃过夜。次日,将孔板用PBST洗3次后用5%脱脂牛奶封闭2h,用PBST洗板3次后,加入不同浓度的候选双特异性抗体、对照抗体D21-4孵育1h。之后,用PBST清洗3次后加入二抗Anti-human-IgG-Fc-HRP(abcam,ab79225)并孵育1h。孵育完成后,PBST洗板六次,加TMB(SurModics,TMBS-1000-01)显色。根据显色结果,加入2M HCl终止反应,通过酶标仪(Molecular Devices,SpecterMax 190)在OD450下读板。利用PRISMTM(GraphPad Software,San Diego,CA)分析数据,并且计算EC50值。Coat the recombinant protein PD-L1-His on a 96-well ELISA plate overnight at 4°C. The next day, the well plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 h. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibody and control antibody D21-4 were added and incubated for 1 h. Afterwards, after washing with PBST for 3 times, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 h. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop the color. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ™ (GraphPad Software, San Diego, CA), and EC50 values were calculated.
ELISA结合测定结果如图5和表4所示,候选双特异性抗体均表现出与对照抗体D21-4相当的与PD-L1结合的能力。The results of the ELISA binding assay are shown in Figure 5 and Table 4, and the candidate bispecific antibodies all showed comparable ability to bind to PD-L1 as the control antibody D21-4.
4.3基于FACS方法检测候选双特异性抗体对huPD-L1-CHO-K的结合能力4.3 Detection of the binding ability of candidate bispecific antibodies to huPD-L1-CHO-K based on FACS method
收集指数生长期的huPD-L1-CHO-K细胞,300g离心去上清,将细胞用FACS缓冲液(含有1%BSA的PBS)重悬,计数并将细胞悬液密度调整为2×106个/mL。随后,将huPD-L1-CHO-K细胞以每孔100μL加入96孔圆底板中,300g离心去上清。向对应孔中加入不同浓度的候选双特异性抗体和对照抗体D21-4稀释液,将细胞重悬后放置于4℃孵育30min。将孵育后的细胞混合液洗涤3次后加入PE标记的anti-human-IgG-Fc流式抗体(Abcam,98596),重悬后放置于4℃孵育30min。将孵育后的细胞混合液洗涤3次后加入200μL的FACS缓冲液重悬细胞,通过流式细胞仪(Beckman,CytoFLEX AOO-1-1102)上机检测分析。利用PRISMTM(GraphPad Software,San Diego,CA)分析数据,并且计算EC50值。Collect the huPD-L1-CHO-K cells in the exponential growth phase, centrifuge at 300g to remove the supernatant, resuspend the cells in FACS buffer (PBS containing 1% BSA), count and adjust the density of the cell suspension to 2×10 6 individual/mL. Subsequently, huPD-L1-CHO-K cells were added to a 96-well round bottom plate at 100 μL per well, and centrifuged at 300 g to remove the supernatant. Different concentrations of candidate bispecific antibody and control antibody D21-4 dilutions were added to the corresponding wells, the cells were resuspended and incubated at 4°C for 30 min. After the incubated cell mixture was washed 3 times, a PE-labeled anti-human-IgG-Fc flow antibody (Abcam, 98596) was added, resuspended and incubated at 4°C for 30 min. After the incubated cell mixture was washed three times, 200 μL of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). Data were analyzed using PRISM ™ (GraphPad Software, San Diego, CA), and EC50 values were calculated.
FACS结合测定结果如图6和表4所示,双特异性抗体BsAb10和BsAb12表现出优于对照抗体D21-4的对细胞上表达的PD-L1的结合能力。FACS binding assay results are shown in Figure 6 and Table 4, bispecific antibodies BsAb10 and BsAb12 showed better binding ability to PD-L1 expressed on cells than control antibody D21-4.
4.4基于ELISA方法检测候选双特异性抗体对VEGF和PD-L1同时结合的能力4.4 ELISA-based detection of the ability of candidate bispecific antibodies to simultaneously bind to VEGF and PD-L1
在96孔ELISA板上包被重组蛋白VEGF-Fc,4℃过夜。次日,将孔板用PBST洗3次后用5%脱脂牛奶封闭2h,用PBST洗板3次后,加入不同浓度的候选双特异性抗体孵育1h。之后,用PBST清洗3次后加入重组蛋白PD-L1-His,用PBST洗板3次后,加入Anti-6×His-HRP(Proteintech,HRP-660050),室温孵育50min,完成后PBST洗板12次,加TMB(SurModics,TMBS-1000-01)显色。根据显色结果,加入2M HCl终止反应,通过酶标仪(Molecular Devices,SpecterMax 190)在OD450下读板。利用PRISMTM(GraphPad Software,San Diego,CA)分析数据,并且计算EC50值。Coat the recombinant protein VEGF-Fc on a 96-well ELISA plate, overnight at 4°C. On the next day, the well plate was washed 3 times with PBST and then blocked with 5% skim milk for 2 hours. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibodies were added and incubated for 1 hour. After that, add recombinant protein PD-L1-His after washing with PBST for 3 times, wash the plate with PBST for 3 times, add Anti-6×His-HRP (Proteintech, HRP-660050), incubate at room temperature for 50min, and then wash the plate with PBST Twelve times, add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ™ (GraphPad Software, San Diego, CA), and EC50 values were calculated.
在96孔ELISA板上包被重组蛋白PD-L1-Fc,4℃过夜。次日,将孔板用PBST洗3次后用5%脱脂牛奶封闭2h,用PBST洗板3次后,加入不同浓度的候选双特异性抗体孵育1h。之后,用PBST清洗3次后加入重组蛋白VEGF-His,用PBST洗板3次后,加入Anti-6×His-HRP(Proteintech,HRP-660050),室温孵育50min,完成后,PBST洗板12次,加TMB(SurModics,TMBS-1000-01)显色。根据显色结果,加入2M HCl终止反应,通过酶标仪(Molecular Devices,SpecterMax 190)在OD450下读板。利用
PRISMTM(GraphPad Software,San Diego,CA)分析数据,并且计算EC50值。The recombinant protein PD-L1-Fc was coated on a 96-well ELISA plate overnight at 4°C. On the next day, the well plate was washed 3 times with PBST and then blocked with 5% skim milk for 2 hours. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibodies were added and incubated for 1 hour. Afterwards, wash 3 times with PBST, add recombinant protein VEGF-His, wash the plate 3 times with PBST, add Anti-6×His-HRP (Proteintech, HRP-660050), incubate at room temperature for 50 min, after completion, wash the plate with PBST for 12 The second time, add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). use PRISM ™ (GraphPad Software, San Diego, CA) analyzed data and calculated EC50 values.
ELISA结合测定结果如图7A-7B,双特异性抗体BsAb10表现出能够与PD-L1和VEGF同时结合的能力。The results of the ELISA binding assay are shown in Figures 7A-7B, the bispecific antibody BsAb10 showed the ability to simultaneously bind to PD-L1 and VEGF.
表4抗PD-L1和VEGF双特异性抗体的抗原结合力数据
Table 4 Antigen binding data of anti-PD-L1 and VEGF bispecific antibodies
Table 4 Antigen binding data of anti-PD-L1 and VEGF bispecific antibodies
实施例5基于报告基因法检测抗PD-L1和VEGF双特异性抗体的PD-1/PD-L1阻断活性Example 5 Detection of PD-1/PD-L1 blocking activity of anti-PD-L1 and VEGF bispecific antibody based on reporter gene method
本实施例采用荧光素酶报告基因系统检测候选抗PD-L1和VEGF双特异性抗体阻断PD-1/PD-L1信号途径的活性,PD-1与PD-L1结合可以阻断CD3下游信号转导从而抑制荧光素酶表达的体系,当加入包含抗PD-L1和VEGF双特异性抗体时阻断效应反转,荧光素酶表达,用不同浓度梯度的抗体刺激,会得到具有抗体浓度依赖性的荧光读数曲线,从而可评价抗体的阻断活性。本实施例采用荧光素酶报告基因系统检测候选抗PD-L1和VEGF双特异性抗体阻断PD-1/PD-L1信号途径的活性具体方法如下:In this example, the luciferase reporter gene system was used to detect the activity of candidate anti-PD-L1 and VEGF bispecific antibodies in blocking the PD-1/PD-L1 signaling pathway, and the combination of PD-1 and PD-L1 can block the downstream signal of CD3 Transduced to inhibit luciferase expression system, when adding anti-PD-L1 and VEGF bispecific antibody, the blocking effect is reversed, luciferase expression, stimulated with antibodies with different concentration gradients, will have antibody concentration-dependent A specific fluorescence readout curve allows the evaluation of the blocking activity of the antibody. In this example, the luciferase reporter gene system was used to detect the activity of candidate anti-PD-L1 and VEGF bispecific antibodies in blocking the PD-1/PD-L1 signaling pathway. The specific method is as follows:
在96孔白边底透细胞培养板中加入体积为50μL的2×104个/孔的CHO-PD-L1-CD3L细胞和1×105个/孔的Jurkat-PD-1-NFAT细胞。加入50μL梯度稀释的候选双特异性抗体和对照抗体D21-4,37℃培养箱孵育6h。每孔加入50μL Bright-Lite(vazyme,货号:DD1204-03),避光孵育10min,检测荧光信号。Add 2× 104 /well CHO-PD-L1-CD3L cells and 1× 105 /well Jurkat-PD-1-NFAT cells in a volume of 50 μL in a 96-well permeable cell culture plate with white edges. Add 50 μL of serially diluted candidate bispecific antibody and control antibody D21-4, and incubate in a 37°C incubator for 6h. Add 50 μL Bright-Lite (vazyme, product number: DD1204-03) to each well, incubate in the dark for 10 min, and detect the fluorescent signal.
阻断活性检测结果如下:如图8所示,双特异性抗体BsAb10和BsAb12均表现出与对照抗体D21-4相当的PD-1/PD-L1阻断活性。The results of the blocking activity test are as follows: As shown in Figure 8, both bispecific antibodies BsAb10 and BsAb12 exhibited PD-1/PD-L1 blocking activity comparable to that of the control antibody D21-4.
实施例6基于报告基因法检测抗PD-L1和VEGF双特异性抗体的VEGF中和活性Example 6 Detection of VEGF neutralizing activity of anti-PD-L1 and VEGF bispecific antibody based on reporter gene method
本实施例中,为确定候选双特异性抗体中和VEGF-VEGFR2信号通路功能,采用HEK293-VEGFR2-NFAT细胞株(在此细胞株培养体系中加入VEGF重组蛋白,通过VEGF-VEGFR2信号轴激活胞内NFAT荧光素酶报告基因转录和表达,加入荧光素酶的催化底物产生荧光信号)作为材料,检测候选双特异性抗体中和VEGF-VEGFR2结合从而阻断下游NFAT荧光素酶报告基因表达的能力。具体实施方式如下:In this example, in order to determine the function of the candidate bispecific antibody neutralizing the VEGF-VEGFR2 signaling pathway, the HEK293-VEGFR2-NFAT cell line was used (the VEGF recombinant protein was added to the cell line culture system to activate the cell line through the VEGF-VEGFR2 signaling axis). Transcription and expression of the internal NFAT luciferase reporter gene, adding the catalytic substrate of luciferase to generate a fluorescent signal) as a material to detect the binding of the candidate bispecific antibody to VEGF-VEGFR2 to block the expression of the downstream NFAT luciferase reporter gene ability. The specific implementation is as follows:
将HEK293-VEGFR2-NFAT细胞株调整成4×105个/mL,以每孔100μL加至新的96孔细胞培养板中,置于37℃细胞培养箱中。同时,使用DMEM培养基梯度稀释候选双特异性抗体和对照抗体P30-10-26,加入60ng/mL VEGF-Fc,混合后室温孵育30min。随后,将共孵育后的梯度稀释抗体和VEGF-Fc混合液加入至96孔细胞培养板,在37℃培养箱中培养18h。培养结束后,每孔加入30μL荧光素酶底物Bright-Lite(Vazyme,DD1204-03),震荡2min后检测96孔板荧光值。
Adjust the HEK293-VEGFR2-NFAT cell line to 4×10 5 cells/mL, add 100 μL per well to a new 96-well cell culture plate, and place it in a 37°C cell culture incubator. At the same time, the candidate bispecific antibody and control antibody P30-10-26 were serially diluted in DMEM medium, 60 ng/mL VEGF-Fc was added, mixed and incubated at room temperature for 30 min. Subsequently, the co-incubated serially diluted antibody and VEGF-Fc mixture was added to a 96-well cell culture plate, and cultured in a 37° C. incubator for 18 hours. After the incubation, 30 μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and the fluorescence value of the 96-well plate was detected after shaking for 2 minutes.
结果如图9和表5所示,双特异性抗体BsAb10中和VEGF的效果显著优于对照抗体P30-10-26,双特异性抗体BsAb11、BsAb12和BsAb13中和VEGF的效果与对照抗体P30-10-26相当。The results are shown in Figure 9 and Table 5, the neutralization effect of bispecific antibody BsAb10 on VEGF was significantly better than that of control antibody P30-10-26, and the neutralization effect of bispecific antibody BsAb11, BsAb12 and BsAb13 on VEGF was comparable to that of control antibody P30- 10-26 is equivalent.
表5抗PD-L1和VEGF双特异性抗体的VEGF中和活性
Table 5 VEGF neutralizing activity of anti-PD-L1 and VEGF bispecific antibodies
Table 5 VEGF neutralizing activity of anti-PD-L1 and VEGF bispecific antibodies
实施例7抗PD-L1和VEGF双特异性抗体的体内抑瘤实验(COLO205小鼠荷瘤模型)Example 7 In vivo tumor suppression experiment of anti-PD-L1 and VEGF bispecific antibody (COLO205 mouse tumor-bearing model)
使用6-8周雌性NOG小鼠(购于上海维通利华,品系:NOD.Cg-PrkdcscidIl2rgtm1Sug/JicCrl),实验小鼠饲养在恒温恒湿的独立通风盒内,饲养室温度21-24℃,湿度30-53%。将huPD-L1-COLO205细胞(稳定转染PD-L1(Gene ID:29126)至COLO205细胞(购于北纳生物,目录号:BNCC338682)获得的过表达人PD-L1的稳转细胞株)以3×106个/每只小鼠进行右侧背部皮下注射(第0天),然后进行随机分组(每组6只小鼠):分别是PBS处理组、Atezolizumab给药组、Bevacizumab给药组、Atezolizumab+Bevacizumab联合给药组、W3256给药组、AK112给药组和双特异性抗体BsAb10给药组,每个给药组均设置高剂量组,其中双特异性抗体BsAb10给药组和AK112给药组还设置了中低剂量组。荷瘤后第二天,通过尾静脉注射PBMC(C2106025),每只小鼠注射3×106个PBMC细胞,2h后进行首次给药,每个星期两次给药,腹腔注射(i.p.)给药,共给药3周。6-8 weeks old female NOG mice (purchased from Shanghai Weitong Lihua, strain: NOD.Cg-Prkdc scid Il2rg tm1Sug /JicCrl) were used, and the experimental mice were kept in an independent ventilated box with constant temperature and humidity, and the temperature of the feeding room was 21 -24°C, humidity 30-53%. The huPD-L1-COLO205 cells (a stably transfected cell line overexpressing human PD-L1 obtained by stably transfecting PD-L1 (Gene ID: 29126) into COLO205 cells (purchased from Beina Biotech, catalog number: BNCC338682)) were 3×10 6 mice/mouse were subcutaneously injected into the right back (day 0), and then randomly divided into groups (6 mice per group): PBS treatment group, Atezolizumab administration group, Bevacizumab administration group , Atezolizumab+Bevacizumab combined administration group, W3256 administration group, AK112 administration group and bispecific antibody BsAb10 administration group, each administration group has a high-dose group, in which the bispecific antibody BsAb10 administration group and AK112 The administration group also set up a middle and low dose group. On the second day after tumor bearing, PBMC (C2106025) was injected through the tail vein, and each mouse was injected with 3×10 6 PBMC cells. The first administration was performed 2 hours later, twice a week, intraperitoneal injection (ip) medicine for a total of 3 weeks.
随时观察和记录肿瘤长(mm)和宽(mm),计算其肿瘤体积(V),计算方式为V=(长×宽2)/2,抑瘤率TGI(%)=(1-给药组肿瘤平均体积/PBS处理组肿瘤平均体积)×100%。抑瘤结果如图10和表6所示。从结果可以看出:所有给药组相对于PBS处理组均呈现出肿瘤生长抑制;Atezolizumab+Bevacizumab联用组优于Atezolizumab单药组和Bevacizumab单药组,证明在该模型中,抗PD-L1和抗VEGF能够呈现联合药效;在1.87μM高剂量下,双特异性抗体BsAb10给药组在第13天时,全部小鼠均展现出肿瘤完全缓解趋势,且抑瘤率为89.75%,显著性优于对照抗体W3256给药组(抑瘤率为80.74%)、对照抗体AK112给药组(抑瘤率为54.79%)以及Atezolizumab+Bevacizumab联用给药组(抑瘤率为76.52%);在0.47μM中剂量下,双特异性抗体BsAb10给药组在第13天时,全部小鼠也均展现出肿瘤完全缓解趋势,且抑瘤率为83.68%,显著优于对照抗体AK112给药组(抑瘤率为35.69%);在0.094μM低剂量下,双特异性抗体BsAb10到第23天时呈现出36.26%抑瘤效果,显著优于对照双特异性抗体AK112(抑瘤率为14.25%)。Observe and record the tumor length (mm) and width (mm) at any time, calculate its tumor volume (V), the calculation method is V=(length×width 2 )/2, tumor inhibition rate TGI (%)=(1-administration The average tumor volume of the group/the average tumor volume of the PBS-treated group)×100%. The tumor suppression results are shown in Figure 10 and Table 6. It can be seen from the results that all drug groups exhibited tumor growth inhibition relative to the PBS treatment group; the Atezolizumab+Bevacizumab combination group was superior to the Atezolizumab single drug group and Bevacizumab single drug group, proving that in this model, anti-PD-L1 and anti-VEGF can present a combined drug effect; at a high dose of 1.87 μM, all mice in the bispecific antibody BsAb10 administration group showed a trend of complete tumor remission on the 13th day, and the tumor inhibition rate was 89.75%, which was significant It was better than the control antibody W3256 administration group (the tumor inhibition rate was 80.74%), the control antibody AK112 administration group (the tumor inhibition rate was 54.79%) and the combination administration group of Atezolizumab+Bevacizumab (the tumor inhibition rate was 76.52%); At the medium dose of 0.47μM, all mice in the bispecific antibody BsAb10-administered group showed a trend of complete tumor remission on the 13th day, and the tumor inhibition rate was 83.68%, which was significantly better than that of the control antibody AK112-administered group (inhibition The tumor rate was 35.69%); at a low dose of 0.094 μM, the bispecific antibody BsAb10 showed a 36.26% tumor inhibitory effect on the 23rd day, which was significantly better than the control bispecific antibody AK112 (the tumor inhibitory rate was 14.25%).
表6不同给药组的抑瘤率TGI(%)
The tumor inhibition rate TGI (%) of different administration groups of table 6
The tumor inhibition rate TGI (%) of different administration groups of table 6
实施例8抗PD-L1和VEGF双特异性抗体的体内抑瘤实验(A431小鼠荷瘤模型)Example 8 In vivo tumor suppression experiment of anti-PD-L1 and VEGF bispecific antibody (A431 mouse tumor-bearing model)
使用6-8周雌性NSG小鼠(购于上海南方模式生物,品系:M-NSG),实验小鼠饲养在恒温恒湿的独立通风盒内,饲养室温度21-24℃,湿度30-53%。将A431细胞(人表皮癌细胞)以5×106个/每只小鼠进行右侧背部皮下注射(第0天),然后进行随机分组(每组8只小鼠):分别是PBS处理组、D21-4低剂量给药组(0.37mpk(mg/kg))、P30-10-26低剂量给药组(0.37mpk)、双特异性抗体BsAb10高剂量给药组(2mpk),双特异性抗体BsAb10低剂量给药组(0.5mpk),D21-4和P30-10-26高剂量联合用药组(1.48+1.48mpk),D21-4和P30-10-26低剂量联合用药组(0.37+0.37mpk)。荷瘤后第二天,通过尾静脉注射PBMC(C2106025),每只小鼠注射5×106个PBMC细胞,2h后进行首次给药,每个星期两次给药,腹腔注射(i.p.)给药,共给药5周。6-8 weeks old female NSG mice (purchased from Shanghai Nanfang Model Organisms, strain: M-NSG) were used, and the experimental mice were kept in an independent ventilation box with constant temperature and humidity. %. A431 cells (human epidermal cancer cells) were subcutaneously injected into the right back of each mouse at 5×10 6 cells (day 0), and then randomly divided into groups (8 mice per group): PBS treatment group respectively , D21-4 low-dose administration group (0.37mpk (mg/kg)), P30-10-26 low-dose administration group (0.37mpk), bispecific antibody BsAb10 high-dose administration group (2mpk), bispecific Antibody BsAb10 low-dose administration group (0.5mpk), D21-4 and P30-10-26 high-dose combination treatment group (1.48+1.48mpk), D21-4 and P30-10-26 low-dose combination treatment group (0.37 +0.37mpk). On the second day after bearing the tumor, inject PBMC (C2106025) through the tail vein, inject 5×10 6 PBMC cells per mouse, and give the first administration 2 hours later, twice a week, intraperitoneal injection (ip) medicine for a total of 5 weeks.
随时观察和记录肿瘤长(mm)和宽(mm),计算其肿瘤体积(V),计算方式为V=(长×宽2)/2,抑瘤率TGI(%)=(1-给药组肿瘤平均体积/PBS处理组肿瘤平均体积)×100%。抑瘤结果如图11和表7所示。从结果可以看出:所有给药组相对于PBS处理组均呈现出肿瘤生长抑制;高剂量下BsAb10(抑瘤率为75.94%)相比于D21-4和P30-10-26联合用药组(抑瘤率为66.1%)展现出更好的抗肿瘤药效;低剂量下BsAb10(抑瘤率为43.14%)相比于D21-4和P30-10-26联合用药组(抑瘤率为36.06%)展现出更好的抗肿瘤药效。Observe and record the tumor length (mm) and width (mm) at any time, calculate its tumor volume (V), the calculation method is V=(length×width 2 )/2, tumor inhibition rate TGI (%)=(1-administration The average tumor volume of the group/the average tumor volume of the PBS-treated group)×100%. The tumor suppression results are shown in Figure 11 and Table 7. As can be seen from the results: all administration groups exhibited tumor growth inhibition relative to the PBS treatment group; BsAb10 (tumor inhibition rate of 75.94%) at high doses compared to D21-4 and P30-10-26 combined treatment group ( Inhibition rate of 66.1%) showed better antitumor efficacy; BsAb10 (inhibition rate of 43.14%) at low doses compared with D21-4 and P30-10-26 combination group (inhibition rate of 36.06 %) showed better antitumor efficacy.
表7不同给药组的抑瘤率TGI(%)
The tumor inhibition rate TGI (%) of different administration groups of table 7
The tumor inhibition rate TGI (%) of different administration groups of table 7
序列表概述Sequence Listing Overview
本申请附带有包含许多核酸和氨基酸序列的序列表。下表提供了所包含的序列的概述。
Accompanying this application is a Sequence Listing containing a number of nucleic acid and amino acid sequences. The table below provides an overview of the sequences included.
Accompanying this application is a Sequence Listing containing a number of nucleic acid and amino acid sequences. The table below provides an overview of the sequences included.
Claims (18)
- 一种特异性结合PD-L1和VEGF的双特异性抗体,其中所述抗体包括特异性结合第一抗原的VHH结构域和特异性结合第二抗原的VHH结构域,其中第一抗原和第二抗原彼此不同并独立地选自PD-L1和VEGF;A bispecific antibody specifically binding to PD-L1 and VEGF, wherein the antibody comprises a VHH domain specifically binding to a first antigen and a VHH domain specifically binding to a second antigen, wherein the first antigen and the second the antigens are different from each other and are independently selected from PD-L1 and VEGF;其中,特异性结合VEGF的VHH结构域包含SEQ ID NO:2所示的VHH结构域的CDR1-3序列;Wherein, the VHH structural domain specifically binding to VEGF comprises the CDR1-3 sequence of the VHH structural domain shown in SEQ ID NO:2;且优选地,其中特异性结合PD-L1的VHH结构域包含SEQ ID NO:1所示的VHH结构域的CDR1-3序列。And preferably, wherein the VHH domain specifically binding to PD-L1 comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:1.
- 权利要求1的双特异性抗体,其中,特异性结合VEGF的VHH结构域包含SEQ ID NO:2所示的氨基酸序列,或与SEQ ID NO:2具有至少80%、85%、90%、95%或99%同一性的氨基酸序列,或与SEQ ID NO:2相比具有一个或多个(优选地1-10个,更优选地1-5个)氨基酸的添加、缺失和/或取代的氨基酸序列,The bispecific antibody of claim 1, wherein the VHH domain specifically binding to VEGF comprises the amino acid sequence shown in SEQ ID NO: 2, or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 2 Amino acid sequence with % or 99% identity, or with one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and/or substitutions compared to SEQ ID NO: 2 amino acid sequence,最优选地,所述VHH结构域包含SEQ ID NO:2的氨基酸序列,或由SEQ ID NO:2所示的氨基酸序列组成。Most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence shown in SEQ ID NO: 2.
- 权利要求1-2任一项的双特异性抗体,其中,特异性结合PD-L1的VHH结构域包含SEQ ID NO:1所示的氨基酸序列,或与SEQ ID NO:1具有至少80%、85%、90%、95%或99%同一性的氨基酸序列,或与SEQ ID NO:1相比具有一个或多个(优选地1-10个,更优选地1-5个)氨基酸的添加、缺失和/或取代的氨基酸序列,The bispecific antibody according to any one of claims 1-2, wherein the VHH domain specifically binding to PD-L1 comprises the amino acid sequence shown in SEQ ID NO: 1, or has at least 80%, An amino acid sequence that is 85%, 90%, 95% or 99% identical, or has one or more (preferably 1-10, more preferably 1-5) amino acid additions compared to SEQ ID NO: 1 , deleted and/or substituted amino acid sequences,最优选地,所述VHH结构域包含SEQ ID NO:1的氨基酸序列,或由SEQ ID NO:1所示的氨基酸序列组成。Most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.
- 权利要求1-3任一项的双特异性抗体,其中,其中所述双特异性抗体包含连接子,所述连接子包含10-20个氨基酸长度,优选地,包含氨基酸序列(G4S)3。The bispecific antibody according to any one of claims 1-3, wherein said bispecific antibody comprises a linker comprising 10-20 amino acids in length, preferably comprising an amino acid sequence (G 4 S) 3 .
- 权利要求1-4任一项的双特异性抗体,其中所述双特异性抗体包含免疫球蛋白的Fc区,其中,Fc区为IgG Fc区,例如,人IgG1、IgG2、IgG3或IgG4的Fc区,优选人IgG1的Fc区,更优选所述Fc区在N端带有免疫球蛋白铰链区序列。The bispecific antibody according to any one of claims 1-4, wherein the bispecific antibody comprises an Fc region of an immunoglobulin, wherein the Fc region is an IgG Fc region, for example, the Fc of human IgG1, IgG2, IgG3 or IgG4 Region, preferably the Fc region of human IgG1, more preferably the Fc region has an immunoglobulin hinge region sequence at the N-terminus.
- 根据权利要求1-5任一项的双特异性抗体,其中所述双特异性抗体包含从N端至C端包含第一VHH结构域、Fc区、连接子和第二VHH结构域的多肽链,The bispecific antibody according to any one of claims 1-5, wherein said bispecific antibody comprises a polypeptide chain comprising a first VHH domain, an Fc region, a linker and a second VHH domain from the N-terminus to the C-terminus ,其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen, respectively,其中所述第一抗原和第二抗原彼此不同并独立地选自PD-L1和VEGF,优选地,第一抗原是PD-L1且第二抗原是VEGF。Wherein said first antigen and second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF.
- 根据权利要求1-5任一项的双特异性抗体,其中所述双特异性抗体包含从N端至C端包含第一VHH结构域、连接子、第二VHH结构域和Fc区的多肽链,The bispecific antibody according to any one of claims 1-5, wherein said bispecific antibody comprises a polypeptide chain comprising a first VHH domain, a linker, a second VHH domain and an Fc region from the N-terminus to the C-terminus ,其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen, respectively,其中所述第一抗原和第二抗原彼此不同并独立地选自PD-L1和VEGF,优选地,第一抗原是PD-L1且第二抗原是VEGF。 Wherein said first antigen and second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF.
- 根据权利要求1-5任一项的双特异性抗体,其中所述双特异性抗体包含第一和第二多肽链,其中,所述第一多肽链从N端至C端包含第一VHH结构域、CH1结构域和Fc区;所述第二多肽链从N端至C端包含第二VHH结构域和CL结构域;The bispecific antibody according to any one of claims 1-5, wherein said bispecific antibody comprises a first and a second polypeptide chain, wherein said first polypeptide chain comprises a first polypeptide chain from N-terminus to C-terminus VHH domain, CH1 domain and Fc region; the second polypeptide chain comprises a second VHH domain and a CL domain from N-terminal to C-terminal;其中第一多肽链中的第一VHH结构域和CH1结构域与第二多肽链中的第二VHH结构域和CL结构域配对形成Fab样结构;wherein the first VHH domain and the CH1 domain in the first polypeptide chain are paired with the second VHH domain and the CL domain in the second polypeptide chain to form a Fab-like structure;其中第一VHH结构域和第二VHH结构域分别特异性结合第一抗原和第二抗原,wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen, respectively,其中所述第一抗原和第二抗原彼此不同且独立地选自PD-L1和VEGF,优选地,第一抗原是PD-L1且第二抗原是VEGF。Wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF.
- 权利要求1-8任一项的双特异性抗体,其中所述双特异性抗体包含SEQ ID NO:7所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列;或The bispecific antibody according to any one of claims 1-8, wherein said bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 7 or has at least 90%, 91%, 92%, 93%, 94% thereof , 95%, 96%, 97%, 98% or 99% identical amino acid sequences; or包含SEQ ID NO:8所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。Comprising the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
- 权利要求1-8任一项的双特异性抗体,其中所述双特异性抗体包含第一和第二多肽链,其中:The bispecific antibody of any one of claims 1-8, wherein said bispecific antibody comprises a first and a second polypeptide chain, wherein:第一多肽链包含SEQ ID NO:9所示的氨基酸序列或与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列;且The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 9 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto a specific amino acid sequence; and第二多肽链包含SEQ ID NO:10所示的氨基酸序列与其具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的氨基酸序列。The second polypeptide chain comprises an amino acid sequence shown in SEQ ID NO: 10 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith amino acid sequence.
- 多核苷酸,其编码权利要求1-10中任一项所述的双特异性抗体。A polynucleotide encoding the bispecific antibody of any one of claims 1-10.
- 载体,优选地表达载体,其包含权利要求11的多核苷酸。A vector, preferably an expression vector, comprising the polynucleotide of claim 11.
- 宿主细胞,其包含权利要求11所述的多核苷酸或权利要求12所述的载体,例如,所述宿主细胞是哺乳动物细胞。A host cell comprising the polynucleotide of claim 11 or the vector of claim 12, for example, the host cell is a mammalian cell.
- 用于生产权利要求1-10中任一项所述的双特异性抗体的方法,所述方法包括:A method for producing the bispecific antibody of any one of claims 1-10, said method comprising:在适于表达所述抗体的多肽链的条件下培养包含编码所述多肽链的宿主细胞;和在适于所述多肽链装配为所述抗体的条件下使多肽链装配产生所述抗体。culturing a host cell comprising a polypeptide chain encoding said antibody under conditions suitable for expressing said polypeptide chain; and allowing assembly of said polypeptide chain into said antibody to produce said antibody under conditions suitable for assembly of said polypeptide chain into said antibody.
- 药物组合物,其包含权利要求1-10中任一项所述的双特异性抗体和可药用载体。A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1-10 and a pharmaceutically acceptable carrier.
- 根据权利要求1-10中任一项所述的双特异性抗体或权利要求15所述的药物组合物的用途,用于体内或体外Use of the bispecific antibody according to any one of claims 1-10 or the pharmaceutical composition of claim 15, for in vivo or in vitro-结合人PD-L1和人VEGF;或- binds human PD-L1 and human VEGF; or-阻断人PD-L1/PD-1信号途径;或- Block the human PD-L1/PD-1 signaling pathway; or-中和VEGF活性;或- neutralizes VEGF activity; or-抑制肿瘤细胞生长,- inhibition of tumor cell growth,或用于制备用于上述任一项用途的药物。Or for the preparation of medicines for any of the above purposes.
- 权利要求16的用途,其中所述双特异性抗体或药物组合物用作在个体中治疗和/或预防疾病的药 物或用作疾病的诊断工具,优选地,所述个体是哺乳动物,更优选地是人。The use of claim 16, wherein the bispecific antibody or pharmaceutical composition is used as a drug for treating and/or preventing diseases in an individual or as a diagnostic tool for a disease, preferably said individual is a mammal, more preferably a human.
- 权利要求17的用途,其中所述疾病为PD-L1阳性癌症,优选PD-L1阳性实体瘤或血液瘤,例如头颈鳞癌,黑色素瘤,肾细胞癌、非小细胞肺癌、膀胱癌、尿路上皮癌、胃癌、结肠癌、结直肠癌、卵巢癌、乳腺癌、肺癌、宫颈癌、胶质母细胞癌、胰腺癌、前列腺癌、食管癌、淋巴瘤、肝癌、微卫星不稳定型实体瘤。 The use according to claim 17, wherein the disease is PD-L1 positive cancer, preferably PD-L1 positive solid tumor or blood tumor, such as head and neck squamous cell carcinoma, melanoma, renal cell carcinoma, non-small cell lung cancer, bladder cancer, urinary tract cancer Skin cancer, gastric cancer, colon cancer, colorectal cancer, ovarian cancer, breast cancer, lung cancer, cervical cancer, glioblastoma cancer, pancreatic cancer, prostate cancer, esophageal cancer, lymphoma, liver cancer, microsatellite unstable solid tumors .
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CN103965363A (en) * | 2013-02-06 | 2014-08-06 | 上海白泽生物科技有限公司 | Fusion protein efficiently combined with PD-1 and VEGF, and coding sequence and use thereof |
CN109942712A (en) * | 2019-04-01 | 2019-06-28 | 华博生物医药技术(上海)有限公司 | Anti- PD-L1/VEGF bifunctional antibody and application thereof |
WO2020181221A1 (en) * | 2019-03-07 | 2020-09-10 | Children's Medical Center Corporation | Targeted delivery of immune-modulating vhh and vhh-fusion protein |
CN111848800A (en) * | 2020-07-31 | 2020-10-30 | 三优生物医药(上海)有限公司 | PD-L1 single domain antibodies and uses thereof |
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AR110040A1 (en) * | 2016-05-27 | 2019-02-20 | Abbvie Biotherapeutics Inc | BISPECIFIC UNION PROTEINS |
WO2021104302A1 (en) * | 2019-11-25 | 2021-06-03 | 中山康方生物医药有限公司 | Anti-pd-1-anti-vegfa bispecific antibody, pharmaceutical composition and use thereof |
CN114106190A (en) * | 2020-08-31 | 2022-03-01 | 普米斯生物技术(珠海)有限公司 | anti-VEGF/PD-L1 bispecific antibody and application thereof |
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CN103965363A (en) * | 2013-02-06 | 2014-08-06 | 上海白泽生物科技有限公司 | Fusion protein efficiently combined with PD-1 and VEGF, and coding sequence and use thereof |
WO2020181221A1 (en) * | 2019-03-07 | 2020-09-10 | Children's Medical Center Corporation | Targeted delivery of immune-modulating vhh and vhh-fusion protein |
CN109942712A (en) * | 2019-04-01 | 2019-06-28 | 华博生物医药技术(上海)有限公司 | Anti- PD-L1/VEGF bifunctional antibody and application thereof |
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