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WO2025014896A1 - Humanized 40h3 antibody - Google Patents

Humanized 40h3 antibody Download PDF

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Publication number
WO2025014896A1
WO2025014896A1 PCT/US2024/037091 US2024037091W WO2025014896A1 WO 2025014896 A1 WO2025014896 A1 WO 2025014896A1 US 2024037091 W US2024037091 W US 2024037091W WO 2025014896 A1 WO2025014896 A1 WO 2025014896A1
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seq
antibody
antigen binding
binding fragment
cell
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PCT/US2024/037091
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French (fr)
Inventor
David J. Fitzgerald
Antonella Antignani
Robert Sarnovsky
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2025014896A1 publication Critical patent/WO2025014896A1/en

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    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
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    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
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    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
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    • A61K40/4204—Epidermal growth factor receptors [EGFR]
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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Abstract

Disclosed are antibodies (e.g., humanized monoclonal antibodies and antigen binding fragments) that specifically bind epidermal growth factor receptor variant III (EGFRvIII) and/or gene-amplified EGFR, as well as conjugates and chimeric antigen receptors (CARs) including such antibodies. Also provided are nucleic acid molecules encoding an antibody, conjugate, or CAR disclosed herein, and host cells expressing the nucleic acid molecules. Further disclosed are methods of using the disclosed compositions, for example, for treating or detecting a tumor, such as a tumor expressing EGFRvIII and/or gene-amplified EGFR.

Description

HUMANIZED 40H3 ANTIBODY
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 63/525,493, filed July 7, 2023, which is incorporated by reference in its entirety.
FIELD OF THE DISCLOSURE
This application is related to the field of cancer biology, specifically to humanized monoclonal antibodies and antigen binding fragments that specifically bind epidermal growth factor receptor mutants/variants expressed by tumor cells (e.g., EGFRvIII and/or gene-amplified EGFR).
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with Government support under project number Z01#: Z1 A BC 008757 by the National Institutes of Health, National Cancer Institute. The United States Government has certain rights in the invention.
SEQUENCE LISTING REFERENCE
The Sequence Listing is submitted as an XML file named “Sequence. xml,” created on July 8, 2024, (1,386,226 bytes), which is incorporated herein by reference.
BACKGROUND
EGFR is frequently involved in the oncogenic progression of human cancer. Various alterations in expression including gene amplifications and activating mutations contribute to oncogenesis. The human form of this large receptor has an external domain (ECD) of 621 amino acids, a single pass transmembrane domain (TM) of 23 amino acids and an enzymatically active intracellular domain (ICD) of 542 amino acids. EGFR is a member of the receptor tyrosine kinase family and was the first receptor shown to be associated positively with human cancer. Ligand binding leads to receptor dimer formation and the activation of the kinase domain which signals to one of several pathways that can promote the growth, survival and spread of mammalian cells. Activating mutations can occur in either the ECD or the ICD. Gene amplifications and deletions can also activate EGFR. Exemplary deletions include the loss of exons 2-7, which produces EGFR variant III (EGFRvIII), and the loss of exon 19 generates a constitutively active enzyme mutant. Gene amplification of EGFR, expression of EGFRvIII, or the loss of exon 19 of EGFR are reported only in cancer cells.
Antibodies generally bind their target antigen tightly and specifically, making them usefill therapeutics for treating a wide range of diseases characterized by altered protein expression, such as the expression of EGFRvIII or gene-amplified EGFR. For many therapeutic applications, however, the efficacy and safety of non-human antibodies is compromised because non-human immunoglobulin (Ig) molecules are themselves immunogenic, and thus are capable of inducing an anti-Ig immune response, making repeat dosing highly compromised. Thus, for therapeutic use, it can be beneficial to modify an antibody to reduce or eliminate immunogenicity. A need remains for humanized antibodies that can bind EGFRvIII or gene-amplified EGFR, but not wild-type EGFR, to exhibit cancer cell specificity.
SUMMARY OF THE DISCLOSURE
Disclosed are isolated humanized monoclonal antibodies and antigen binding fragments thereof, wherein the monoclonal antibody specifically binds to EGFRvIII and/or gene-amplified EGFR. In some aspects, the monoclonal antibody or antigen binding fragment includes a heavy chain variable region (VH) and a light chain variable region (Vj comprising: a) SEQ ID NO: 5 and SEQ ID NO: 8, respectively (A10, VH3+VL3); b) SEQ ID NO: 3 and SEQ ID NO: 6, respectively (A2, VH1+VL1); c) SEQ ID NO: 3 and SEQ ID NO: 7, respectively (A3, VH1+VL2); d) SEQ ID NO: 3 and SEQ ID NO: 8, respectively (A4, VH1+VL3); e) SEQ ID NO: 4 and SEQ ID NO: 6, respectively (A5, VH2+VL1); f) SEQ ID NO: 4 and SEQ ID NO: 7, respectively (A6, VH2+VL2); g) SEQ ID NO: 4 and SEQ ID NO: 8, respectively (A7, VH2+VL3); h) SEQ ID NO: 5 and SEQ ID NO: 6, respectively (A8, VH3+VL1); i) SEQ ID NO: 5 and SEQ ID NO: 7, respectively (A9, VH3+VL2); j) SEQ ID NO: 1 and SEQ ID NO: 9, respectively (Bl, 40H3 VH+VL-EG); k) SEQ ID NO: 1 and SEQ ID NO: 10, respectively (B2, 40H3 VH+VL-DA); l) SEQ ID NO: 4 and SEQ ID NO: 11, respectively (B3, VH2+VL1-DA); m) SEQ ID NO: 4 and SEQ ID NO: 12, respectively (B4, VH2+VL2-DA); n) SEQ ID NO: 5 and SEQ ID NO: 11, respectively (B5, VH3 and VL1-DA); o) SEQ ID NO: 31 and SEQ ID NO: 32, respectively (CIO); p) SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2); or q) SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3).
Conjugates of these antibodies and antigen binding fragments are also disclosed. Further disclosed are chimeric antigen receptors (CARs) including a disclosed antibody or antigen binding fragment, and cells (e.g., immune cells) expressing such chimeric antigen receptors (e.g., CAR T cells).
Also disclosed are nucleic acid molecules encoding a VH and/or a VL of any of the antibodies disclosed herein, vectors including these nucleic acids, and host cells transformed with these nucleic acids and/or vectors.
In further aspects, disclosed is the use of a monoclonal antibody or antigen binding fragment disclosed herein for inhibiting a tumor that expresses EGFRvIII or gene-amplified EGFR in a subject. In some implementations, a subject having a tumor or cancer that expresses EGFRvIII or gene-amplified EGFR is selected for treatment. In other aspects, disclosed is the use of a monoclonal antibody or antigen binding fragment disclosed herein for detecting EGFRvIII or gene-amplified EGFR. The foregoing and other features will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
FIGs. 1A-1B: Antibody binding to rat glioma cells expressing human EGFRvIII. A10 exhibited highest MFI when compared to other candidates including the chimeric antibody (mouse VH and VL from 40H3 antibody + human IgGICH/human IgkappaCL). Cetuximab is used as a universal positive control with binding to both WT and cancer-expressed EGFR (EGFRvIII).
FIGs. 2A-2B: Antibody binding to human tumor cells expressing amplified EGFR, specifically MDA-MB-468 (FIG. 2A) and A431 (FIG. 2B) cells. The antibody A10 exhibited the highest MFI.
FIGs. 3A-3B: Antibody binding to cells expressing WT EGFR, specifically WI-38 (FIG. 3A) or U87MG (FIG. 3B) cells. The A10 antibody does not bind cells expressing WT EGFR. However, evidence of surface WT EGFR is confirmed via cetuximab binding.
FIGs. 4A-4B: Table showing properties of antibodies including variable region combinations, dissociation constant (KD), cell binding, and ECso.
FIGs. 5A-5B: Comparative antibody binding data for 40H3, A10, and cetuximab. FIG. 5A shows 40H3 & A10 bind strongly to cells expressing amplified EGFR (MDA-MB-468) but weakly to cells with EGFR-WT (WI-38). In contrast, cetuximab is shown to strongly bind cells with EGFR-WT. FIG. 5B shows both A10 and cetuximab bind strongly to cells with EGFRvIII.
FIGs. 6A-6C: Crystal structures of EGFR loop-AlO Fab. FIGs. 6B and 6C are close-ups of FIG. 6A to show greater detail of the EGRF loop-AlO interaction.
FIG. 7: A summary of information for the indicated cell lines (see, column 1).
FIGs. 8A-8B: Vector maps of pMH289-A10-l (FIG. 8A, SEQ ID NO: 51) and pMH289-A10-2 (FIG. 8B, SEQ ID NO: 52).
FIG. 9: Cell viability of A431-luc cells 24 or 48 hours after the indicated treatments. “CAR-1” and “CAR-2” are A10 scFv-CAR-T-cells (see, FIG. 8A and 8B, respectively). Controls are activated T-cells lacking a transduced chimeric antigen receptor.
FIG. 10: Cell viability of MDA-MB-468 cells following treatment with two A10 antibody -drug conjugates (ADCs): A10 conjugated to deruxtecan (AlO-Dxd) or A10 conjugated to monomethyl auristatin E (A10-MMAE).
SEQUENCES
The nucleic and amino acid sequences listed are shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by reference to the displayed strand. In the heavy and light chain variable domain sequences, the CDR sequences (as determined by the Kabat, IM GT, or Chothia numbering scheme) are underlined. In SEQ ID NOs: 3-32, framework residues that are bolded are residues that have been humanized, specifically in comparison to SEQ ID NO: 1 (VH sequences) or SEQ ID NO: 2 (VL sequences), respectively.
SEQ ID NO: 1 is the amino acid sequence of the 40H3 heavy chain variable domain.
OVOLKOSGPGLVQPSOSLSITCTVSGFSLTNYGIHWLROSPGKGLEWLGMMWRGGGTDYNAAFIS
RLTITKDTSKSOVFFRMNNLOTNDTAIYYCARKGVGMGLGYWGOGTSVTVSS
SEQ ID NO: 2 is the amino acid sequence of the 40H3 light chain variable domain.
DIOMTOSPASOSASLGESVTITCLASOTIGTWVAWYOOKPGRSPOLLIYGATNLADGVPSRFSGSGS
GTKFSFKISSLOAEDFVSYYCOOLYSNPYTFGGGTKLEIK
SEQ ID NO: 3 is the amino acid sequence of the VH1 heavy chain variable domain.
OVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWIROPPGKALEWLAMMWRGGGTDYNAAFIS
RETISKDTSKSQVVLTMTNMDPVDTATYYCARKGVGMGEGYWGQGTLVTVSS
SEQ ID NO: 4 is the amino acid sequence of the VH2 heavy chain variable domain.
OVTEKESGPVEVKPTETETLTCTVSGFSETNYGIHWIROPPGKAEEWEGMMWRGGGTDYNAAFIS
RETITKDTSKSOVVLTMTNMDPVDTATYYCARKGVGMGEGYWGOGTLVTVSS
SEQ ID NO: 5 is the amino acid sequence of the VH3 heavy chain variable domain, which is also the D2 and D3 heavy chain variable domain.
OVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLROPPGKALEWLGMMWRGGGTDYNAAFI
SRLTITKDTSKSOVVFTMTNMDPVDTATYYCARKGVGMGLGYWGOGTLVTVSS
SEQ ID NO: 6 is the amino acid sequence of the VL1 light chain variable domain.
DIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKAPKLLIYGATNLADGVPSRFSGSG
SGTDFTLTISSLOPEDFATYYCOOLYSNPYTFGGGTKLEIK
SEQ ID NO: 7 is the amino acid sequence of the VL2 light chain variable domain.
DIOMTOSPSSVSASVGDRVTITCLASOTIGTWVAWYOQKPGKSPOLLIYGATNLADGVPSRFSGSGS
GTDFTLTISSLQPEDFATYYCQQLYSNPYTFGGGTKLEIK
SEQ ID NO: 8 is the amino acid sequence of the VL3 light chain variable domain, which is also the DI light chain variable domain.
DIOMTOSPSSVSASVGDRVTITCLASOTIGTWVAWYOQKPGKSPOLLIYGATNLADGVPSRFSGSGS
GTKFTLTISSLQPEDFATYYCOQLYSNPYTFGGGTKLEIK
SEQ ID NO: 9 is the amino acid sequence of the VL-EG light chain variable domain.
DIOMTOSPASOSASLGESVTITCLASOTIGTWVAWYOOKPGRSPOLLIYGATNLAEGVPSRFSGSGS
GTKFSFKISSLOAEDFVSYYCOOLYSNPYTFGGGTKLEIK
SEQ ID NO: 10 is the amino acid sequence of the VE-DA light chain variable domain.
DIQMTQSPASOSASEGESVTITCEASOTIGTWVAWYOQKPGRSPQEEIYGATNEADAVPSRFSGSGS
GTKFSFKISSLOAEDFVSYYCOOEYSNPYTFGGGTKLEIK SEQ ID NO: 11 is the amino acid sequence of the VL1-DA light chain variable domain.
DIOMTOSPSSVSASVGDRVTITCLASOTIGTWVAWYOQKPGKAPKLLIYGATNLADAVPSRFSGSG
SGTDFTLTISSLOPEDFATYYCOQLYSNPYTFGGGTKLEIK
SEQ ID NO: 12 is the amino acid sequence of the VL2-DA light chain variable domain.
DIOMTOSPSSVSASVGDRVTITCLASQTIGTWVAWYOQKPGKSPQLLIYGATNLADAVPSRFSGSGS
GTDFTLTISSLOPEDFATYYCOOLYSNPYTFGGGTKLEIK
SEQ ID NO: 13 is the amino acid sequence of the Cl heavy chain variable domain, which is also the DI heavy chain variable domain.
OVOLOESGPGLVKPSETLSLTCTVSGGSISNYGIHWIRQPPGKGLEWIGMMWRGGGTDYNAAFIS
RVTISVDTSKNOFSLKLSSVTAADTAVYYCARKGVGMGLGYWGOGTLVTVSS
SEQ ID NO: 14 is the amino acid sequence of the Cl light chain variable domain, which is also the D2 light chain variable domain.
DIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKAPKLLIYGATNLADGVPSRFSGSG
SGTDFTLTISSLOPEDFATYYCOOLYSNPYTFGOGTKLEIK
SEQ ID NO: 15 is the amino acid sequence of the C2 heavy chain variable domain.
OVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLGMMWRGGGTDYNAAFI
SRVTISVDTSKNQFSLKLSSVTAADTAVYYCARKGVGMGEGYWGQGTLVTVSS
SEQ ID NO: 16 is the amino acid sequence of the C2 light chain variable domain.
DIOMTOSPSSVSASVGDRVTITCLASOTIGTWVAWYOQKPGKAPKLLIYGATNLADGVPSRFSGSG
SGTDFTLTISSLOPEDFATYYCOOLYSNPYTFGOGTKLEIK
SEQ ID NO: 17 is the amino acid sequence of the C3 heavy chain variable domain.
OVOLQESGPGLVKPSETLSLTCTVSGGSISNYGIHWIRQPPGKGLEWIGMMWRGGGTDYNAAFIS
RLTITKDTSKSQVVFTMTNMDPVDTATYYCARKGVGMGLGYWGQGTLVTVSS
SEQ ID NO: 18 is the amino acid sequence of the C3 light chain variable domain.
DIOMTOSPSSVSASVGDRVTITCLASOTIGTWVAWYOQKPGKAPKLLIYGATNLADGVPSRFSGSG
SGTDFTLTISSLOPEDFATYYCOOLYSNPYTFGOGTKLEIK
SEQ ID NO: 19 is the amino acid sequence of the C4 heavy chain variable domain.
QVQLQESGPGLVKPSETLSLTCTVSGGSISNYGIHWIRQPPGKGLEWIGMMWRGGGTDYNAAFIS
RLTISKDTSKNOVSLKLSSVTAADTAVYYCARKGVGMGLGYWGOGTLVTVSS
SEQ ID NO: 20 is the amino acid sequence of the C4 light chain variable domain.
DIOMTOSPSSVSASVGDRVTITCLASOTIGTWVAWYOQKPGKAPKLLIYGATNLADGVPSRFSGSG
SGTDFTLTISSLOPEDFATYYCOOLYSNPYTFGOGTKLEIK
SEQ ID NO: 21 is the amino acid sequence of the C5 heavy chain variable domain. OVOLOESGPGLVKPSETLSLTCTVSGFSLTNYGIHWLRQPPGKGLEWIGMMWRGGGTDYNAAFIS
RVTISVDTSKNQFSLKLSSVTAADTAVYYCARKGVGMGLGYWGQGTLVTVSS
SEQ ID NO: 22 is the amino acid sequence of the C5 light chain variable domain.
DIOMTOSPSSVSASVGDRVTITCLASOTIGTWVAWYOQKPGKAPKLLIYGATNLADGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCOOLYSNPYTFGOGTKLEIK
SEQ ID NO: 23 is the amino acid sequence of the C6 heavy chain variable domain.
OVOLOESGPGLVKPSETLSLTCTVSGFSLTNYGWSWIROPPGKGLEWIGYMWRGGGTNYNPSLK
SRVTISVDTSKNOFSLKLSSVTAADTAVYYCARKGVGMGLGYWGOGTLVTVSS
SEQ ID NO: 24 is the amino acid sequence of the C6 light chain variable domain, which is also the light chain variable domain of D3.
DIQMTQSPSSVSASVGDRVTITCRASQTIGTWLAWYQQKPGKAPKLLIYGATSLQSGVPSRFSGSGS
GTDFTLTISSLQPEDFATYYCOOLYSNPYTFGOGTKLEIK
SEQ ID NO: 25 is the amino acid sequence of the C7 heavy chain variable domain.
OVOLOESGPGLVKPSETLSLTCTVSGFSLTNYYWSWIROPPGKGLEWIGYIWRGGGTNYNPSLKS
RVTISVDTSKNOFSLKLSSVTAADTAVYYCARKGVGMGLGYWGOGTLVTVSS
SEQ ID NO: 26 is the amino acid sequence of the C7 light chain variable domain.
DIOMTOSPSSVSASVGDRVTITCLASOTIGTWVAWYOQKPGKAPKLLIYGATNLADGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCOOLYSNPYTFGOGTKLEIK
SEQ ID NO: 27 is the amino acid sequence of the C8 heavy chain variable domain.
OVOLQESGPGLVKPSOTLSLTCTVSGGSISNYGIHWIROSPGKGLEWIGYMMWRGGGTDYNAAFI
SRLTISKDTSKSQVVLTMTNMDPVDTATYYCARKGVGMGLGYFGQGTRLTVSS
SEQ ID NO: 28 is the amino acid sequence of the C8 light chain variable domain.
DIOMTOSPSSLSASVGDRVTITCLASOTIGTWVAWYLOKPGOSPOLLIYGATNLADGVPSRFSGSGS
GTEFTLTISSLQPEDFATYYCOOLYSNPYTFGOGTKLEIK
SEQ ID NO: 29 is the amino acid sequence of the C9 heavy chain variable domain.
OVTLKESGPVLVKPTETLTLTCTVSGFSLSNYGIHWIRQPPGKALEWLAMMWRGGGTDYNAAFIS
RLTISKDTSKSOVVLTMTNMDPVDTATYYCARKGVGMGLGYWGOGTLVTVSS
SEQ ID NO: 30 is the amino acid sequence of the C9 light chain variable domain.
DIQMTQSPSSVSASVGDRVTITCLASOTIGTWVAWYQQKPGKAPKLLIYGATNLADGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCQQLYSNPYTFGQGTKLEIK
SEQ ID NO: 31 is the amino acid sequence of the CIO heavy chain variable domain.
OVOLOESGPGLVKPSETLSLTCTVSGFSLTNYGIHWLROPPGKGLEWIGMMWRGGGTDYNAAFIS
RLTISKDTSKNQVSLKLSSVTAADTAVYYCARKGVGMGLGYWGQGTLVTVSS
SEQ ID NO: 32 is the amino acid sequence of the CIO light chain variable domain. DIQMTOSPSSVSASVGDRVTITCLASOTIGTWVAWYOQKPGKAPKLLIYGATNLADGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCOQLYSNPYTFGOGTKLEIK
SEQ ID NOs: 33-42 are the amino acid sequences of components of chimeric antigen receptors.
SEQ ID NO: 43 is an amino acid sequence of A10 VH-VL scFv.
QVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLGMMWRGGGTDYNAAFIS
RLTITKDTSKSQVVFTMTNMDPVDTATYYCARKGVGMGLGYWGQGTLVTVSSGGGGSGGGGSG
GGGSDIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKSPQLLIYGATNLADGVPSRF
SGSGSGTKFTLTISSLQPEDFATYYCQQLYSNPYTFGGGTKLEIK
SEQ ID NO: 44 is an exemplary nucleic acid sequence encoding an A10 VH-VL scFv.
CAAGTCACCCTGAAAGAATCCGGTCCGGTGCTCGTGAAGCCGACTGAGACCCTGACTCTGACT
TGTACTGTATCCGGCTTCAGTCTGACCAACTATGGCATTCATTGGCTGCGCCAGCCGCCGGGCA
AAGCCCTCGAGTGGCTGGGTATGATGTGGAGGGGCGGCGGCACCGATTACAATGCGGCTTTCA
TCTCCAGGCTCACTATTACCAAAGACACCTCCAAGAGCCAGGTTGTCTTCACCATGACCAACAT
GGACCCGGTAGACACCGCGACCTATTATTGCGCACGCAAAGGCGTGGGTATGGGCCTGGGCTA
CTGGGGCCAGGGTACTCTGGTTACGGTATCTTCCGGTGGTGGTGGCTCAGGCGGTGGTGGCTCC
GGCGGCGGTGGCTCCGACATCCAGATGACCCAATCCCCGTCTTCTGTTTCCGCGAGCGTAGGCG
ATCGGGTAACCATCACTTGTCTGGCTTCCCAAACCATTGGCACGTGGGTTGCCTGGTACCAGCA
GAAACCCGGGAAATCCCCACAGCTGCTGATCTATGGTGCCACCAACCTGGCTGACGGTGTTCCT
TCCCGATTTAGTGGTTCCGGTAGCGGCACCAAATTTACCCTGACCATCTCCTCCCTGCAGCCGG
AAGACTTTGCAACTTACTATTGTCAACAGTTGTACTCCAACCCTTACACTTTCGGTGGTGGTAC
CAAACTGGAAATCAAA
SEQ ID NO: 45 is an amino acid sequence of A10 VL-VH scFv.
DIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKSPQLLIYGATNLADGVPSRFSGSGS
GTKFTLTISSLQPEDFATYYCQQLYSNPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVTLKESGPVLV
KPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLGMMWRGGGTDYNAAFISRLTITKDTSKSQV
VFTMTNMDPVDTATYYCARKGVGMGLGYWGQGTLVTVSS
SEQ ID NO: 46 is an exemplary nucleic acid sequence encoding an A10 VL-VH scFv.
GACATCCAGATGACCCAATCCCCGTCTTCTGTTTCCGCGAGCGTAGGCGATCGGGTAACCATCA
CTTGTCTGGCTTCCCAAACCATTGGCACGTGGGTTGCCTGGTACCAGCAGAAACCCGGGAAATC
CCCACAGCTGCTGATCTATGGTGCCACCAACCTGGCTGACGGTGTTCCTTCCCGATTTAGTGGT
TCCGGTAGCGGCACCAAATTTACCCTGACCATCTCCTCCCTGCAGCCGGAAGACTTTGCAACTT
ACTATTGTCAACAGTTGTACTCCAACCCTTACACTTTCGGTGGTGGTACCAAACTGGAAATCAA
AGGTGGTGGTGGCTCAGGCGGTGGTGGCTCCGGCGGCGGTGGCTCCCAAGTCACCCTGAAAGA
ATCCGGTCCGGTGCTCGTGAAGCCGACTGAGACCCTGACTCTGACTTGTACTGTATCCGGCTTC
AGTCTGACCAACTATGGCATTCATTGGCTGCGCCAGCCGCCGGGCAAAGCCCTCGAGTGGCTG
GGTATGATGTGGAGGGGCGGCGGCACCGATTACAATGCGGCTTTCATCTCCAGGCTCACTATTA
CCAAAGACACCTCCAAGAGCCAGGTTGTCTTCACCATGACCAACATGGACCCGGTAGACACCG
CGACCTATTATTGCGCACGCAAAGGCGTGGGTATGGGCCTGGGCTACTGGGGCCAGGGTACTC
TGGTTACGGTATCTTCC
SEQ ID NO: 47 is an amino acid sequence of an A10 Fab (heavy chain). The sequence ends where papain cleavage occurs.
QVTLKESGPVLVKPTETLTLTCTVSGFSLTNYGIHWLRQPPGKALEWLGMMWRGGGTDYNAAFIS
RLTITKDTSKSQVVFTMTNMDPVDTATYYCARKGVGMGLGYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTH SEQ ID NO: 48 is an exemplary nucleic acid sequence encoding an A10 Fab (heavy chain).
CAAGTCACCCTGAAGGAGAGTGGGCCAGTTTTAGTTAAGCCCACCGAGACACTGACCCTGACC
TGTACCGTGTCCGGCTTCTCTCTGACCAACTATGGCATCCACTGGCTGCGGCAGCCTCCTGGCA
AGGCACTGGAATGGCTGGGCATGATGTGGCGGGGCGGCGGCACAGACTACAATGCCGCCTTCA
TCAGCCGGCTGACCATCACCAAGGACACCTCCAAGAGCCAGGTGGTGTTCACCATGACCAACA
TGGACCCTGTGGACACCGCCACCTACTACTGTGCCCGGAAGGGCGTGGGCATGGGCCTGGGCT
ACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCCAGTACCAAGGGACCCTCTGTGTTTCC
TCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGAC
TACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTT
TCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCT
CTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACA
AGAAGGTGGAACCCAAGTCCTGCGACAAGACCCAC
SEQ ID NO: 49 is an amino acid sequence of an A10 Fab (light chain).
DIQMTQSPSSVSASVGDRVTITCLASQTIGTWVAWYQQKPGKSPQLLIYGATNLADGVPSRFSGSGS
GTKFTLTISSLQPEDFATYYCQQLYSNPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
SEQ ID NO: 50 is an exemplary nucleic acid sequence encoding an A10 Fab (light chain).
GACATCCAGATGACCCAGTCTCCTTCCTCCGTGTCTGCTTCTGTGGGCGACAGAGTGACCATCA
CCTGCCTGGCCTCTCAGACCATCGGCACCTGGGTGGCCTGGTATCAGCAGAAGCCTGGCAAGT
CCCCCCAGCTGCTGATCTATGGCGCCACCAACCTGGCAGATGGAGTGCCCTCCAGATTCTCTGG
CTCTGGCTCTGGCACCAAGTTCACCCTGACCATCTCCTCCCTGCAGCCTGAGGACTTCGCCACC
TACTACTGCCAGCAGCTGTACAGCAACCCTTACACCTTTGGCGGCGGCACCAAGCTGGAGATC
AAGAGAACAGTGGCTGCTCCTTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCG
GCACAGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGA
AGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGG
ACAGCACCTACAGCCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGG
TGTACGCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTTCAACCGGGG
CGAGTGC
SEQ ID NO: 51 is an exemplary sequence encoding the pMH289-A10-l construct.
TTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACA
CAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACC
TTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGA
GAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTG
TTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAG
TACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGA
GGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTT
TTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAG
GGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCT
GTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC
AGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGG
ACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAA
AATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGG
GGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAA
TTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTA
GAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCA
GAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAG
ATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCAC
CGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTG
GAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAA GGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTG
GGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCA
GACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAAC
AGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGG
AAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCA
CCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACAC
GACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGA
AGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAA
GTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT
AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAG
GGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAA
GGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC
TCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAA
GAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAA
ATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGATCGATGGCTCCGGTGCCCGT
CAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTG
AACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCG
CCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTT
CGCAACGGGTTTGCCGCCAGAACACAGGTGTCGTGACGCGGATCCAGGCCTAAGCTTACGCGT
CCTAGCGCTACCGGTCGCCACCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCA
CACCCAGCATTCCTCCTGATCCCACATATGCAGGTTACCCTGAAGGAGAGCGGCCCTGTGCTGG
TGAAGCCTACAGAGACACTGACACTGACCTGCACCGTGAGCGGCTTTTCTCTGACAAATTACG
GCATCCACTGGCTGAGACAGCCACCTGGCAAGGCCCTGGAATGGCTGGGCATGATGTGGCGGG
GCGGCGGAACAGATTACAACGCCGCCTTTATCAGCAGACTGACAATCACCAAGGACACCTCTA
AGAGCCAGGTGGTGTTCACCATGACAAACATGGACCCCGTGGACACCGCCACCTACTATTGTG
CCAGAAAGGGCGTCGGCATGGGCCTGGGCTACTGGGGACAGGGCACCCTGGTGACCGTGTCCT
CTGGCGGAGGTGGCTCTGGAGGCGGTGGATCTGGTGGTGGCGGATCAGACATCCAGATGACCC
AGAGCCCTAGCTCTGTGTCCGCCAGCGTGGGCGACAGAGTGACCATCACCTGCCTGGCCAGCC
AAACAATCGGCACCTGGGTCGCCTGGTACCAGCAGAAACCTGGAAAAAGCCCACAGCTGCTGA
TCTACGGCGCCACAAACCTGGCTGATGGCGTGCCCAGCCGGTTCAGCGGATCTGGCTCCGGAA
CCAAGTTCACACTGACCATTAGCAGCCTCCAGCCCGAGGATTTCGCTACCTACTACTGCCAGCA
ACTGTACAGCAACCCTTACACCTTCGGCGGCGGCACCAAGCTGGAAATCAAGACTAGTACCAC
GACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG
CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCT
GTGACATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTAT
CACCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACA
AACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTG
AACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG
CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGC
CGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGA
ACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA
GGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACG
CCCTTCACATGCAGGCCCTGCCCCCTCGCGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACG
TGGAGGAGAATCCCGGCCCTATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACA
CCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGAC
TCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATC
TCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACA
GGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCT
GAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCA
ACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTC
AAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACA
ATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGT
GAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGG
GGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGAC
AAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGC
CACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGT ATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATG
GGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCAT
CCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAG
ATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGA
TCGGCCTCTTCATGTGAAAGCTTATCGCGATACCGTCGACCTCGAGGGAATTCCGATAATCAAC
CTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTA
TGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCC
TCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTG
GCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCA
GCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCC
TTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAA
GCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCT
GCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCG
GCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC
ATCGGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCC
ACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATGGG
ATCAATTCACCATGGGAATAACTTCGTATAGCATACATTATACGAAGTTATGCTGCTTTTTGCTT
GTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCC
ACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGT
GACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGCATCT
AGAATTAATTCCGTGTATTCTATAGTGTCACCTAAATCGTATGTGTATGATACATAAGGTTATG
TATTAATTGTAGCCGCGTTCTAACGACAATATGTACAAGCCTAATTGTGTAGCATCTGGCTTAC
TGAAGCAGACCCTATCATCTCTCTCGTAAACTGCCGTCAGAGTCGGTTTGGTTGGACGAACCTT
CTGAGTTTCTGGTAACGCCGTCCCGCACCCGGAAATGGTCAGCGAACCAATCAGCAGGGTCAT
CGCTAGCCAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGT
TGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCAT
GAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCAT
CTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGC
TTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAATGGTGCACTCTCAGTACAATCTGCTCT
GATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT
TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGA
GGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATA
GGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGC
GGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACC
CTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC
CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT
AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGG
TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTG
CTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACT
ATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGA
CAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT
GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAAC
TCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCAC
GATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT
TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCG
GCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTA
TCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGA
GTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGC
ATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA
TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGT
TTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTT
TCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG
GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT
ACTGTCTTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACAT
ACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGG
GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATG
AGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCG
GAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCG
GGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG
GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG
TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATAC
CGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCC
CAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGTGGAATGTGTGT
CAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTC
AATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGC
ATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTC
CGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG
GCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTT
GCAAAAAGCTTGGACACAAGACAGGCTTGCGAGATATGTTTGAGAATACCACTTTATCCCGCG
TCAGGGAGAGGCAGTGCGTAAAAAGACGCGGACTCATGTGAAATACTGGTTTTTAGTGCGCCA
GATCTCTATAATCTCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGATAAACAGG
CTGGGACACTTCACATGAGCGAAAAATACATCGTCACCTGGGACATGTTGCAGATCCATGCAC
GTAAACTCGCAAGCCGACTGATGCCTTCTGAACAATGGAAAGGCATTATTGCCGTAAGCCGTG
GCGGTCTGTACCGGGTGCGTTACTGGCGCGTGAACTGGGTATTCGTCATGTCGATACCGTTTGT
ATTTCCAGCTACGATCACGACAACCAGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAGGCGAT
GGCGAAGGCTTCATCGTTATTGATGACCTGGTGGATACCGGTGGTACTGCGGTTGCGATTCGTG
AAATGTATCCAAAAGCGCACTTTGTCACCATCTTCGCAAAACCGGCTGGTCGTCCGCTGGTTGA
TGACTATGTTGTTGATATCCCGCAAGATACCTGGATTGAACAGCCGTGGGATATGGGCGTCGTA
TTCGTCCCGCCAATCTCCGGTCGCTAATCTTTTCAACGCCTGGCACTGCCGGGCGTTGTTCTTTT
TAACTTCAGGCGGGTTACAATAGTTTCCAGTAAGTATTCTGGAGGCTGCATCCATGACACAGGC
AAACCTGAGCGAAACCCTGTTCAAACCCCGCTTTAAACATCCTGAAACCTCGACGCTAGTCCGC
CGCTTTAATCACGGCGCACAACCGCCTGTGCAGTCGGCCCTTGATGGTAAAACCATCCCTCACT
GGTATCGCATGATTAACCGTCTGATGTGGATCTGGCGCGGCATTGACCCACGCGAAATCCTCGA
CGTCCAGGCACGTATTGTGATGAGCGATGCCGAACGTACCGACGATGATTTATACGATACGGT
GATTGGCTACCGTGGCGGCAACTGGATTTATGAGTGGGCCCCGGATCTTTGTGAAGGAACCTTA
CTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATA
AAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAAC
CTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAACCTGTTTTGCTC
AGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAA
AAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAGTCAT
GCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCAC
TGCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAAGTAGGCATAACAGTTATAA
TCATAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTC
AAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTTGATGTATAG
TGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAA
CCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTT
ATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTT
TTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAAC
TGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCCA
ATTACCTAGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAA
TTGTATTTGTTAAATATGTACTACAAACTTAGTAG
SEQ ID NO: 52 is an exemplary sequence encoding the pMH289-A10-2 construct.
TTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACA
CAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACC
TTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGA
GAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTG
TTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAG
TACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGA
GGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTT TTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAG
GGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCT
GTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC
AGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGG
ACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAA
AATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGG
GGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAA
TTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTA
GAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCA
GAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAG
ATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCAC
CGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTG
GAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAA
GGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTG
GGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCA
GACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAAC
AGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGG
AAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCA
CCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACAC
GACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGA
AGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAA
GTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT
AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAG
GGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAA
GGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC
TCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAA
GAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAA
ATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGATCGATGGCTCCGGTGCCCGT
CAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTG
AACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCG
CCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTT
CGCAACGGGTTTGCCGCCAGAACACAGGTGTCGTGACGCGGATCCAGGCCTAAGCTTACGCGT
CCTAGCGCTACCGGTCGCCACCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCA
CACCCAGCATTCCTCCTGATCCCACATATGGATATCCAGATGACACAGAGCCCTAGCAGCGTGT
CCGCCTCTGTGGGCGACAGAGTGACCATTACCTGCCTGGCCAGCCAGACCATCGGAACCTGGG
TCGCCTGGTACCAGCAGAAACCTGGTAAAAGCCCTCAGCTGCTGATCTACGGCGCCACAAACC
TGGCTGATGGCGTTCCTAGCCGGTTCAGCGGCAGCGGCAGCGGAACCAAGTTCACCCTCACAA
TCAGCTCTCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAACTGTACAGCAACCCCTA
CACCTTCGGCGGCGGAACAAAGCTGGAGATCAAGGGCGGAGGTGGCTCTGGAGGCGGTGGAT
CTGGTGGTGGCGGATCACAGGTGACCCTGAAGGAAAGCGGCCCAGTGCTGGTGAAGCCTACAG
AGACACTGACCCTGACCTGTACCGTGTCCGGCTTTAGCCTGACAAACTACGGCATCCACTGGCT
GAGACAGCCTCCAGGCAAGGCCCTGGAATGGCTGGGAATGATGTGGCGGGGCGGAGGAACTG
ATTACAACGCCGCTTTTATCTCCAGACTGACAATCACCAAGGACACCAGCAAGAGCCAAGTGG
TGTTCACCATGACCAATATGGACCCCGTGGACACCGCCACATATTACTGCGCCAGAAAGGGCG
TGGGAATGGGCCTGGGCTACTGGGGCCAGGGCACACTGGTCACCGTGTCTTCTACTAGTACCA
CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGC
GCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCC
TGTGACATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTAT
CACCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACA
AACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTG
AACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG
CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGC
CGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGA
ACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA
GGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACG
CCCTTCACATGCAGGCCCTGCCCCCTCGCGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACG TGGAGGAGAATCCCGGCCCTATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACA
CCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGAC
TCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATC
TCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACA
GGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCT
GAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCA
ACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTC
AAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACA
ATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGT
GAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGG
GGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGAC
AAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGC
CACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGT
ATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATG
GGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCAT
CCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAG
ATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGA
TCGGCCTCTTCATGTGAAAGCTTATCGCGATACCGTCGACCTCGAGGGAATTCCGATAATCAAC
CTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTA
TGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCC
TCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTG
GCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCA
GCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCC
TTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAA
GCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCT
GCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCG
GCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC
ATCGGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCC
ACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATGGG
ATCAATTCACCATGGGAATAACTTCGTATAGCATACATTATACGAAGTTATGCTGCTTTTTGCTT
GTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCC
ACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGT
GACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGCATCT
AGAATTAATTCCGTGTATTCTATAGTGTCACCTAAATCGTATGTGTATGATACATAAGGTTATG
TATTAATTGTAGCCGCGTTCTAACGACAATATGTACAAGCCTAATTGTGTAGCATCTGGCTTAC
TGAAGCAGACCCTATCATCTCTCTCGTAAACTGCCGTCAGAGTCGGTTTGGTTGGACGAACCTT
CTGAGTTTCTGGTAACGCCGTCCCGCACCCGGAAATGGTCAGCGAACCAATCAGCAGGGTCAT
CGCTAGCCAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGT
TGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCAT
GAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCAT
CTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGC
TTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAATGGTGCACTCTCAGTACAATCTGCTCT
GATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT
TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGA
GGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATA
GGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGC
GGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACC
CTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC
CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT
AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGG
TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTG
CTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACT
ATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGA
CAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT
GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAAC
TCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCAC GATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT
TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCG
GCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTA
TCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGA
GTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGC
ATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA
TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGT
TTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTT
TCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG
GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT
ACTGTCTTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACAT
ACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGG
GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG
CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATG
AGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCG
GAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCG
GGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG
GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG
TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATAC
CGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCC
CAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGTGGAATGTGTGT
CAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTC
AATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGC
ATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTC
CGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG
GCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTT
GCAAAAAGCTTGGACACAAGACAGGCTTGCGAGATATGTTTGAGAATACCACTTTATCCCGCG
TCAGGGAGAGGCAGTGCGTAAAAAGACGCGGACTCATGTGAAATACTGGTTTTTAGTGCGCCA
GATCTCTATAATCTCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGATAAACAGG
CTGGGACACTTCACATGAGCGAAAAATACATCGTCACCTGGGACATGTTGCAGATCCATGCAC
GTAAACTCGCAAGCCGACTGATGCCTTCTGAACAATGGAAAGGCATTATTGCCGTAAGCCGTG
GCGGTCTGTACCGGGTGCGTTACTGGCGCGTGAACTGGGTATTCGTCATGTCGATACCGTTTGT
ATTTCCAGCTACGATCACGACAACCAGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAGGCGAT
GGCGAAGGCTTCATCGTTATTGATGACCTGGTGGATACCGGTGGTACTGCGGTTGCGATTCGTG
AAATGTATCCAAAAGCGCACTTTGTCACCATCTTCGCAAAACCGGCTGGTCGTCCGCTGGTTGA
TGACTATGTTGTTGATATCCCGCAAGATACCTGGATTGAACAGCCGTGGGATATGGGCGTCGTA
TTCGTCCCGCCAATCTCCGGTCGCTAATCTTTTCAACGCCTGGCACTGCCGGGCGTTGTTCTTTT
TAACTTCAGGCGGGTTACAATAGTTTCCAGTAAGTATTCTGGAGGCTGCATCCATGACACAGGC
AAACCTGAGCGAAACCCTGTTCAAACCCCGCTTTAAACATCCTGAAACCTCGACGCTAGTCCGC
CGCTTTAATCACGGCGCACAACCGCCTGTGCAGTCGGCCCTTGATGGTAAAACCATCCCTCACT
GGTATCGCATGATTAACCGTCTGATGTGGATCTGGCGCGGCATTGACCCACGCGAAATCCTCGA
CGTCCAGGCACGTATTGTGATGAGCGATGCCGAACGTACCGACGATGATTTATACGATACGGT
GATTGGCTACCGTGGCGGCAACTGGATTTATGAGTGGGCCCCGGATCTTTGTGAAGGAACCTTA
CTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATA
AAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAAC
CTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAACCTGTTTTGCTC
AGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAA
AAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAGTCAT
GCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCAC
TGCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAAGTAGGCATAACAGTTATAA
TCATAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTC
AAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTTGATGTATAG
TGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAA
CCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTT
ATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTT
TTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAAC TGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCCA
ATTACCTAGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAA
TTGTATTTGTTAAATATGTACTACAAACTTAGTAG
DETAILED DESCRIPTION
I. Summary
Disclosed herein are humanized monoclonal antibodies and antigen binding fragments thereof that specifically bind a mutant/variant EGFR expressed by cancer cells (e.g., EGFRvIII or gene-amplified EGFR), but do not bind wild-type EGFR expressed on normal cells. Not only are these humanized monoclonal antibodies less immunogenic, but they also have increased binding to target EGFRvIII and/or gene-amplified EGFR.
Activation of wild-type EGFR involves dimerization which requires ligand binding and a monomer to dimer transition with attendant changes in receptor conformation. There are several structures reported for the extracellular domain of EGFR both in monomer and dimer conformations. Analyses of these structures indicate the presence of residues that are not exposed in the wild-type receptor. However, under oncogenic conditions, where receptors are highly expressed and may not be folded correctly or where mutant versions of the receptor are expressed, cryptic structures may become exposed. One structural element that is sterically unavailable under normal conditions is the 287-302 (numbering of mature receptor - or 301-326 of full-length receptor) disulfide-limited loop. This loop is exposed in EGFRvIII and may become exposed when receptor expression is very high or when ECD mutations alter wild type structure. In several examples, an antibody or antigen binding fragment disclosed herein bind to the EGFRisv-stnloop. Also provided are methods of inhibiting or treating a tumor that has the EGFR287-302 loop exposed on cells. Methods of inhibiting a tumor over-expressing EGFR in a subject are also provided.
II. Terms
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers el al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.
As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
To facilitate review of the various aspects, the following explanations of terms are provided:
About: Unless context indicated otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.
Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition (such as a composition including a disclosed humanized monoclonal antibody or antigen binding fragment, etc.) is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes. In one example administration is systemic. In one example, administration is local, for example directly to a tumor.
Agent: Any substance or any combination of substances that is useful for achieving an end or result; for example, a substance or combination of substances useful for inhibiting tumor growth or metastasis in a subject. Agents include proteins, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. An agent can include a therapeutic agent (such as a chemotherapeutic agent), a diagnostic agent or a pharmaceutical agent. In some examples disclosed herein, the agent is a humanized monoclonal antibody that specifically binds EGFRvIII and/or gene-amplified EGFR, an antigen binding fragment thereof, a conjugate thereof, or a chimeric antigen receptor (CAR) including the antibody or antigen binding fragment. A skilled artisan will understand that particular agents may be useful to achieve more than one result.
Amino Acid Substitutions: The replacement of one amino acid in a polypeptide with a different amino acid or with no amino acid (z.e., a deletion). In some examples, an amino acid in a polypeptide is substituted with an amino acid from a homologous polypeptide, for example, an amino acid in a humanized monoclonal antibody that specifically binds EGFRvIII or antigen binding fragment thereof can be substituted with the corresponding amino acid from another antibody that specifically binds EGFRvIII or antigen binding fragment thereof.
Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen), for example, EGFRvIII. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit the desired antigen-binding activity.
Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and antigen binding fragments thereof that retain binding affinity for the antigen. Examples of antigen binding fragments include but are not limited to Fv, Fab, dsFv. Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and ds-scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer- Verlag, 2010).
Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies).
An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally -occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.
Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (E) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). In combination, the heavy and the light chain variable regions specifically bind the antigen.
References to “VH” or “VH” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab. References to “VL” or “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, ds-scFv or Fab.
The VH and VL contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991; “Kabat” numbering scheme), Al-Eazikani et al., (“Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Bio., 273(4):927-948, 1997; “Chothia” numbering scheme), and Eefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27(l):55-77, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the VH of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the VL of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.
In some examples, a disclosed humanized monoclonal antibody includes a heterologous constant domain. For example, the antibody includes a constant domain that is different from a native constant domain, such as a constant domain including one or more modifications (such as the “LS” mutations) to increase half-life.
A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies are described herein. In some examples monoclonal antibodies are isolated from a subject, such as a mammal, such as a human. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions (see, for example, Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press, 2014).
A “humanized” antibody or antigen binding fragment includes amino acid substitutions, usually in a human framework region, as compared to a corresponding non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. In some examples of the present disclosure, a non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In some examples of the present disclosure, all the CDRs and many of the framework residues are from a donor immunoglobulin, but some of the framework residues at particular positions are substituted with an amino acid from the corresponding position in a human framework region. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical.
A “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species. In some examples, a chimeric antibody includes one or more CDRs and/or framework regions from one antibody (e.g., a mouse antibody) and CDRs and/or framework regions from another antibody (e.g., a human antibody).
A “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species. A human antibody can, for example, include CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.: Lonberg, Nat. Biotech., 23: 1117-1125, 2005: Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).
Biological Sample: A sample obtained from a subject. Biological samples include all clinical samples useful for detection of disease or a tumor (for example, a head and neck cancer, breast cancer pancreatic cancer, colorectal cancer, bladder cancer, or a glioma) in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as blood, derivatives and fractions of blood (such as serum), cerebrospinal fluid; as well as biopsied or surgically removed tissue, for example tissues that are unfixed, frozen, or fixed in formalin or paraffin. In a particular example, a biological sample is obtained from a subject having or suspected of having a tumor, such as, but not limited to, head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, glioma, or bladder cancer.
Bispecific Antibody: A recombinant molecule composed of two different antigen binding domains that consequently binds to two different antigenic epitopes. Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv, ds-scFv), or combinations thereof. A bispecific antibody can include one or more constant domains but does not necessarily include a constant domain.
Carcinoma: A malignant tumor including transformed epithelial cells. Non-limiting examples of carcinomas include adenocarcinoma, squamous cell carcinoma, anaplastic carcinoma and large and small cell carcinoma. In some examples, a carcinoma is a breast carcinoma, head and neck carcinoma, pancreatic carcinoma, colorectal carcinoma, glioma carcinoma, or bladder carcinoma.
Chemotherapeutic Agent: Any chemical agent with therapeutic usefulness in the treatment of a disease characterized by abnormal cell growth. For example, chemotherapeutic agents are useful for treating cancer, including, but not limited to, head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, bladder cancer, and glioma. In a non-limiting example, a chemotherapeutic agent is an agent of use in treating a carcinoma. Particular examples of additional therapeutic agents that can be used in combination with the disclosed humanized antibodies include one or more of microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. In a non-limiting example, a chemotherapeutic agent is a radioactive compound. Other examples include the use of anti-neoplastic drugs 5-fluoro uracil (5-FU) and IRT. A practitioner can readily identify a chemotherapeutic agent of use (see, e.g., Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993; Chabner and Longo, Cancer Chemotherapy and Biotherapy: Principles and Practice (4th ed.). Philadelphia: Lippincott Willians & Wilkins, 2005; Skeel, Handbook of Cancer Chemotherapy (6th ed.). Lippincott Williams & Wilkins, 2003). Combination chemotherapy is the administration of more than one agent to treat cancer, such as one or more of the humanized antibodies and another chemotherapeutic agent. Such agents can be administered sequentially, or at the same time.
Chimeric Antigen Receptor (CAR): An engineered T cell receptor having an extracellular antibody-derived targeting domain (such as an scFv) joined to one or more intracellular signaling domains of a T cell receptor. A “chimeric antigen receptor T cell” is a T cell expressing a CAR, and has antigen specificity determined by the antibody-derived targeting domain of the CAR. Methods of making CARs (e.g., for treatment of cancer) have been described (see, e.g., Park et al., Trends Biotechnol., 29:550-557, 201 1 ; Grupp et al., N Engl J Med., 368: 1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT Pubs. W02012/079000, WO2013/059593; and U.S. Pub. 2012/0213783).
Conditions Sufficient to Form an Immune Complex: Conditions that allow an antibody or antigen binding fragment to bind to its cognate epitope to a detectably greater degree than, and/or to the substantial exclusion of, all other epitopes. Conditions sufficient to form an immune complex are dependent upon the format of the binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013) for a description of immunoassay formats and conditions. The conditions employed in the methods are “physiological conditions” which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.
Conjugate: A complex of two molecules linked together, for example, linked together by a covalent bond. In one example, an antibody is linked to an effector molecule; for example, an antibody or antigen binding fragment disclosed herein is covalently linked to an effector molecule. The linkage can be by chemical or recombinant means. In one example, the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.”
Conservative Variants: A sequence variant due to conservative substitutions. “Conservative” amino acid substitutions do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein. For example, an EGFRvIII-specific and/or gene-amplified EGFR-specific humanized monoclonal antibody disclosed herein can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity for EGFRvIII or gene-amplified EGFR, respectively. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.
Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for example less than 5%, less than 3%, less than 1%, etc.) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.
The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
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 (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Non-conservative substitutions are those that reduce an activity or function of an antibody, such as the ability to specifically bind to EGFRvIII and/or gene-amplified EGFR, or bind to a cancer cell expressing EGFRvIII and/or gene -amplified EGFR. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.
Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as a peptide, that contacts another polypeptide. Contacting can also include contacting a cell for example by placing a polypeptide in direct physical association with a cell.
Control: A reference standard. In some examples, the control is a negative control sample obtained from a healthy subject (e.g., one not having a tumor) or a subject not having a tumor that expresses EGFRvIII or gene-amplified EGFR. In other examples, the control is a positive control sample obtained from a subject diagnosed with a tumor that expresses EGFRvIII and/or gene-amplified EGFR, or recombinantly produced purified EGFRvIII. In still other examples, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of subjects with known prognosis or outcome, or group of samples that represent baseline or normal values). A practitioner can readily determine an appropriate control.
A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%. In some examples, tumor growth, volume and/or metastasis is decreased following treatment with a disclosed antibody.
Decrease or Reduce: To reduce in quality, amount, or strength; for example a reduction in tumor burden. In one example, a therapy reduces a tumor (such as the size of a tumor, the number of tumors, the metastasis of a tumor, or combinations thereof), or one or more symptoms associated with a tumor, for example as compared to the response in the absence of the therapy. In a particular example, a therapy decreases the size of a tumor, the number of tumors, the metastasis of a tumor, or combinations thereof, subsequent to the therapy, such as a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Such decreases can be measured using the methods disclosed herein.
Degenerate Variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide sequence variant due to redundancy of the genetic code (there are 20 natural amino acids, most of which are specified by more than one codon). Thus, while the nucleotide sequence of degenerate variants vary, the encoded peptide is the same.
Detectable Marker: A detectable molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, such as a humanized monoclonal antibody, to facilitate detection of the second molecule. For example, the detectable marker can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance in the choice of detectable markers appropriate for various purposes are discussed for example in Green and Sambrook (Molecular Cloning: A Laboratory’ Manual, 4th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).
Detecting: To identify the existence, presence, or fact of something.
Effective Amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject to whom the substance is administered, such as a therapeutically effective amount for treatment of a disease. For instance, this can be the amount of a humanized monoclonal antibody necessary to inhibit tumor growth and/or metastasis, or to measurably alter outward symptoms of the tumor.
In some aspects, administration of an effective amount of a disclosed humanized monoclonal antibody or antigen binding fragment that binds to EGFRvIII and/or gene-amplified EGFR reduces or inhibits tumor growth, tumor metastasis, or tumor volume by a desired amount, for example by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of the tumor), as compared to a suitable control.
The effective amount of a humanized monoclonal antibody or antigen binding fragment that specifically binds to EGFRvIII and/or gene-amplified EGFR that is administered to a subject may vary depending on a number of factors associated with that subject, for example the overall health and/or weight of the subject. An effective amount can be determined by varying the dosage and measuring the resulting response, such as, for example, a reduction in tumor burden. Effective amounts also can be determined through various in vitro, in vivo, or in situ immunoassays or clinical trials.
An effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining an effective response. For example, an effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment lasting several days or weeks. However, the effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in an amount, or in multiples of the effective amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.
Effector Molecule: A molecule intended to have or produce a desired effect; for example, a desired effect on a cell to which the effector molecule is targeted. Effector molecules include such molecules as chemical compounds, polypeptides, radioisotopes and small molecules. Non-limiting examples of effector molecules include toxins, chemotherapeutic agents and anti-angiogenic agents. The skilled artisan will understand that some effector molecules may have or produce more than one desired effect. In one example, an effector molecule includes a disclosed humanized monoclonal antibody or fragment thereof. In another example, an effector molecule is a chimeric molecule that includes a disclosed humanized monoclonal antibody or fragment thereof, that is intended to have a desired effect on a cell to which the chimeric molecule is targeted.
Epidermal Growth Factor Receptor (EGFR): EGFR (also known as HER1 or ERBB1) is a receptor belonging to the ERBB family of the receptor thymidine kinases (RTKs). In vivo, ligand-binding by EGF results in the activation of the RTK/RAS/PI(3)K pathway via receptor phosphorylation, and results in cellular proliferation, angiogenesis, and increased local tissue invasion as well as resistance to apoptosis. A nucleic acid sequence for human EGFR can be found at GENBANK® Accession No. NM_005228.5, June 18, 2019, and GENBANK® Accession No. NC_000007.14 (EGFR in the chromosome), June 14, 2019, both incorporated herein by reference. EGFR has an external domain (ECD) of 621 amino acids, a single pass transmembrane domain (TM) of 23 amino acids and an enzymatically active intracellular domain (ICD) of 542 amino acids. Ligand binding leads to receptor dimer formation and the activation of the kinase domain which signals to one of several pathways that can promote the growth, survival and spread of mammalian cells. In tumors, the loss of exons 2-7 to produces EGFR variant III (EGFRvIII), which is constitutively active. A cDNA sequence for EGFRvIII can be found at GENBANK® Accession No. NM_001346941, June 18, 2019, incorporated herein by reference, and an amino acid sequence can be found at NP_001333870.1, June 14, 2019, incorporated herein by reference. Activation of wild-type EGFR involves dimerization which requires ligand binding and a monomer to dimer transition with attendant changes in receptor conformation. Activating mutations can occur in either the ECD or the ICD. The loss of exon 19 generates a constitutively active enzyme mutant. EGFR can be overexpressed either by gene amplification or by reduced transcriptional control. High level expression (such as greater than about 50,000 receptors per cell) leads to either misfolding of the receptor or mutations in one of more of the gene copies. Overexpression can result in a two-fold or greater increase in EGFR present in the cell, as compared to a wild-type control. One structural element that is sterically unavailable under normal conditions is the 287- 302 (numbering of mature receptor - or 301-326 of full-length receptor) disulfide-limited loop. This loop is exposed in EGFRvIII and may become exposed when receptor expression is very high or when ECD mutations alter wild type structure.
Over-expressed EGFR and EGFRvIII is also known to occur in cancers of non-human animals (see, e.g., Cho et al. Oncology 58(4): 674-682, 2021). EGFR of non-human primates has the same reactive loop sequence of human EGFR (EGFR287-302 loop), where the antibodies disclosed herein may bind. In some examples, the antibodies disclosed herein bind to mammalian EGFRvIII or gene-amplified EGFR. In some examples, the antibodies disclosed herein bind to human, non-human primate, canine, or feline EGFRvIII or gene-amplified EGFR. In a non-limiting example, the antibodies disclosed herein bind to human, non- human primate, canine, or feline EGFRvIII.
Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and/or T cells respond. An antibody can bind to a particular antigenic epitope, such as an epitope on EGFRvIII.
Expression: Transcription or translation of a nucleic acid sequence. For example, a gene is expressed when its DNA is transcribed into an RNA or RNA fragment, which in some examples is processed to become mRNA. A gene may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. The term “expression” is used herein to denote either transcription or translation. Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced. Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.
A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for celltype specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, exemplary inducible promoters include μL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. When cloning in mammalian cell systems, non-limiting exemplary promoters include those derived from the genome of mammalian cells (such as metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter). Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of a nucleic acid sequence.
A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates transcription of the inserted genetic sequence in a host cell. Expression vectors typically contain an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.
Expression Vector: A vector that includes expression control sequences (e.g., promoter, enhancer, terminator, etc.) operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, for example, cosmids, plasmids (e.g., naked or contained in liposomes) and viral vectors (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
Fc Region: The constant region of an antibody excluding the first heavy chain constant domain. Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains Cy2 and Cy3 and optionally the lower part of the hinge between Cyl and Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues following C226 or P230 to the Fc carboxyl-terminus, wherein the numbering is according to Kabat. For IgA, the Fc region includes immunoglobulin domains Ca2 and Ca3 and optionally the lower part of the hinge between Cal and Ca2.
Framework Region: Amino acid sequences interposed between CDRs in a heavy or light variable region of an antibody. Includes variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation.
Gene- Amplified EGFR: An increased copy number of an EGFR gene in a cell (e.g., a tumor cell) relative to a normal cell (e.g., non-tumor cell) that results in over-expression of EGFR. Typically, a cell (e.g., a tumor cell) that includes a copy number of EGFR greater than 6 is considered to have gene-amplified EGFR. Gene-amplified EGFR has been described, for example, in French et al. “Defining EGFR amplification status” Neuro-Oncology, 21(19): 1263-1272, 2019.
In some examples, gene-amplified EGFR includes a copy number of an EGFR gene greater than 6, for example, a copy number of at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more of an EGFR gene. In a non-limiting example, gene-amplified EGFR includes an EGFR gene copy number of at least 7. In some examples, gene-amplified EGFR includes a copy number of 6 to 200 of an EGFR gene, such as 6 to 150, 6 to 100, 6 to 75, 6 to 50, 6 to 25, 6 to 10, 7 to t50, 7 to tOO, 7 to 75, 7 to 50, 7 to 25, 7 to 10, 8 to 150, 8 to 100, 8 to 75, 8 to 50, 8 to 25, 8 to 10, 10 to 150, 10 to 100, 10 to 75, 10 to 50, 10 to 25, 15 to 150, 15 to 100, 15 to 75, 15 to 50, 15 to 25, 20 to 150, 20 to 100, 20 to 75, or 20 to 50 copies of an EGFR gene. In a non-limiting example, gene-amplified EGFR includes a copy number of 7 to 100 of an EGFR gene. Gene amplification results in over-expression of EGFR in a cell (e.g., a tumor cell). In some examples, at least one copy of EGFR includes an activating mutation (e.g., a substitution, insertion, or deletion that activates EGFR).
Heterologous: Originating from a different genetic source. A nucleic acid molecule that is heterologous to a cell originated from a genetic source other than that cell. In a specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein, such as a disclosed humanized monoclonal antibody or fragment thereof, is expressed in a cell, such as a mammalian cell. Methods for introducing a heterologous nucleic acid molecule in a cell or organism are known in the art, for example transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.
Host Cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.
IgG: A polypeptide belonging to the class or isotype of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class comprises IgGi, IgG 2, IgG , and IgG i. Immune Complex: The binding of antibody or antigen binding fragment (such as a scFv) to a soluble antigen forms an immune complex. The formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, radiography, and affinity chromatography.
Immune Response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. The response can be specific for a particular antigen (an “antigen-specific response”). In some examples, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In other examples, the response is a B cell response, and results in the production of specific antibodies.
Immunogen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal, such as EGFRvIII coupled to a carrier. An immunogen can be used to produce antibodies, such as those disclosed herein.
Inhibiting or Treating a Tumor: A therapeutic intervention (for example, administration of a therapeutically effective amount of a humanized monoclonal antibody disclosed herein) that reduces a sign or symptom of a tumor. In some examples, treatment reduces the size of a tumor, number of tumors, or induces remission. In particular examples, treatment includes inhibiting metastasis.
The term “reduces” is a relative term, such that an agent reduces a disease or condition if the disease or condition is quantitatively diminished following administration of the agent, or if it is diminished following administration of the agent, as compared to a reference agent. Reducing a sign or symptom refers to any observable beneficial effect of the treatment. Reducing a sign or symptom associated with a tumor can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject (such as a subject having a tumor which has not yet metastasized), a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease (for example by prolonging the life of a subject having tumor), a reduction in the number of tumors or the time between removal of a tumor and the reappearance of the tumor, an improvement in the overall health or well-being of the subject, or by other known parameters specific to the particular tumor.
A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a tumor, but has a genetic predisposition to the tumor, or exhibits only early signs, such as a pre-cancerous lesion, for the purpose of decreasing the risk of developing the tumor. The term “prevents” does not necessarily mean that an agent completely eliminates the disease or condition, so long as at least one characteristic of the disease or condition is eliminated. Thus, a composition that reduces or prevents a tumor, can, but does not necessarily completely, prevent risk for developing a tumor.
Isolated: A biological component (such as a nucleic acid, peptide, protein or protein complex, for example an antibody) that has been substantially separated, produced apart from, or purified away from, other components of a cell or sample. Thus, isolated nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as, chemically synthesized nucleic acids. An isolated nucleic acid, peptide or protein, for example an antibody, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
Kabat Position: A position of a residue in an amino acid sequence that follows the numbering convention delineated by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIH Publication No. 91-3242, 1991).
Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link an effector molecule to an antibody, or a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine- serine linkers.
The terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule or detectable marker radionuclide or other molecule to a polypeptide, such as an scFv. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.
Neoplasia, Cancer, or Tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue or can metastasize (or both) is referred to as “malignant.”
Tumors of the same tissue type are primary tumors originating in a particular organ or part of the body (such as head and neck, breast, pancreas, colon or rectum, central nervous system (CNS), or bladder). Tumors of the same tissue type may be divided into tumors of different sub-types. For examples, lung carcinomas can be divided into adenocarcinomas, small cell, squamous cell, or non-small cell tumors.
Examples of solid tumors, such as sarcomas (connective tissue cancer) and carcinomas (epithelial cell cancer), include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, head and neck carcinoma, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).
Nucleic Acid: A polymer composed of nucleotide units (e.g., ribonucleotides, deoxyribonucleotides) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”
“Nucleotide” includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.
Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a singlestranded nucleotide sequence is the 5'-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as “upstream sequences;” sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as “downstream sequences.”
“cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.
“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate variants of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. A first sequence is an “antisense” with respect to a second sequence if a polynucleotide whose sequence is the first sequence specifically hybridizes with a polynucleotide whose sequence is the second sequence.
Operably Linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Pharmaceutically Acceptable Carrier: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed humanized monoclonal antibodies and antigen binding fragments thereof.
The nature of the carrier may depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the carrier is sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.
Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” includes naturally occurring amino acid polymers and non-naturally occurring amino acid polymer, such as those in which one or more amino acid residue is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.
Polypeptide Modifications: Polypeptides and peptides, such as the antibodies disclosed herein, can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of formula NR1R2 wherein Ri and R2 are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C1-C16 alkyl or dialkyl amino or further converted to an amide.
Hydroxyl groups of the peptide side chains can be converted to C1-C16 alkoxy or to a C1-C16 ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains can be substituted with one or more halogen atoms, such as F, Cl, Br or I, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well- recognized protecting groups, such as acetamide groups. Methods for introducing cyclic structures into the peptides, for example to select and provide conformational constraints to the structure that result in enhanced stability, have been described. For example, a C- or N-terminal cysteine can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, generating a cyclic peptide. Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody or fragment thereof) is more enriched than the peptide or protein is in its natural environment within a cell. In one example, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or more. In a non-limiting example, a preparation is purified such that a protein (e.g., an antibody disclosed herein) represents at least 70% of the total protein content of the preparation.
Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished for example by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant protein has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several examples, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of inducing expression of the encoded protein in a host cell. In some examples, a recombinant nucleic acid can be integrated into a host cell chromosome.
Sequence Identity: The degree of similarity between amino acid or nucleic acid sequences. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology ); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
Methods of alignment of sequences for comparison are known. Various programs and alignment algorithms are described, for example, in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166-^1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.1 1 , 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. In some examples, sequence identity is determined by using a BLAST program with default parameters.
Homologs and variants of a polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. A practitioner will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are typically used.
As used herein, reference to “at least 80% identity” refers to “at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence. As used herein, reference to “at least 90% identity” refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.
Specifically Bind: When referring to an antibody or antigen binding fragment, refers to a binding reaction which determines the presence of a target protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated conditions, an antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as an antigen present on the surface of a tumor cell, for example EGFRvIII) and does not bind in a significant amount to other proteins present in the sample or subject, including wild-type EGFR expressed on wild-type (non-tumor) cells from the same tissue. An antibody, such as an antibody disclosed herein, can specifically bind EGFRvITI and/or forms of EGFR that are overexpressed on tumor cells (e.g., gene- amplified EGFR), but not bind wild-type EGFR expressed on wild-type (non-tumor) cells of the same tissue. Specific binding can be determined by standard methods. See Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed., Cold Spring Harbor Publications, New York (2013), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
With reference to an antibody-antigen complex, specific binding of the antigen and antibody has a KD of less than about 10'7 Molar, such as less than about 10'8 Molar, 10'9, or even less than about IO 10 Molar. KD refers to the dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction. For example, for the bimolecular interaction of an antibody or antigen binding fragment and an antigen it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.
A humanized monoclonal antibody that specifically binds to an epitope on EGFRvIII is a humanized monoclonal antibody that binds substantially to EGFRvIII protein, including cells or tissue expressing EGFRvIII, substrate to which EGFRvIII is attached, or EGFRvIII in a biological specimen or isolated from a biological specimen. It is, of course, recognized that a certain degree of non-specific interaction may occur between an antibody and a non-target (such as a cell of the same tissue type that does not express wild-type EGFR). Typically, specific binding results in a much stronger association between the antibody and protein or cells bearing the antigen than between the antibody and protein or cells lacking the antigen. Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody (per unit time) to a protein including the epitope or cell or tissue expressing the target epitope as compared to a protein or cell or tissue lacking this epitope. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.
Subject: Living multi-cellular vertebrate organisms, a category that includes human and nonhuman mammals. In some examples, the subject is a mammalian subject, for example, a human, non-human primate, canine, or feline. In a non-limiting example, the subject is a human. In a particular example, the subject has a cancer. In an additional example, a subject is selected that is in need of inhibiting of growth of a tumor or metastasis. For example, the subject can be one who has been diagnosed with a tumor that expresses EGFRvIII and/or gene-amplified EGFR, such as a head and neck, breast, pancreas, colon, CNS, or bladder carcinoma, and is in need of treatment.
T Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4+ T cells and CD8+ T cells. A CD4+ T lymphocyte is an immune cell that expresses CD4 on its surface. These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. Thl and Th2 cells are functional subsets of helper T cells. Thl cells secrete a set of cytokines, including interferon-gamma, and whose principal function is to stimulate phagocyte-mediated defense against infections, especially related to intracellular microbes. Th2 cells secrete a set of cytokines, including interleukin (IL)-4 and IL-5, and whose principal functions are to stimulate IgE and eosinophil/mast cell-mediated immune reactions and to downregulate Thl responses.
Therapeutic Agent: Used in a generic sense, it includes treating agents, prophylactic agents, and replacement agents. A therapeutic agent is used to ameliorate a specific set of conditions in a subject with a disease or a disorder, such as cancer.
Toxin: An effector molecule that induces cytotoxicity when it contacts a cell. Specific, nonlimiting examples of toxins include, but are not limited to, abrin, ricin, auristatins (such as monomethyl auristatin E (MMAE; see, for example, Francisco et al., Blood, 102: 1458-1465, 2003)) and monomethyl auristatin F (MMAF; see, for example, Doronina et al., BioConjugate Chem., 17: 114-124, 2006), maytansinoids (such as DM1; see, for example, Phillips et al., Cancer Res., 68:9280-9290, 2008), Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin or gelonin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as the domain la of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.
Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.
Treating or Preventing a Disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk of or has a disease such as a tumor. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters specific to a particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
Vector: A nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. In some examples, a viral vector includes a nucleic acid molecule encoding a disclosed humanized monoclonal antibody or antigen binding fragment.
Under Conditions Sufficient For: A phrase that is used to describe any environment that permits a desired activity.
III. Overview of Several Aspects of the Disclosure
Isolated humanized monoclonal antibodies and antigen binding fragments thereof that specifically bind a mutant/variant EGFR expressed by cancer/tumor cells (e.g., EGFRvIII and/or gene-amplified EGFR) are provided. In some aspects, a humanized monoclonal antibody or antigen binding fragment disclosed herein specifically binds EGFRvIII. In some aspects, a humanized monoclonal antibody or antigen binding fragment disclosed herein specifically binds gene-amplified EGFR. In some aspects, a humanized monoclonal antibody or antigen binding fragment disclosed herein specifically binds EGFRvIII, but not to gene-amplified EGFR. In some aspects, a humanized monoclonal antibody or antigen binding fragment disclosed herein specifically binds gene-amplified EGFR, but not to EGFRvIII. In some aspects, a humanized monoclonal antibody or antigen binding fragment disclosed herein specifically bind cells expressing EGFRvIII and/or gene-amplified EGFR, but not wild-type EGFR. In some aspects, an antibody disclosed herein binds cells (e.g., tumor cells) that have the EGFR287-302 loop exposed on the cell surface.
In some aspects, the antibodies disclosed herein bind mammalian EGFRvIII and/or mammalian gene-amplified EGFR, for example, EGFRvIII and/or gene-amplified EGFR of a human, non-human primate (e.g., simians (Siniiiformes), chimps (Pan troglodytes), bonobos (Pan pa.ni.scus)), canine (e.g., Canis lupus familiaris), or feline (e.g., Fells catus). In a non-limiting example, the antibodies disclosed herein bind human EGFRvIII and/or human gene-amplified EGFR.
In several aspects, the monoclonal antibodies and antigen binding fragments provided herein can be used to treat a tumor that expresses EGFRvIII, such as, but not limited to a head and neck carcinoma, breast carcinoma, pancreatic carcinoma, colon or rectal carcinoma, CNS carcinoma, or bladder carcinoma. In several aspects, the monoclonal antibodies and antigen binding fragments can be used to treat a tumor that expresses gene-amplified EGFR, such as, but not limited to a head and neck carcinoma, breast carcinoma, pancreatic carcinoma, colon or rectal carcinoma, CNS carcinoma, or bladder carcinoma. The monoclonal antibodies and antigen binding fragments are useful for treating a tumor in a human or veterinary subject (e.g., non-human primate, dog, cat).
Also disclosed are compositions including an antibody or antigen binding fragment disclosed herein, and a pharmaceutically acceptable carrier. Nucleic acids encoding the antibodies or antigen binding fragments, expression vectors including these nucleic acids, and isolated host cells that express the nucleic acids are also provided.
Compositions including the monoclonal antibodies or antigen binding fragments disclosed herein can be used, for example, for research, diagnostic, and/or therapeutic purposes. For example, the monoclonal antibodies can be used to diagnose and/or treat a subject that has a tumor that expresses EGFRvIII and/or gene -amplified EGFR. The subject can be a human or veterinary subject (e.g., non-human primate, dog, cat).
A. Antibodies and Antigen Binding Fragments
Isolated humanized monoclonal antibodies and antigen binding fragments that specifically bind an epitope of EGFRvIII and/or gene-amplified EGFR are provided. These antibodies are humanized and/or variants of parental monoclonal antibody 40H3 (see, US Patent Publication No. 2022-0380474). In some aspects, an isolated humanized monoclonal antibody or antigen binding fragment disclosed herein specifically binds an epitope of EGFRvIII. In some aspects, an isolated humanized monoclonal antibody or antigen binding fragment disclosed herein specifically binds an epitope of gene-amplified EGFR. The disclosed antibodies include one or more humanized residues, and thus include one or more changes in the framework regions as compared to the parental mouse monoclonal antibody 40H3. In some aspects, the disclosed humanized monoclonal antibodies are less immunogenic than parental monoclonal antibody 40H3. The disclosed monoclonal antibodies surprisingly have increased binding to EGFRvIII, as compared to parental monoclonal antibody 40H3. In several aspects, the disclosed humanized antibodies and antigen binding fragments can inhibit a biological function or property of EGFRvIII protein in vivo, including, but not limited to, a reduction and/or inhibition of tumor growth, or a reduction and/or inhibition of tumor metastasis. The disclosed humanized monoclonal antibodies and antigen binding fragments preferentially bind EGFRvIII and overexpressed EGFR expressed on tumor cells (e.g., gene-amplified EGFR), but do not substantially bind EGFR expressed on wild- type (not cancerous cells). In some aspects, the disclosed humanized monoclonal antibodies and antigen binding fragments bind the EGFR287-302 loop.
One structural element of EGFR that is sterically unavailable under normal conditions (e.g., when expressed on a healthy, non-cancerous cell) is the disulfide-limited loop at residues 287-302 (numbering refers to the mature receptor; alternatively, residues 301-326 of the full-length receptor). The EGFR287-302 loop is exposed in EGFRvIII. In some aspects, the disclosed humanized monoclonal antibodies bind the EGFR287-302 loop. In some examples, the EGFR287-302 loop includes R300. The disclosed antibodies are humanized, and thus include one or more changes in a framework region as compared to the parental mouse monoclonal antibody 40H3.
The discussion of monoclonal antibodies below refers to isolated monoclonal antibodies that include a VH and a VL, which include the HCDRs and LCDRs, respectively. A practitioner will understand that various CDR numbering schemes (such as the Kabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions. The amino acid sequence and the CDR positions of the heavy and light chain of the disclosed monoclonal antibodies are shown herein using Kabat, IGMT, or Chothia numbering.
However, a practitioner will readily understand use of various CDR numbering schemes when referencing particular amino acids of the antibodies disclosed herein. Programs for the identification of CDRs using Chothia, IGMT, or Kabat are publicly available.
The antibodies disclosed herein include a heavy chain variable region (VH) and a light chain variable region (VL). In some examples, the antibodies disclosed herein include a VH and a VL combination described in Table 1 (excluding Al).
Table 1: Exemplary antibodies, corresponding VH and VL, and respective cell binding.
Figure imgf000039_0001
Figure imgf000040_0001
1 MDA-MB-468
* Chimera: mouse VH and VL from 40H3 antibody + human IgGICH/human IgkappaCL
In some implementations, the antibodies disclosed herein include a heavy chain variable region (VH) and a light chain variable region (VL) including: a) SEQ ID NO: 5 and SEQ ID NO: 8, respectively (A10, VH3+VL3); b) SEQ ID NO: 3 and SEQ ID NO: 6, respectively (A2, VH1+VL1); c) SEQ ID NO: 3 and SEQ ID NO: 7, respectively (A3, VH1+VL2); d) SEQ ID NO: 3 and SEQ ID NO: 8, respectively (A4, VH1+VL3); e) SEQ ID NO: 4 and SEQ ID NO: 6, respectively (A5, VH2+VL1); f) SEQ ID NO: 4 and SEQ ID NO: 7, respectively (A6, VH2+VL2); g) SEQ ID NO: 4 and SEQ ID NO: 8, respectively (A7, VH2+VL3); h) SEQ ID NO: 5 and SEQ ID NO: 6, respectively (A8, VH3+VL1); i) SEQ ID NO: 5 and SEQ ID NO: 7, respectively (A9, VH3+VL2); j) SEQ ID NO: 1 and SEQ ID NO: 9, respectively (Bl, 40H3 VH+VL-EG); k) SEQ ID NO: 1 and SEQ ID NO: 10, respectively (B2, 40H3 VH+VL-DA); l) SEQ ID NO: 4 and SEQ ID NO: 1 1 , respectively (B3, VH2+VL1-DA); m) SEQ ID NO: 4 and SEQ ID NO: 12, respectively (B4, VH2+VL2-DA); n) SEQ ID NO: 5 and SEQ ID NO: 11, respectively (B5, VH3 and VL1-DA); o) SEQ ID NO: 31 and SEQ ID NO: 32, respectively (CIO); p) SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2); or q) SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 5 and SEQ ID NO: 8, respectively (A10, VH3+VL3). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 3 and SEQ ID NO: 6, respectively (VH1+VL1). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 3 and SEQ ID NO: 7, respectively (VH1+VL2). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 3 and SEQ ID NO: 8, respectively (VH1+VL3). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 4 and SEQ ID NO: 6, respectively (VH2+VL1). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 4 and SEQ ID NO: 7, respectively (VH2+VL2). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 4 and SEQ ID NO: 8, respectively (VH2+VL3). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 5 and SEQ ID NO: 6, respectively (VH3+VL1). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 5 and SEQ ID NO: 7, respectively (VH3+VL2). The humanized monoclonal antibody can include a VH and a Vi, including the amino acids set forth as SEQ ID NO: 1 and SEQ ID NO: 9, respectively (40H3 VH+VL-EG). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 1 and SEQ ID NO: 10, respectively (40H3 VH+VL-DA). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 4 and SEQ ID NO: 11, respectively (VH2+VL1-DA). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 4 and SEQ ID NO: 12, respectively (VH2+VL2-DA). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 5 and SEQ ID NO: 11, respectively (VH3 and VL1-DA). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 31 and SEQ ID NO: 32, respectively (CIO). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2). The humanized monoclonal antibody can include a VH and a VL including the amino acids set forth as SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3). In several implementations, the antibodies disclosed herein bind EGFRvIII and/or gene-amplified EGFR. In some examples, the antibodies disclosed herein bind EGFRvIII. In some examples, the antibodies disclosed herein bind gene-amplified EGFR.
The presently disclosed monoclonal antibodies and antigen binding fragments are humanized. Human framework regions, and mutations that can be made in a human antibody framework regions, can be introduced into antibodies using various methods (see, for example, in U.S. Patent No. 5,585,089, Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239: 1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:431 , 1992; and Singer et al., J. Immunol. 150:2844, 1993). 1. Additional Description of Antibodies and Antigen Binding Fragments
The humanized monoclonal antibody can be of any isotype. The monoclonal antibody can be, for example, an IgM or an IgG antibody, such as IgGi, IgG2, IgG;,, or IgG4. The class of a humanized monoclonal antibody disclosed herein can be switched with another. In one aspect, a nucleic acid molecule encoding VL or VH is isolated, such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively. A nucleic acid molecule encoding VL or VH is then operatively linked to a nucleic acid sequence encoding a CL or CH from a different class of immunoglobulin molecule. This can be achieved using a vector or nucleic acid molecule that includes a CL or CH chain, as known in the art. For example, a humanized monoclonal antibody that was originally IgG may be class switched. Class switching can be used to convert one IgG subclass to another, such as from IgGi to IgGz, IgG i. or IgG4.
In some examples, the disclosed humanized antibodies can be prepared as oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, septamers, octomers and so on.
(a) Binding affinity
In several aspects of the disclosure, the antibody or antigen binding fragment specifically binds EGFRvIII or gene-amplified EGFR, for example, with an affinity (e.g., measured by Kd) of no more than 1 .0 x 10'8 M, no more than 5.0 x 10'8 M, no more than 1.0 x 10'9M, no more than 5.0 x 10'9 M, no more than 1.0 x 10 10M, no more than 5.0 x 10 10M, or no more than 1.0 x 10'11 M. In a non-limiting example, an antibody or antigen binding fragment disclosed herein specifically binds EGFRvIII or gene-amplified EGFR with an affinity (Kd) of less than 6 nm. In a non-limiting example, an antibody or antigen binding fragment disclosed herein specifically binds EGFRvIII protein with an affinity (Kd) of less than 4 nm. In some aspects, an antibody or antigen binding fragment disclosed herein specifically binds EGFRvIII or gene- amplified EGFR with an affinity (Kd) of 0.5 to 10 nanomolar, for example, 0.5 to 9 nm, 1 to 9 nm, 1 to 8 nm, 1 to 7 nm, 1 to 6 nm, 1 to 5 nm, 1 to 4 nm, 1 to 3 nm, 2 to 9 nm, 2 to 8 nm, 2 to 7 nm, 2 to 6 nm, 2 to 5 nm, 2 to 4 nm, 2 to 3 nm, 3 to 9 nm, 3 to 8 nm, 3 to 7 nm, 3 to 6 nm, 3 to 5 nm, or 3 to 4 nm. In a non-limiting example, an antibody or antigen binding fragment disclosed herein specifically binds EGFRvIII or gene- amplified EGFR with an affinity (Kd) of 1 nm to 6 nm. In a non-limiting example, an antibody or antigen binding fragment disclosed herein specifically binds EGFRvIII or gene-amplified EGFR with an affinity (Kd) of 2 nm to 6 nm. In some examples, affinity is measured using the Octet® Bio-Layer Interferometry (BLI) Platform. In several aspects of the disclosure, the antibody or antigen binding fragment disclosed herein specifically binds a product of gene-amplified EGFR, for example, with an affinity (e.g., measured by Kd) of no more than 1.0 x 10'8 M, no more than 5.0 x 10'8 M, no more than 1.0 x 10'9 M, no more than 5.0 x 10'9 M, no more than 1.0 x 10‘10M, no more than 5.0 x 1010M, or no more than 1.0 x 10'11 M. In some examples, the binding affinity of an antibody or antigen binding fragment disclosed herein for EGFRvIII is 2 nm to 5 nm (e.g., 2 nm to 4 nm, 2 nm to 3 nm, 3 nm to 5 nm, or 3 nm to 4 nm). In other examples, Kd can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with the Fab version of a humanized monoclonal antibody of interest and its antigen using known methods. In one assay, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( 1251) -labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see. e.g., Chen et al., J. Mol. Biol. 293:865- 881 (1999)). To establish conditions for the assay, MICROTITER® multi- well plates (Thermo Scientific) are coated overnight with 5 pg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [12’I]- antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed, and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 pl/well of scintillant (MICROSCINT-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.
In another assay, Kd can be measured using surface plasmon resonance assays using a BIACORE®- 2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE®, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (-0.2 pM) before injection at a flow rate of 5 1/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C at a flow rate of approximately 25 1/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k0ff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M s-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette. (b) Multispecific antibodies
In some aspects, a disclosed humanized monoclonal antibody or antigen binding fragment is included on a multispecific antibody, such as a bi-specific antibody. Such multispecific antibodies can be produced by known methods, such as crosslinking two or more antibodies, antigen binding fragments (such as scFvs) of the same type or of different types. Exemplary methods of making multispecific antibodies include those described in PCT Pub. No. WO2013/163427. Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m- maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.
In some aspects, the disclosed humanized monoclonal antibody or antigen binding fragment is included on a bispecific antibody that that specifically binds to EGFRvIII and/or gene- amplified EGFR, and further specifically binds to a second tumor antigen, such as Her-2, or a checkpoint inhibitor, for example, programmed death (PD)-l, or PD ligand (PD-L1) or PD-L2. In some examples, a bispecific antibody further binds a MET oncogene antigen (see, e.g., Comoglio et al., “Known and novel roles of the MET oncogene in cancer: a coherent approach to targeted therapy,” Nature Reviews Cancer, 18:341-358, 2018).
Various types of multi-specific antibodies are known. Bispecific single chain antibodies can be encoded by a single nucleic acid molecule. Examples of bispecific single chain antibodies, as well as methods of constructing such antibodies have been described (see, e.g., U.S. Pat. Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538). Additional examples of bispccific single chain antibodies can be found in PCT application No. WO 99/54440; Mack, J. Immunol., 158:3965-3970, 1997; Mack, PNAS, 92:7021-7025, 1995; Kufer, Cancer Immunol. Immunother., 45: 193-197, 1997; Loffler, Blood, 95:2098-2103, 2000; and Bruhl, J. Immunol., 166:2420-2426, 2001. Production of bispecific Fab-scFv (“bibody”) molecules are described, for example, in Schoonjans et al. (J. Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786:161-76, 2003). For bibodies, a scFv molecule can be fused to one of the VL-CL (L) or VH-CH1 chains, e.g., to produce a bibody one scFv is fused to the C-term of a Fab chain.
(c) Antigen Binding Fragments
Also disclosed are antigen binding fragments (e.g., Fab or scFv) of any monoclonal antibody disclosed herein (e.g., A10). Antigen binding fragments disclosed herein include a heavy chain and light chain variable region and specifically bind EGFRvIII and/or gene-amplified EGFR. Antibody fragments retain the ability to selectively bind with an antigen (e.g., EGFRvIII and/or gene-amplified EGFR) and include, for example: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
(3) (Fab'h, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(abr>2 is a dimer of two Fab' fragments held together by two disulfide bonds;
(4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains and disulfide linked forms thereof (dsFV); and
(5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. A scFv is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH-domain and the VL-domain in a scFv, is not decisive for the provided antibodies (e.g., for the provided multispecific antibodies). Thus, scFvs with both possible arrangements (Vu-domain-linker domain-V, .-domain; Vu-domain-linker domain-Vu-domain) may be used. Other forms, such as ds-scFv are also of use.
(6) A dimer of a single chain antibody (scFVi), defined as a dimer of a scFV. This has also been termed a “miniantibody.” Methods of making these fragments have been described (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, 2nd, Cold Spring Harbor Laboratory, New York, 2013).
In further aspects, the antibody binding fragment can be an Fv antibody, which is typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain. To produce Fv antibodies, the VH and the VL can be expressed from two individual nucleic acid constructs in a host cell. If the VH and the VL are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
In an additional example, the Fv fragments include VH and VL chains connected by a peptide linker. These single -chain antigen binding proteins (scFv) are prepared by constructing a nucleic acid molecule encoding the VH and VL domains connected by an oligonucleotide. The nucleic acid molecule is inserted into an expression vector, which is subsequently introduced into a host cell such as a mammalian cell. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs have been described (see, e.g., Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio/Technology 11:1271, 1993; Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). Dimers of a single chain antibody (scFVz), are also contemplated.
Antigen binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as an E. coli cell) of DNA encoding the fragment. Antigen binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see, e.g., U.S. Patent No. 4,036,945 and U.S. Patent No. 4,331,647; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1 -2.8.10 and 2.10.1 -2.10.4).
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent lightheavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Antigen binding single Vn domains, called domain antibodies (dAb), have also been identified from a library of murine VH genes amplified from genomic DNA of immunized mice (Ward et al. Nature 341 :544-546, 1989). Human single immunoglobulin variable domain polypeptides capable of binding antigen with high affinity have also been described (see, for example, PCT Publication Nos. WO 2005/035572 and WO 2003/002609). The CDRs disclosed herein can also be included in a dAb.
In some aspects, one or more of the heavy and/or light chain complementarity determining regions (CDRs) from a disclosed humanized monoclonal antibody is expressed on the surface of another protein, such as a scaffold protein. The expression of domains of antibodies on the surface of a scaffolding protein have been described (see e.g., Liu et al., J. Virology 85(17): 8467-8476, 2011). Such expression creates a chimeric protein that retains the binding for EGFRvIII and/or gene- amplified EGFR. In a specific nonlimiting example, one or more of the heavy chain CDRs is grafted onto a scaffold protein, such as one or more of heavy chain CDR1, CDR2, and/or CDR3. One or more CDRs can also be included in a diabody or another type of single chain antibody molecule.
In some aspects, the antigen binding fragment disclosed herein is an scFv. In some examples, the antigen binding fragment includes an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to SEQ ID NO: 43 or 45. In some examples, the antigen binding fragment includes or consists of SEQ ID NO: 43 or 45. In some aspects, the antigen binding fragment disclosed herein is an Fab. In some examples, the antigen binding fragment includes an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to SEQ ID NO: 47 and/or 49. In some examples, the antigen binding fragment includes or consists of SEQ ID NO: 47 and/or 49.
(d) Variants
Amino acid sequence variants of the humanized antibodies provided herein are included in the disclosure. For example, it may be desirable to further improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of a humanized monoclonal antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., specific binding to EGFRvIII and/or gene -amplified EGFR.
In certain aspects, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and the framework regions. Amino acid substitutions may be introduced into a humanized monoclonal antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
The variants typically retain amino acid residues necessary for correct folding and stabilizing between the Vn and the VL regions and retain the charge characteristics of the residues to preserve the low pl and low toxicity of the molecules. Amino acid substitutions can be made in the VH and the VL regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are known, for example, the following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
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 (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
In some aspects, the heavy chain of the antibody includes up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs: 3, 4, 5, and/or 31. In some aspects, the light chain of the antibody includes up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 14, 24 or 32.
In some aspects, the antibody or antigen binding fragment can include up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) in the framework regions of the heavy chain of the antibody, and/or the light chain of the antibody, or the heavy and light chains of the antibody, compared to a known framework region, or compared to the framework regions of the antibodies as disclosed herein, and maintain the specific binding activity for EGFRvIII protein and/or a product of gene-amplified EGFR.
In certain aspects, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind a target antigen (e.g., EGFRvIII and/or gene-amplified EGFR). For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. In certain examples of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions. In some examples, amino acid modifications can be made the L-CDR2.
To increase binding affinity of the antibody, the Vi and VH segments can be randomly mutated, such as within H-CDR3 region or the L-CDR3 region, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. Thus in vitro affinity maturation can be accomplished by amplifying VH and VL regions using PCR primers complementary to the H-CDR3 or L-CDR3, respectively. In this process, the primers have been "spiked" with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VH and VL segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL segments can be tested to determine the binding affinity for EGFRvIII and/or a product of gene-amplified EGFR. Methods of in vitro affinity maturation have been described (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).)
A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties. In certain aspects, a humanized monoclonal antibody or antigen binding fragment is altered to increase or decrease the extent to which the antibody or antigen binding fragment is glycosylated. Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in a humanized monoclonal antibody may be made in order to create variants with certain improved properties.
In one aspect, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose -deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533- 545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and W02003/085107).
Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.y, and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function.
Such antibody variants are described, e.g., in WO 1997/30087 (Patel et ally, WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
In several aspects, the constant region of the antibody includes one or more amino acid substitutions to optimize in vivo half-life of the antibody. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in several aspects, the antibody includes an amino acid substitution that increases binding to the FcRn. Several such substitutions are known, such as substitutions at IgG constant regions T250Q and M428L (see, e.g., Hinton et al., J Immunol.. 176:346-356, 2006): M428L and N434S (the “LS” mutation, see, e.g., Zalevsky, et al., Nature Biotechnology, 28:157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769, 2006); and M252Y, S254T, and T256E (see. e.g., Dall’Acqua et al.. J. Biol. Chem., 281:23514-23524, 2006).
In some aspects, the constant region of the antibody includes one or more amino acid substitutions to optimize antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is mediated primarily through a set of closely related Fey receptors. In some aspects, the antibody includes one or more amino acid substitutions that increase binding to FcyRHIa. Several such substitutions are known, such as substitutions at IgG constant regions S239D and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. U.S.A., 103:4005- 4010, 2006); and S239D, A330L, and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. U.S.A., 103:4005- 4010, 2006).
Combinations of the above substitutions are also included, to generate an IgG constant region with increased binding to FcRn and FcyRIIIa. The combinations increase antibody half-life and ADCC. For example, such combinations can include antibodies with the following amino acid substitution in the Fc region: (1) S239D/I332E and T250Q/M428L; (2) S239D/I332E and M428L/N434S; (3) S239D/I332E and N434A; (4) S239D/I332E and T307A/E380A/N434A; (5) S239D/I332E and M252Y / S254T/T256E; (6) S239D/A330L/I332E and T250Q/M428L; (7) S239D/A330L/I332E and M428L/N434S; (8) S239D/A330L/I332E and N434A; (9) S239D/A330L/I332E and T307A/E380A/N434A; or (10) S239D/A330L/I332E and M252Y/S254T/T256E.
In some examples, the antibodies, or an antigen binding fragment thereof is modified such that it is directly cytotoxic to infected cells, or uses natural defenses such as complement, antibody dependent cellular cytotoxicity (ADCC), or phagocytosis by macrophages.
A humanized monoclonal antibody provided herein may be further modified to contain additional nonproteinaceous moieties. Moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n- vinyl pyrrolidonejpolyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
The humanized antibody or antigen binding fragment can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibody or antigen binding fragment is derivatized such that the binding to a desired target is not affected adversely by the derivatization or labeling. For example, the humanized antibody or antigen binding fragment can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bi-specific antibody or a diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate association of the humanized antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
Also included are humanized antibodies that bind to the same epitope on EGFRvIII to which the disclosed humanized monoclonal antibodies provided herein bind. Humanized monoclonal antibodies that bind to such an epitope on the EGFR287-302 loop can be identified based on their ability to cross-compete (for example, to competitively inhibit the binding of, in a statistically significant manner) with the specific antibodies provided herein in binding assays (such as those described in the Examples). A humanized antibody “competes” for binding when the competing antibody inhibits EGFR287-302 loop binding of a humanized monoclonal antibody of the present disclosure by more than 50%, in the presence of competing antibody concentrations higher than 106 x KD of the competing antibody. In a non-limiting example, the humanized antibody that binds to the same epitope on the EGFR287-302 loop as the antibodies of the present disclosure is a human monoclonal antibody. Such humanized monoclonal antibodies can be prepared and isolated as described herein.
B. Conjugates
Humanized monoclonal antibodies or antigen binding fragments disclosed herein, can be conjugated to an agent, such as an effector molecule or detectable marker, using any number of means known in the art. Covalent or non-covalent attachment can be used. Conjugates include, but are not limited to, molecules in which there is a covalent linkage of an effector molecule or a detectable marker to a humanized monoclonal antibody or antigen binding fragment disclosed herein. A practitioner will appreciate that various effector molecules and detectable markers can be used, including (but not limited to) chemotherapeutic agents, anti- angiogenic agents, toxins, radioactive agents such as 1251, 32P, 14C, 3H and 35S and other labels, target moieties and ligands, etc.
The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin that is used to bring about the death of a particular target cell (such as a tumor cell expressing EGFRvIII and/or gene-amplified EGFR).
The procedure for attaching an effector molecule or detectable marker to an antibody or antigen binding fragment varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen binding fragment is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of a number of known linker molecules such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to join the humanized antibody or antigen binding fragment to the effector molecule or detectable marker. The linker is capable of forming covalent bonds to both the humanized antibody or antigen binding fragment and to the effector molecule or detectable marker. Suitable linkers are known and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the humanized antibody or antigen binding fragment and the effector molecule or detectable marker are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
In addition, the linker can include a spacer element, which, when present, increases the size of the linker such that the distance between the effector molecule or the detectable marker and the humanized antibody or antigen binding fragment is increased. Exemplary spacers are known, and include those listed in U.S. Pat. Nos. 7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well as U.S. Pat. Pub. Nos. 20110212088 and 20110070248.
In some examples, the conjugate includes a linker that connects the effector molecule or detectable marker to the humanized monoclonal antibody or antigen binding fragment disclosed herein. In some examples, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the humanized antibody or antigen binding fragment in the intracellular environment. In other examples, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some examples, the linker is cleavable by a cleaving agent that is present in the intracellular environment (for example, within a lysosome or endosome or caveolea). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some aspects, the peptide linker is at least two amino acids long or at least three amino acids long. In some examples, the linker is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long, such as 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, 1-15, amino acids long. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker that is cleavable by the thiol-dependent protease cathepsin-B, can be used (for example, a Phenylalanine -Leucine or a Glycine- Phenylalanine -Leucine-Glycine linker). Other examples of linkers are described, for example, in U.S. Pat. No. 6,214,345. In a specific example, the peptide linker cleavable by an intracellular protease is a Valine-Citruline linker or a Phenylalanine-Lysine linker (see, for example, U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the Valine-Citruline linker).
In other aspects, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid- labile linker that is hydrolyzable in the lysosome (for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used (see, for example, U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67- 123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain examples, a hydrolyzable linker is a thioether linker (such as, for example, a thioether attached to the therapeutic agent via an acylhydrazone bond (see, for example, U.S. Pat. No. 5,622,929)).
In yet other aspects, the linker is cleavable under reducing conditions (for example, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyLoxycarbonyl-alpha-methyl- alpha-(2-pyridyl-dithio)toluene)- , SPDB and SMPT (see, for example, Thorpe et aL, 1987, Cancer Res. 47:5924-5931; Wawrzynczak et a/., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987); Phillips et aL, Cancer Res. 68:92809290, 2008); see also U.S. Pat. No. 4,880,935).
In yet other examples, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299- 1304), or a 3'-N- amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).
In several examples, the linker is resistant to cleavage in an extracellular environment. For example, no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of a conjugate disclosed herein, are cleaved when the conjugate is present in an extracellular environment (for example, in plasma).
Whether or not a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined time period (for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free effector molecule or detectable marker present in the plasma. A variety of exemplary linkers that can be used in conjugates are described in WO 2004-010957, U.S. Publication No. 2006/0074008, U.S. Publication No. 2005/0238649, and U.S. Publication No. 2006/0024317.
The humanized monoclonal antibodies or antigen binding fragments disclosed herein can be derivatized, for example, by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate). Such linkers are commercially available.
A humanized antibody or antigen binding fragment disclosed herein can be conjugated with small molecular weight drugs, for example, Monomethyl Auristatin E (MMAE), Monomethyl Auristatin F (MMAF), maytansine, maytansine derivatives, including the derivative of may tansine known as DM1 (also known as mertansine), deruxtecan, or other chemotherapeutic agents to make an antibody drug conjugate (ADC). Chemotherapeutic agents described herein can be conjugated to the provided antibodies to generate an ADC.
A humanized monoclonal antibody or antigen binding fragment disclosed herein can be conjugated with one or more small molecule toxins, such as a calicheamicin, maytansinoids, dolastatins, auristatins, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity. Maytansine compounds suitable for use as maytansinoid toxin moieties are available and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see, Yu et al (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogues prepared synthetically according to known methods. Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896, 111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533. Conjugates containing maytansinoids, methods of making same, and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0425 235 Bl.
In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies, a practitioner will be able to determine a suitable method for attaching a given agent to a humanized monoclonal antibody or antigen binding fragment disclosed herein.
In a specific, non-limiting example, a conjugate includes a humanized monoclonal antibody that specifically binds EGFRvIII or a product of gene amplified EGFR (or antigen binding fragment thereof) disclosed herein, a non-reducible thioester linker, and the maytansinoid toxin DM1; for example the conjugate can include the structure set forth below (wherein “mAh” refers to the humanized monoclonal antibody or antigen binding fragment thereof):
Figure imgf000055_0001
In some examples, the effector molecule is an auristatin, such as auristatin E (also known as a derivative of dolastatin-10) or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoyl valeric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Application Publication No. 2003/0083263; International Patent Publication No. WO 04/010957, International Patent Publication No. WO 02/088172, and U.S. Pat. Nos. 7,498,298, 6,884,869, 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414. Additional description of antibody drug conjugates including the auristatin MMAE, and methods of making such conjugates, is provided in, e.g., U.S. Pub. Nos. 2011/0268751, 2008/0305044, 2007/0258987. Auristatins have been shown to interfere with microtubule dynamics and nuclear and cellular division and have anticancer activity. Auristatins bind tubulin and can exert a cytotoxic or cytostatic effect on cells. There are a number of different assays known in the art, which can be used for determining whether an auristatin or resultant conjugate exerts a cytostatic or cytotoxic effect on a desired cell line.
In one example, the conj ugate includes a humanized monoclonal antibody (or antigen binding fragment thereof) that specifically binds EGFRvIII or a product of gene amplified EGFR disclosed herein, a cleavable linker including a Valine-Citruline (Val-Cit) peptide cleavage site, a spacer, and the toxin MMAE; for example the conjugate can include the structure set forth below (wherein “mAb” refers to the monoclonal antibody or antigen binding fragment thereof):
Figure imgf000055_0002
In another non-limiting example, the conjugate is
Figure imgf000056_0001
where n is an integer (such as an even integer) from 0 to 10 (such as 0 to 8, 0 to 4, 2 to 4, 2 to 8, 1 to 10, 1 to 8, or 1 to 4, or 2, 4, 6, or 8), A is a humanized monoclonal antibody or antigen binding fragment thereof as disclosed herein, and S is a sulfur atom from the humanized antibody. In one example, preferably n is an even integer from 0 to 8, for example, an even integer from 0 to 4. The S moiety can be exposed by reduction or partial reduction of the inter-chain disulfides of the humanized antibody (e.g., by treatment with a reducing agent such as DTT or TCEP).
In one non-limiting example, the conjugate is:
Figure imgf000056_0002
where n is 4, and A is a monoclonal antibody or antigen binding fragment thereof as disclosed herein.
Additional toxins can be employed with humanized monoclonal antibodies that specifically bind EGFRvIII and/or gene-amplified EGFR, and antigen binding fragment of these antibodies. Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and subunits thereof, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A through F. These toxins are known and many are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins (see, for example, see, U.S. Patent Nos. 5,079,163 and 4,689,401). In some aspects, a conjugate disclosed herein is of use for the treatment of a carcinoma, for example a head and neck carcinoma, a breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or a bladder carcinoma.
Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio/Technology, 10:405-412, 1992). However, the toxin has no mechanism for specific entry into cells, and therefore requires conjugation to an antibody or antigen binding fragment that recognizes a cell-surface protein that is internalized in order to be efficiently taken up by cells. Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or to eliminate non-specific toxicity. A mutant known as CRM107, which has full enzymatic activity but markedly reduced non-specific toxicity, has been known since the 1970’s (Laird and Groman, J. Virol. 19:220, 1976), and has been used in human clinical trials. See, U.S. Patent No. 5,792,458 and U.S. Patent No. 5,208,021.
Ricin is the lectin RCA60 from Ricinus communis (Castor bean). For examples of ricin, see, U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) occurs in two forms designated RCAeo and RCA120 according to their molecular weights of approximately 65 and 120 kD, respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to cell-surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).
Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see, Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins such as a-sarcin and restrictocin are discussed in, for example Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247- 54, 2000. Calicheamicins were first isolated from Micromonospora echinospora and are members of the enediyne antitumor antibiotic family that cause double strand breaks in DNA that lead to apoptosis (see, for example Lee et al., J. Antibiot. 42:1070-87,1989). The drug is the toxic moiety of an immunotoxin in clinical trials (see, for example, Gillespie et al., Ann. Oncol. 11 :735-41, 2000).
Abrin includes toxic lectins from Abrus precatorius. The toxic principles, abrin a, b, c, and d, have a molecular weight of from about 63 and 67 kD and are composed of two disulfide-linked polypeptide chains A and B. The A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem. 52: 1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).
In one example, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, PE includes full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al., J. Biol. Chem. 264:14256-14261, 1989). PE employed with the provided antibodies can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell. Cytotoxic fragments of PE include PE25, PE40, PE38, and PE35. For additional description of PE and variants thereof, see for example, U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; PCT Publication No. WO 99/51643; Pai et al., Proc. Natl. Acad. Sci. USA, 88:3358-3362, 1991; Kondo et al., J. Biol. Chem., 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta, 1333:C1- C6, 1997. Also contemplated are protease -resistant PE variants and PE variants with reduced immunogenicity, such as, but not limited to PE-LR, PE-6X, PE-8X, PE-LR/6X and PE-LR/8X (see, for example, Weldon et al., Blood 113(161:3792-3800, 2009; Onda et a/., Proc. Natl. Acad. Sci. USA, 105(32): 11311-11316, 2008; and PCT Publication Nos. WO 2007/016150, WO 2009/032954 and WO 2011/032022). The PE variant can be PE25 (see, Weldon et al., Blood 2009; 113:3792-3800).
In some examples, the PE is a variant that is resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; PCT Publication No. WO 2009/032954). In other examples, the PE is a variant designated PE-LR/6X (PCT Publication No. WO 2011/032022). In further examples, the PE is a variant designated PE-LR/8M (PCT Publication No. WO 2011/032022).
A humanized monoclonal antibody (or antigen binding fragment thereof) that specifically binds EGFRvIII and/or gene-amplified EGFR can also be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), etc. A humanized monoclonal antibody or antigen binding fragment can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, 0- galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When a humanized monoclonal antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. A humanized monoclonal antibody or antigen binding fragment may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. The avidin itself can also be conjugated with an enzyme or detectable marker.
A humanized monoclonal antibody or antigen binding fragment may be conjugated with a paramagnetic agent, such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also of use as labels. Antibodies can also be conjugated with lanthanides (such as europium and dysprosium), and manganese. A humanized monoclonal antibody or antigen binding fragment may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). A humanized monoclonal antibody or antigen binding fragment can also be conjugated with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect EGFRvIII and/or gene-amplified EGFR, and EGFRvIII and/or gene-amplified EGFR expressing cells by x-ray, emission spectra, or other diagnostic techniques. Further, the radiolabel may be used therapeutically as a toxin for treatment of tumors in a subject, for example for treatment of any tumor that expresses EGFRvIII and/or gene-amplified EGFR, such as a carcinoma, for example a head and neck carcinoma, a breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or a bladder carcinoma. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, "Tc, 1 HIn, 125I, 131I.
Means of detecting detectable markers have been described, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
A humanized monoclonal antibody or antigen binding fragment disclosed herein can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the humanized antibody or antigen binding fragment, such as to increase serum half-life or to increase tissue binding.
The average number of effector molecule or detectable marker moieties per humanized monoclonal antibody or antigen binding fragment in a conjugate can range, for example, from 1 to 20 moieties per antibody or antigen binding fragment. For some conjugates, the average number of effector molecule or detectable marker moieties per antibody or antigen binding fragment may be limited by the number of attachment sites on the humanized monoclonal antibody or antigen binding fragment. For example, where the attachment is a cysteine thiol, a humanized monoclonal antibody or antigen binding fragment may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. In certain examples, the average number of effector molecule or detectable marker moieties per humanized monoclonal antibody or antigen binding fragment in a conjugate range from 1 to 10; for example, from 2 to 6; from 2 to 8, from 3 to 5; from 3 to 4; from 3.1 to 3.9; from 3.2 to 3.8; from 3.2 to 3.7; from 3.2 to 3.6; from 3.3 to 3.8; or from 3.3 to 3.7. In further examples, the average number of effector molecule or detectable marker moieties per humanized monoclonal antibody or antigen binding fragment is about 1, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9. The average number of effector molecule or detectable marker moieties per humanized monoclonal antibody or antigen binding fragment in preparations of conjugates may be characterized by conventional means such as mass spectroscopy or ELISA assay. The loading (for example, effector molecule/antibody ratio) of an conjugate may be controlled in different ways, for example, by: (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the humanized antibody such that the number and position of cysteine residues is modified for control of the number or position of linker-effector molecule attachments (such as thioMab or thioFab prepared as disclosed in W02006/03448).
C. Chimeric Antigen Receptors (CARs)
Also disclosed herein are chimeric antigen receptor (CARs) including an antibody disclosed herein (e.g., a humanized monoclonal antibody, or antigen binding fragment thereof, specific for EGFRvIII and/or gene-amplified EGFR). CARs are artificially constructed chimeric receptor proteins that include an extracellular antigen binding domain (e.g., single chain variable fragment (scFv)) that specifically binds to a target (e.g., EGFRvIII), linked to a transmembrane domain, linked to one or more intracellular T-cell signaling domains. Characteristics of the disclosed CARs include the ability to redirect T-cell specificity and reactivity towards EGFRvIII and/or gene-amplified EGFR expressing cells in a non-MHC-restricted manner. The non-MHC-restricted EGFRvIII and/or gene-amplified EGFR recognition gives immune cells (e.g., T cells) expressing a disclosed CAR the ability to recognize an antigen independent of antigen processing.
The intracellular T cell signaling domains can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of the CAR including the intracellular domain of a T cell receptor, such as the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain refers to a portion of the CAR including the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or their ligands that are required for an efficient response of lymphocytes to antigen.
1. Extracellular Region
The CAR includes an antigen binding domain that specifically binds to EGFRvIII and/or gene- amplified EGFR. For example, the antigen binding domain can be a scFv including the heavy chain variable region and the light chain variable region of any of the humanized monoclonal antibodies or antigen binding fragments thereof disclosed herein.
In some aspects, the EGFRvIII and/or gene-amplified EGFR specific antibodies include a variable heavy chain region (VH) and a variable light chain region (VL) and specifically bind EGFRvIII and/or gene- amplified EGFR. In some examples, the heavy and light chain included in the CAR include the amino acid sequences set forth as: a) SEQ ID NO: 5 and SEQ ID NO: 8, respectively (A10, VH3+VL3); b) SEQ ID NO: 3 and SEQ ID NO: 6, respectively (A2, VH1+VL1); c) SEQ ID NO: 3 and SEQ ID NO: 7, respectively (A3, VH1+VL2); d) SEQ ID NO: 3 and SEQ ID NO: 8, respectively (A4, VH1+VL3); e) SEQ ID NO: 4 and SEQ ID NO: 6, respectively (A5, VH2+VL1); f) SEQ ID NO: 4 and SEQ ID NO: 7, respectively (A6, VH2+VL2); g) SEQ ID NO: 4 and SEQ ID NO: 8, respectively (A7, VH2+VL3); h) SEQ ID NO: 5 and SEQ ID NO: 6, respectively (A8, VH3+VL1); i) SEQ ID NO: 5 and SEQ ID NO: 7, respectively (A9, VH3+VL2); j) SEQ ID NO: 1 and SEQ ID NO: 9, respectively (Bl, 40H3 VH+VL-EG); k) SEQ ID NO: 1 and SEQ ID NO: 10, respectively (B2, 40H3 VH+VL-DA); l) SEQ ID NO: 4 and SEQ ID NO: 11, respectively (B3, VH2+VL1-DA); m) SEQ ID NO: 4 and SEQ ID NO: 12, respectively (B4, VH2+VL2-DA); n) SEQ ID NO: 5 and SEQ ID NO: 11, respectively (B5, VH3 and VL1-DA); o) SEQ ID NO: 31 and SEQ ID NO: 32, respectively (CIO); p) SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2); or q) SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3).
Any of the antibodies or antigen binding fragments disclosed herein can be used in the CAR. The disclosed antibody or antigen binding fragment is humanized. In a non-limiting example, the heavy and light chain included in the CAR includes SEQ ID NO: 5 and SEQ ID NO: 8, respectively.
In several aspects, the antigen binding fragment is a scFv. In some aspects, the scFv includes a heavy chain variable region and a light chain variable region joined by a peptide linker, such as a linker including the amino acid sequence set forth as GGGGSGGGGSGGGGS (SEQ ID NO: 33).
The CAR can include a signal peptide sequence, e.g., N-terminal to the antigen binding domain. The signal peptide sequence may comprise any suitable signal peptide sequence. In an example, the signal peptide sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor sequence, such as an amino acid sequence including or consisting of LLVTSLLLCELPHPAFLLIPDT (SEQ ID NO: 34). While the signal peptide sequence may facilitate expression of the CAR on the surface of the cell, the presence of the signal peptide sequence in an expressed CAR is not necessary in order for the CAR to function. Upon expression of the CAR on the cell surface, the signal peptide sequence may be cleaved off of the CAR. Accordingly, in some examples, the CAR lacks a signal peptide sequence.
Between the antigen binding domain and the transmembrane domain of the CAR, there may be a spacer domain, which includes a polypeptide sequence. The spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. In some aspects, the spacer domain can include an immunoglobulin domain, such as a human immunoglobulin sequence. In an example, the immunoglobulin domain comprises an immunoglobulin CH2 and CH3 immunoglobulin G (IgGl) domain sequence (CH2CH3). In this regard, the spacer domain can include an immunoglobulin domain comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 35 : EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK Without being bound to any particular theory, it is believed that the CH2CH3 domain extends the antigen binding domain of the CAR away from the membrane of CAR-expressing cells and may more accurately mimic the size and domain structure of a native TCR.
2. Transmembrane Domain
With respect to the transmembrane domain, the CAR can be designed to include a transmembrane domain that is fused to the extracellular domain of the CAR. In one example, the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains for use in the disclosed CARs can include at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In several examples, a triplet of phenylalanine, tryptophan and valine is found at each end of a synthetic transmembrane domain.
Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular T cell signaling domain and/or T cell costimulatory domain of the CAR. An exemplary linker sequence includes one or more glycine-serine doublets.
In some examples, the transmembrane domain comprises the transmembrane domain of a T cell receptor, such as a CD8 transmembrane domain. Thus, the CAR can include a CD8 transmembrane domain including or consisting of SEQ ID NO: 36: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY C
In other examples, the transmembrane domain comprises the transmembrane domain of a T cell costimulatory molecule, such as CD 137 or CD28. Thus, the CAR can include a CD28 transmembrane domain including or consisting of SEQ ID NO: 37: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR
3. Intracellular Region
The intracellular region of the CAR includes one or more intracellular T cell signaling domains responsible for activation of at least one of the normal effector functions of a T cell in which the CAR is expressed or placed in. Exemplary T cell signaling domains are provided herein, and have been described.
While an entire intracellular T cell signaling domain can be employed in a CAR, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular T cell signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the relevant T cell effector function signal.
Examples of intracellular T cell signaling domains for use in the CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-stimulatory molecules that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
T cell receptor signaling domains regulate primary activation of the T cell receptor complex either in a stimulatory way, or in an inhibitory way. The disclosed CARs can include primary cytoplasmic signaling sequences that act in a stimulatory manner, which may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences that can be included in a disclosed CAR include those from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d proteins. In some examples, the cytoplasmic signaling molecule in the CAR includes an intracellular T cell signaling domain from CD3 zeta.
The intracellular region of the CAR can include the ITAM containing primary cytoplasmic signaling domain (such as CD3-zeta) by itself or combined with any other desired cytoplasmic domain(s) useful in the context of a CAR. For example, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and an intracellular costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR including the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. An additional example of a signaling domain that can be included in a disclosed CAR is a Tumor necrosis factor receptor superfamily member 18 (TNFRSF18; also known as glucocorticoid-induced TNFR -related protein, GITR) signaling domain.
In some examples, the CAR includes a CD3 zeta signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD 137 signaling domain or a combination of two or more thereof. In another example, the cytoplasmic domain includes the signaling domain of CD3-zeta and the signaling domain of CD28. In further examples, the cytoplasmic domain includes the signaling domain of CD3 zeta and the signaling domain of CD137. In yet another example, the cytoplasmic domain includes the signaling domain of CD3-zeta and the signaling domain of CD28 and CD137. The order of the one or more T cell signaling domains on the CAR can be varied as needed by the practitioner. Exemplary amino acid sequences for such T cell signaling domains are provided. For example, the CD3 zeta signaling domain can include or consist of the amino acid sequence set forth as SEQ ID NO: 38: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. The CD8 signaling domain can include or consist of the amino acid sequence set forth as SEQ ID NO: 39: FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV LLLSLVITLYCNHRNR. The CD28 signaling domain can include or consist of the amino acid sequence set forth as SEQ ID NO:40:SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS. The CD137 signaling domain can include or consist of the amino acid sequences set forth as SEQ ID NO: 41 : KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, or SEQ ID NO: 42: RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.
The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CARs disclosed herein may be linked to each other in a random or specified order. Optionally, a short polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides a particularly suitable linker. Further, between the signaling domain and the transmembrane domain of the CAR, there may be a spacer domain, which includes a polypeptide sequence. The spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
4. Additional Description of CARs
Also provided are functional portions of the CARs described herein. The term "functional portion" when used in reference to a CAR refers to any part or fragment of the CAR, which part or fragment retains the biological activity of the CAR of which it is a part (the parent CAR). Functional portions encompass, for example, those parts of a CAR that retain the ability to recognize target cells, or detect, treat, or prevent a disease, to a similar extent, the same extent, or to a higher extent, as the parent CAR. In reference to the parent CAR, the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent CAR.
The CAR or functional portion thereof, can include additional amino acids at the amino or carboxy terminus, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the CAR or functional portion, e.g., recognize target cells, detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent CAR.
Also provided are functional variants of the CARs described herein, which have substantial or significant sequence identity or similarity to a parent CAR, which retain the biological activity of the CAR of which it is a variant. Functional variants encompass, for example, variants of a CAR described herein (the parent CAR) that retain the ability to recognize target cells to a similar extent, the same extent, or to a higher extent, as the parent CAR. In reference to the parent CAR, the functional variant can, for instance, be at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to a parent CAR. A functional variant can, for example, include the amino acid sequence of the parent CAR with at least one conservative amino acid substitution. Alternatively, or additionally, functional variants can comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR.
The CARs (including functional portions and functional variants) can be of any length, i.e., can comprise any number of amino acids, provided that the CARs (or functional portions or functional variants thereof) retain their biological activity, e.g., the ability to specifically bind to antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal, etc. In some examples, the CAR is about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.
The CARs disclosed herein (including functional portions and functional variants of the CARs disclosed herein) can include synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4- hydroxyproline, 4- aminophenylalanine, 4- nitrophenylalanine, 4-chlorophenylalanine, 4- carboxyphenylalanine, P-phenylserine [3-hydroxyphenylalanine, phenylglycine, a -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 ,2,3,4- tetrahydroisoquinoline-3- carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'- dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, oc- aminocycloheptane carboxylic acid, -(2-amino-2-norbornane)-carboxylic acid, y- diaminobutyric acid, a,P-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.
The CARs disclosed herein (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
Methods of generating chimeric antigen receptors, T cells including such receptors, and their use (e.g., for treatment of cancer) are known and further described, for example, in Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1 -9; Till et al., 2008, Blood, 1 12:2261 -2271; Park etal., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT Pub. W02012/079000, WO2013/126726; and U.S. Pub. 2012/0213783).
In some examples, a nucleic acid molecule encoding a CAR disclosed herein is included in an expression vector (such as a lentiviral vector) for expression in a host cell, such as a T cell, to make the disclosed CAR. In some aspects, methods of using the chimeric antigen receptor include isolating immune cells from a subject (e.g., T cells), transforming the isolated cells with an expression vector (such as a lentiviral or AAV vector) encoding a CAR disclosed herein, and administering the engineered cells expressing the CAR to a subject for treatment, for example, treatment of a tumor in the subject.
D. Polynucleotides and Expression
Nucleic acids encoding a humanized monoclonal antibody, antibody binding fragment, conjugate, or CAR, that specifically binds EGFRvIII and/or gene-amplified EGFR as disclosed herein, are provided. Nucleic acids encoding these molecules can readily be produced by a practitioner using the amino acid sequences provided herein (such as the CDR sequences, heavy chain and light chain sequences), sequences available in the art (such as framework sequences), and the genetic code. A practitioner can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same antibody sequence, or encode a conjugate or fusion protein including the VL and/or VH nucleic acid sequence.
Nucleic acid sequences encoding the disclosed humanized monoclonal antibodies, antibody binding fragments, conjugates, and CARs, that specifically bind EGFRvIII and/or gene-amplified EGFR, can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al.. Meth. Enzymol. 68:90- 99, 1979; the phosphodiester method of Brown et cd., Meth. Enzymol. 68: 109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automated synthesizer as described in, for example, Needham- VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. A practitioner would recognize that while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
Nucleic acids of the disclosure can be prepared using standard molecular biology techniques. Examples of suitable cloning and sequencing techniques are known (see, e.g, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources. Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known.
In a non-limiting example, provided is a nucleic acid molecule encoding a CAR disclosed herein expressed in an immune cell, for example, a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell. In some examples, a CAR disclosed herein is for expression in a T cell, for example, to generate a chimeric antigen receptor T cell (CAR T-cell). The nucleic acid molecule encoding the CAR can be included in a vector (such as a lentiviral vector). Methods of generating nucleic acid molecules encoding chimeric antigen receptors and cells including such receptors are known (see, e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1 -9; Till et al., 2008, Blood, 1 12:2261 -2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al.. N Engl J Med., 368: 1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT Pub. WO2012/079000, WO2013/126726; and U.S. Pub. 2012/0213783.)
The nucleic acid molecules can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. The antibodies, antigen binding fragments, and conjugates can be expressed as individual VH and/or VL chain (linked to an effector molecule or detectable marker as needed), or can be expressed as a fusion protein. Methods of expressing and purifying antibodies and antigen binding fragments are known and further described herein (see, e.g., Al-Rubeai (ed), Antibody Expression and Production, Springer Press, 2011). An immunoadhesin can also be expressed. Thus, in some examples, nucleic acids encoding a Vn and VL, and immunoadhesin are provided. The nucleic acid sequences can optionally encode a leader sequence.
To create a scFv the VH- and VL-encoding DNA fragments can be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)j, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker (see, e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990; Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd Ed., Springer Press, 2010; Harlow and Lane, Antibodies: A Laboratory Manual, 2nd, Cold Spring Harbor Laboratory, New York, 2013). Optionally, a cleavage site can be included in a linker, such as a furin cleavage site.
The nucleic acid encoding a VH and/or the VL optionally can encode an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM or IgG Fc domain. The Fc domain can be an optimized Fc domain, as described in U.S. Published Patent Application No. 2010/093979. In one example, the immunoadhesin is an IgGi Fc.
The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to, for example, EGFRvIII, and another antigen, such as, but not limited to, CD3. The encoded VH and VL optionally can include a furin cleavage site between the VH and VL domains.
Numerous expression systems are known and available for expressing nucleic acids in hosts, such as E. coli, other bacterial hosts, yeast, or various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.
One or more DNA sequences encoding a disclosed humanized monoclonal antibody, antibody binding fragment, conjugate, or CAR can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Hybridomas expressing the humanized monoclonal antibodies are also encompassed by this disclosure.
Polynucleotide sequences encoding a disclosed humanized monoclonal antibody, antibody binding fragment, conjugate, or CAR, can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to: promoters, enhancers, transcription terminators, a start codon (e.g., ATG) in front of a protein-encoding gene, splicing signal for introns, spacers for maintaining the correct reading frame and permit proper translation of mRNA, and stop codons.
To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a transcription/translation terminator. For E. coli, this includes a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences). Expression cassettes can be transferred into a chosen host cell by known methods, such as transformation or electroporation for E. coli, or calcium phosphate treatment, electroporation, or lipofection for mammalian cells. Cells transformed by expression cassettes or vectors can be selected by resistance to antibiotics conferred by genes contained in such cassettes or vectors, such as the amp, gpt, neo and hyg genes.
The polynucleotide sequences encoding a disclosed humanized monoclonal antibody, antigen binding fragment, conjugate, or CAR can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are known. Numerous viral and plasmid DNA vectors capable of expression and replication in host cells have been described, many are commercially available.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding, for example, a humanized antibody or antigen binding fragment disclosed herein, and a second foreign DNA molecule encoding, for example, a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see, for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011). A practitioner can readily use an expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.
For purposes of producing a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. In some examples, the host cell is a peripheral blood lymphocyte (PBL), a peripheral blood mononuclear cell (PBMC), or a T cell. The T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g.. Jurkat, SupTl, etc., or a T cell obtained from a mammal (such as a T cell obtained from a human subject). If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells, e.g., Th] and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like. The T cell may be a CD8+ T cell or a CD4+ T cell.
Also provided is a population of cells including at least one host cell described herein. The population of cells can be a heterogeneous population including the host cell including any of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) including the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell including a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one example, the population of cells is a clonal population including host cells including a recombinant expression vector as described herein.
Modifications can be made to a nucleic acid encoding a humanized monoclonal antibody or antigen binding fragment described herein, without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are known and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps. In addition to recombinant methods, the immunoconjugates, effector moieties, and antibodies of the present disclosure can also be constructed in whole or in part using standard peptide synthesis.
Once expressed, the humanized monoclonal antibodies, antigen binding fragments, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson ed., Basic methods in Protein Purification and Analysis: A laboratory Manual, Cold Harbor Press, 2008). The antibodies, antigen binding fragment, and conjugates need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.
Methods for expression of the humanized monoclonal antibodies, antigen binding fragments, and conjugates, and/or refolding to an appropriate active form, from mammalian cells, and bacteria such as E. coli have been described and are applicable to the antibodies disclosed herein. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, 2nd, Cold Spring Harbor Laboratory, New York, 2013, Simpson ed., Basic methods in Protein Purification and Analysis: A laboratory Manual, Cold Harbor Press, 2008, and Ward et al., Nature 341 :544, 1989.
Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, a reducing agent is present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (di thioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, and especially as described by Buchner et al., supra.
In addition to recombinant methods, the humanized monoclonal antibodies, antigen binding fragments, and/or conjugates can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al.. Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicylohexylcarbodimide) are known. E. Methods of Detection
Methods are provided for detecting the presence of a cell that expresses EGFRvIII and/or gene- amplified EGFR in a subject, such as a tumor cells that expresses EGFRvIII and/or gene-amplified EGFR. In some aspects, the methods include contacting a cell (e.g., a tumor cell) from a subject with one or more of the humanized monoclonal antibodies that specifically bind EGFRvIII and/or gene-amplified EGFR, or conjugate thereof to form an immune complex. The presence (or absence) of the immune complex is then detected. The presence of the immune complex indicates the presence of a cell that expresses EGFRvIII and/or gene-amplified EGFR in the subject.
In some examples, the methods include contacting a cell (e.g., a tumor cell) from a subject with one or more of the humanized monoclonal antibodies that specifically bind EGFRvIII or a conjugate thereof to form an immune complex. The presence (or absence) of the immune complex is then detected, and the presence of the immune complex indicates the presence of a cell that expresses EGFRvIII in the subject.
The detection methods can involve in vivo detection or in vitro detection of the immune complex. In several aspects, detection of a cell that expresses EGFRvIII includes detecting cell-surface expression of EGFRvIII on the tumor cell. In several aspects of the provided methods, detecting a cell that expresses EGFRvIII and/or gene-amplified EGFR in a subject detects a tumor. In several non-limiting examples, the tumor is a carcinoma, such as a head and neck carcinoma, a breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or a bladder carcinoma. In some examples, the methods detect a tumor cell that overexpresses EGFRvIII (e.g., gene-amplified EGFR). In several examples, the humanized monoclonal antibodies binds to the EGRF287-302 loop.
A variety of formats are of use for detecting a cell (e.g., a tumor cell) that expresses EGFRvIII and/or gene-amplified EGFR. In some aspects, a subject is selected who has, is suspected of having, or is at risk of developing, a tumor, for example, a carcinoma. For example, the subject has, is suspected of having, or is at risk of developing head and neck carcinoma, breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or bladder carcinoma. In some examples the subject has, is suspected of having, or is at risk of developing, head and neck carcinoma, breast carcinoma, or bladder carcinoma. Thus, the presence of a cell expressing EGFRvIII and/or gene-amplified EGFR can be detected in these subjects.
The disclosed methods can also detect overexpression of EGFR. Overexpression can be measured, such that any cell with more than about 50,00 receptors, more than about 60,00 receptors, more than about 70,00 receptors, more than about 80,00 receptors, more than about 90,00 receptors, or more than about more than about 100,00 receptors is detected. For example, such methods include contacting a tumor cell in a biological sample from the subject with one or more of the conjugates or antibodies provided herein or an antigen binding fragment thereof to form an immune complex. The presence (or absence) of the immune complex is then detected and/or quantified. The presence (or amount) of the immune complex on the cell from the subject indicates the presence of a tumor cell that overexpresses EGFR in the subject. In one non-limiting example, a sample is obtained from a subject, and the presence of a tumor cell that expresses EGFRvIII is assessed in vitro. For example, such methods include contacting a tumor cell in a biological sample from the subject with one or more of the conjugates or humanized monoclonal antibodies provided herein that specifically bind EGFRvIII or an antigen binding fragment thereof to form an immune complex. The presence (or absence) of the immune complex is then detected. The presence of the immune complex on the cell from the subject indicates the presence of a tumor cell that expresses EGFRvIII in the subject. For example, an increase in the presence of the immune complex in the sample as compared to formation of the immune complex in a control sample indicates the presence of a tumor cell that expresses EGFRvIII in the subject. In some examples, a control is utilized. Similar methods can be used to detect the presence of a tumor cell that expresses gene-amplified EGFR using one or more of the humanized monoclonal antibodies or antigen binding fragments disclosed herein that specifically bind gene- amplified EGFR, or a conjugate thereof.
A biological sample is typically obtained from a mammalian subject of interest, such as human. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes.
In some examples of the disclosed methods, the humanized monoclonal antibody or antigen binding fragment disclosed herein is conjugated to a detectable marker. In some examples, the methods further include contacting a second antibody that specifically binds the antibody, antigen binding fragment thereof, or a conjugate disclosed herein, for a sufficient amount of time to form an immune complex and detecting this immune complex. An increase in the presence of this immune complex in a biological sample from a selected subject (as described above) compared to the presence of the immune complex in a control sample or other standard detects the presence of an endothelial cell that expresses EGFRvIII and/or gene-amplified EGFR in the biological sample. In some examples, the second antibody is conjugated to a detectable marker.
Suitable detectable markers for the humanized monoclonal antibody or secondary antibody are described and known to a practitioner. For example, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125I, 131I, 35S or 3H. The disclosed humanized monoclonal antibodies disclosed herein and conjugates thereof can be used in immunohistochemical assays. These assays are known and have been described (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed., Cold Spring Harbor Publications, New York (2013)).
The humanized monoclonal antibodies disclosed herein can also be used to detect tumor cells that express EGFRvIII and/or gene-amplified EGFR in vivo. In some examples, in vivo detection diagnoses the presence of the tumor in the subject. Thus, methods are disclosed for detecting pathological conditions in a subject, such as a tumor, such as a carcinoma; for example, head and neck carcinoma, breast carcinoma, pancreas carcinoma, colon or rectal carcinoma, CNS carcinoma, or bladder carcinoma. In one example, an effective amount of a disclosed humanized monoclonal antibody (or antigen binding fragment thereof) or a conjugate thereof is administered to the subject for a sufficient amount of time for the humanized monoclonal antibody or antigen binding fragment to form an immune complex, which can then be detected. Detection of the immune complex in the subject determines the presence of a tumor cell that expresses EGFRvIII and/or gene-amplified EGFR. In a specific, non-limiting example, detection of an immune complex is performed by immunoscintography. Other specific, non-limiting examples of immune complex detection include radiolocalization, radioimaging, magnetic resonance imaging (such as using a biotinylated antibody and avidin-iron oxide), or fluorescence imaging (such as using luciferase or green fluorescent protein labeled antibodies) (see, e.g., Paty et al., Transplantation., 77:1 133-1 137, 2004). In several examples, the disclosed methods detect, for example, head and neck carcinoma, breast carcinoma, pancreas carcinoma, colon or rectal carcinoma, CNS carcinoma, or bladder carcinoma.
In the setting of magnetic resonance imaging, contrast agent detection can be greatly impacted by magnetic resonance scanner field strength. Increased field strengths provide improvements by orders of magnitude in the ability to detect contrast agents (Hu et al., Ann. Rev. Biomed. Eng., 6:157-184, 2004; Wedeking et al., Magn. Reson. Imaging., 17:569-575, 1999). For example, the limit of detection of gadolinium at 2 tesla (T) is -30 uM. At 4T the limit of detection is reduced to -1 pM. With newly available 7 to 12T scanners one would expect to detect low (10-100) nM concentrations of this contrast agent. Similar sensitivity can also be identified using contrast agents such as iron oxide. Once detected the test results can be used to assist in or guide surgical or other excision of a tumor.
In one example, an effective amount of a humanized monoclonal antibody or antigen binding fragment that specifically binds to EGFRvIII or a conjugate thereof disclosed herein is administered to a subject having a tumor following anti-cancer treatment. After a sufficient amount of time has elapsed to allow for the administered antibody or antigen binding fragment or conjugate to form an immune complex with EGFRvIII on a tumor cell, the immune complex is detected. For example, a humanized monoclonal antibody that specifically binds to EGFRvIII or conjugate thereof can be administered to a subject prior to, or following, treatment of a tumor. The tumor can be (but is not limited to) a carcinoma, such as a head and neck carcinoma, a breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or a bladder carcinoma. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in the immune complex compared to a control taken prior to the treatment indicates that the treatment is not effective, whereas a decrease in the immune complex compared to a control taken prior to the treatment indicates that the treatment is effective. Similar methods using a humanized monoclonal antibody or antigen binding fragment that specifically binds to gene-amplified EGFR, or a conjugate thereof, disclosed herein can be used to detect gene-amplified EGFR, with the presence (or absence) of the immune complex indicating the effectiveness of the treatment.
F. Methods of Treatment
A therapeutically effective amount of a humanized monoclonal antibody, antigen binding fragment that specifically binds EGFRvIII, conjugate thereof, or immune cell expressing a CAR disclosed herein (e.g., a CAR T cell or CAR NK cell), can be administered to a subject to treat a tumor that expresses EGFRvIII, for example a carcinoma, such as a head and neck carcinoma, a breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or a bladder carcinoma. In some examples, administration of a therapeutically effective amount of a humanized monoclonal antibody, antigen binding fragment that specifically binds EGFRvIII, conjugate thereof, or immune cell expressing a CAR disclosed herein, decreases a sign or symptom of a tumor that expresses EGFRvIII. The tumor can be a carcinoma, for example, a head and neck carcinoma, breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or bladder carcinoma. Thus, a subject can be selected for treatment that has, is suspected of having or is at risk of developing the tumor that expresses EGFRvIII. A therapeutically effective amount of the nucleic acid molecules and vectors disclosed herein are also of use. In other examples, the tumor can overexpress EGFR, and/or express misfolded EGFR. In some examples, a subject is selected that has a tumor that overexpresses EGFR and/or expresses misfolded EGFR. The compositions disclosed herein are of use for treating these tumors in a subject.
In further examples, a therapeutically effective amount of a humanized antibody or antigen binding fragment, as disclosed herein, can also be used in method of inhibiting a tumor over-expressing EGFR in a subject (e.g., gene-amplified EGFR). The method includes administering an effective amount of the humanized antibody, antigen binding fragment, nucleic acid molecule, vector, T cell or a pharmaceutical composition disclosed herein to the subject having the tumor overexpressing EGFR. In non-limiting examples, the humanized monoclonal antibody or antigen binding fragment include a VH and VL that include the amino acid sequences set forth as: a) SEQ ID NO: 5 and SEQ ID NO: 8, respectively (A10, VH3+VL3); b) SEQ ID NO: 3 and SEQ ID NO: 6, respectively (A2, VH1+VL1); c) SEQ ID NO: 3 and SEQ ID NO: 7, respectively (A3, VH1+VL2); d) SEQ ID NO: 3 and SEQ ID NO: 8, respectively (A4, VH1+VL3); e) SEQ ID NO: 4 and SEQ ID NO: 6, respectively (A5, VH2+VL1); f) SEQ ID NO: 4 and SEQ ID NO: 7, respectively (A6, VH2+VL2); g) SEQ ID NO: 4 and SEQ ID NO: 8, respectively (A7, VH2+VL3); h) SEQ ID NO: 5 and SEQ ID NO: 6, respectively (A8, VH3+VL1); i) SEQ ID NO: 5 and SEQ ID NO: 7, respectively (A9, VH3+VL2); j) SEQ ID NO: 1 and SEQ ID NO: 9, respectively (Bl, 40H3 VH+VL-EG); k) SEQ ID NO: 1 and SEQ ID NO: 10, respectively (B2, 40H3 VH+VL-DA); l) SEQ ID NO: 4 and SEQ ID NO: 11, respectively (B3, VH2+VL1-DA); m) SEQ ID NO: 4 and SEQ ID NO: 12, respectively (B4, VH2+VL2-DA); n) SEQ ID NO: 5 and SEQ ID NO: 11, respectively (B5, VH3 and VL1-DA); o) SEQ ID NO: 31 and SEQ ID NO: 32, respectively (CIO); p) SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2); or q) SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3).
Nucleic acid molecules, vectors, and immune cells expressing a CAR that includes these antigen binding fragments are also of use. The tumor can be a carcinoma, for example, a head and neck carcinoma, breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or bladder carcinoma. Thus, a subject can be selected for treatment that has, is suspected of having or is at risk of developing the tumor that overexpresses EGFR (e.g., expresses gene-amplified EGFR).
In some examples, the humanized monoclonal antibodies, antigen binding fragments, immune cells expressing a CAR (e.g., CAR T cells), compositions and conjugates disclosed herein can be administered to a subject to slow or inhibit the growth or metastasis of a tumor, reduce tumor volume or reduce metastasis. In these applications, a therapeutically effective amount of a humanized monoclonal antibody or antigen binding fragment that specifically binds EGFRvIII and/or gene-amplified EGFR or a conjugate or CAR T cells or composition disclosed herein is administered to a subject in an amount and under conditions sufficient to form an immune complex with EGFRvIII and/or gene-amplified EGFR, thereby slowing or inhibiting the growth or the metastasis of a tumor, to reduce tumor volume, or to inhibit a sign or a symptom of a cancer. Examples of suitable subjects include those diagnosed with or suspecting of having a tumor that expresses EGFRvIII and/or gene-amplified EGFR, for example subjects having a carcinoma, such as a head and neck carcinoma, breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or a bladder carcinoma.
The therapeutically effective amount will depend upon the severity of the disease and the general state of the patient’s health. A therapeutically effective amount is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. In one example, a therapeutically effective amount is the amount necessary to inhibit tumor growth (such as growth of a carcinoma, such as a head and neck carcinoma, breast carcinoma, a pancreas carcinoma, a colon or rectal carcinoma, a CNS carcinoma, or a bladder carcinoma), inhibit metastasis, reduce tumor volume, or the amount that is effective at reducing a sign or a symptom of the tumor. The therapeutically effective amount of the agents administered can vary depending upon the desired effects and the subject to be treated. In some examples, therapeutic amounts are amounts which eliminate or reduce the patient's tumor burden, or which prevent or reduce the proliferation of metastatic cells, or reduce a symptom of the tumor. Subjects that can benefit from the disclosed methods include human and veterinary subjects (e.g., cats, dogs, non-human primates, etc.). In some examples, the subject is a mammalian subject, for example, a human, non-human primate, canine, or feline. In some examples, the subject is a human, non-human primate, domestic dog (Cani.s lupus familiaris), or domestic cat (e.g., Felis catus). In a non-limiting example, the subject is a human. Subjects can be screened prior to initiating the disclosed therapies, for example to determine whether the subject has a tumor. The presence of a tumor that expresses EGFRvIII and/or gene-amplified EGFR indicates that the composition can be treated using the methods provided herein.
Any method of administration can be used, including local or systemic administration. For example, topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, intratumoral, and subcutaneous administration can be used. The particular mode of administration and the dosage regimen can be selected by the attending clinician, taking into account the particulars of the case (for example the subject, the disease, the disease state involved, and whether the treatment is prophylactic). In cases in which more than one agent or composition is being administered, one or more routes of administration may be used; for example, a chemotherapeutic agent may be administered orally and a humanized monoclonal antibody or antigen binding fragment or conjugate or composition may be administered intravenously. Methods of administration include injection for which the conjugates, humanized monoclonal antibodies, antigen binding fragments, immune cells comprising a CAR, nucleic acid molecules, or compositions provided herein, in a nontoxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl oleate, or liposomes. In some examples, local administration can be used, for instance by applying a humanized antibody or antigen binding fragment disclosed herein to a region of tissue from which a tumor has been removed, or a region suspected of being prone to tumor development. In some examples, sustained intra-tumoral (or near- tumoral) release of a pharmaceutical preparation that includes a therapeutically effective amount of the humanized antibody or antigen binding fragment (or conjugate thereof) may be beneficial.
Compositions that include a humanized monoclonal antibody or antigen binding fragment or conjugate thereof, or an immune cell comprising a CAR disclosed herein, can be formulated in unit dosage form suitable for individual administration of precise dosages. In addition, the compositions may be administered in a single dose or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, for instance 1-10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions. Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years. Thus, the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgment of the administering practitioner.
Exemplary dosages of the humanized monoclonal antibodies, antigen binding fragments, conjugates, compositions or additional agents can range from about 0.01 to about 30 mg/kg (of the subject’s weight), such as from about 0.1 to about 10 mg/kg, or about 5 to about 15 mg/kg. In some examples, the dosage is at least about 0.1 mg/kg, at least about 0.2 mg/kg, at least about 0.3 mg/kg, at least about 0.4 mg/kg, at least about 0.5 mg/kg, at least about 1 mg/kg, at least about 4 mg/kg, at least about 3 mg/kg, at least about 5 mg/kg, at least about 6 mg/kg, at least about 7 mg/kg, at least about 8 mg/kg is at least about 9 mg/kg, at least about 10 mg/kg, at least about 11 mg/kg, at least about 12 mg/kg, at least about 13 mg/kg, at least about 14 mg/kg, at least about 15 mg/kg, at least about 16 mg/kg, at least about 17 mg/kg, at least about 18 mg/kg, at least about 19 mg/kg, at least about 20 mg/kg, at least about 21 mg/kg, at least about 22 mg/kg, at least about 23 mg/kg, at least about 24 mg/kg at least about 25 mg/kg, at least about 26 mg/kg, at least about 27 mg/kg, at least about 28 mg/kg, at least about 29 mg/kg, or at least about 30 mg/kg. In some examples, the dosage is no more than about Img/kg, 5mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, or 30 mg/kg. In some examples, the dosage of the humanized monoclonal antibodies, antigen binding fragments, conjugates, compositions or additional agents disclosed herein is 1 mg/kg to 20 mg/kg, for example, 2 mg/kg to 20 mg/kg, 2 mg/kg to 15 mg/kg, 2 mg/kg to 10 mg/kg, 2 mg/kg to 5 mg/kg, 5 mg/kg to 20 mg/kg, 5 mg/kg to 15 mg/kg, 5 mg/kg to 10 mg/kg, 8 mg/kg to 20 mg/kg, 8 mg/kg to 15 mg/kg, 8 mg/kg to 12 mg/kg, or 8 mg/kg to 10 mg/kg. In a non-limiting example, the dosage of a humanized monoclonal antibody or antigen binding fragment disclosed herein is 5 mg/kg to 15 mg/kg. In another non-limiting example, the dosage of a humanized monoclonal antibody or antigen binding fragment disclosed herein is 8 mg/kg to 12 mg/kg.
In some examples, the dosage of a humanized monoclonal antibody, antigen binding fragment, conjugate, composition or additional agent disclosed herein ranges from 100 mg/m2 to 700 mg/m2, for example, 200 mg/m2 to 700 mg/m2, 200 mg/m2 to 600 mg/m2, 200 mg/m2 to 500 mg/m2, 200 mg/m2 to 400 mg/m2, 300 mg/m2 to 700 mg/m2, 300 mg/m2 to 600 mg/m2, 300 mg/m2 to 500 mg/m2, 300 mg/m2 to 400 mg/m2, 350 mg/m2 to 700 mg/m2, 350 mg/m2 to 600 mg/m2, 350 mg/m2 to 500 mg/m2, 350 mg/m2 to 400 mg/m2. In a non-limiting example, the dosage of a humanized monoclonal antibody or antigen binding fragment disclosed herein is 200 mg/m2 to 500 mg/m2. In another non-limiting example, the dosage of a humanized monoclonal antibody or antigen binding fragment disclosed herein is 300 mg/m2 to 400 mg/m2.
In some aspects, 104-1012 immune cells comprising a CAR (e.g., CAR T cells) per kg of the subject’s weight are administered to a subject, for example, 104-108, 1O6-1O7, 1O6-1O8, 1O6-1O10, 106-1012, 108-1010, or 108-1012 cells per kg. In some examples, 5 x 106 to 5 x 108 immune cells comprising a CAR (e.g., CAR T cells) per kg of the subject’s weight are administered to a subject. In some examples, 1 x 107 to 5 x 107 immune cells comprising a CAR (e.g., CAR T cells) per kg of the subject’s weight are administered to a subject. In some examples, about 106 to 108 cells per kg are administered to the subject, for example, 0.2 to 5.0 x 106 per kg, or 0. 1 to 2.5 x 108 per kg. In some examples, at least 104, 105, 106, 107, or 108 cells per kg are administered to the subject. In further examples, no more than 104, 105, 106, 107 108, 109, or 1010 cells per kg are administered to the subject. Multiple doses can be administered, for example, immune cells comprising a CAR (e.g., CAR T cells) can be administered daily, every other day, twice per week, weekly, every other week, every three weeks, monthly, or less frequently. The dosage and frequency of administration can be affected by a number of factors, for example, the condition being treated, severity of symptoms, the subject’s age or overall condition, etc. Typically, treatment regimens are determined by clinical trials.
Any suitable route of administration can be used to administer immune cells comprising a CAR (e.g., CAR T cells). In some examples, immune cells comprising a CAR are administered parenterally, for example intravenously; however, injection or infusion in close proximity to a target tumor (e.g., local administration) or administration to the peritoneal cavity can also be used. In some examples immune cells comprising a CAR disclosed herein are delivered locally to a tumor by injection or a catheter. Appropriate routes of administration can be determined by a practitioner based on factors, for example, the condition being treated, severity of symptoms, the subject’s age or overall condition, or other factors.
In particular examples, the subject is administered a therapeutic composition that includes one or more of the conjugates, humanized monoclonal antibodies, antigen binding fragments, compositions, CAR T cells or additional agents, on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years. In one example, the subject is administered the conjugates, antibodies, compositions or additional agents for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.
In some examples, a disclosed therapeutic agent is administered intravenously, subcutaneously or by another mode daily or multiple times per week for a period of time, followed by a period of no treatment, then the cycle is repeated. In some examples, the initial period of treatment (e.g., administration of the therapeutic agent daily or multiple times per week) is for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In some examples, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. In certain examples, the dosing regimen of the therapeutic agent is daily for 3 days followed by 3 days off; or daily or multiple times per week for 1 week followed by 3 days or 1 week off; or daily or multiple times per week for 2 weeks followed by 1 or 2 weeks off; or daily or multiple times per week for 3 weeks followed by 1, 2 or 3 weeks off; or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks followed by 1, 2, 3 or 4 weeks off.
Administration of the humanized monoclonal antibodies, antigen binding fragments, conjugates, immune cells comprising a CAR, or compositions can be accompanied by administration of other anti-cancer or anti-angiogenesis agents or therapeutic treatments (such as surgical resection of a tumor or radiation therapy). For example, prior to, during, or following administration of a therapeutic amount of the antibodies or conjugates, the subject can receive one or more additional therapies. In one example, the subject receives one or more treatments to remove or reduce the tumor prior to administration of a therapeutic amount of one or more agents for treatment of the tumor. For example, the additional agent may include, but is not limited to, a chemotherapeutic agent, an anti-angiogenic agent, or a combination thereof. In another example, at least part of the tumor is surgically or otherwise excised or reduced in size or volume prior to administering the therapeutically effective amount of the humanized antibody or antigen binding fragment or conjugate. Particular examples of additional therapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenic agent can be administered in combination with the antibodies, conjugates disclosed herein. Methods and therapeutic dosages of such agents are known, and can be determined by a practitioner. In one example the chemotherapeutic agent includes 5-FU or IRT, or both.
Microtubule binding agents interact with tubulin to stabilize or destabilize microtubule formation thereby inhibiting cell division. Examples of microtubule binding agents that can be used in conjunction with the disclosed therapy include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin. Analogs and derivatives of such compounds also can be used. For example, suitable epothilones and epothilone analogs are described in International Publication No. WO 2004/018478. Taxoids, such as paclitaxel and docetaxel, as well as the analogs of paclitaxel taught by U.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264, can be used.
Suitable DNA and RNA transcription regulators, including, without limitation, actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof also are suitable for use in combination with the disclosed therapies. DNA intercalators and cross-linking agents that can be administered to a subject include, without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof. DNA synthesis inhibitors suitable for use as therapeutic agents include, without limitation, methotrexate, 5-fluoro-5'-deoxyuridine, 5-FU and analogs thereof. Examples of suitable enzyme inhibitors include, without limitation, camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof. Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.
Examples of the commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, IRT (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI- 571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.
Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).
Thus, non-limiting examples of chemotherapeutic agents for use in combination with the antibodies, antigen binding fragments, conjugates thereof, immune cells comprising a CAR , and nucleic acid molecules disclosed herein include chemotherapeutic agents such as erlotinib (TARCEVA®, Genentech/OSI Phami.), bortezomib (VELCADE®, Millenium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), sutent (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; antifolate antineoplastic such as pemetrexed (ALIMTA® Eli Lilly), aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics, calicheamicin, calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, for example, paclitaxel (TAXOL®, Bristol- Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin, nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone- Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Non-limiting examples of anti-angiogenic agents include molecules, such as proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, and recombinant vectors, and small molecules that function to reduce or even inhibit blood vessel growth. Examples of suitable angiogenesis inhibitors include, without limitation, angiostatin KI -3, staurosporine, genistein, fumagillin, medroxyprogesterone, suramin, interferonalpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, thromobospondin, endostatin, thalidomide, and derivatives and analogs thereof. For example, in some examples the anti-angiogenesis agent is an antibody that specifically binds to VEGF (for example, AVASTIN®, Roche) or a VEGF receptor (for example, a VEGFR2 antibody). In one example the anti-angiogenic agent includes a VEGFR2 antibody, or DMXAA (also known as Vadimezan or ASA404; available commercially, for example, from Sigma Corp., St. Louis, MO) or both. Exemplary kinase inhibitors include GLEEVAC®, IRESSA®, and TARCEVA ® that prevent phosphorylation and activation of growth factors. Antibodies that can be used include HERCEPTIN® and AVASTIN® that block growth factors and the angiogenic pathway.
In some examples, the additional agent is a monoclonal antibody, for example, 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab , Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Bavituximab, Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicin, Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomab estafenatox, Necitumumab, Nimotuzumab, Nofetumomab merpentan, Ofatumumab, Olaratumab, Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, sorafenib, sunitinib, Tacatuzumab tetraxetan, Taplitumomab paptox, Tenatumomab, TGN1412, Ticilimumab (tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab, and Zalutumumab.
Another common treatment for some types of cancer is surgical treatment, for example surgical resection of the cancer or a portion of it. Another example of a treatment is radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.
Other therapeutic agents, for example anti-tumor agents, that may or may not fall under one or more of the classifications above, also are suitable for administration in combination with the disclosed therapies. By way of example, such agents include adriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.
Preparation and dosing schedules for the additional agent may be used according to manufacturer's instructions or as determined empirically by a practitioner. Preparation and dosing schedules for such chemotherapy are also described, for example, in Chemotherapy Service, (1992) Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md.
The combination therapy may provide synergy and prove synergistic, that is, the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1) coformulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation, a synergistic effect may be attained when the compounds are administered or delivered sequentially, for example by different injections in separate syringes. In general, during alternation, an effective dosage of each active ingredient is administered sequentially, e.g. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
G. Compositions
Compositions are provided that include one or more humanized monoclonal antibodies, antigen binding fragments, conjugates, immune cells comprising a CAR (e.g., CAR T cells), or nucleic acid molecules, disclosed herein in a carrier (such as a pharmaceutically acceptable carrier). In some examples, the composition is a pharmaceutical composition. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome. The compositions can be formulated for systemic (such as intravenous) or local (such as intra-tumor) administration. In one example, a humanized monoclonal antibody or antigen binding fragment thereof, conjugate, nucleic acid molecule, or immune cell comprising a CAR, disclosed herein, is/are formulated for parenteral administration, such as intravenous administration. The disclosed compositions are of use, for example, for the treatment and/or detection of a tumor, for example a tumor occurring in the head and/or neck, breast, pancreas, colon or rectum, central nervous system (CNS), or bladder. In some examples, the compositions are useful for the treatment or detection of a carcinoma.
Compositions for administration to a subject can include a solution of a therapeutic agent dissolved in (or cells suspended in) a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of humanized monoclonal antibody, antigen binding fragment, conjugate, nucleic acid molecule, or immune cell comprising a CAR, in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs. Actual methods of preparing such dosage forms are known, or will be apparent, a practitioner.
An exemplary composition for intravenous administration includes, for example, about 0.01 to about 30 mg/kg of antibody or antigen binding fragment or conjugate per subject per day (or the corresponding dose of a conjugate including the humanized antibody or antigen binding fragment). Actual methods for preparing administrable compositions will be known or apparent to a practitioner and are described in more detail in such publications, such as, Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995). In some examples, the composition is a liquid formulation including one or more antibodies, antigen binding fragments (such as a humanized monoclonal antibody or antigen binding fragment that specifically binds to EGFRvIII), in a concentration range from about 0.1 mg/ml to about 20 mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/ml to about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg/ml.
The compositions disclosed herein may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. In one example, a lyophilized humanized monoclonal antibody or antigen binding fragment or conjugate is suspended and added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art in the administration of antibody or antigen binding fragment and conjugate drugs; for example, antibody drugs have been marketed in the U.S. since the approval of RlTUXAN® in 1997. Antibodies, antigen binding fragments and conjugates can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg antibody or antigen binding fragment (or the corresponding dose of a conjugate including the humanized monoclonal antibody or antigen binding fragment) may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated.
Any of the compositions disclosed herein can be formulated for controlled release. Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, e.g., Banga, A. J., Therapeutic Peptides and Proteins: Formulation. Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).
Polymers can be used for ion-controlled release of a composition disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known and have been described (see, e.g., Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al.. J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342 and U.S. Patent No. 5,534,496).
In some examples, a subject is administered DNA encoding a humanized monoclonal antibody, antigen binding fragments thereof, or conjugate (such as with a toxin) disclosed herein to provide in vivo antibody production, for example using the cellular machinery of the subject. Immunization by nucleic acid constructs is known and taught, for example, in U.S. Patent No. 5,643,578, and U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103. The methods can include liposomal delivery of nucleic acids. Such methods can be applied to the production of a humanized monoclonal antibody, or antibody binding fragments thereof, by a practitioner.
One approach for administration of nucleic acids is direct administration of a vector, such as administering a mammalian expression plasmid (or viral vector). The nucleotide sequence encoding the disclosed humanized monoclonal antibody, or antibody binding fragments thereof, can be included in a vector and placed under the control of a promoter to increase expression.
In another approach to using nucleic acids, a disclosed humanized monoclonal antibody or antibody binding fragments, or conjugate thereof can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors can be used to express the humanized antibody. For example, vaccinia vectors and methods useful protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the disclosed humanized monoclonal antibodies (see Stover, Nature 351:456-460, 1991).
In one example, a nucleic acid encoding a disclosed humanized monoclonal antibody, or antibody binding fragments thereof, is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 pg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).
H. Kits
Kits are also provided. For example, kits are provided for detecting a tumor cell that expresses EGFRvIII and/or gene-amplified EGFR in a subject, and/or treating a tumor in a subject. The kits will typically include a humanized monoclonal antibody or antigen binding fragment that specifically binds EGFRvIII and/or gene-amplified EGFR; and/or a conjugate thereof disclosed herein. In some examples, components of the kit are of useful for both detecting and/or treating a tumor.
More than one of the conjugates, humanized antibodies, antigen binding fragments, immune cells comprising a CAR, nucleic acids, or compositions disclosed herein can be included in the kit. Thus, the kit can include two or more humanized monoclonal antibodies that specifically bind EGFRvIII and/or gene- amplified EGFR. The kit can include at least two of a humanized monoclonal antibody or antigen binding fragment that specifically binds EGFRvIII and/or gene-amplified EGFR, a conjugate thereof, or a combination thereof. In some examples, an antigen binding fragment or conjugate including an antigen binding fragment, such as an Fv fragment, is included in the kit. In one example, such as for in vivo uses, the fragment can be a scFv fragment.
The kit can include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container typically holds a composition including one or more of the disclosed antibodies, antigen binding fragments, or conjugates. In several examples the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). A label or package insert indicates that the composition is used for treating the particular condition.
The label or package insert typically will further include instructions for use of kit components (e.g., an antibody or fragment thereof, conjugate, nucleic acid, immune cells comprising a CAR, or compositions disclosed herein), for example, in a method of treating, preventing, or detecting a tumor. The package insert typically includes instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents used for the practice of a particular method.
EXAMPLES
The mouse monoclonal antibody, 40H3, binds human amplified EGFR when this receptor is overexpressed on cancer cells. 40H3 also binds immobilized EGFRvIII or EGFRvIII when expressed on transfected cells. To utilize 40H3 as a clinical antibody, the variable portions of the heavy and light chains of 40H3 were humanized.
Each CDR was tentatively identified via alignment with the KABAT or similar databases. The variable regions of 40H3, heavy and light chains, were compared to the closest human immunoglobulin families and then residues were altered to produce candidate humanized antibodies. Candidates contained different number of human replacement amino acids and in slightly different locations. Not only were several heavy and light chain candidates generated but combinations were generated where a given humanized heavy chain was matched with several light chains - and vice versa. Thus, optimal pairings were established. Humanizing mouse antibodies is a tunable enterprise. Substituting multiple human residues into a mouse framework will generate a less immunogenic protein but risks the loss of binding activity. All candidates were assayed for retention of binding activity, both to immobilized EGFRvIII (via ELISA) or to cells expressing high levels of EGFR or EGFRvIII (via flow cytometry).
To evaluate the binding activity of humanized candidates, either the immobilized extracellular domain of EGFRvIII (assayed in ELISA or Octet format), various cancer cells expressing EGFR (assayed by flow cytometry), or cancer cells expressing transfected EGFRvIII (also assayed by flow cytometry) were used. All the disclosed humanized sequences retained binding activity to immobilized EGFRvIII but only a subset of them bound to cells that overexpressed EGFR or cells with transfected EGFRvIII. Antibodies were identified with superior cell binding activity.
Example 1 Materials and Methods
ELISA
The binding of candidate humanized antibodies was assessed via an ELISA-type assay. The extracellular domain (ECD) of human EGFRvIII with a C-terminal His tag (EGLH52H4, ACROB iosystems, Newark, DE, USA) was bound to clear nickel-coated 96-well plates (Thermo Fisher Scientific) at 3.5 ng/rnl for 1 h at room temperature. The plates were washed three times with PBS-Tween 20 (PBST) containing 0.05% TWEEN®. Primary antibodies at various concentrations were added to immobilized EGFRvIII on a rocking platform at room temperature for 1 h. Antibodies were diluted in PBS with 5% BSA (Sigma- Aldrich) to the desired concentration. After the addition of the primary antibody, plates were washed three times with PBST. Peroxidase-labeled AffiniPure Donkey Anti-Human IgG (H+L), (Cat# 709035149; Jackson ImmunoResearch, ME, USA) was diluted to 1:20,000 and incubated with rocking at room temperature for 1 h. Plates were washed three times with PBST and the horseradish peroxidase (HRP)- labeled secondary antibody was quantified with the addition of a 3, 3’, 5, 5 ’-tetramethylbenzidine substrate solution (Thermo Fisher Scientific). The reaction was stopped with the addition of sulfuric acid 1 M (Sigma- Aldrich). Horseradish peroxidase activity was analyzed with a VersaMax plate reader using SoftMax Pro software (Molecular Devices, CA, USA).
Flow cytometry'
Antibodies were incubated with suspended cells (2.5 x!05 cells per tube) in 500 ul of FACS buffer consisting of PBS (KD Medical, MD, USA), 2 mM EDTA (KD Medical, MD, USA), 1% BSA (Sigma- Aldrich, MO, USA) and 0.1% sodium azide (Sigma-Aldrich, MO, USA) on ice for 30 min. Bound antibodies were detected with R-Phycoerythrin AffiniPure F(ab')2 Fragment Goat Anti-Human IgG (H+L) (Cat # 109116088; Jackson ImmunoResearch, ME, USA) at 1:250 dilution for 30 min on ice. Afterward, the viability dye, efluor R780 (cat# 65086514, Invitrogen,), was added to each tube and incubated on ice for 15 min (dilution 1/700). Antibody binding was characterized using a BD FACSCANTO™II System (BD Bioscience, San Jose, CA, USA) and the data were analyzed with FlowJo (Tree Star, Inc., Ashland, OR, USA) and displayed in histogram format with the median fluorescence intensity plotted. ECso values for binding to cells were calculated using GraphPad Prism Version 9.4.
Example 2 Results
When assessed for binding to cells or immobilized EGFRvIII, the A10 antibody was the best binder (of all candidates) and was superior to the chimeric antibody with the original mouse VH and mouse VL (see, e.g., FIGs. 1A-1B, 2A-2B, and 4A-4B for the assay results). A10 exhibited an increase in binding affinity (i.e., tighter binding) for both cells and immobilized antigen. The A10 antibody is the combination of candidate heavy chain ‘VH3’ matched with candidate light chain ‘VL3’. Other combinations produced candidates that retained binding activity, albeit with a lower binding affinity. The superior binding activity of all candidates could not be predicted by sequence analysis alone.
All antibody binding data are summarized in FIGs. 4A-4B, while specific cell line results are presented in separate figures. FIGS. 1A-1B show binding to EGFRvIII when transfected F98 cells are analyzed by flow cytometry with increasing concentration of various antibodies. FIG. 2A-2B show antibody binding to EGFR-amplified cell lines, MDA-MB-468 (FIG. 2A) and A431 (FIG. 2B). 40H3 does not bind WT EGFR. To check for any gain of binding activity to WT EGFR, WI-38 and U87MG cells (which express only WT EGFR) were incubated with several humanized candidates. FIGs. 3A-3B show no increased binding to WT EGFR by any of the humanized candidates. However, the presence of WT EGFR was confirmed by cetuximab binding.
FIGS. 4A-4B provide four series of humanized antibodies. The A series consists of VH1-3 candidates paired with VL1-3. All candidates except Al have amino acid substitutions in the framework portions of the variable heavy and light chains. The Al candidate is the original mouse VH and VL fused with human constant sequences for CHI-3 and a constant Kappa chain (pFUSE vectors from Invivogen (pFUSE2ss-CLTg-hK and pFUSEss-hchgl).
The B series included two light chain variants (DA and EG - see sequence information) that changed the last residue of light chain CDR2. These changes were intended to ‘protect’ the antibody from possible post translational modifications. These variants were-not superior to the A10 candidate and were not characterized further.
The C series included sequences that were generated using the BioPhi platforms (Prihoda el al. MABs, 2022). Surprisingly only CIO had beneficial properties for binding cells. C1-C9 did not show binding to cells, but bound antigen. The D series included the variable light chain from antibody Cl paired with VH3 of series A or the light chain from antibody C6 paired with VH3. DI (VH from Cl with VL3) did not bind cells, while D2 and D3 bound cells.
For cell binding experiments, cetuximab is used as a positive control to detect all surface expressed EGFR. The 40H3 antibody (and all humanized versions of 40H3) bind a subset of EGFR that is only expressed on cancer cells whereas cetuximab binds to all cells with surface-expressed EGFR (see, e.g., FIGS. 5A-5B). Using 40H3 and humanized versions in vivo permits the specificity for binding cancer cells while NOT binding normal cells, thus reducing possible side effects.
Example 3 A10 Crystal Structure
The crystal structure of EGFR loop-AlO fab was determined (see, FIGs. 6A-6C). The crystallization conditions were as follows:
1357 Wizard Gil
NaH2PO4 (0.8M) KH2PO4 (1.2M)
Na Acetate (0.1M, pH 4.5)
No cryoprotectant
Table 2: Data Collection.
Figure imgf000090_0001
Table 3: Contact residues between the EGFR target peptide and the A10 antibody.
Figure imgf000090_0002
Example 4
A10 CAR-T Cells
CAR vectors were prepared as shown in FIGS. 8A-8B. CAR T cells were generated via lentiviral transduction and activation of T cells using anti-CD3 and IL-2. FIG. 9 shows cell viability of A431-luc cell line 24 or 48 hours following the indicated treatments. “CAR-1” and “CAR-2” refer to T cells including/expressing the A10 scFv-CAR construct of FIG. 8A or 8B, respectively.
For in vivo administration, after transformation cells are grown for a week and then infused by IV, IP, or locally administered to a tumor site. Transduction of T-cells are monitored via the inclusion of a surface marker (e.g., truncated EGFR) which can be detected using an antibody (e.g., cetuximab). A preparation of T cells that is 35-50% for cetuximab is suitable for infusion.
Example 5
A10 Antibody-Drug Conjugates
Purified A10 antibody was conjugated to a deruxtecan (AlO-Dxd) or monomethyl auristatin E (A10- MMAE) payload. Conjugation reactions were performed commercially by NJBio®, Princeton, NJ. In brief, for conjugation via internal and reduced disulfides, antibody preparations were subjected to mild reduction (4.5 molar excess of TCEP to antibody in PBS). Samples were desalted using Zeba® columns (ThermoFisher® Scientific, NY, USA) according to the manufacturer’s instructions and then conjugated via maleimide groups on the linker-payload composition. Following the conjugation reaction, ADCs were fractionated to remove both under- and over-modified antibody. Drug antibody ratios (DARS) for each ADC were determined. ADCs with 2-8 drugs per antibody (without aggregation) were used. FIG. 10 shows cell viability of MDA-MB-468 cells following treatment with AlO-Dxd or A10-MMAE.
Example 6
Additional Studies
An antibody or fragment thereof disclosed herein (see, e.g., Table 1) is further modified either by attaching drug-based cytotoxic payloads, engineering a CAR construct comprising the antibody or fragment thereof, or combining with a T-cell engager to produce novel antitumor agents. In each case, cancer cells with amplified EGFR or EGFRvIII are killed. Further, cells that express low or normal levels of WT EGFR (non-cancerous cells) are not killed.
It will be apparent that the precise details of the methods or compositions described herein may be varied or modified without departing from the spirit of this disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim:
1. An isolated humanized monoclonal antibody or antigen binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL) comprising: a) SEQ ID NO: 5 and SEQ ID NO: 8, respectively (A10, VH3+VL3); b) SEQ ID NO: 3 and SEQ ID NO: 6, respectively (A2, VH1+VL1); c) SEQ ID NO: 3 and SEQ ID NO: 7, respectively (A3, VH1+VL2); d) SEQ ID NO: 3 and SEQ ID NO: 8, respectively (A4, VH1+VL3); e) SEQ ID NO: 4 and SEQ ID NO: 6, respectively (A5, VH2+VL1); f) SEQ ID NO: 4 and SEQ ID NO: 7, respectively (A6, VH2+VL2); g) SEQ ID NO: 4 and SEQ ID NO: 8, respectively (A7, VH2+VL3); h) SEQ ID NO: 5 and SEQ ID NO: 6, respectively (A8, VH3+VL1); i) SEQ ID NO: 5 and SEQ ID NO: 7, respectively (A9, VH3+VL2); j) SEQ ID NO: 1 and SEQ ID NO: 9, respectively (Bl, 40H3 VH+VL-EG); k) SEQ ID NO: 1 and SEQ ID NO: 10, respectively (B2, 40H3 VH+VL-DA); l) SEQ ID NO: 4 and SEQ ID NO: 11, respectively (B3, VH2+VL1-DA); m) SEQ ID NO: 4 and SEQ ID NO: 12, respectively (B4, VH2+VL2-DA); n) SEQ ID NO: 5 and SEQ ID NO: 11, respectively (B5, VH3 and VL1-DA); o) SEQ ID NO: 31 and SEQ ID NO: 32, respectively (CIO); p) SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2); or q) SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3).
2. The isolated humanized monoclonal antibody of claim 1, wherein the antibody comprises a human constant domain.
3. The isolated humanized monoclonal antibody of claim 1 or claim 2, wherein the antibody is an IgG.
4. The isolated humanized monoclonal antibody of any one of the prior claims, comprising a recombinant constant domain comprising a modification that increases the half-life of the antibody.
5. The isolated humanized monoclonal antibody or antigen binding fragment of any one of the prior claims, conjugated to a toxin or a chemotherapeutic agent.
6. The isolated humanized monoclonal antibody or antigen binding fragment of claim 5, wherein the toxin is a Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin, ribotoxin, ribonuclease, saporin, calicheamicin, or a botulinum toxin.
7. The isolated humanized monoclonal antibody or antigen binding fragment of claim 5 or claim 6, wherein the toxin is the PE, and wherein the PE is PE25, PE38 or PE40.
8. The isolated humanized monoclonal antibody or antigen binding fragment of a claim 5, wherein the chemotherapeutic agent is monomethyl auristatin E or a maytansinoid.
9. The antigen binding fragment of any one of the prior claims.
10. The antigen binding fragment of any one of the prior claims, wherein the antigen binding fragment is a Fv, dsFV, ds-scvFV, Fab, F(ab')2, scFV or a scFVi fragment.
11. The antigen binding fragment of any one of the prior claims, wherein the antigen binding fragment is a Fab or scFV.
12. The isolated humanized monoclonal antibody or antigen binding fragment of any one of the prior claims, conjugated to a detectable marker.
13. A chimeric antigen receptor (CAR) comprising the antigen binding fragment of any one of the prior claims.
14. A bispecific antibody comprising the humanized monoclonal antibody or antigen binding fragment of any one of claims 1-11.
15. An isolated nucleic acid molecule encoding the humanized antibody or antigen binding fragment of any one of claims 1-12, or a VH or VL of the antibody or antigen binding fragment, the CAR of claim 13, or the bispecific antibody of claim 14.
16. The isolated nucleic acid molecule of claim 15, wherein the nucleic acid molecule is a cDNA sequence.
17. The isolated nucleic acid molecule of claim 15 or claim 16, operably linked to a promoter.
18. A vector comprising the nucleic acid molecule of any one of claims 15-17.
19. The vector of claim 18, wherein the vector is a viral vector.
20. An isolated host cell comprising the nucleic acid molecule of any one of claims 15-17, or the vector of claim 18 or 19.
21. An isolated T cell expressing the CAR of claim 13.
22. A pharmaceutical composition for use in treating a cancer that expresses EGFRvIII, comprising an effective amount of the humanized monoclonal antibody or antigen binding fragment of any one of claims 1-12, the bispecific antibody of claim 14, the nucleic acid molecule of any one of claims 15- 17, the vector of claim 18 or claim 19, the host cell of claim 20, or the T cell of claim 21; and a pharmaceutically acceptable carrier.
23. A method of producing a monoclonal antibody or antigen binding fragment that specifically binds to EGFRvIII or a bispecific antibody comprising the monoclonal antibody or antigen binding fragment, the method comprising: expressing one or more nucleic acid molecules of claims 15-17 in a host cell; and purifying the antibody, antigen binding fragment, or the bispecific antibody.
24. A method of detecting the presence of EGFRvIII in a biological sample from a subject, comprising: contacting the biological sample with an effective amount of the humanized monoclonal antibody or antigen binding fragment of any one of claims 1-12 or the bispecific antibody of claim 14 under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the biological sample, wherein the presence of the immune complex in the biological sample indicates the presence of EGFRvIII in the sample.
25. The method of claim 24, wherein the biological sample is a biopsy from a glioma, head and neck cancer, breast cancer, pancreatic cancer, colorectal cancer, or bladder cancer.
26. A method of inhibiting a tumor expressing EGFRvIII and/or expressing gene-amplified EGFR in a subject, comprising administering an effective amount of the humanized monoclonal antibody or antigen binding fragment of any one of claims 1-12, the bispecific antibody of claim 14, the nucleic acid molecule of any one of claims 15-17, the vector of claim 18 or claim 19, the host cell of claim 20, the T cell of claim 21, or the pharmaceutical composition of claim 22, to the subject, wherein the subject has a tumor expressing EGFRvIII or gene-amplified EGFR, respectively.
27. The method of claim 26, wherein the method inhibits the tumor expressing EGFRvIII and a subject having a tumor expressing EGFRvIII is selected for treatment.
28. A method of inhibiting a tumor over-expressing EGFR in a subject, comprising: administering an effective amount of the humanized monoclonal antibody or antigen binding fragment of any one of claims 1-12, the bispecific antibody of claim 14, the nucleic acid molecule of any one of claims 15-17, the vector of claim 18 or claim 19, the T cell of claim 21, or the pharmaceutical composition of claim 22, to the subject having the tumor overexpressing EGFR, wherein the VH of the monoclonal antibody or antigen binding fragment thereof comprises SEQ ID NO: 5 and the VL of the monoclonal antibody or antigen binding fragment thereof comprises SEQ ID NO: 8.
29. The method of any one of claims 24 to 28, wherein the tumor is a glioma, a head and neck cancer, a breast cancer, a pancreatic cancer, a colorectal cancer, or a bladder cancer.
30. The method of any one of claims 24 to 29, wherein the subject is human.
31 . The method of any one of claims 26-33, wherein inhibiting the tumor comprises reducing the growth, size, or metastasis of the tumor.
32. Use of the humanized monoclonal antibody or antigen binding fragment of any one of claims 1-12, the bispecific antibody of claim 14, the nucleic acid molecule of any one of claims 15-17, the vector of claim 18 or claim 19, the T cell of claim 21, or the pharmaceutical composition of claim 22, to inhibit a tumor expressing EGFRvIII in a subject or to detect the presence of EGFRvIII in a biological sample.
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