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WO2025064951A1 - Anti-tumor antibodies - Google Patents

Anti-tumor antibodies Download PDF

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Publication number
WO2025064951A1
WO2025064951A1 PCT/US2024/047873 US2024047873W WO2025064951A1 WO 2025064951 A1 WO2025064951 A1 WO 2025064951A1 US 2024047873 W US2024047873 W US 2024047873W WO 2025064951 A1 WO2025064951 A1 WO 2025064951A1
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WIPO (PCT)
Prior art keywords
antibody
amino acid
tumor
acid sequence
seq
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French (fr)
Inventor
Jessica FINN
Shaun M. Lippow
Erin WECHSLER
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Immunome Inc
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Immunome Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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

Definitions

  • This application relates to therapeutic antibodies for the treatment of various cancers.
  • Protein glycosylation is one of the most complex and common post-translational modifications representing the enzymatic addition of carbohydrate chains called glycans.
  • Glycan-specific antibodies can be detected early in life without immunization, as through infections and vaccinations. The expression of glycans and glycan-specific antibodies may change during the cancer progression. Glycans and glycan-specific antibodies have been suggested to serve as cancer diagnostic and prognostic markers (Tikhonov et al., Glycan- specific antibodies as potential cancer biomarkers: a focus on microarray application, Clinical Chemistry and Laboratory Medicine (CCLM) (2019) Vol. 58: Issue 10). However, since most known glycans are found in cancer patients and healthy individuals, the utility of glycans in cancer therapeutics has not been fully explored.
  • antibodies that bind to a tumor or a fragment thereof comprising: a heavy chain variable region (VH) comprising (i) a complementarity determining region (HCDR) 1 having an amino acid sequence according to any one of SEQ ID NOs: 16, 17, 18, 19, and 20; (ii) a HCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 25, 26, 27, 28, 29, 30, and 31-62; and (iii) a HCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 65-85; and a light chain variable region (VL) comprising (i) a LCDR1 having an amino acid sequence according to any one of SEQ ID NOs: 86, 87, 88, and 91-111; (ii) a LCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 112-122; and (iii) a LCDR3 having an amino acid sequence according to any one of S
  • the VL sequence comprises (i) a LCDR1 having an amino acid sequence according to any one of SEQ ID NOs: 89 and 91-111; (ii) a LCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 1 16-122; and (iii) a LCDR3 having an amino acid sequence according to SEQ ID NO: 126.
  • the VH sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 131-208 and 252.
  • the VL sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 214-251.
  • the VH sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 131-208 and 252, and the VL sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 214-251.
  • antibodies that bind to a tumor or a fragment thereof comprising: a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 127- 208 and 252 and at least one modification in a position selected from the group consisting of: 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 111, 112, 113, 114, 115, and 118; and/or a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 209-251 and at least one modification in a position selected from the group consisting of: 27, 28, 31, 57, and 97.
  • the modification is
  • antibodies that bind to a tumor or fragment thereof comprising: a heavy chain variable region (VH) comprising (i) a complementarity determining region (HCDR) 1 having an amino acid sequence according to any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, and 20; (ii) a HCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31-62; and (iii) a HCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 63-85; and at least one modification in a position selected from the group consisting of: 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 11 1, 112, 113, 114, 115, and 118
  • immunoconjugates comprising an antibody disclosed herein, and a cytotoxic agent conjugated to the antibody using a linker.
  • the cytotoxic agent is selected from the group consisting of: ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, methotrexact, actinomycin, a diphtheria toxin, extotoxin A from Pseudomonas, Pseudomonas exotoxin 40, abrin, abrin A chain, modeccin A chain, alpha sarcin, gelonin, mitogellin, restrictocin, cobran venom factor, a ribonuclease, engineered
  • antibodies that bind to a tumor comprising, a heavy chain variable region comprising an HCDR1, HCDR2, and/or HCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB
  • the heavy chain variable region of the antibody comprises an HCDR3 variant of an HCDR3 of any one of AB- 012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB- 012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB- 012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB- 012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB- 012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB- 012214, AB-01-01
  • the heavy chain variable region of the antibody comprises an HCDR3 variant comprising SEQ ID NO: 84.
  • the antibody comprises all six CDRs of an antibody selected from the group consisting of designated as AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB
  • antibodies comprising a VH region comprising a VH amino acid sequence in Table 3 or an amino sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the VH amino acid sequence in Table 3, and/or wherein the antibody comprises a VL region comprising a VL amino acid sequence in Table 3; and an amino sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the VL amino acid sequence in Table 3.
  • the antibody comprises: a VH region having an amino acid sequence at least 80% identical to the amino acid sequence according to any one of SEQ ID NOs: 127-208 or 252; and/or a VL region having an amino acid sequence at least 80% identical to the amino acid sequence according to any one of SEQ ID NOs: 209-251.
  • the antibody comprises both the VH and VL of an antibody selected from the group consisting of designated as AB-012179, AB-012180, AB- 012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB- 012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB- 012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB- 012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB- 012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215,
  • the antibody comprises both the VH and VL of an antibody selected from the group consisting of designated as AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216,
  • antibodies that competes for binding with an antibody disclosed above.
  • the binding of the antibody or the immunoconjugate to the tumor is dependent on the expression of one or more glycosyltransferases in the tumor.
  • one of the one or more glycosyltransferases has N-acetyl- galactosaminyltransferase activity.
  • one of the one or more glycosyltransferases has fucosyltransferase activity.
  • the glycosyltransferase that has N-acetyl-galactosaminyltransferase activity is selected from the group consisting of B4GALNT3 and B4GALNT4.
  • the glycosyltransferase is B4GALNT3.
  • one of the one or more glycosyltransferases that has fucosyltransferase activity is selected from the group consisting of FUT4, FUT5, FUT6, and FUT9.
  • the glycosyltransferase is FUT9.
  • the tumor expresses a tumor-associated glycan.
  • the tumor- associated glycan is an extracellular glycan.
  • the presence of the tumor- associated glycan is dependent on the expression of B4GALNT3 and FUT9 in the tumor.
  • the antibody preferentially binds to a tumor tissue relative to a normal tissue.
  • the antibody is internalized by the tumor cells upon contacting the tumor.
  • a polynucleotide encoding a polypeptide comprising a VH sequence of an antibody or an immunoconjugate disclosed herein, and /or a VL sequence of an antibody or an immunoconjugate disclosed herein.
  • an expression vector comprising a polynucleotide encoding the VH region and/or the VL region of the antibody or the immunoconjugate.
  • a host cell that comprises the expression vector.
  • compositions comprising the antibody or the immunoconjugate disclosed above and a pharmaceutically acceptable carrier.
  • methods of treating a cancer patient comprising administering the antibody or the immunoconjugate to the patient.
  • the cancer is selected from the group consisting of colorectal, lung, endometrial, breast, stomach and esophageal cancer.
  • the antibody or the immunoconjugate for a method of treating cancer.
  • the cancer is selected from the group consisting of colorectal, lung, endometrial, breast, stomach and esophageal cancer.
  • polypeptides comprising a heavy chain variable region, wherein the heavy chain variable region comprises an HCDR1, HCDR2, and/or HCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-0122
  • the heavy chain variable region of the antibody comprises an HCDR3 variant of an HCDR3 of any one of AB-012179, AB- 012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB- 012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB- 012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB- 012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB- 012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214,
  • polypeptides comprising a light chain variable region, wherein the light chain variable region comprises an LCDR1, LCDR2, and/or LCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211,
  • FIGs. 1A-1D show binding of anti-tumor antibodies to lysate from the colorectal cancer (CRC) cell line LoVo and human serum as determined by ELISA.
  • FIG. 1A shows binding of AB-011110 variants to LoVo lysate.
  • FIG. IB shows binding of AB-022788 variants to LoVo lysate.
  • FIG. 1C shows binding of AB-011628 variants to LoVo lysate.
  • FIG. ID shows binding to human serum.
  • FIG. 2 shows flow cytometry analysis of the binding of anti-tumor antibodies to the CRC cell line LoVo.
  • the x axis is R-phycoerythrin (R-PE) median fluorescence intensity (MedFI) levels and the y axis indicates log antibody concentration (log Ab [C]) nm.
  • FIG. 3 shows the ADC activity of anti-tumor antibodies in LoVo cells using a secondary, monomethyl auristatin F (MMAF) toxin-conjugated antibody assay.
  • the x axis is cytotoxicity and the y axis indicates log antibody concentration (log[Ab]) M.
  • FIG. 4 shows the ADC activity of anti-tumor antibodies in NUGC4 cells using a secondary, monomethyl auristatin F (MMAF) toxin-conjugated antibody assay.
  • the x axis is cytotoxicity and the y axis indicates log antibody concentration (log[AB]) M.
  • FIG. 5 shows results of immunohistochemistry staining of anti-tumor antibodies binding to colorectal cancer tissue sections.
  • FIG. 6A and FIG. 6B show results of immunohistochemistry staining of anti-tumor antibodies binding to cancer tissue sections.
  • FIG. 6A shows binding to tissue sections from colorectal cancer (top panels), lung cancer (middle panels), and endometrial cancer (bottom panels).
  • FIG. 6B shows binding to tissue sections from breast cancer (top panels), stomach cancer (middle panels), and esophageal cancer (bottom panels).
  • FIGs. 7A-7D show alignments and CDR designations for various anti-tumor antibodies.
  • FIG. 7A shows alignments for HCDR1 (top) and HCDR2 (bottom).
  • FIG. 7B shows alignments for HCDR3.
  • FIG. 7C shows alignments for LCDR1 (top) and LCDR2 (bottom).
  • FIG. 7D shows alignments for LCDR3.
  • an “antibody” means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of an antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and specifically covers a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies e.g., bispecific antibodies), antibody fragments including but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.
  • antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds.
  • a light chain consists of one variable region (VL) and one constant region (CL).
  • the heavy chain consists of one variable region (VH) and at least three constant regions (CHI, CH2 and CH3). The variable regions determine the binding specificity of the antibody.
  • Each variable region contains three hypervariable regions known as complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the extent of the FRs and CDRs has been defined (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).
  • each variable region e.g., light chain variable region or heavy chain variable region, with six CDRs total in a typical antibody format
  • CDR1 , CDR2, and CDR3 collectively contribute to antibody binding specificity.
  • Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies.
  • examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies).
  • An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.
  • Antibody fragments comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies (e. . , Zapata et al. , Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab’)2 fragment with two antigen combining sites and is still capable of cross-linking antigen.
  • an anti-tumor antibody refers to an antibody that binds preferentially to a tumor tissue than normal tissue.
  • the normal tissue is the tissue that is adjacent to the tumor, referred to as tumor- adjacent tissue or TAT.
  • an anti-tumor antibody also decreases the rate of tumor growth, tumor size, invasion, and/or metastasis, via direct or indirect effects on tumor cells.
  • V-region refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4.
  • the heavy chain V-region, VH is a consequence of the rearrangement of a V-gene (HV), a D-gene (HD), and a J-gene (HJ), in what is termed V(D)J recombination during B-cell differentiation.
  • the light chain V-region, VL is a consequence of the rearrangement of a V-gene (LV) and a J-gene (LJ).
  • CDR complementarity-determining region
  • VH CDR3 is in the variable domain of the heavy chain of the antibody in which it is found
  • VL CDR3 is the CDR3 from the variable domain of the light chain of the antibody in which it is located.
  • CDR is used interchangeably with “HVR” when referring to CDR sequences.
  • the amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, Structural repertoire of the human VH segments J. Mol. Biol.
  • Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • CDRs as shown in Tables 1 and 2 are defined by IMGT and Kabat.
  • the VH CDRS as listed in Table 1, are defined as follows: HCDR1 is defined by combining Kabat and IMGT; HCDR2 is defined by Kabat, and the HCDR3 is defined by IMGT.
  • the VL CDRS as listed in Table 2 are defined by Kabat.
  • FIGs. 7A-7D show alignment of certain anti-tumor antibody VH and VL sequences with CDRs designated by Kabat and IMGT.
  • the CDRs of other antitumor antibodies provided herein can be similarly designated.
  • numbering and placement of the CDRs can differ depending on the numbering system employed. It is understood that disclosure of a variable heavy and/or variable light sequence includes the disclosure of the associated CDRs, regardless of the numbering system employed.
  • an “Fc region” refers to the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy2.
  • Fc region may vary, however, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxylterminus, using the numbering according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.).
  • the term “Fc region” may refer to this region in isolation or this region in the context of an antibody or antibody fragment. “Fc region” includes naturally occurring allelic variants of the Fc region as well as modified Fc regions, e.g., that are modified to modulate effector function or other properties such as pharmacokinetics, stability or production properties of an antibody.
  • KD Equilibrium dissociation constant
  • kd dissociation rate constant
  • association rate constant k a , time -1 M 1
  • Equilibrium dissociation constants can be measured using any method.
  • antibodies of the present disclosure have a KD of less than about 50 nM, typically less than about 25 nM, or less than 10 nM, e.g., less than about 5 nM or than about 1 nM and often less than about 10 nM as determined by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37°C.
  • an antibody of the present disclosure has a KD of less than 5 x 10’ 5 M, less than 10’ 5 M, less than 5 x 10' 6 M, less than 10" 6 M, less than 5 x 10" 7 M, less than 10" 7 M, less than 5 x 10' 8 M, less than 10' 8 M, less than 5 x 10" 9 M, less than 10" 9 M, less than 5 xlO 10 M, less than 10 lo M, less than 5 x 10 11 M, less than 10 11 M, less than 5 x 10’ 12 M, less than 10" 12 M, less than 5 x 10' 13 M, less than 10‘ 13 M, less than 5 x 10‘ 14 M, less than 10 14 M, less than 5 x 10' 15 M, or less than 10‘ 13 M or lower as measured as a bivalent antibody.
  • an anti-tumor antibody of the present disclosure has KD less than 100 pM, e.g., or less than 75 pM, e.g., in the range of 1 to 100 pM, when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37°C.
  • an anti-tumor antibody of the present disclosure has KD of greater than 100 pM, e.g., in the range of 100-1000 pM or 500-1000 pM when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37°C.
  • the term “monovalent molecule” refers to a molecule having one antigen-binding site, e.g., a Fab or scFv.
  • bivalent molecule refers to a molecule having two antigen-binding sites.
  • a bivalent molecule of the present disclosure is a bivalent antibody or a bivalent fragment thereof.
  • a bivalent molecule of the present disclosure is a bivalent antibody.
  • a bivalent molecule of the present disclosure is an IgG.
  • monoclonal antibodies have a bivalent basic structure.
  • IgG and IgE have only one bivalent unit, while IgA and IgM consist of multiple bivalent units (2 and 5, respectively) and thus have higher valencies. This bivalency increases the avidity of antibodies for antigens.
  • bivalent binding or “bivalently binds to” refer to the binding of both antigen-binding sites of a bivalent molecule to its antigen. In some embodiments, both antigen-binding sites of a bivalent molecule share the same antigen specificity.
  • valency refers to the number of different binding sites of an antibody for an antigen.
  • a monovalent antibody comprises one binding site for an antigen.
  • a bivalent antibody comprises two binding sites for the same antigen.
  • antibody binding in the context of antibody binding to an antigen refers to the combined binding strength of multiple binding sites of the antibody.
  • bivalent avidity refers to the combined strength of two binding sites.
  • nucleotide or amino acid residues refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same e.g., 100% identity) or have a specified percentage of nucleotides or amino acid residues are the same e.g.
  • BLAST 2.0 can be used with the default parameters to determine percent sequence identity.
  • the terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence.
  • the polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.
  • a “conservative” substitution refers to a substitution of an amino acid such that charge, polarity, hydropathy (hydrophobic, neutral, or hydrophilic), and/or size of the side group chain is maintained.
  • Illustrative sets of amino acids that may be substituted for one another include (i) positively-charged amino acids Lys and Arg; and His at pH of about 6; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic hydrophobic amino acids Ala, Vai, Leu and He; (vi) hydrophobic sulfur-containing amino acids Met and Cys, which are not as hydrophobic as Vai, Leu, and He; (vii) small polar uncharged amino acids Ser, Thr, Asp, and Asn (viii) small hydrophobic or neutral amino acids Gly, Ala, and Pro; (ix) amide- comprising amino acids Asn and Gin; and
  • polynucleotide oligonucleotide
  • nucleic acid are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form.
  • Contemplated polynucleotides include a gene or fragment thereof.
  • Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or noncoding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers.
  • loci locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-
  • a T means U (Uracil) in RNA and T (Thymine) in DNA.
  • a polynucleotide can be exogenous or endogenous to a cell and/or exist in a cell-free environment.
  • the term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure are imparted before or after assembly of the polymer.
  • Non-limiting examples of modifications include: 5 -bromouracil, peptide nucleic acid, xeno nucleic acid, morpholines, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • a “vector” as used herein refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector.
  • Certain vectors can direct the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
  • substitution denotes the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • isolated nucleic acid encoding an antibody or fragment thereof refers to one or more nucleic acid molecules encoding antibody heavy or light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • a host cell is a recombinant host cell and includes the primary transformed cell and progeny derived therefrom without regard to the number of passages.
  • a polypeptide “variant” is a polypeptide that typically differs from one or more polypeptide sequences specifically disclosed herein in one or more substitutions, deletions, additions, and/or insertions.
  • cancer cell or “tumor cell” as used herein refers to a neoplastic cell.
  • the term includes cells from tumors that are benign as well as malignant. Neoplastic transformation is associated with phenotypic changes of the tumor cell relative to the cell type from which it is derived. The changes can include loss of contact inhibition, morphological changes, and unregulated cell growth,
  • inhibiting growth of a tumor and “inhibiting growth of a cancer” are interchangeable and refer to slowing growth and/or reducing the cancer cell burden of a patient that has cancer. “Inhibiting growth of a cancer” thus includes killing cancer cells, as well as decreasing the rate of tumor growth, tumor size, invasion, and/or metastasis by direct or indirect effects on tumor cells.
  • treatment refers to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of affecting a partial or complete cure for a disease and/or symptoms of the disease.
  • Treatment may include treatment of a disease or disorder (e.g., cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g.
  • Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms; or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • the treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician.
  • treating includes the administration of the compounds or agents of the present disclosure to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with diseases (e.g., cancer).
  • therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
  • the terms “recipient,” “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and in some embodiments, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the mammal is human. None of these terms require the supervision of medical personnel.
  • “Expression of a glycan by a cell” or a “glycan expressed by a cell” means that the glycan is present in or on that cell.
  • a “tumor overexpressing glycans”, or a “tumor that overexpresses glycans”, or a “cancer overexpressing glycans” or a “cancer that overexpresses glycans”, refers to a tumor or cancer that expresses specific glycans at a level that higher than the level of those glycans expressed in normal tissue (e.g., tumor adjacent tissues or TAT) or otherwise has an increased amount of those glycans as compared to normal tissue.
  • normal tissue e.g., tumor adjacent tissues or TAT
  • a tumor or cancer that overexpresses glycans expresses glycan at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 300% higher or more than the normal tissue (e.g., tumor-adjacent tissues or TAT).
  • the normal tissue e.g., tumor-adjacent tissues or TAT
  • tumor-associated glycan refers to a glycan expressed by a tumor cell.
  • the tumor-associated glycan is not expressed by normal tissue cells.
  • the tumor-associated glycan is expressed by a tumor at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 300% higher or more as compared to normal tissue (e.g., tumor- adjacent tissues or TAT),
  • the tumor-associated glycan is expressed on the cell surface, i.e. , is an extracellular glycan.
  • a glycan or a tumor-associated glycan disclosed herein may be attached to proteins or lipids, known as glycoproteins and glycolipids.
  • the disclosure additionally provides methods of identifying subjects who are candidates for treatment with an anti-tumor antibody having tumortargeting effects.
  • the present disclosure provides a method of identifying a patient who can benefit from treatment with an anti-tumor antibody of the present disclosure.
  • the patient has tumor that expresses glycans.
  • the patient has tumor expressing a tumor- associated glycan.
  • the tumor sample is from a primary tumor.
  • the tumor sample is a metastatic lesion. Binding of antibody to tumor cells through a binding interaction with the glycans can be measured using any assay, such as immunohistochemistry or flow cytometry.
  • binding of antibody to at least 0.2%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, or at least 50%, of the tumor cells in a sample may be used as a selection criterion for determining a patient to be treated with an anti-tumor antibody as described herein.
  • analysis of components of the blood is used to identify a patient whose tumor cells are expressing a tumor-associated glycan.
  • An anti-tumor antibody disclosed herein can be used to treat several different cancers.
  • a cancer patient who can benefit from the treatment of the anti-tumor antibody has a cancer expressing glycans.
  • a cancer patient who can benefit from the treatment of the anti-tumor antibody has a tumor expressing a tumor- associated glycan.
  • the cancer is a carcinoma, a melanoma, or a sarcoma.
  • a glycan binder refers to a molecule, for example, an antibody or antibody binding domain, that binds to a tumor and the binding is dependent on the activity of one or more glycosyltransferases.
  • the glycan binder binds to a tumor-associated glycan under permissible conditions (e.g., in a suitable buffer), and the detected signal resulted from the binding is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 150 fold, or at least 200 fold above a reference level.
  • the reference level is a detected signal produced by contacting a control antibody with the glycans, or by contacting the antibody with a control protein.
  • a “variant” of a reference antibody refers to an antibody that typically differs from the reference antibody in one or more substitutions, deletions, additions, and/or insertions in the amino acid sequence of the heavy and/or light chain.
  • the term “internalize,” or “internalization” refer to the phenomenon that an antibody molecule crosses the cell membrane and reaches the cytoplasm and/or the nucleus.
  • tumor-targeting, or anti-tumor antibodies that bind to tumors and the binding is dependent on the activity one or more glycosyltransferases.
  • the anti-tumor antibody binds to a tumor-associated glycan.
  • These antibodies are referred to glycan binders in this disclosure.
  • the antibodies bind preferentially to tumor tissues relative to normal tissue and demonstrate cytotoxicity in various antibody-toxin or antibody-immunomodulating agent constructs towards tumor cells. Thus, these antibodies show therapeutic potential in treating cancers.
  • the anti-tumor antibodies are also useful in detecting tumors suitable for treatment with an anti-tumor antibody in diagnostic applications.
  • Glycans are a complex group of monosaccharide or polysaccharide compounds comprised of diverse monosaccharide residues linked glycosidically.
  • Glycosidic bonds are covalent linkages of carbohydrate moieties to another group which may or may not be another carbohydrate (e.g., C-, O-, N-). Through these glycosidic bonds, glycolipids, glycoproteins, and polysaccharides are formed.
  • glycocan refers to a polysaccharide or oligosaccharide, or the carbohydrate portion of all glycol conjugate such as glycoprotein, glycolipid, glycopeptide, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans can be homo or heteropolymers of monosaccharide residues.
  • Forming glycosidic bonds typically require glycosyltransferases.
  • Glycosyltransferases modify glycans in the ER and Golgi apparatus during their biosynthesis. Glycosyltransferases catalyze the transfer of saccharide moieties from an activated nucleotide sugar (also known as the "glycosyl donor") to a nucleophilic glycosyl acceptor molecule, the nucleophile of which can be oxygen- carbon-, nitrogen-, or sulfurbased.
  • an activated nucleotide sugar also known as the "glycosyl donor”
  • nucleophilic glycosyl acceptor molecule the nucleophile of which can be oxygen- carbon-, nitrogen-, or sulfurbased.
  • B4GALNT3 (beta-l,4-N-acetyl-galactosaminyltransferase 3) is responsible for transfer of N-acetylgalactosamine (GalNAc) to N-acetylglucosamine-beta (GlcNAc) to form N, N'-diacetyllactosediamine with betal,4-linkage.
  • the product of this reaction is GalNAcbetal,4GlcNAc (LacdiNAc).
  • B4GALNT4 is reported to have similar activity to B4GALNT3.
  • Examples of human B4GALNT3 and B4GALNT4 and their murine homologs include human B4GALNT3 (GenBank accession no 1 . AB089940; UniProt
  • accession numbers throughout the application refer to the versions that were current in the database as of the filing date of the application.
  • Fucosyltransferases are glycosyltransferases involved in the synthesis of cellsurface antigens through catalyzing the transfer of fucose from GDP-fucose to acceptor sugars on biomolecules.
  • the FUT family includes enzymes catalyzing al, 2-, al, 3/4-, al, 6- and protein O-FUT linkages.
  • the al ,3/4-FUT group includes at least eight members: FUT3, FUT4, FUT5, FUT6, FUT7, FUT9, FUT10, and FUT11.
  • FUT4 is a known to catalyze the alpha (l->3) linkage of beta-L-fucose to the GlcNAc of type 2 lactosmaines (LacNAc, Gal- beta (l->4) GlcNAc). See Lowe JB et al. “Molecular cloning of a human fucosyltransferase gene that determines expression of the Lewis x and VIM-2 epitopes but not ELAM-1- dependent cell adhesion”. J Biol Chem. 1991 Sep 15;266(26):17467-77. Examples of human FUTs include FUT3 (GenBank accession no.
  • FUT10 GenBank accession no. AJ582015; UniProt ID: Q6P4F1; and FUT11 (GenBank accession no. BC036037 UniProt ID: Q495W5).
  • murine FUTs include FUT4 (GenBank accession no. NM_010242; UniProt ID: B2RPT3); FUT7 (GenBank accession no. NM_013524; UniProt ID: QI 1131); FUT9 (GenBank accession no. NM_010243; UniProt ID: 088819); FUT10 (GenBank accession no. AJ880009; UniProt ID: Q5F2L2); and FUT11 (GenBank accession no. NM_028428.2; UniProt ID: Q8BHC9).
  • Anti-tumor antibody AB -006410 was discovered in antibody repertoires generated by Immune Repertoire Capture® (IRC®) technology from plasmablast B cells isolated from a melanoma patient who had undergone treatment with a pembrolizumab. The patient exhibited an active anti-tumor immune response evidenced by tumor-selective antibodies derived from their plasmablast B cells.
  • IRC® Immune Repertoire Capture®
  • AB-006410 and other anti-tumor antibodies provided herein, bind to tumors and the binding is dependent on the expression of one or more specific glycosyltransferases in the tumor, that is, in the absence of expression of the one or more glycosyltransferases, the anti- tumor antibody will not show detectable binding to the tumor.
  • one of the one or more glycosyltransferases has N-acetyl-galactosaminyltransferase activity, such as the activity of B4GALNT3 or B4GALNT4.
  • the one of the one or more glycosyltransferases has fucosyltransferase activity, such as the activity of FUT3, FUT4, FUT5, FUT6, FUT7, FUT9, FUT10, and FUT11.
  • the one of the one or more glycosyltransferases has fucosyltransferase activity, such as the activity of FUT4, FUT5, FUT6, and FUT9.
  • the one of the one or more glycosyltransferases has N-acetyl-galactosaminyltransferase activity, such as the activity of B4GALNT3 or fucosyltransferase activity, such as the activity of FUT9.
  • the one of the one or more glycosyltransferases has N-acetyl- galactosaminyltransferase activity, such as the activity of B4GALNT3 and one of the one or more glycosyltransferases has fucosyltransferase activity, such as the activity of FUT9.
  • the binding of the anti-tumor antibody to the tumor is dependent on the expression one or more specific glycosyltransferases in the tumor, that is, in the absence of the expression of the one or more glycosyltransferases, the anti-tumor antibody will not show detectable binding to the tumor.
  • each of the one or more glycosyltransferases is selected from the group consisting of B4GALNT3, B4GALNT4, FUT3, FUT4, FUT5, FUT6, FUT7, FUT9, FUT10, and FUT11.
  • the binding is dependent on the expression of the glycosyltransferase B4GALNT3 or the glycosyltransferase FUT9.
  • the anti-tumor antibody binds to a tumor cell that expresses a glycan comprising GalNAcbetal,4GlcNAc (LacdiNAc).
  • the anti-tumor antibody binds to a tumor cell that expresses GalNAcbetal,4GlcNAc (LacdiNAc).
  • the LacdiNAc is fucosylated.
  • an anti-tumor antibody disclosed herein binds to an extracellular, tumor- ssociated glycan.
  • the presence of the glycan is dependent on the expression of one or more specific glycosyltransferases in the tumor, that is, in the absence of expression of the one or more glycosyltransferases, the glycan will not be produced in the tumor cells or will not be displayed on the surface of tumor cells.
  • each of the one or more glycosyltransferases is selected from the group consisting of B4GALNT3, B4GALNT4, FUT3, FUT4, FUT5, FUT6, FUT7, FUT9, FUT10, and FUT11.
  • each of the one or more glycosyltransferases is selected from the group consisting of B4GALNT3, B4GALNT4, FUT4, FUT5, FUT6, and FUT9.
  • the glycan is dependent on the expression of the glycosyltransferase B4GALNT3 or the glycosyltransferase FUT9. In some embodiments, the glycan is dependent on the expression of B4GALNT3 and FUT9.
  • the anti-tumor antibody binds to a glycan comprising GalNAcbetal,4GlcNAc (LacdiNAc). In some embodiments, the anti-tumor antibody binds to GalNAcbetal,4GlcNAc (LacdiNAc). In some embodiments, the antitumor antibody binds to a tumor cell that expresses a glycan comprising GalNAcbetal,4GlcNAc (LacdiNAc). In some embodiments, the anti-tumor antibody binds to a tumor cell that expresses GalNAcbetal ,4GlcNAc (LacdiNAc). In some embodiments, the LacdiNAc is fucosylated.
  • an anti-tumor antibody comprises an HCDR1 having an amino acid sequence according to any one of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20 or a variant HCDR1 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence; an HCDR2 having an amino acid sequence according to any one of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-62, or a variant HCDR2 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence; and an HCDR3 having an amino acid sequence according to any one of SEQ ID NO: 63-85 or a variant HCDR3 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence.
  • an anti-tumor antibody comprises a light chain variable region comprising: an LCDR1 having an amino acid sequence according to any one of SEQ ID NO: 86, 87, 88, 91-11 1, or a variant LCDR1 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence; an LCDR2 having an amino acid sequence according to any one of SEQ ID NO: 112-122, or variant LCDR2 in which 1, 2, or 3 amino acids are substituted relative to the sequence; and an LCDR3 having an amino acid sequence according to any one of SEQ ID NO: 123-126, or a variant LCDR3 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence.
  • an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 70% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 75% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 80% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 85% identical to any one of SEQ ID NO: 127-208.
  • an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 95% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 96% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 97% identical to any one of SEQ ID NO: 127-208.
  • an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 98% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 99% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid identical to any one of SEQ ID NO: 127-208.
  • an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 70% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 75% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 80% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 85% identical to any one of SEQ ID NO: 209-251.
  • an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 95% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 96% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 97% identical to any one of SEQ ID NO: 209-251.
  • an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 98% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 99% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence identical to any one of SEQ ID NOs: 209-251. [0080] In some embodiments an anti-tumor antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region.
  • VH heavy chain variable
  • VL light chain variable
  • the VH region has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 127-208; and comprises an HCDR1 having an amino acid sequence according to any one of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, or the HCDR1 of any one of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20 in which 1 , 2, 3, 4, or 5 amino acids are substituted; an HCDR2 having an amino acid sequence according to any one of SEQ ID NO:
  • HCDR3 having an amino acid sequence according to SEQ ID NO: 63-85 or the HCDR3 of SEQ ID NO: 63-85 in which 1, 2, 3, 4, or 5 amino acids are substituted.
  • the VL region has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 209-251, and comprises an LCDR1 having an amino acid sequence according to SEQ ID NO: 86, 87, 88, 91-111 or the LCDR1 of SEQ ID NO: 86, 87, 88, 91-111 in which 1, 2, 3, 4, or 5 amino acids are substituted; an LCDR2 having an amino acid sequence according to SEQ ID NO: 112-122, or the LCDR2 of SEQ ID NO: 112-122 in which 1, 2, or 3 amino acids are substituted; an LCDR3 having an amino acid sequence according to SEQ ID NO: 123-126 or the LCDR3 of SEQ ID NO: 123-126 in which 1, 2, 3, 4, or 5 amino acids are substituted.
  • an anti-tumor antibody comprises: a Vn region comprising amino acid sequence SEQ ID NO: 127-208 or 252 and a VL region comprising amino acid sequence SEQ ID NO: 209-251.
  • an anti-tumor antibody of the present disclosure has one, two, or three CDRs of a VL sequence (LCDRs) having an amino acid sequence according to a sequence set forth in Table 2.
  • the anti-tumor antibody has at least one mutation and no more than 10, 20, 30, 40 or 50 mutations in the VL amino acid sequences compared to a VL sequence set forth in Table 3.
  • the anti-tumor antibody has a VL amino acid sequence set forth in Table 3.
  • the VL amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions compared to a VL sequence set forth in Table 3.
  • the VL amino acid sequence may comprise a deletion or insertion, e.g., a 1, 2, 3, 4, 5, 6, or 7 amino acid deletion or insertion, relative to an LCDR sequence shown in Table 2.
  • the VL region comprises an LCDR1 having 1 or 2 substitutions in relative to an LCDR1 sequence shown in Table 2.
  • an LCDR1 has 3, 4, or 5 substitutions relative to a CDR1 sequence shown in Table 2.
  • the VL region comprises an LCDR2 that has 1 or 2; or 1, 2, or 3; substitutions relative to the LCDR2 sequence shown in Table 2.
  • the VL region comprises an LCDR3 that has 1, 2, or 3; or 1, 2, 3, or 4; substitutions relative to an LCDR3 sequence shown in Table 2.
  • an anti-tumor antibody of the present disclosure comprises a CDR1 , CDR2, and CDR3, each having at least 70% identity to an LCDR1 , LCDR2, and LCDR3 as shown in Table 2.
  • an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 75% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2.
  • an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 80% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 85% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 90% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2.
  • an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 95% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having 100% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2.
  • an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3 of an antibody designated as AB-011110, AB-011367, AB-011622, Ab-011788, AB-011861, AB-011263, AB-011628, AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB
  • an anti-tumor antibody of the present disclosure has one, two, or three CDRs of a VH sequence (HCDRs) having an amino acid sequence according to a sequence set forth in Table 1.
  • the anti-tumor antibody has at least one mutation and no more than 10, 20, 30, 40 or 50 mutations in the VH amino acid sequences compared to a VH sequence set forth in Table 3.
  • the anti-tumor antibody has a VH amino acid sequence set forth in Table 3.
  • the VH amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions compared to a VH sequence set forth in Table 3.
  • the VH amino acid sequence may comprise a deletion or insertion, e.g., a 1, 2, 3, 4, 5, 6, or 7 amino acid deletion or insertion, relative to an HCDR sequence shown in Table 1.
  • the VH region comprises an HCDR1 having 1 or 2 substitutions in relative to an HCDR1 sequence shown in Table 1.
  • an HCDR1 has 3, 4, or 5 substitutions relative to an HCDR1 sequence shown in Table 1.
  • the VH region comprises an HCDR2 that has 1 or 2; or 1, 2, or 3; substitutions relative to the HCDR2 sequence shown in Table 1.
  • the VH region comprises an HCDR3 that has 1, 2, or 3; or 1, 2, 3, or 4; substitutions relative to an HCDR3 sequence shown in Table 1.
  • an anti-tumor antibody of the present disclosure comprises an HCDR1, HCDR2, and HCDR3, each having at least 70% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1.
  • an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 75% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1.
  • an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 80% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 85% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 90% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1.
  • an anti-tumor antibody of the present disclosure comprises a CDR1 , CDR2, and CDR3, each having at least 95% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having 100% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1.
  • an anti-tumor antibody of the present disclosure comprises an HCDR1 , HCDR2, and HCDR3 of an antibody designated as AB-011110, AB- 011367, AB-011622, Ab-011788, AB-011861, AB-011263, AB-011628, AB-012179, AB- 012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB- 012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB- 012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB- 012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207
  • variants of any of the anti-tumor antibodies disclosed herein can be generated by introducing mutations to the heavy chain and/or light chain sequences.
  • the mutation(s) are introduced into one or more of the CDRs of an anti-tumor antibody disclosed herein, e.g., AB-006410, AB-01110, AB-0011367, AB- 011788, AB-001 1628 or other antibodies disclosed in Table 1 or 2.
  • the mutation(s) are introduced in the framework regions.
  • a variant is engineered to be as much like self as possible to minimize immunogenicity. One approach to do so is to identify a close germline sequence and mutate one of the anti-tumor antibodies at as many mismatched positions (also known as “germline deviations”) to the germline residue type as possible.
  • the name indicates the antibody ID of the parental antibody (e.g., 11110 indicates parental antibody AB- 11110), whether the mutation is a heavy chain or light chain, the position in the heavy chain or light chain of the mutation (with the numbering based on the sequences as provided in Table 3 and not on Kabat or other amino acid sequence numbering conventions), the amino acid residue at the position before introduction of the mutation, and the amino acid at the position after introduction of the mutation.
  • L26NS refers to that the asparagine (N) in the light chain position 26 is mutated to a serine (S).
  • the anti-tumor antibodies comprise amino acid residue mutation(s) of the heavy chain at position 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 111 , 1 12, 113, 114, 115, and/or 1 18.
  • the anti-tumor antibodies comprise an amino acid residue mutation of the heavy chain at position 107.
  • the anti-tumor antibodies comprise amino acid residue mutaiton(s) of the light chain at position 27, 28, 31, 57, and/or 97.
  • the anti-tumor antibodies comprise amino acid residue mutation(s) of the heavy chain at position 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 111, 112, 113, 114, 115, and/or 118 and amino acid residue mutation(s) of the light chain at position 27, 28, 31, 57, and/or 97.
  • the anti-tumor antibodies comprise amino acid residue mutation(s) selected from Table 6.
  • the mutation(s) are in a CDR region.
  • the mutation(s) are in a framework region.
  • one or more mutations are selected from the group consisting of L74ND, H54DK, H57HY, H31NV, H32AT, H33WY, H34VI, H35NH, H50RK, H52RH, H52RN, H53SV, H53SA, H54DN, H54DH, H54DR, H54DS, H56ES, H56ED, H56EN, H57GY, H57GK, H57GS, H58WY, H59TA, H59TW, H59TN, H61DN, H61DY, H61DS, H61DF, H62YW, H73SY, H74RA, H75EA, H76EA, H99NE, H101GA, H102YW, H103WY, H106SY, H106SA, H107AN, H107AS, H107AV, H108FY, H108FW, Hl 1 IND, Hl 1 INS, Hl 12R
  • one or more mutations are selected from the group consisting of L74ND, H54DK, H57HY, H31NV, H32AT, H33WY, H34VI, H35NH, H50RK, H52RH, H52RN, H53SV, H53SA, H54DN, H54DH, H54DR, H54DS, H56ES, H56ED, H56EN, H57GY, H57GK, H57GS, H58WY, H59TA, H59TW, H59TN, H61DN, H61DY, H61DS, H61DF, H62YW, H73SY, H74RA, H75EA, H76EA, H99NE, H101 GA, H102YW, H103WY, H106SY, H106SA, H107AN, H107AS, H107AV, H108FY, H108FW, Hl 1 IND, Hl 1 INS, H112RK, Hl BAE, H114YW, H114YP,
  • entire CDR regions or entire framework regions of parental antibodies are replaced with an entire CDR region or entire framework region from a different antibody (e.g., those listed in Tables 1-3).
  • the one or more of the CDRs of the anti-tumor antibodies disclosed in Table 1 or 2 are mutated to generate variants with improved properties.
  • the anti-tumor antibodies disclosed herein comprise a modified Fc region (as further explained herein).
  • Fc regions typically comprises one or more Fc chains.
  • An IgG Fc chain typically contains two constant heavy domains (Cu2 and CH3) and a hinge region connected to the CH2 domain.
  • Fc regions may typically comprise two Fc chains which dimerize with one another; however, an Fc region may have a single chain or more than two Fc chains, e.g., as may be present in some antibody formats.
  • the anti-tumor antibodies comprise an IgGl Fc region (e.g., human IgGl Fc region), that is, except for having particular residue(s) at certain positions as noted herein, the Fc region has an amino acid sequence that is substantially similar to that of the Fc region within a wild type IgGl Fc.
  • the wild type IgGl Fc is a human IgGl.
  • the anti-tumor antibodies comprise an Fc region, each Fc chain of which has an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to that of an Fc chain within a wild-type IgGl Fc.
  • the anti-tumor antibodies comprise an IgG2 Fc region (e.g., human IgG2 Fc region), that is, except for having particular residue(s) at certain positions as noted herein, the Fc region has an amino acid sequence that is substantially similar to that of the Fc region within a wild type IgG2 Fc.
  • the wild type IgG2 Fc is a human IgG2 Fc.
  • the anti-tumor antibodies comprise an Fc region, each Fc chain of which has an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to that of an Fc chain within a wild-type IgG2 Fc.
  • the anti-tumor antibodies comprise an IgG4 Fc region (e.g., human IgG4 Fc region), that is, except for having particular residue(s) at certain positions as noted herein, the Fc region has an amino acid sequence that is substantially similar to that of the Fc region within a wild type IgG4 Fc.
  • the wild type IgG4 Fc is a human IgG4 Fc.
  • the anti-tumor antibodies comprise an Fc region, each Fc chain of which has an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to that of an Fc chain within a wild-type IgG4 Fc.
  • Fc regions are modified (e.g., substituted) at one more amino acid residues.
  • modifications alter the half-life of a molecule e.g., binding protein) which comprises the Fc region by altering (e.g., enhancing) binding to an Fc receptor such as the neonatal Fc receptor (FcRn.)
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • one or more modifications in the modified Fc region is selected from the group consisting of: S298A, E333A, K334A, K326A, F243L, R292P, Y300L, V305I, P396L, F243L, R292P, Y300L, L235V, P396L, F243L, S239D, I332E, A330L, S267E, L328F, D265S, S239E, K326A, A327H, G237F, K326E, G236A, D270L, H268D, S324T, L234F, N325L, V266L, and S267D.
  • one or more modifications in the modified Fc region is selected from the group consisting of S228P, M252Y, S254T, T256E, T256D, T250Q, H285D, T307A, T307Q, T307R, T307W, L309D, Q411H, Q311V, A378V, E38OA, M428L, N434A, N434S, N297A, D265A, L234A, L235A, and N434W.
  • the modified Fc region comprises a specific combination of amino acid substitutions selected from the group consisting of: L234A/L235A; V234A/G237A; L235A/G237A/E318A; S228P/L236E; H268Q/V309L/A330S/A331S; C220S/C226S/C229S/P238S; C226S/C229S/E3233P/L235V/L235A; L234F/L235E/P331S; C226S/P230S; L234A/G237A; L234A/L235A/G237A; Q311R/M428L;
  • the modified Fc region comprises a specific combination of amino acid substitutions consisting of L234A/L235 A/P329A (LALAPA).
  • the modified Fc region comprises a specific combination of amino acid substitutions selected from the group consisting of M428L/N434S (LS); M252Y/S254T/T256E (YTE); T250Q/M428L; T307A/E380A/N434A; T256D/T307Q (DQ); T256D/T307W (DW); M252Y/T256D (YD); T307Q/Q311V/A378V (QVV);
  • T256D/H285D/T307R/Q311V/A378V DDRVV
  • L309D/Q311H/N434S DHS
  • S228P/L235E SPLE
  • L234A/L235A LALA
  • M428L/N434A LA
  • L234A/G237A LAGA
  • L234A/L235A/G237A LALAGA
  • L234A/L235A/P329G LALAPG
  • L234A/L235A/P329A LALAPA
  • N297A/YTE D265A/YTE; LALA/YTE; LAGA/YTE; LALAGA/YTE; LALAPG/YTE; N297A/LS; D265A/LS; LALA/LS; LAGA/LS;
  • LALAPG/N434A N297A/N434W; D265A/N434W; LALA/N434W; LAGA/N434W;
  • LALAGA/N434W LALAPG/N434W; N297A/DQ; D265A/DQ; LALA/DQ; LAGA/DQ; LALAGA/DQ; LALAPG/DQ; N297A/DW; D265A/DW; LALA/DW; LAGA/DW;
  • LALAGA/DW LALAPG/DW; N297A/YD; D265A/YD; LALA/YD; LAGA/YD;
  • LALA/Q311R/M428L LAGA/Q311R/M428L; LALAGA/Q311R/M428L; and LALAPG/Q311R/M428L.
  • the antibodies provided herein can include such amino acid modifications with or without the heterodimerization variants outlined herein (e.g., the pl variants and steric variants).
  • Each set of variants can be independently and optionally included or excluded from any heterodimeric protein.
  • the subject antibody includes modifications that alter the binding to one or more FcyR receptors (/. ⁇ ?., “FcyR variants”).
  • FcyR variants substitutions that result in increased binding as well as decreased binding can be useful.
  • ADCC antibody dependent cell-mediated cytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcyRIIb an inhibitory receptor
  • Amino acid substitutions that find use in the antibodies described herein include those listed in US Patent Nos. 8,188,321 (particularly Figure 41) and 8,084,582, and US Publ. App. Nos.
  • Fc substitutions that find use in increased binding to the FcRn receptor and increased serum half-life, as specifically disclosed in USSN 12/341,769 including, but not limited to, 434S, 434 A, 428L, 308F, 2591, 428L/434S, 259U308F, 436U428L, 4361 or V/434S, 436V/428L and 259I/308F/428L.
  • Such modification may be included in one or both Fc domains of the subject antibody.
  • the anti-tumor antibodies comprising Fc substitutions that have increased binding to the FcRn receptor and increased serum half-life have an increased half-life that is about 10,000-fold, 1,000-fold, 500-fold, 100-fold, 50-fold, 20-fold, 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4.5-fold, 4-fold, 3.5-fold, 3-fold, 2.5-fold, 2-fold, 1.95-fold, 1.9-fold, 1.85-fold, 1.8-fold, 1.75-fold, 1.7-fold, 1.65-fold, 1.6-fold, 1.55-fold, 1.50-fold, 1.45-fold, 1.4-fold, 1.35-fold, 1.3-fold, 1.25-fold, 1.2-fold, 1.15-fold, 1.1-fold, or 1.05-fold longer compared to a binding protein comprising a wild-type Fc region.
  • an anti-tumor antibody disclosed herein, including antibody fragments, of the present disclosure comprises an Fc region that has effector function, e.g., exhibits antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC).
  • the Fc region may be an Fc region engineered to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or ADCC.
  • an Fc region can comprise additional mutations to increase or decrease effector functions, i.e., the ability to induce certain biological functions upon binding to an Fc receptor expressed on an immune cell.
  • Immune cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans’ cells, natural killer (NK) cells, and cytotoxic T cells.
  • an Fc region described herein can include additional modifications that modulate effector function.
  • Fc region amino acid mutations that modulate an effector function include, but are not limited to, one or more substitutions at positions 228, 233, 234, 235, 236, 237, 238, 239, 243, 265, 269, 270, 297, 298, 318, 326, 327 , 329, 330, 331, 332, 333, and 334 (EU numbering scheme) of an Fc region.
  • Illustrative substitutions that decrease effector functions include the following: position 329 may have a mutation in which proline is substituted with a glycine or arginine or an amino acid residue large enough to destroy the Fc/Fcy receptor interface that is formed between proline 329 of the Fc and tryptophan residues Trp 87 and Trp 110 of FcyRIII. Additional illustrative substitutions that decrease effector functions include S228P, E233P, L235E, N297A, N297D, and P331S.
  • L234A and L235A of a human IgGl Fc region may also be present, e.g., L234A and L235A of a human IgGl Fc region; L234A, L235A, and P329G of a human IgGl Fc region; S228P and L235E of a human IgG4 Fc region; L234A and G237A of a human IgGl Fc region; L234A, L235A, and G237A of a human IgGl Fc region; V234A and G237A of a human IgG2 Fc region; L235A, G237A, and E318A of a human IgG4 Fc region; and S228P and L236E of a human IgG4 Fc region, to decrease effectors functions.
  • substitutions that increase effector functions include, e.g., E333A, K326W/E333S, S239D/I332E/G236A, S239D/A330L/I332E, G236A/S239D/A330L/I332E, F243L, G236A, and S298A/E333A/K334A.
  • the Fc mutations include P329G, L234A, L235A, or a combination thereof. Descriptions of amino acid mutations in an Fc region that can increase or decrease effector functions can be found in, e.g., Wang et al., Protein Cell.
  • an Fc region may have one or more amino acid substitutions that modulate ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region, according to the EU numbering scheme.
  • S298A, E333A, and K334A can be introduced to an Fc region to increase the affinity of the Fc region to FcyRIIIa and decrease the affinity of the Fc region to FcyRIIa and FcyRIIb.
  • An Fc region can also comprise additional mutations to increase serum half-life. Through enhanced binding to the neonatal Fc receptor (FcRn), such mutations in an Fc region can improve the pharmacokinetics of the antibody. Examples of substitutions in an Fc region that increase the serum half-life of an antibody include, e.g., M252Y/S254T/T256E, T250Q/M428L, N434A, N434H, T307A/E380A/N434A, M428L/N434S, M252Y/M428L, D259I/V308F, N434S, V308W, V308Y, and V3O8F.
  • FcRn neonatal Fc receptor
  • an anti-tumor antibody described herein comprise an Fc region having altered glycosylation that increases the ability of the antibody to recruit NK cells and/or increase ADCC.
  • the Fc region comprises glycan containing no fucose (i.e. , the Fc region is afucosylated).
  • Afucosylated antibodies can be produced using cell lines that express a heterologous enzyme that depletes the fucose pool inside the cell (e.g., GlymaxX® by ProBioGen AG, Berlin, Germany).
  • Non-fucosylated antibodies can also be produced using a host cell line in which the endogenous a-1,6- fucosyltransferase (FUT8) gene is deleted. See Satoh, M. et al., “Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies,” Expert Opinion on Biological Therapy, 6:11, 1161-1173, DOI: 10.1517/14712598.6.11.1161.
  • an antibody of the disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified, e.g., produced in cell lines and/or in cell culture conditions to alter its glycosylation (e.g., hypofucosylation, afucosylation, or increased sialylation), to alter one or more functional properties of the antibody.
  • the antibody can be linked to one of a variety of polymers, for example, polyethylene glycol.
  • an antibody may comprise mutations to facilitate linkage to a chemical moiety and/or to alter residues that are subject to post-translational modifications, e.g., glycosylation. ACTIVITY
  • the activity of the anti-tumor antibodies as described herein can be assessed for binding in binding assays.
  • suitable assays include surface plasmon resonance analysis using a biosensor system such as a Biacore® system or a flow cytometry assay, which are further described in the EXAMPLES section.
  • binding to glycans protein is assessed in a competitive assay format with a reference antibody AB-006410 or a reference antibody having the variable regions of AB-006410.
  • a variant anti-tumor antibody in accordance with the present disclosure may block binding of the reference antibody in a competition assay by about 50% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 80% or more, or about 90% or more.
  • binding assays to assess variant activity are performed on tumor tissues or tumor cells ex vivo, e.g., on tumor cells that were grown as a tumor graft in a syngeneic (immune-matched) mouse in vivo then harvested and processed within 24-48 hrs. Binding can be assessed by any number of means including flow cytometry and immunohistochemistry or immunofluorescence-based assays.
  • the antibody is added to a cancer cell line and the binding is analyzed by flow cytometry.
  • AB-006410 was shown to bind A549 cells and the binding of AB-006410 diminished in A549 cells in which one or more selected glycosyltransferase have been knocked out.
  • the glycosyltransferase is B4GALNT3 or FUT9. In some embodiments, the glycosyltransferase is B4GALNT3 and FUT9.
  • the binding of the antibodies to bind to tumor cells are assessed by immunofluorescence methods, as described in the EXAMPLES.
  • the anti-tumor antibodies preferentially bind to various tumors but not to normal human tissues.
  • the AB-006410 showed preferential binding to ovarian, lung, pancreatic, and esophageal cancer tissues relative to the respective tumor adjacent tissues (TATs).
  • the binding activity of the antibodies is assessed by determining ECso values, and in some embodiments additionally determining delta activity, i.e. , the difference in specific activity between lower and upper plateaus of the activation curve expressed as percent of activity of a selected antibody having known in vitro activity.
  • ECso values are compared to a reference antibody.
  • an antibody comprising the VH and VL regions of an anti-tumor antibody disclosed herein and a mouse IgG2a Fc region when testing ex vivo binding using a mouse tumor model, is employed as a reference antibody and included in an assay to assess variant activity relative to the reference antibody.
  • an anti-tumor antibody of the present disclosure comprises an Fc region that has effector function.
  • effector functions include, but are not limited to, Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding (e.g., FcyR binding), ADCC, antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptor), and B-cell activation. Effector functions may vary with the antibody class.
  • native human IgGl and lgG3 antibodies can elicit ADCC and CDC activities upon binding to an appropriate Fc receptor present on an immune system cell; and native human IgGl, IgG2, IgG3, and IgG4 can elicit ADCP functions upon binding to the appropriate Fc receptor present on an immune cell.
  • the Fc region of an anti-tumor antibody disclosed herein may be an Fc region engineered to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or ADCC.
  • an Fc region can comprise additional mutations to increase or decrease effector functions, i.e., the ability to induce certain biological functions upon binding to an Fc receptor expressed on an immune cell.
  • Immune cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans’ cells, natural killer (NK) cells, and cytotoxic T cells.
  • an antibody of the present disclosure has enhanced ADCC and/or serum stability compared to antibody AB-006410 when the antibody is assayed in a human IgGl isotype format.
  • the anti-tumor antibodies of the present disclosure may be evaluated in various assays for their ability to mediate FcR-dependent activity.
  • the binding activity of an anti-tumor antibody is evaluated in an Fc receptor engagement assay.
  • “engagement” of an Fc receptor occurs when a variant antibody binds to both a target tumor cell via its Fv region and an FcyR present on an immune cell via the antibody Fc region in such as manner to transduce a signal. If the Fc region is kept constant among variants that differ in their Fv regions, then the assay allows an evaluation of tumor binding activity across such variants in the context of potential signal transduction through a particular Fc region binding a particular Fc receptor.
  • binding of the antibody Fc region can result in clustering and/or internalization of the FcR, resulting in a luminescence signal in cells harboring a NFAT-RE-Lucif erase reporter construct.
  • an anti-tumor antibody of the present disclosure has ADCC when the antibodies are assayed in a mouse IgG2a isotype format.
  • ADCP activity of an anti-tumor antibody is assessed using fluorescently labeled, in vitro cultured tumor cells and Raw264.7 murine macrophages.
  • opsonization of the tumor cell by the antibody leads to phagocytosis detected by flow cytometry. Variations of this assay have been described and can include co-labeling of tumor and effector cells or assessment of phagocytosis through FcyRIIa engagement (e.g., FcyRIIa-H ADCP Reporter Bioassay from Promega).
  • An anti-tumor antibody is deemed to have ADC activity if, when the antibody is conjugated to a drug molecule (toxin) to form an antibody drug conjugate (ADC), said ADC can kill target cells.
  • the antibody is deemed to have ADC activity if the ECso of the assay measuring the cell killing activity of the ADC is less than 1 x 10' 8 .
  • the ADC activity of an anti-tumor antibody is evaluated using a drug- conjugated secondary antibody.
  • the antibody-drug conjugate assay involves tumor target cells, primary antibodies of interest (the antibody to be tested), and a secondary antibody that is conjugated to a drug molecule, where the secondary antibody recognizes the primary antibody.
  • primary antibody dilutions are incubated with target cells at room temperature for a first period (for example, 10-30 minutes).
  • the drug-conjugated secondary antibody is then added to the incubation mixture containing the target cells and the primary antibody.
  • the mixture is then incubated for second period before measuring the extent of target cell lysis.
  • the assay generates a 100% cell lysis value by adding cell lysis buffer directly to target cell sample, which are not treated by the primary or drug-conjugated second antibody, and cell killing data from samples treated the antibody mixtures as disclosed above can be normalized to the value of 100% cell lysis.
  • the results of the assay can be used to predict whether an ADC produced by conjugating a particular antibody and the drug molecule can kill target cells.
  • activity of an anti-tumor antibody is evaluated in vivo in a suitable animal tumor model.
  • a reduction in tumor load of a subject treated with a test article relative to the tumor load of a subject treated with a control article reflects the antitumor function of an antibody.
  • An anti-tumor antibody, or anti-tumor antibody immunoconjugate, disclosed herein can reduce tumor load of a subject by at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60%, or at least 70%, or greater relative to the tumor load of a control subject.
  • a variant of an antibody as described herein has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or greater, of the antitumor activity of a reference antibody as shown in Tables 1-3 when evaluated under the same assay conditions to measure the anti-tumor activity in vivo.
  • an anti-tumor antibody exhibits improved activity, i.e., greater than 100% activity, compared to the reference antibody.
  • an anti-tumor antibody in accordance with the present disclosure is in a monovalent format.
  • the tumor-targeting antibody is in a fragment format, e.g., a Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment.
  • an anti-tumor antibody in accordance with the disclosure may be an antibody fragment, e.g., a Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment.
  • the antibody is a substantially full-length antibody, e.g., an IgG antibody or other antibody class or isotype as defined herein.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.
  • an anti-tumor antibody according to the present disclosure that is administered to a patient is an IgG of the IgGl subclass.
  • such an antibody is an IgG of the IgG2, IgG3, or IgG4 subclass.
  • such an antibody is an IgM.
  • such an antibody has a lambda light chain constant region.
  • such an antibody has a kappa light chain constant region.
  • an anti-tumor antibody of the present disclosure is employed in a bispecific or multi- specific format, e.g., a tri-specific format.
  • the antibody may be incorporated into a bispecific or multi-specific antibody that comprises a further binding domain that binds to the same or a different antigen.
  • the formats can vary elements such as the number of binding arms, the format of each binding arm (e.g., Fab, scFv, scFab, or Vu-only), the number of antigen binding domains present on the binding arms, the connectivity and geometry of each arm with respect to each other, the presence or absence of an Fc domain, the Ig class (e.g. , IgG or IgM), the Fc subclass (e.g., hlgGl, hIgG2, or hIgG4), and any mutations to the Fc (e.g., mutations to reduce or increase effector function or extend serum half-life).
  • Ig class e.g. , IgG or IgM
  • the Fc subclass e.g., hlgGl, hIgG2, or hIgG4
  • any mutations to the Fc e.g., mutations to reduce or increase effector function or extend serum half-life.
  • the tumor-targeting binding domain comprises all six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) sequences from the individual antibodies disclosed in Tables 1 and 2.
  • the tumor-targeting binding domain comprises the VH and VL sequences from the individual antibodies disclosed in Tables 3.
  • the tumor-targeting binding domain comprises all six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) sequences from any one of antibodies AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210
  • the tumor-targeting binding domain comprises the Vu and VL sequences of AB- 012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB- 012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB- 012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB- 012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB- 012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213,
  • an anti-tumor antibody disclosed herein is constructed as a multivalent antibody.
  • an anti-tumor antibody is constructed as a tetravalent molecule, comprising four glycan-binding arms per molecule. Such constructs exhibit increased ADCC activity, as well as increased binding to tumor cells a measured by flow cytometry.
  • an anti-tumor antibody of the present disclosure is employed in a bispecific or multi-specific format, e.g., a tri-specific format.
  • the antibody may be incorporated into a bispecific or multi-specific antibody that comprises a further binding domain that binds to the same or a different antigen.
  • Illustrative antigens that can be targeted by a further binding domain in a bispecific or multi- specific antibody that comprises an antigen binding domain of an anti-tumor antibody described herein include, but are not limited to, antigens on T cells to enhance T cell engagement and/or activate T cells.
  • Illustrative examples of such an antigen include, but are not limited to, CD3, CD2, CD4, CD5, CD6, CD8, CD28, CD40L, CD44, IL-15Ra, CD122, CD132, or CD25.
  • the antigen is CD3.
  • any collection of anti-CD3 CDRs, anti- CD3 variable light and variable heavy domains, Fabs and scFvs as depicted in any of the Figures can be used.
  • any of the anti-glycans antigen binding domains can be used, whether CDRs, variable light and variable heavy domains, Fabs and scFvs, can be used, optionally and independently combined in any combination.
  • an anti-tumor antibody is incorporated into a multi- specific antibody that comprises a binding domain from an agonist antibody that binds to 4-1BB.
  • the 4- IBB agonist antibody is a bispecific antibody that is capable of binding to both glycans and 4- IBB.
  • the term “4- IBB engager,” refers to the portion of a molecule (e.g. , a bispecific antibody capable of binding to both 4-1BB and glycans) that binds to 4-1 BB.
  • the 4-1BB engager is an antibody or an antibody fragment e.g. , scFv) that binds to 4- IBB.
  • the 4- IBB engager is a multimeric 4- IBB ligand (“4-1BBL”), for example, a 4-1BBL trimer.
  • the bispecific antibody comprises one or more scFv fragments of an anti-4- IBB antibody and an anti-tumor antibody disclosed herein.
  • the 4- IBB agonist antibody is a trispecific antibody.
  • the fusion molecule comprises a silenced human IgGl with three human 4- IBB ligand ectodomains attached via flexible linkers.
  • an anti-tumor antibody is incorporated into a fusion molecule comprising one or more 4- 1BB ligands (4-1 BBL).
  • a trimer of 4-1 BBL is fused to the C-terminal of either the light chain or heavy chain of an anti-tumor antibody.
  • one or more individual 4-1 BBL domains are connected via linkers, with one of the domains additionally fused to the anti-tumor antibodies via a linker.
  • the 4-1 BBL domains comprise the entire ECD portion of the molecule or truncated forms that can still bind and activate 4- 1BB.
  • the anti-tumor antibodies can be produced using vectors and recombinant methodology well known in the art. Reagents, cloning vectors, and kits for genetic manipulation are available from commercial vendors. Accordingly, in some embodiments of the disclosure, provided herein are isolated nucleic acids encoding a VH and/or VL region, or fragment thereof, of any of the tumor-targeting antibodies as described herein; vectors comprising such nucleic acids and host cells into which the nucleic acids are introduced that are used to replicate the antibody-encoding nucleic acids and/or to express the antibodies.
  • nucleic acids may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of the tumor-targeting antibody (e.g., the light and/or heavy chains of the antibody).
  • the host cell contains (1) a vector containing a polynucleotide that encodes the VL amino acid sequence and a polynucleotide that encodes the VH amino acid sequence, or (2) a first vector containing a polynucleotide that encodes the VL amino acid sequence and a second vector containing a polynucleotide that encodes the VH amino acid sequence.
  • the disclosure provides a method of making an anti-tumor antibody as described herein.
  • the method includes culturing a host cell as described in the preceding paragraph under conditions suitable for expression of the antibody.
  • the antibody is subsequently recovered from the host cell (or host cell culture medium).
  • Suitable vectors containing polynucleotides encoding antibodies of the present disclosure, or fragments thereof include cloning vectors and expression vectors. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally can self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g.
  • Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure.
  • the expression vector can be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA.
  • Suitable expression vectors include but are not limited to plasmids and viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and any other vector.
  • Suitable host cells for expressing an anti-tumor antibody as described herein include both prokaryotic and eukaryotic cells.
  • an anti-tumor antibody may be produced in bacteria when glycosylation and Fc effector function are not needed.
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • the host cell may be a eukaryotic host cell, including eukaryotic microorganisms, such as filamentous fungi or yeast, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern, vertebrate, invertebrate, and plant cells.
  • invertebrate cells include insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells. Plant cell cultures can also be utilized as host cells.
  • vertebrate host cells are used for producing an anti-tumor antibody of the present disclosure.
  • mammalian cell lines such as a monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59,1977; baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
  • monkey kidney cells (CV 1); African green monkey kidney cells (VERO- 76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g. , in Mather et al. , Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC 5 cells; and FS4 cells may be used to express an tumor-targeting antibodies.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc.
  • Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells.
  • isolated cells in vitro cultured cells
  • ex vivo cultured cells for a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268, 2003.
  • an anti-tumor antibody of the present disclosure is produced by a CHO cell line, e.g., the CHO-K1 cell line.
  • One or more expression plasmids can be introduced that encode heavy and light chain sequences.
  • an expression plasmid encoding a heavy chain disclosed herein, and an expression plasmid encoding a light chain disclosed herein are transfected into host cells.
  • the expression plasmids can be introduced as linearized plasmids at a ratio of 1:1 in the CHO-K1 host cell line using reagents such as Freestyle Max reagent. Fluorescence- activated cell sorting (FACS) coupled with single cell imaging can be used as a cloning method to obtain a production cell line.
  • FACS Fluorescence- activated cell sorting
  • a host cell transfected with an expression vector encoding an anti-tumor antibody of the present disclosure, or fragment thereof, can be cultured under appropriate conditions to allow expression of the polypeptide to occur.
  • the polypeptides may be secreted and isolated from a mixture of cells and medium containing the polypeptides. Alternatively, the polypeptide may be retained in the cytoplasm or in a membrane fraction and the cells harvested, lysed, and the polypeptide isolated using a desired method.
  • an anti-tumor antibody of the present disclosure can be produced by in vitro synthesis (see, e.g., Sutro Biopharma biochemical protein synthesis platform).
  • a method of generating variants of an antitumor antibody as disclosed herein.
  • a construct encoding a variant of a VH CDR3 as described herein can be modified and the VH region encoded by the modified construct can be tested for binding activity to LoVo cells and/or in vivo tumor-targeting activity in the context of a VH region as described herein, that is paired with a VL region or variant region as described herein.
  • a construct encoding a variant of a VL CDR3 as described herein can be modified and the VL region encoded by the modified construct can be tested for binding to CT26 cells, or other tumor cells, and/or in vivo tumor- targeting activity efficacy.
  • Such an analysis can also be performed with other CDRs or framework regions and an antibody having the desired activity can then be selected.
  • an anti-tumor antibody disclosed herein may be conjugated or linked to therapeutic, imaging/detectable moieties, or enzymes.
  • the anti-tumor antibody may be conjugated to a detectable marker, a cytotoxic agent, an immunomodulating agent, an imaging agent, a therapeutic agent, an oligonucleotide, or an enzyme.
  • Methods for conjugating or linking antibodies to a desired molecule are well known in the art.
  • the moiety may be linked to the antibody covalently or by non-covalent linkages.
  • the antibody is conjugated, either directly or via a cleavable or non-cleavable linker or spacer, to a cytotoxic moiety or other moiety that exerts their effects on critical cellular processes required for survival (“payload”) to form an antibodydrug conjugate (“ADC”).
  • ADC antibodydrug conjugate
  • the linker is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, the linker is an enzymatic cleavable linker. In some embodiments, the linker is a pH-sensitive linker. In some embodiments, the linker is a reducible linker (e.g., sulfo-SPDB).
  • the linker is cleaved in response to changes in pH or redox potential. In some embodiments, the linker is cleaved when contacted with lysosomal enzymes.
  • MC Maleimidocaproyl
  • MC-VC-PAB Maleimidocaproyl-Valine-Citrulline- p-amino-benzyloxy carbonyl
  • SMCC Maleimidomethyl cyclohexane- 1 -carboxylate
  • SPDB N-succinimidyl-4- (2-pyridyldithio)butanoate
  • the anti-tumor antibody is conjugated to an auristatin to form an ADC.
  • the ADC comprises an anti-tumor antibody conjugated to a ZymeLinkTM Auristatin (ZLA) payload.
  • ZLA ZymeLinkTM Auristatin
  • an ADC of the present disclosure is conjugated to a microtubule inhibitor that induces apoptosis in cells undergoing mitosis by, for example, causing cell cycle arrest at G2/M.
  • microtubule inhibitors that can be used include maytansine derivatives (DM1/DM4), or auristatins (monomethyl auristatin E (MMAE)/ monomethyl auristatin F (MMAF)) and variants thereof, such as monomethyl auristatin D, PF-06380101, duostatin5, AS269, Tapl8Hrl, AGD-0182, HPA-Auristatin F.
  • the payload is a tubulin-targeting agent, for example, hemiasterlin, tubulysin, or eribulin.
  • the payloads are DNA-damaging payloads, which include enediynes (calicheamicin), duocarmycin derivatives, pyrrolobenzodiazepine dimers (PBD dimers), and indolinobenzodiazepine pseudo-dimers.
  • the antibody is conjugated to a cytotoxic agent including, but not limited to, e.g., ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, methotrexact, actinomycin, a diphtheria toxin, extotoxin A from Pseudomonas, Pseudomonas exotoxin 40, abrin, abrin A chain, modeccin A chain, alpha sarcin, gelonin, mitogellin, restrictocin, cobran venom factor, a ribonuclease, engineered Shiga toxin, phenomycin, enomycin, curicin, crotin, calicheamicin
  • a cytotoxic agent
  • the antibody may be linked to an agent such as an enzyme inhibitor, a proliferation inhibitor, a lytic agent, a DNA or RNA synthesis inhibitors, a membrane permeability modifier, a DNA metabolite, a dichloroethylsulfide derivative, a protein production inhibitor, a ribosome inhibitor, or an inducer of apoptosis.
  • the antibody is conjugated to a drug such as a topoisomeriase Inhibitor, e.g., a topoisomeraise I inhibitor.
  • Topoisomeraise I inhibitors include but are not limited to quinoline alkaloids (e.g., SN-38, DXd).
  • the antibody is conjugated to one or more of the cytotoxic and/or anti-mitotic compounds.
  • an anti-tumor antibody as described herein is joined to a molecule that facilitates transport of the antibody across a biological membrane, e.g. , by enhancing penetration of the membrane, facilitating protein translocation across membranes.
  • the antibody may be linked to a cell penetration agent, such as a cellpenetrating peptide.
  • cell penetration agent such as a cellpenetrating peptide.
  • cell penetrating peptides include TAT, penetrating, polyarginine molecules, Kunitz domain-derived peptides, e.g., angiopep-2, SynB, buforin, transportan, amphiphathic peptides and others.
  • the antibody may be conjugated with a cationic molecule such as a polyamine.
  • the antibody may be conjugated to an agent that facilitates transport across the blood brain barrier, e.g., transcytosis.
  • the antibody may be conjugated to an agent that binds to endothelial cell receptors that are internalized, e.g., transferrin receptor, insulin receptor, insulin-like growth factor receptor, or a low-density lipoprotein receptor, and the like.
  • the antibody may be conjugated to a toxin facilitating entry of the antibody into the cytoplasm, e.g., Shiga toxin.
  • an anti-tumor antibody as described herein can be conjugated to an engineered toxin body (ETBs) to facilitate internalization of the antibody into a cell.
  • ETBs engineered toxin body
  • an anti-tumor antibody described herein is conjugated or administered with a polypeptide immunomodulating agent, e.g., an adjuvant.
  • immunomodulating agents include, but are not limited to, cytokines e.g., transforming growth factor- p (TGF0)), growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL- 15, an IL-15/IL-15Ra, e.g., sushi domain, complex, IL- 18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF), interferons (e.g., interferon-a, -0 or -y, erythrop
  • G-CSF gran
  • the antibody is linked or administered with a compound that stimulates the innate immune system, such as an adjuvant, a Toll-like receptor (TLR) agonist, a C-type lectin receptor (CLR) agonist, a retinoic acid-inducible gene I-like receptor (RLR) agonist, a saponin, a polysaccharide such as chitin, chitosan, P-glucan, an ISCOM, QS-21, a stimulator of interferon genes (STING) agonist, or another immunopotentiating agent.
  • TLR Toll-like receptor
  • CLR C-type lectin receptor
  • RLR retinoic acid-inducible gene I-like receptor
  • an anti-tumor antibody described herein is conjugated to or administered with an IL- 15 receptor agonist, such as an IL- 15 fusion construct, an IL-15:IL- 15Ra fusion construct or a single-chain IL-15:IL-15Ra (sushi) fusion construct.
  • an IL- 15 receptor agonist such as an IL- 15 fusion construct, an IL-15:IL- 15Ra fusion construct or a single-chain IL-15:IL-15Ra (sushi) fusion construct.
  • the tumor-targeting antibody conjugated to an IL- 15 receptor agonist is a bispecific or multispecific antibody.
  • the antibody is a bispecific or multispecific antibody comprising an antigen binding domain described herein that further comprises an IL- 15 receptor agonist.
  • an anti-tumor antibody described herein is administered with a single-chain IL-15:IL-15Ra (sushi) fusion construct.
  • an anti-tumor antibody is administered with a polymer-conjugated IL-15 construct, such as NKTR-255.
  • the !L-15:lL-15Ra single chain constructs can be administered to a subject in a therapeutically effective dose, for example, a dose in a range from less than about 0.01 mg/kg body weight to about 25 mg/kg body weight, for example, 0.1 - 10 mg/kg body weight.
  • the dose is about 0.1 to about 25 mg/kg, about 1 to about 20 mg/kg, about 2 to about 15 mg/kg, about 3 to about 10 mg/kg, about 4 to about 5 mg/kg body weight
  • the constructs can be administered in a dose of approximately 1 mg - 2 g per patient, or approximately 50 mg - 1000 mg per patient, or approximately 100 mg - 500 mg per patient.
  • the single-chain IL- 15 fusion construct comprises IL- 15 joined to IL-15Ra (sushi) with a polypeptide linker.
  • the single-chain IL- 15 fusion construct is joined via a polypeptide linker to another protein, such as an Fc for long half-life. See, for example, FIG. 9B in W02018071919A1 (corresponding to U.S.
  • the IL- 15 is joined or fused to the N-terminus of the heavy chain of an Fc, and IL- 15Ra( sushi) is joined or fused to the other Fc heavy chain N-terminus, using a heavy chain heterodimerization technology to form the desired hybrid Fc.
  • the IL-15:IL-15Ra (sushi) single chain constructs are fused to the C-terminus of an antibody light chain, or the C-terminus of an antibody heavy chain, in both cases producing a molecule with two tumor-targeting binding sites (the Fab arms), and two IL15:IL15Ra units.
  • one copy of an IL 15 :IL 15 Rot fusion construct is fused to an anti-tumor antibody, thereby producing an antibody molecule comprising two tumor-targeting binding sites (the Fab arms) and only one ILI 5:ILI 5Rcx unit, for example using a knob-in-holes approach to heavy chain heterodimerization, or other heterodimerization technology.
  • the IL-15:IL-15Ra (sushi) fusion constructs or the antibodies comprising the fusion constructs comprise a low affinity IL- 15 variant having improved pharmacokinetics (PK).
  • the IL-15:IL-15Ra (sushi) fusion constructs comprise a high affinity IL- 15 variant having increased agonist activity.
  • the high affinity IL- 15 variant has an N72D mutation.
  • the high affinity variant is fused to a dimeric IL-15Ra sushi domain-IgGl Fc fusion protein.
  • the IL-15:IL-15Ra (sushi) fusion construct is ALT- 803.
  • antibodies comprising the IL15:IL15Ra fusion construct comprise one or more mutations in the Fc region described herein, for example E333A, K326W/E333S, S239D/I332E/G236A, S239D/A330L/I332E, G236A/S239D/A330L/I332E, F243L, G236A, and S298A/E333A/K334A.
  • antibodies comprising the IL15:IL15Ra fusion comprise one or more mutations in the Fc region that increase binding of the antibody to tumor cells, for example the mutations P329G, L234A, L235A, or a combination thereof.
  • an anti-tumor antibody described herein is conjugated to or administered with an IL-2 receptor agonist.
  • the tumor-targeting antibody conjugated to an IL-2 receptor agonist is a bispecific or multispecific antibody.
  • the antibody is a bispecific or multispecific antibody comprising an antigen binding domain of an antibody described herein (e.g., AB-006410) that further comprises an IL-2 receptor agonist.
  • the IL-2 receptor agonist is pegylated IL-2.
  • an anti-tumor antibody described herein is conjugated to or administered with a construct that can act as a trap for transforming growth factor- ⁇ (TGFp).
  • TGFp trap comprises the extracellular domain (ECD) of TGFp.
  • TGFP trap comprises the extracellular domain (ECD) of TGFPRII.
  • the TGF trap is in the form of a bispecific antibody.
  • the TGF RII ECD can preferably trap TGFP 1, and its low affinity to TGFp2 may mitigate potential cardiac toxicity.
  • an anti-tumor antibody described herein comprises an extracellular domain (ECD) of the TGFP Receptor fused to the C-terminus of the heavy chain or to the C-terminus of the light chain.
  • the TGFP trap is a single trap construct.
  • the single TGFP trap is a bispecific tumortargeting TGF trap comprising a TGFp RII ECD fused to any one of the antibodies disclosed herein via a flexible linker to the C-terminus of the heavy chain or to the C- terminus of the light chain.
  • the TGFP trap is a tandem trap construct.
  • the tandem TGFP trap comprises an IgG fused to two TGF RII ECDs.
  • the tandem TGFP trap comprises two TGFP2RII ECDs.
  • the two TGFP2RII ECDs are fused in series and are linked by a short linker (for example LIO or L25).
  • the two TGFP2RII ECDs are fused directly in series without a linker (L0).
  • the tandem TGFPRII ECDs are fused to the C-terminus of the heavy chain (HC-Cter), and the heavy chains were designed as an asymmetric pair such that the tandem-Trap is on only one heavy chain.
  • the asymmetric pair of heavy chains comprise knob-in-hole mutation that promote pairing of the heavy chains.
  • one heavy chain comprises the amino acid substitutions T366S+L368A+Y407V (and optionally Y349C), and the other heavy chain comprises the amino acid substitution T336W (and optionally S354C).
  • the asymmetric single heavy chain C-ter fusion improves steric access of the Fc region to Fc gamma receptors and thereby improve function.
  • tandem TGFP trap is fused to the C-terminus of the light chain (LC-Cter), such that both light chains comprise two TGFPRII ECDs.
  • the net molecule exhibits twice the TGFP trapping capacity per molecule, and therefore may exhibit improved function.
  • the bispecific TGFP trap construct comprises human variable regions.
  • the bispecific TGFP trap construct comprises a IgGl or IgG2 constant region.
  • the bispecific TGFP trap construct comprises a human IgGl constant region.
  • the bispecific TGFP trap construct comprises a mouse IgG2a constant region.
  • the variable regions of the TGFP trap construct are fused in frame to the IgG constant regions.
  • Binding of the TGFP trap construct can be determined using an ELISA assay, as described in the Examples.
  • the ability of TGF trap constructs to bind to target tumor cells can be determined, for example, using flow-cytometry, as described in the Examples.
  • the ability of TGFP trap constructs to engage and stimulate Fc-gamma Receptor in the presence of target tumor cells can be determined using a reporter bioassay, as described in the Examples.
  • the ability of TGFP trap constructs to inhibit tumor growth can be determined, for example, in a syngeneic mouse model, as described in the Examples.
  • the antibody may be linked to a radionuclide, an iron-related compound, a dye, a fluorescent agent, or an imaging agent.
  • an antibody may be linked to agents, such as, but not limited to, metals; metal chelators; lanthanides; lanthanide chelators; radiometals; radiometal chelators; positron-emitting nuclei; microbubbles (for ultrasound); liposomes; molecules microencapsulated in liposomes or nanosphere; monocrystalline iron oxide nanocompounds; magnetic resonance imaging contrast agents; light absorbing, reflecting and/or scattering agents; colloidal particles; fluorophores, such as near-infrared fluorophores.
  • the anti-tumor antibody comprises a modified Fc region comprising mutations described herein.
  • the antibody comprises an Fc mutation that increases effector function selected from E333A, K326W/E333S, S239D/I332E/G236A, S239D/A330L/I332E, G236A/S239D/A330L/I332E, F243L, G236A, S298A/E333A/K334A, and P329G/L234A/L235A, or a combination thereof.
  • the antibody comprises a modified Fc region that is afucosylated.
  • the antibody is conjugated to or administered with an IL- 15 receptor agonist, a TGF trap, a TLR agonist, or an agonist anti-4- IBB antibody.
  • the antibody is a bispecific or multispecific antibody described herein.
  • binding of antibody to at least 0.2%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, or at least 50%, of the tumor cells in a sample may be used as a selection criterion for determining a patient to be treated with an anti-tumor antibody as described herein.
  • an anti-tumor antibody or an anti-tumor antibody immunoconjugate e.g., an antibody- drug conjugate
  • a cancer patient who can benefit from the treatment of the anti-tumor antibody or antibody immunoconjugate has a cancer expressing a tumor-associated glycan.
  • the cancer is a carcinoma, a melanoma, or a sarcoma.
  • the cancer is colorectal, pancreatic, gastric, or uterine cancer.
  • the cancer is a hematological cancer.
  • the cancer is breast cancer, prostate cancer, testicular cancer, renal cell cancer, bladder cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, colorectal cancer, anal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, a brain cancer, e.g., glioblastoma, melanoma, or a bone or soft tissue sarcoma.
  • the cancer is acral melanoma.
  • the cancer is acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, astrocytoma, basal-cell carcinoma, bile duct cancer, bone tumor, brainstem glioma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, bronchial adenomas, Burkitt’s lymphoma, central nervous system lymphoma, cerebellar astrocytoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, epithelioid hemangioend
  • Plasma cell neoplasia pleuropulmonary blastoma, primary central nervous system lymphoma, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing sarcoma, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, melanoma,, small intestine cancer, squamous cell carcinoma, squamous neck cancer, stomach cancer, cutaneous T-Cell lymphoma, throat cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, gestational, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms tumor.
  • the cancer is lung cancer, e.g., non-small cell lung adenocarcinoma or squamous cell carcinoma; breast cancer, e.g., Triple-, ER/PR + Her2-, ER/PR Her2 + , or Triple-; colorectal cancer, e.g., adenocarcinoma, mucinous adenocarcinoma, or papillary adenocarcinoma; esophageal cancer; stomach cancer; kidney cancer, e.g., kidney clear cell cancer; ovarian cancer, e.g., ovarian endometrioid carcinoma, ovarian mucinous cystadenocarcinoma, or ovarian serous cystadenomcarcinoma; melanoma, e.g., acral melanoma, cutaneous melanoma, or mucosal melanoma; uterine or cervical cancer; liver cancer, e.
  • lung cancer e.g
  • the cancer is pancreatic adenocarcinoma, esophageal adenocarcinoma, NSCLC adenocarcinoma, or ovarian mucinous adenocarcinoma.
  • methods of the disclosure comprise administering an antitumor antibody disclosed herein, or a variant thereof, as a pharmaceutical composition to a cancer patient in a therapeutically effective amount using a dosing regimen suitable for treatment of the cancer.
  • the composition can be formulated for use in a variety of drug delivery systems.
  • One or more physiologically acceptable excipients or carriers can also be included in the compositions for proper formulation.
  • the anti-tumor antibody is provided in a solution suitable for administration to the patient, such as a sterile isotonic aqueous solution for injection.
  • the antibody is dissolved or suspended at a suitable concentration in an acceptable carrier.
  • the carrier is aqueous, e.g., water, saline, phosphate buffered saline, and the like.
  • the compositions may contain auxiliary pharmaceutical substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and the like.
  • the pharmaceutical compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the disease or symptoms of the disease and its complications.
  • An amount adequate to accomplish this is defined as a “therapeutically effective dose.”
  • a therapeutically effective dose is determined by monitoring a patient’ s response to therapy. Typical benchmarks indicative of a therapeutically effective dose includes the amelioration of symptoms of the disease in the patient. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient’s health, including other factors such as age, weight, gender, administration route, and the like Single or multiple administrations of the antibody may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the methods provide a sufficient quantity of tumor-targeting antibody to effectively treat the patient.
  • An anti-tumor antibody can be administered by any suitable means, including, for example, parenteral, intrapulmonary, and intranasal, administration, as well as local administration, such as intratumor administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the antibody may be administered by insufflation.
  • the antibody may be stored at 10 mg/ml in sterile isotonic aqueous saline solution for injection at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient.
  • the antibody is administered by intravenous infusion over the course of 1 hour at a dose of between 0.01 and 25 mg/kg.
  • the dose is about 0.1 to about 25 mg/kg, about 1 to about 20 mg/kg, about 2 to about 15 mg/kg, about 3 to about 10 mg/kg, about 4 to about 5 mg/kg body weight,
  • the constructs can be administered in a dose of approximately 1 mg - 2 g per patient, or approximately 50 mg - 1000 mg per patient, or approximately 100 mg - 500 mg per patient.
  • the antibody is administered by intravenous infusion over a period of between 15 minutes and 2 hours.
  • the dose is administered over a period of about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 100 minutes, about 110 minutes, or about 120 minutes. In some embodiments the dose is administered over a period of about 0.5 hours, about 1 hour, about 1.5 hours or about 2 hours. In still other embodiments, the administration procedure is via sub-cutaneous bolus injection.
  • the dose of antibody is chosen to provide effective therapy for the patient and is in the range of less than 0.01 mg/kg body weight to about 25 mg/kg body weight or in the range 1 mg - 2 g per patient. Preferably the dose is in the range 0. 1 - 10 mg/kg or approximately 50 mg - 1000 mg / patient.
  • the dose may be repeated at an appropriate frequency which may be in the range once per day to once every three months, or every six months, depending on the pharmacokinetics of the antibody (e.g., half-life of the antibody in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect of the antibody).
  • the in vivo half-life of the antibody is between about 7 and about 25 days and antibody dosing is repeated between once per week and once every 3 months or once every 6 months.
  • the antibody is administered approximately once per month.
  • the antibody may be stored at 10 mg/ml or 20 mg/ml in a sterile isotonic aqueous solution.
  • the solution can comprise agents such as buffering agents and stabilizing agents.
  • a buffering agent such as histidine is included to maintain a formulation pH of about 5.5.
  • Additional reagents such as sucrose or alternatives can be added to prevent aggregation and fragmentation in solution and during freezing and thawing.
  • Agents such as polysorbate 80 or an alternative can be included to lower surface tension and stabilizes the antibody against agitation-induced denaturation and air- liquid and ice- liquid surface denaturation.
  • the solution for injection is stored at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient.
  • An anti-tumor antibody may be administered with one or more additional therapeutic agents, e.g., radiation therapy, chemotherapeutic agents and/or immunotherapeutic agents.
  • an anti-tumor antibody can be administered in conjunction with an agent that targets an immune checkpoint antigen.
  • the agent is a biologic therapeutic or a small molecule.
  • the agent is a monoclonal antibody, a humanized antibody, a human antibody, a fusion protein, or a combination thereof.
  • the agents inhibit, e.g., by blocking ligand binding to receptor, a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDO1, IDO2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, GITR, HAVCR2, LAG3, KIR, LAIR1 , LIGHT, MARCO, OX-40, SLAM, , 2B4, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137 (4-1BB), CD160, CD39, VISTA, TIGIT, a SIGLEC, CGEN-15049, 2B4, CHK1, CHK2, A2aR, B-7 family ligands or their receptors, or a combination thereof.
  • a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDO1, IDO2, B7-H3, B7-H4, BTLA, HVEM,
  • the agent targets PD-1, e.g., an antibody that blocks PD-L1 binding to PD-1 or otherwise inhibits PD-1.
  • the agent targets CTLA-4.
  • the agent targets LAG3.
  • the agent targets TIM3.
  • the agents target ICOS.
  • an anti-tumor antibody can be administered in conjunction with a therapeutic antibody, such as an antibody that targets a tumor cell antigen.
  • therapeutic antibodies include as rituximab, trastuzumab, tositumomab, ibritumomab, alemtuzumab, atezolizumab, avelumab, durvalumab, pidilizumab, AMP-224, AMP-514, PDR001, cemiplimab, BMS-936559, CK-301, epratuzumab, bevacizumab, elotuzumab, necitumumab, blinatumomab, brentuximab, cetuximab, daratumumab, denosumab, dinutuximab, gemtuzumab ibritumomab ipilimumab, nivolumab,
  • an anti-tumor antibody is administered with a chemotherapeutic agent.
  • cancer chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofo
  • alkylating agents such
  • paclitaxel and doxetaxel paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; docetaxel, platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as bexarotene, alitretinoin; denileukin diftitox; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, mifepristone, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 1 17018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • cancer therapeutic agents include sorafenib and other protein kinase inhibitors such as afatinib, axitinib, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, vandetanib, vemurafenib, and sunitinib; sirolimus (rapamycin), everolimus and other mTOR inhibitors.
  • additional chemotherapeutic agents include topoisomerase I inhibitors (e.g.
  • irinotecan topotecan, camptothecin and analogs or metabolites thereof, and doxorubicin
  • topoisomerase II inhibitors e.g., etoposide, teniposide, and daunorubicin
  • alkylating agents e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, decarbazine, methotrexate, mitomycin C, and cyclophosphamide
  • DNA intercalators e.g., cisplatin, oxaliplatin, and carboplatin
  • DNA intercalators and free radical generators such as bleomycin
  • nucleoside mimetics e.g., 5-fhiorouracil, capecitibine, gemcitabine, fludarabine, cytarabine, mercaptopurine
  • Illustrative chemotherapeutic agents additionally include paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, and related analogs; thalidomide, lenalidomide, and related analogs (e.g., CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g. , imatinib mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-KB inhibitors, including inhibitors of IKB kinasei and other inhibitors of proteins or enzymes known to be upregulated, over-expressed or activated in cancers, the inhibition of which down regulates cell replication. Additional agents include asparaginase and a Bacillus Calmete- Guerin preparation.
  • an anti-tumor antibody as described herein is administered after, or at the same time, as a therapeutic agent, e.g., a chemotherapeutic agent, such as doxorubicin, that induces stress granules (“SG-inducing agent”).
  • a therapeutic agent e.g., a chemotherapeutic agent, such as doxorubicin
  • SG-inducing agent that induces stress granules
  • Increasing the amount of stress granules in cancer cells can promote targeting the tumor cells by the tumor-targeting antibody.
  • exemplary therapeutic agents that can induce stress granules include pyrimidine analogs (e.g., 5-FU, under trade names of Adrucil®, Carac®, Efudex®, Efudix®); protease inhibitors (e.g., Bortezomib, under the trade name of Velcade®); kinase inhibitors (e.g, Sorafenib and Imatinib, under the trade names of Nexavar® and Gleevec® , respectively); Arsenic compounds (e.g.
  • Various combinations with the anti-tumor antibody and the SG-inducing agent (or a combination of such agents) described herein may be employed to treat a cancer patient.
  • the tumor- targeting antibody and the SG-inducing agent can be administered following the same or different dosing regimen. In some embodiments, the tumor-targeting antibody and the SG-inducing agent is administered sequentially in any order during the entire or portions of the treatment period.
  • the tumor-targeting antibody and the SG- inducing agent is administered simultaneously or approximately simultaneously (e.g., within about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or about 30 minutes of each other).
  • the SG-inducing agent may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days before administration of the tumortargeting antibody.
  • the SG-inducing agent is administered from 1 to 4 weeks, 2 to 8 weeks, 3 to 12 weeks, 4 to 16 weeks or longer, before the tumor-targeting antibody is administered.
  • An anti-tumor antibody may also be administered to a cancer patient in conjunction with a cell-based therapy, such as natural killer (NK) cell therapy or a cancer vaccine.
  • a cancer vaccine is a peptide-based vaccine, a nucleic acid-based vaccine, a cell-based vaccine, a virus-based or viral fragment-based vaccine or an antigen presenting cell (APC) based vaccine e.g., dendritic cell-based vaccine).
  • APC antigen presenting cell
  • Cancer vaccines include Gardasil®, Cervarix®, sipuleucel-T (Provenge®), NeuVaxTM, HER-2 ICD peptide-based vaccine, HER-2/neu peptide vaccine, AdHER2/neu dendritic cell vaccine, HER-2 pulsed DC1 vaccine, Ad-sig-hMUC-l/ecdCD40L fusion protein vaccine, MVX-0NC0-1, hTERT/ survivin /CMV multipeptide vaccine, E39, J65, PlOs-PADRE, rV-CEA-Tricom, GVAX®, Lucanix®, HER2 VRP, AVX901, ONT-10, ISA1O1, ADXS1 1-001, VGX-3100, INO-9012, GSK1437173A, BPX-501, AGS-003, IDC-G3O5, HyperAcute®-Renal (HAR) immunotherapy, Prevenarl3, MAGER-3.
  • Gardasil® Cervarix®, si
  • NCT02115126 latent membrane protein-2 (LMP2)-loaded dendritic cell vaccine
  • HS410-101 NCT02010203, Heat Biologies
  • EAU RF 2010-01 NCT01435356, GSK
  • 140036 NCT02015104, Rutgers Cancer Institute of New Jersey
  • 130016 NCTO 1730118, National Cancer Institute
  • MVX-201101 NCT02193503, Maxivax SA
  • ITL-007-ATCR- MBC NCT01741038, Immuno vative Therapies, Limited
  • CDR0000644921 NCT00923143, Abramson cancer center of the University of Pennsylvania
  • SuMo-Sec-01 NCT00108875, Julius Maximilians Universitaet Hospital
  • MCC-15651 NCT01176474, Medarex, Inc, BMS
  • the anti-tumor antibody can be administered with an agent, e.g., a corticosteroid, that mitigates side-effects resulting from stimulation of the immune system.
  • an agent e.g., a corticosteroid
  • a therapeutic agent that is administered in conjunction with an anti-tumor antibody of the present disclosure can be administered prior to administrations of the tumor-targeting antibody or after administration of the tumortargeting antibody.
  • an anti-tumor antibody may be administered at the same time as the additional therapeutic agent.
  • an anti-tumor antibody and an additional therapeutic agent described above can be administered following the same or different dosing regimens.
  • the tumor-targeting antibody and the therapeutic agent are administered sequentially in any order during the entire treatment period or portions thereof.
  • the tumor-targeting antibody and the therapeutic agent are administered simultaneously or approximately simultaneously e.g., within about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or about 30 minutes of each other).
  • the therapeutic agent may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days before the administration of the tumor-targeting antibody.
  • the therapeutic agent may be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the administration of the tumortargeting antibody.
  • the assay measures antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), or complement-dependent cytoxicity (CDC).
  • ADCC antibody dependent cellular cytotoxicity
  • ADCP antibody dependent cellular phagocytosis
  • CDC complement-dependent cytoxicity
  • binding and activation of FcyRIII by an antibody described herein is determined.
  • binding and activation of FcyRIIa by an antibody described herein is determined.
  • binding and activation of the FcR is determined using an ex vivo bioluminescent cell-based assay.
  • a Jurkat cell line that stably expresses human a FcyR and a NFAT-induced luciferase.
  • the effector cells expressing FcyRIIa or FcyRIII transduce intracellular signals resulting in NFAT- mediated luciferase activity that can be easily quantified.
  • the activity of the antibodies is evaluated in vivo in an animal model that is known for specific human tumors.
  • One exemplary model is the CT26 mouse model.
  • Tumor-targeting activity of these antibodies in vivo may be assessed by using several assays, including but not limited to using flow cytometry to analyze the immune profiling of the blood and tumor, monitoring tumor growth, and performing immunofluorescence to semi-quantitative estimate tumor infiltration.
  • the effect of the antibody can be assessed using Survival, a normalized area above the curve metric (NAAC), and a normalized growth rate metric (NGRM), where NAAC and NGRM were both developed at Atreca.
  • NAAC normalized area above the curve metric
  • NGRM normalized growth rate metric
  • an “in vivo active’- determination can be based on the in vivo activity was assessed by a p- value ⁇ 0.05 in at least one of the analyses of survival, NAAC, and NGRM, i.e., if an antibody exhibited a p-value of less than or equal to 0.05 for survival, NAAC, and/or NGRM (any one alone being sufficient), the antibody is considered “in vivo active”.
  • antibodies that exhibit inhibitory effects on tumors, including decreasing rate of tumor growth, size, tumor invasion and/or metastasis.
  • Such antibodies exhibit tumor-targeting effects in vivo, e.g., when administered to subjects that has a tumor expressing a tumor-associated glycan.
  • an antibody or variant thereof described herein is modified to have improved developability (i.e., reduced development liabilities), including but not limited to, decreased heterogeneity, increased yield, increased stability, improved net charges to improve pharmacokinetics, and or/reduced immunogenicity.
  • improved developability i.e., reduced development liabilities
  • antibodies having improved developability can be obtained by introducing mutations to reduce or eliminate potential development liabilities.
  • antibodies having improved developability possess modifications as compared to a reference or control antibody in their amino acid sequence.
  • the antibodies or variants thereof disclosed herein have improved developability while maintaining comparable or improved binding affinity to the target antigen as compared to a reference or control (unmodified) antibody. In some embodiments, the antibodies or variants thereof disclosed herein have improved developability while maintaining activities similar to a reference or control (unmodified) antibody.
  • the antibodies or variants thereof have improved developability, e.g., as identified through various in vitro assays, such as aggregation assessment by HPLC or UPLC, hydrophobic interaction chromatography (HIC), polyspecificity assays (e.g., baculovirus particle binding), self-interaction nanoparticle spectroscopy (SINS), or mass spec analysis after incubation in an accelerated degradation condition such as high temperature, low pH, high pH, or oxidative H2O2. Mutations are successful if activity is maintained (or enhanced) while removing or reducing the severity of the liability.
  • HIC hydrophobic interaction chromatography
  • polyspecificity assays e.g., baculovirus particle binding
  • SINS self-interaction nanoparticle spectroscopy
  • mass spec analysis after incubation in an accelerated degradation condition such as high temperature, low pH, high pH, or oxidative H2O2. Mutations are successful if activity is maintained (or enhanced) while removing or reducing the severity of the liability
  • This motif consists of a K or R, followed by a K or R. Stated differently, the motif can be KK, KR, RK, or RR.
  • the dipeptide NG poses a medium risk of development liability.
  • the dipeptides NA, NN, NS, and NT pose a low risk of development liability.
  • N may also exhibit low risk of liability for other successor residues, e.g., D, H, or P.
  • dipeptide ND, NH, or NP poses a low risk of development liability.
  • the dipeptide DG poses a medium risk of development liability.
  • D may also exhibit low risk of development liability for other successor residues, e.g., N, H, or P.
  • Free cysteine refers to a cysteine that does not form a disulfide bond with another cysteine and thus is left “free” as thiols.
  • the presence of free cysteines in the antibody can be a potential development liability.
  • an odd net number of cysteines in the protein shows a likelihood there is a free cysteine.
  • antibodies described herein comprise improved efficacy and/or reduced immunogenicity.
  • CDR3 is modified to improve efficacy and/or reduce immunogenicity.
  • Development liabilities can be removed or reduced by one or more mutations. Mutations are designed to preserve antibody structure and function while removing or reducing development liabilities and to improve function. In some embodiments, mutations to chemically similar residues can be identified that maintain size, shape, charge, and/or polarity. Illustrative mutations are described in Table 5.
  • the antibodies described herein comprise an Fc region having altered glycosylation that increase the ability of the antibody to recruit NK cells and/or increase ADCC.
  • the Fc region comprises glycan containing no fucose (i.e. the Fc region is afucosylated).
  • Fucosylated antibodies can be produced using cell lines that express a heterologous enzyme that depletes the fucose pool inside the cell (e.g., GlymaxX® by ProBioGen AG, Berlin, Germany).
  • Non-fucosylated antibodies can also be produced using a host cell line in which the endogenous a-l,6-fucosyltransferase (FUT8) gene is deleted.
  • AB-006410 discovered in antibody repertoires generated by Immune Repertoire Capture® (IRC®) technology from plasmablast B cells isolated from a melanoma patient who had undergone treatment with a pembrolizumab, was previously mutated to generate antibodies with improved properties.
  • AB -006410 was mutated to remove an N-linked glycosylation site in the light chain CDR1 to generate AB-011110 and AB-011111.
  • AB- 011110 was then used as the basis for generation of additional variants designed to address other potential liabilities.
  • One of these variants, AB-011622 contained the mutations R30Q in the light chain CDR1 and D97N in the light chain CDR3 designed to remove two liabilities.
  • AB-011367 was generated using a consensus of three sibling antibodies with a light chain CDR1 N-glycosylation site and a light chain CDR3 free cysteine removed. Additional variants were generated based on AB-011367, including AB-011628, which contains the mutation R57N in the light chain CDR2 and AB-011861 which contains the mutation D54N in the heavy chain HCDR2. The variants tested retained the thermostability of the parental antibodies. These variants were optimized further as shown in Table 6.
  • the name of the mutation in Table 6 indicates the antibody ID of the parental antibody (e.g., 11110 indicates parental antibody AB-11110), whether the mutation is a heavy chain or light chain, the position in the heavy chain or light chain of the mutation (with the numbering based on the sequences as provided in Table 3 and not on Kabat or other amino acid sequence numbering conventions), the amino acid residue at the position before introduction of the mutation, and the amino acid at the position after introduction of the mutation.
  • L26NS refers to that the asparagine (N) in the light chain position 26 is mutated to a serine (S).
  • S serine
  • certain of the new variants showed improved properties over their parental antibody.
  • those antibodies that contain the change in position 107 of the heavy chain to a tryptophan H107VW and H107AW) showed more potent binding and reduced polyreactivity as compared to the parental antibody that did not contain that mutation.
  • Results of the assay indicated that all of the variants tested, AB-012909, AB-012918, AB-012921, AB-012926, AB-012929 and AB-012934, showed improved binding to the cell line as compared to the parental antibodies AB-01 110, AB-011628, and AB-01 1861 (FIG. 2).
  • ADC antibody-drug conjugate activity of the anti-tumor antibodies were assessed on LoVo and NUGC4 cells. Briefly, the antibodies were tested for ADC activity using a secondary, toxin-conjugated antibody. Target cells were detached from the culture plate and cell concentration was adjusted to 31,250 cells/mL in assay media. 2,500 cells were added to each well of a 96 well plate and incubated with different concentrations of primary antibody for 15 min at room temperature. Following, secondary Fab anti-mouse IgG Fc conjugated to MMAF with a cleavable linker was added at a final concentration of 250 ng/mL. Cells were incubated for 72 h at 37 °C and 5% CO2.
  • Results of the assay showed that the tested variants exhibited improved ADC cytotoxicity in both LoVo (FIG. 3) and NUGC4 cells (FIG. 4) as compared to the parental antibodies AB-01110 and AB-011628.
  • EC50 values, Area Under the Curve (AUC) and maximum % cytotoxicity values from the assays are shown in Table 8 (LoVo) and Table 9 (NUGC4).
  • Antibody-drug conjugate activity can be tested on antibodies directly conjugated with a toxic payload, such as Exatecan.
  • LoVo target cells are detached from the culture plate and cell concentration is adjusted to 31,250 cells/mL in assay media.
  • 2,500 cells are added to each well of a 96 well plate and incubated with different concentrations of directly conjugated primary antibody for 15 min at room temperature. Cells are than incubated for 72 h at 37 °C and 5% CO2.
  • 100 pl CellTiter-Glo® are added to each well and allowed to incubate for 5-10 min at room temperature before reading luminescence. Data is then normalized to a maximum lysis control and plotted using GraphPad Prism.
  • Antibodies in the ADC conjugate construct format can be tested in mice carrying tumors from LoVo tumor cells.
  • 1 x 10 7 LoVo tumor cells are injected subcutaneously into female BALB/c nude mice. Tumors are allowed to establish and randomized at around 150 mm 3 . Dosing is performed at day of randomization.

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Abstract

Provided herein are antibodies that target tumors. These antibodies bind preferentially to tumor tissue as compared to normal tissue. Such antibodies are used in methods of inhibiting tumor cell growth.

Description

ANTI TUMOR ANTIBODIES
FIELD OF CANCER THERAPEUTICS
[0001] This application relates to therapeutic antibodies for the treatment of various cancers.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63/584,162, filed on September 20, 2023 and U.S. Provisional Application No. 63/589,137, filed on October 10, 2023, each of which is incorporated herein by reference.
BACKGROUND
[0003] Protein glycosylation is one of the most complex and common post-translational modifications representing the enzymatic addition of carbohydrate chains called glycans. Glycan-specific antibodies can be detected early in life without immunization, as through infections and vaccinations. The expression of glycans and glycan-specific antibodies may change during the cancer progression. Glycans and glycan-specific antibodies have been suggested to serve as cancer diagnostic and prognostic markers (Tikhonov et al., Glycan- specific antibodies as potential cancer biomarkers: a focus on microarray application, Clinical Chemistry and Laboratory Medicine (CCLM) (2019) Vol. 58: Issue 10). However, since most known glycans are found in cancer patients and healthy individuals, the utility of glycans in cancer therapeutics has not been fully explored.
BRIEF SUMMARY
[0004] Provided herein, in certain embodiments, are antibodies that bind to a tumor or a fragment thereof, comprising: a heavy chain variable region (VH) comprising (i) a complementarity determining region (HCDR) 1 having an amino acid sequence according to any one of SEQ ID NOs: 16, 17, 18, 19, and 20; (ii) a HCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 25, 26, 27, 28, 29, 30, and 31-62; and (iii) a HCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 65-85; and a light chain variable region (VL) comprising (i) a LCDR1 having an amino acid sequence according to any one of SEQ ID NOs: 86, 87, 88, and 91-111; (ii) a LCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 112-122; and (iii) a LCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 123-126; and at least one modification in a position selected from the group consisting of: 27, 28, 31, 57, and 97. In some embodiments, the VL sequence comprises (i) a LCDR1 having an amino acid sequence according to any one of SEQ ID NOs: 89 and 91-111; (ii) a LCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 1 16-122; and (iii) a LCDR3 having an amino acid sequence according to SEQ ID NO: 126. In some embodiments, the VH sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 131-208 and 252. In some embodiments, the VL sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 214-251. In some embodiments, the VH sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 131-208 and 252, and the VL sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 214-251.
[0005] Provided herein, in certain embodiments, are antibodies that bind to a tumor or a fragment thereof, comprising: a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 127- 208 and 252 and at least one modification in a position selected from the group consisting of: 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 111, 112, 113, 114, 115, and 118; and/or a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 209-251 and at least one modification in a position selected from the group consisting of: 27, 28, 31, 57, and 97. In some embodiments, the modification is selected from a modification listed in Table 6.
[0006] Provided herein, in certain embodiments, are antibodies that bind to a tumor or fragment thereof, comprising: a heavy chain variable region (VH) comprising (i) a complementarity determining region (HCDR) 1 having an amino acid sequence according to any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, and 20; (ii) a HCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31-62; and (iii) a HCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 63-85; and at least one modification in a position selected from the group consisting of: 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 11 1, 112, 113, 114, 115, and 118; and/or a light chain variable region (VL) comprising (i) a LCDR1 having an amino acid sequence according to any one of SEQ ID NOs: 86, 87, 88, and 91-111 ; (ii) a LCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 112-122; and (iii) a LCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 123-126; and at least one modification in a position selected from the group consisting of: 27, 28, 31, 57, and 97. In some embodiments, the modification is selected from a modification listed in Table 6.
[0007] Provided herein, in certain embodiments, are immunoconjugates comprising an antibody disclosed herein, and a cytotoxic agent conjugated to the antibody using a linker. In some embodiments, the cytotoxic agent is selected from the group consisting of: ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, methotrexact, actinomycin, a diphtheria toxin, extotoxin A from Pseudomonas, Pseudomonas exotoxin 40, abrin, abrin A chain, modeccin A chain, alpha sarcin, gelonin, mitogellin, restrictocin, cobran venom factor, a ribonuclease, engineered Shiga toxin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolastatin, ccl065, and a cisplatin.
[0008] Provided herein, in certain embodiments, are antibodies that bind to a tumor, wherein the antibody comprises, a heavy chain variable region comprising an HCDR1, HCDR2, and/or HCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213 listed in Table 1, or variants of the HCDR1, HCDR2, and/or HCDR3 amino acid sequence in which 1, 1, 2, 3, 4, 5, or more amino acids are substituted; and at least one modification in a position selected from the group consisting of: 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 11 1 , 112, 1 13, 114, 1 15, and 1 18; and/or a light chain variable region comprising: an LCDR1, LCDR2, and/or LCDR3 amino acid sequence of any one of AB- 012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB- 012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB- 012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB- 012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB- 012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB- 012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB- 012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB- 012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB- 012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB- 012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB- 012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB- 012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB- 012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB- 012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB- 012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB- 012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB- 012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB- 012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB- 012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB- 013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB- 013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB- 013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213, or variants of the LCDR1, LCDR2, and/or LCDR3 amino acid sequence in which 1, 2, 3, 4, 5, or more amino acid are substituted; and at least one modification in a position selected from the group consisting of: 27, 28, 31, 57, and 97. In some embodiments, the heavy chain variable region of the antibody comprises an HCDR3 variant of an HCDR3 of any one of AB- 012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB- 012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB- 012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB- 012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB- 012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB- 012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB- 012221 , AB -012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB- 012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB- 012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB- 012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB- 012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB- 012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB- 012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB- 012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB- 012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB- 012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB- 012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB- 012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB- 012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB- 013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB- 013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB- 013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213, in which the amino acid residue at position 107 has been substituted with a tryptophan. In some embodiments, the heavy chain variable region of the antibody comprises an HCDR3 variant comprising SEQ ID NO: 84. In some embodiments, the antibody comprises all six CDRs of an antibody selected from the group consisting of designated as AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB -012921 , AB -012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, and AB-013213.
[0009] Provided herein, in certain embodiments, are antibodies comprising a VH region comprising a VH amino acid sequence in Table 3 or an amino sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the VH amino acid sequence in Table 3, and/or wherein the antibody comprises a VL region comprising a VL amino acid sequence in Table 3; and an amino sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the VL amino acid sequence in Table 3. In some embodiments, the antibody comprises: a VH region having an amino acid sequence at least 80% identical to the amino acid sequence according to any one of SEQ ID NOs: 127-208 or 252; and/or a VL region having an amino acid sequence at least 80% identical to the amino acid sequence according to any one of SEQ ID NOs: 209-251. In some embodiments, the antibody comprises both the VH and VL of an antibody selected from the group consisting of designated as AB-012179, AB-012180, AB- 012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB- 012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB- 012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB- 012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB- 012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB- 012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB- 012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB- 012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB- 012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB- 012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB- 012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB- 012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB- 012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB- 012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB- 012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921 , AB-012922, AB- 012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB- 012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB- 012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB- 012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB- 013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB- 013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208,, AB-013209, AB- 013210, AB-013211, AB-013212, AB-013213, and a variant thereof. In some embodiments, the antibody comprises both the VH and VL of an antibody selected from the group consisting of designated as AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, and AB-013213.
[0010] Provided herein, in certain embodiments, are antibodies that competes for binding with an antibody disclosed above.
[0011] In some embodiments, the binding of the antibody or the immunoconjugate to the tumor is dependent on the expression of one or more glycosyltransferases in the tumor. In some embodiments, one of the one or more glycosyltransferases has N-acetyl- galactosaminyltransferase activity. In some embodiments, one of the one or more glycosyltransferases has fucosyltransferase activity. In some embodiments, the glycosyltransferase that has N-acetyl-galactosaminyltransferase activity is selected from the group consisting of B4GALNT3 and B4GALNT4. In some embodiments, the glycosyltransferase is B4GALNT3. In some embodiments, one of the one or more glycosyltransferases that has fucosyltransferase activity is selected from the group consisting of FUT4, FUT5, FUT6, and FUT9. In some embodiments, the glycosyltransferase is FUT9. In some embodiments, the tumor expresses a tumor-associated glycan. In some embodiments, the tumor- associated glycan is an extracellular glycan. In some embodiments, the presence of the tumor- associated glycan is dependent on the expression of B4GALNT3 and FUT9 in the tumor. In some embodiments, the antibody preferentially binds to a tumor tissue relative to a normal tissue. In some embodiments, the antibody is internalized by the tumor cells upon contacting the tumor.
[0012] In some embodiments, provided herein is a polynucleotide encoding a polypeptide comprising a VH sequence of an antibody or an immunoconjugate disclosed herein, and /or a VL sequence of an antibody or an immunoconjugate disclosed herein.
[0013] In some embodiments, provided herein is an expression vector comprising a polynucleotide encoding the VH region and/or the VL region of the antibody or the immunoconjugate. In some embodiments, provided herein is a host cell that comprises the expression vector.
[0014] Provided herein, in certain embodiments, are pharmaceutical compositions comprising the antibody or the immunoconjugate disclosed above and a pharmaceutically acceptable carrier. [0015] Provided herein, in certain embodiments, are methods of treating a cancer patient, the method comprising administering the antibody or the immunoconjugate to the patient. In some embodiments, the cancer is selected from the group consisting of colorectal, lung, endometrial, breast, stomach and esophageal cancer.
[0016] Provided herein, in certain embodiments, are uses of the antibody or the immunoconjugate for a method of treating cancer. In some embodiments, the cancer is selected from the group consisting of colorectal, lung, endometrial, breast, stomach and esophageal cancer.
[0017] Provided herein, in certain embodiments, are polypeptides comprising a heavy chain variable region, wherein the heavy chain variable region comprises an HCDR1, HCDR2, and/or HCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213 listed in Table 1, or variants of the HCDR1, HCDR2, and/or HCDR3 amino acid sequence in which 1, 1, 2, 3, 4, 5, or more amino acids are substituted. In some embodiments, provided herein, the heavy chain variable region of the antibody comprises an HCDR3 variant of an HCDR3 of any one of AB-012179, AB- 012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB- 012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB- 012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB- 012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB- 012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB- 012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221 , AB- 012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB- 012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB- 012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB- 012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB- 012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB- 012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB- 012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB- 012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB- 012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB- 012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB- 012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB- 012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB- 012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB- 013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB- 013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB- 013209, AB-013210, AB-013211, AB-013212, or AB-013213, in which the amino acid residue at position 107 has been substituted with a tryptophan. In some embodiments, the heavy chain variable region of the antibody comprises an HCDR3 variant comprising SEQ ID NO: 84.
[0018] Provided herein, in certain embodiments, are polypeptides comprising a light chain variable region, wherein the light chain variable region comprises an LCDR1, LCDR2, and/or LCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261 , AB-012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213 listed in Table 1, or variants of the HCDR1, HCDR2, and/or HCDR3 amino acid sequence in which 1, 1, 2, 3, 4, 5, or more amino acids are substituted. In some embodiments, provided herein is a polypeptide comprising a VH sequence of an antibody or an immunoconjugate disclosed herein, and/or a VL sequence of an antibody or an immunoconjugate disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGs. 1A-1D show binding of anti-tumor antibodies to lysate from the colorectal cancer (CRC) cell line LoVo and human serum as determined by ELISA. FIG. 1A shows binding of AB-011110 variants to LoVo lysate. FIG. IB shows binding of AB-022788 variants to LoVo lysate. FIG. 1C shows binding of AB-011628 variants to LoVo lysate. FIG. ID shows binding to human serum.
[0020] FIG. 2 shows flow cytometry analysis of the binding of anti-tumor antibodies to the CRC cell line LoVo. The x axis is R-phycoerythrin (R-PE) median fluorescence intensity (MedFI) levels and the y axis indicates log antibody concentration (log Ab [C]) nm. [0021] FIG. 3 shows the ADC activity of anti-tumor antibodies in LoVo cells using a secondary, monomethyl auristatin F (MMAF) toxin-conjugated antibody assay. The x axis is cytotoxicity and the y axis indicates log antibody concentration (log[Ab]) M.
[0022] FIG. 4 shows the ADC activity of anti-tumor antibodies in NUGC4 cells using a secondary, monomethyl auristatin F (MMAF) toxin-conjugated antibody assay. The x axis is cytotoxicity and the y axis indicates log antibody concentration (log[AB]) M.
[0023] FIG. 5 shows results of immunohistochemistry staining of anti-tumor antibodies binding to colorectal cancer tissue sections.
[0024] FIG. 6A and FIG. 6B show results of immunohistochemistry staining of anti-tumor antibodies binding to cancer tissue sections. FIG. 6A shows binding to tissue sections from colorectal cancer (top panels), lung cancer (middle panels), and endometrial cancer (bottom panels). FIG. 6B shows binding to tissue sections from breast cancer (top panels), stomach cancer (middle panels), and esophageal cancer (bottom panels).
[0025] FIGs. 7A-7D show alignments and CDR designations for various anti-tumor antibodies. FIG. 7A shows alignments for HCDR1 (top) and HCDR2 (bottom). FIG. 7B shows alignments for HCDR3. FIG. 7C shows alignments for LCDR1 (top) and LCDR2 (bottom). FIG. 7D shows alignments for LCDR3.
DETAILED DESCRIPTION
[0026] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.
[0027] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules and the like.
[0028] The term “about,” as used herein, refers to the usual error range for the respective value readily known to the skilled person in this technical field; for example, + 20%, + 10%, or ± 5%, are within the intended meaning of the recited value.
[0029] As used herein, the term “antibody” means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of an antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and specifically covers a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies e.g., bispecific antibodies), antibody fragments including but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity. In general, antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region (VL) and one constant region (CL). The heavy chain consists of one variable region (VH) and at least three constant regions (CHI, CH2 and CH3). The variable regions determine the binding specificity of the antibody. Each variable region contains three hypervariable regions known as complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The extent of the FRs and CDRs has been defined (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). The three CDRs in each variable region (e.g., light chain variable region or heavy chain variable region, with six CDRs total in a typical antibody format), referred to as CDR1 , CDR2, and CDR3, collectively contribute to antibody binding specificity. Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.
[0030] “Antibody fragments” comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies (e. . , Zapata et al. , Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment with two antigen combining sites and is still capable of cross-linking antigen.
[0031] As used herein, the term “anti-tumor antibody”, “antibody that binds a tumor,” “antibody that targets a tumor,” or “tumor-targeting antibody,” with respect to an antibody, refers to an antibody that binds preferentially to a tumor tissue than normal tissue. In some embodiments, the normal tissue is the tissue that is adjacent to the tumor, referred to as tumor- adjacent tissue or TAT. In some embodiments, an anti-tumor antibody also decreases the rate of tumor growth, tumor size, invasion, and/or metastasis, via direct or indirect effects on tumor cells.
[0032] As used herein, “V-region” refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4. The heavy chain V-region, VH, is a consequence of the rearrangement of a V-gene (HV), a D-gene (HD), and a J-gene (HJ), in what is termed V(D)J recombination during B-cell differentiation. The light chain V-region, VL, is a consequence of the rearrangement of a V-gene (LV) and a J-gene (LJ).
[0033] As used herein, “complementarity-determining region (CDR)” refers to the three hypervariable regions (HVRs) in each chain that interrupt the four “framework” regions established by the light and heavy chain variable regions. The CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of each chain are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also identified by the chain in which the CDR is located. Thus, a VH CDR3 (HCDR3) is in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR3 (LCDR3) is the CDR3 from the variable domain of the light chain of the antibody in which it is located. The term “CDR” is used interchangeably with “HVR” when referring to CDR sequences.
[0034] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, Structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol. 1997, 273(4)). Definitions of antigen combining sites are also described in the following: Ruiz el al. , IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1 ;29(l):207-9 (2001); MacCallum et al., Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin et al., Methods Enzymol., 203, 121— 153, (1991); Pedersen et al., Immunomethods, 1, 126, (1992); and Rees et al., In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996). Reference to CDRs as determined by Kabat numbering is based, for example, on Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
[0035] CDRs as shown in Tables 1 and 2 are defined by IMGT and Kabat. The VH CDRS as listed in Table 1, are defined as follows: HCDR1 is defined by combining Kabat and IMGT; HCDR2 is defined by Kabat, and the HCDR3 is defined by IMGT. The VL CDRS as listed in Table 2 are defined by Kabat. FIGs. 7A-7D show alignment of certain anti-tumor antibody VH and VL sequences with CDRs designated by Kabat and IMGT. The CDRs of other antitumor antibodies provided herein can be similarly designated. As known in the art, numbering and placement of the CDRs can differ depending on the numbering system employed. It is understood that disclosure of a variable heavy and/or variable light sequence includes the disclosure of the associated CDRs, regardless of the numbering system employed.
[0036] An “Fc region” refers to the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, e.g., for human immunoglobulins, “Fc” refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy2. It is understood in the art that the boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxylterminus, using the numbering according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The term “Fc region” may refer to this region in isolation or this region in the context of an antibody or antibody fragment. “Fc region” includes naturally occurring allelic variants of the Fc region as well as modified Fc regions, e.g., that are modified to modulate effector function or other properties such as pharmacokinetics, stability or production properties of an antibody. Fc regions also include variants that do not exhibit alterations in biological function. For example, one or more amino acids can be deleted from the N-terminus or C- terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art to have minimal effect on activity (see, e.g., Bowie et al., Science 247:306-1310, 1990). For example, for IgG4 antibodies, a single amino acid substitution (S228P according to Kabat numbering; designated IgG4Pro) may be introduced to abolish the heterogeneity observed in recombinant IgG4 antibodies (see, e.g., Angal et al., Mol Immunol 30:105-108, 1993).
[0037] An “ECso” as used herein refers to the half-maximal effective concentration, which is the concentration of an antibody that induces a response (signal generated in engagement assay) halfway between the baseline and maximum after a specified exposure time. In some embodiments, the “fold over EC50” is determined by dividing the EC 50 of a reference antibody by the ECso of the test antibody.
[0038] The term “equilibrium dissociation constant” abbreviated (KD), refers to the dissociation rate constant (kd, time 1) divided by the association rate constant (ka, time-1 M 1). Equilibrium dissociation constants can be measured using any method. Thus, in some embodiments antibodies of the present disclosure have a KD of less than about 50 nM, typically less than about 25 nM, or less than 10 nM, e.g., less than about 5 nM or than about 1 nM and often less than about 10 nM as determined by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37°C. In some embodiments, an antibody of the present disclosure has a KD of less than 5 x 10’5 M, less than 10’5 M, less than 5 x 10'6 M, less than 10"6 M, less than 5 x 10"7 M, less than 10"7 M, less than 5 x 10'8 M, less than 10'8 M, less than 5 x 10"9 M, less than 10"9 M, less than 5 xlO 10 M, less than 10 lo M, less than 5 x 10 11 M, less than 10 11 M, less than 5 x 10’12M, less than 10" 12 M, less than 5 x 10'13 M, less than 10‘13 M, less than 5 x 10‘14 M, less than 10 14M, less than 5 x 10'15 M, or less than 10‘13 M or lower as measured as a bivalent antibody. In the context of the present disclosure, an “improved” KD refers to a lower KD. In some embodiments, an antibody of the present disclosure has a KD of less than 5 x 10’5 M, less than 10 s M, less than 5 x 10'6 M, less than 10'6 M, less than 5 x 10’7 M, less than 10’7 M, less than 5 x 10'8 M, less than 10'8 M, less than 5 x 10‘9 M, less than 10’9 M, less than 5 xlO 10 M, less than 10 10 M, less than 5 x 10 11 M, less than 10 11 M, less than 5 x 10 12M, less than 10" 12 M, less than 5 x 10"13 M, less than 10"13 M, less than 5 x 10"14 M, less than 10 14M, less than 5 x 10 15 M, or less than IO 15 M or lower as measured as a monovalent antibody, such as a monovalent Fab. In some embodiments, an anti-tumor antibody of the present disclosure has KD less than 100 pM, e.g., or less than 75 pM, e.g., in the range of 1 to 100 pM, when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37°C. In some embodiments, an anti-tumor antibody of the present disclosure has KD of greater than 100 pM, e.g., in the range of 100-1000 pM or 500-1000 pM when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore® system performed at 37°C.
[0039] The term “monovalent molecule” refers to a molecule having one antigen-binding site, e.g., a Fab or scFv.
[0040] The term “bivalent molecule” as used herein, refers to a molecule having two antigen-binding sites. In some embodiments, a bivalent molecule of the present disclosure is a bivalent antibody or a bivalent fragment thereof. In some embodiments, a bivalent molecule of the present disclosure is a bivalent antibody. In some embodiments, a bivalent molecule of the present disclosure is an IgG. In general, monoclonal antibodies have a bivalent basic structure. IgG and IgE have only one bivalent unit, while IgA and IgM consist of multiple bivalent units (2 and 5, respectively) and thus have higher valencies. This bivalency increases the avidity of antibodies for antigens.
[0041] The terms “monovalent binding” or “monovalently binds to” as used herein refer to the binding of one antigen-binding site to its antigen.
[0042] The terms “bivalent binding” or “bivalently binds to” refer to the binding of both antigen-binding sites of a bivalent molecule to its antigen. In some embodiments, both antigen-binding sites of a bivalent molecule share the same antigen specificity.
[0043] The term “valency” refers to the number of different binding sites of an antibody for an antigen. A monovalent antibody comprises one binding site for an antigen. A bivalent antibody comprises two binding sites for the same antigen.
[0044] The term “avidity” in the context of antibody binding to an antigen refers to the combined binding strength of multiple binding sites of the antibody. Thus, “bivalent avidity” refers to the combined strength of two binding sites.
[0045] The terms “identical” or percent “identity,” in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same e.g., 100% identity) or have a specified percentage of nucleotides or amino acid residues are the same e.g. , at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity; or 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)) identity over a specified region, e.g., the length of the two sequences, when compared and aligned for maximum correspondence over a comparison window or designated region. Alignment for purposes of determining percent amino acid sequence identity can be performed in various methods, including those using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389- 3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). Thus, for purposes of this disclosure, BLAST 2.0 can be used with the default parameters to determine percent sequence identity.
[0046] The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.
[0047] A “conservative” substitution refers to a substitution of an amino acid such that charge, polarity, hydropathy (hydrophobic, neutral, or hydrophilic), and/or size of the side group chain is maintained. Illustrative sets of amino acids that may be substituted for one another include (i) positively-charged amino acids Lys and Arg; and His at pH of about 6; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic hydrophobic amino acids Ala, Vai, Leu and He; (vi) hydrophobic sulfur-containing amino acids Met and Cys, which are not as hydrophobic as Vai, Leu, and He; (vii) small polar uncharged amino acids Ser, Thr, Asp, and Asn (viii) small hydrophobic or neutral amino acids Gly, Ala, and Pro; (ix) amide- comprising amino acids Asn and Gin; and (xi) beta-branched amino acids Thr, Vai, and He. Reference to the charge of an amino acid in this paragraph refers to the charge at pH 6-7. [0048] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. Contemplated polynucleotides include a gene or fragment thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or noncoding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA. A polynucleotide can be exogenous or endogenous to a cell and/or exist in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure are imparted before or after assembly of the polymer. Non-limiting examples of modifications include: 5 -bromouracil, peptide nucleic acid, xeno nucleic acid, morpholines, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. The sequence of nucleotides may be interrupted by non-nucleotide components.
[0049] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. A “vector” as used herein refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors can direct the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
[0050] A “substitution” denotes the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
[0051] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
[0052] “Isolated nucleic acid encoding an antibody or fragment thereof’ refers to one or more nucleic acid molecules encoding antibody heavy or light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
[0053] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Thus, a host cell is a recombinant host cell and includes the primary transformed cell and progeny derived therefrom without regard to the number of passages. [0054] A polypeptide “variant” is a polypeptide that typically differs from one or more polypeptide sequences specifically disclosed herein in one or more substitutions, deletions, additions, and/or insertions.
[0055] The term “cancer cell” or “tumor cell” as used herein refers to a neoplastic cell. The term includes cells from tumors that are benign as well as malignant. Neoplastic transformation is associated with phenotypic changes of the tumor cell relative to the cell type from which it is derived. The changes can include loss of contact inhibition, morphological changes, and unregulated cell growth,
[0056] The terms “inhibiting growth of a tumor” and “inhibiting growth of a cancer” are interchangeable and refer to slowing growth and/or reducing the cancer cell burden of a patient that has cancer. “Inhibiting growth of a cancer” thus includes killing cancer cells, as well as decreasing the rate of tumor growth, tumor size, invasion, and/or metastasis by direct or indirect effects on tumor cells.
[0057] As used herein, the terms “treatment,” “treating,” and the like, in some embodiments, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of affecting a partial or complete cure for a disease and/or symptoms of the disease. “Treatment,” as used herein, may include treatment of a disease or disorder (e.g., cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g. , including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e. , arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms; or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present disclosure to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with diseases (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
[0058] The terms “recipient,” “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and in some embodiments, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the mammal is human. None of these terms require the supervision of medical personnel.
[0059] “Expression of a glycan by a cell” or a “glycan expressed by a cell” means that the glycan is present in or on that cell.
[0060] A “tumor overexpressing glycans”, or a “tumor that overexpresses glycans”, or a “cancer overexpressing glycans” or a “cancer that overexpresses glycans”, refers to a tumor or cancer that expresses specific glycans at a level that higher than the level of those glycans expressed in normal tissue (e.g., tumor adjacent tissues or TAT) or otherwise has an increased amount of those glycans as compared to normal tissue. In certain embodiments, a tumor or cancer that overexpresses glycans expresses glycan at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 300% higher or more than the normal tissue (e.g., tumor-adjacent tissues or TAT).
[0061] The term “tumor-associated glycan” refers to a glycan expressed by a tumor cell. In some embodiments, the tumor-associated glycan is not expressed by normal tissue cells. In some embodiments, the tumor-associated glycan is expressed by a tumor at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 300% higher or more as compared to normal tissue (e.g., tumor- adjacent tissues or TAT), In some embodiments, the tumor-associated glycan is expressed on the cell surface, i.e. , is an extracellular glycan. A glycan or a tumor-associated glycan disclosed herein may be attached to proteins or lipids, known as glycoproteins and glycolipids.
[0062] In some embodiments, the disclosure additionally provides methods of identifying subjects who are candidates for treatment with an anti-tumor antibody having tumortargeting effects. In some embodiments, the present disclosure provides a method of identifying a patient who can benefit from treatment with an anti-tumor antibody of the present disclosure. In some embodiments, the patient has tumor that expresses glycans. In some embodiments, the patient has tumor expressing a tumor- associated glycan. In some embodiments, the tumor sample is from a primary tumor. In alternative embodiments, the tumor sample is a metastatic lesion. Binding of antibody to tumor cells through a binding interaction with the glycans can be measured using any assay, such as immunohistochemistry or flow cytometry. In some embodiments, binding of antibody to at least 0.2%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, or at least 50%, of the tumor cells in a sample may be used as a selection criterion for determining a patient to be treated with an anti-tumor antibody as described herein. In other embodiments, analysis of components of the blood is used to identify a patient whose tumor cells are expressing a tumor-associated glycan.
[0063] An anti-tumor antibody disclosed herein can be used to treat several different cancers. In some embodiments, a cancer patient who can benefit from the treatment of the anti-tumor antibody has a cancer expressing glycans. In some embodiments, a cancer patient who can benefit from the treatment of the anti-tumor antibody has a tumor expressing a tumor- associated glycan. In some embodiments, the cancer is a carcinoma, a melanoma, or a sarcoma.
[0064] As used herein, the term “a glycan binder,” “an antibody that binds to glycans”, or “an anti-glycan antibody” refers to a molecule, for example, an antibody or antibody binding domain, that binds to a tumor and the binding is dependent on the activity of one or more glycosyltransferases. In some embodiments, the glycan binder binds to a tumor-associated glycan under permissible conditions (e.g., in a suitable buffer), and the detected signal resulted from the binding is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 150 fold, or at least 200 fold above a reference level. In some embodiments, the reference level is a detected signal produced by contacting a control antibody with the glycans, or by contacting the antibody with a control protein.
[0065] A “variant” of a reference antibody refers to an antibody that typically differs from the reference antibody in one or more substitutions, deletions, additions, and/or insertions in the amino acid sequence of the heavy and/or light chain.
[0066] As used herein, the term “internalize,” or “internalization” refer to the phenomenon that an antibody molecule crosses the cell membrane and reaches the cytoplasm and/or the nucleus.
[0067] Provided herein are tumor-targeting, or anti-tumor, antibodies that bind to tumors and the binding is dependent on the activity one or more glycosyltransferases. In some embodiments, the anti-tumor antibody binds to a tumor-associated glycan. These antibodies are referred to glycan binders in this disclosure. The antibodies bind preferentially to tumor tissues relative to normal tissue and demonstrate cytotoxicity in various antibody-toxin or antibody-immunomodulating agent constructs towards tumor cells. Thus, these antibodies show therapeutic potential in treating cancers. The anti-tumor antibodies are also useful in detecting tumors suitable for treatment with an anti-tumor antibody in diagnostic applications.
GLYCANS
[0068] Glycans are a complex group of monosaccharide or polysaccharide compounds comprised of diverse monosaccharide residues linked glycosidically. Glycosidic bonds are covalent linkages of carbohydrate moieties to another group which may or may not be another carbohydrate (e.g., C-, O-, N-). Through these glycosidic bonds, glycolipids, glycoproteins, and polysaccharides are formed. For purposes of this disclosure, the term “glycan,” refers to a polysaccharide or oligosaccharide, or the carbohydrate portion of all glycol conjugate such as glycoprotein, glycolipid, glycopeptide, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans can be homo or heteropolymers of monosaccharide residues.
[0069] Forming glycosidic bonds typically require glycosyltransferases.
Glycosyltransferases modify glycans in the ER and Golgi apparatus during their biosynthesis. Glycosyltransferases catalyze the transfer of saccharide moieties from an activated nucleotide sugar (also known as the "glycosyl donor") to a nucleophilic glycosyl acceptor molecule, the nucleophile of which can be oxygen- carbon-, nitrogen-, or sulfurbased.
Glycosyltransferase
[0070] Glycosyltransferase B4GALNT3 (beta-l,4-N-acetyl-galactosaminyltransferase 3) is responsible for transfer of N-acetylgalactosamine (GalNAc) to N-acetylglucosamine-beta (GlcNAc) to form N, N'-diacetyllactosediamine with betal,4-linkage. The product of this reaction is GalNAcbetal,4GlcNAc (LacdiNAc). In humans, B4GALNT4 is reported to have similar activity to B4GALNT3. Examples of human B4GALNT3 and B4GALNT4 and their murine homologs include human B4GALNT3 (GenBank accession no1. AB089940; UniProt
1 All references to accession numbers throughout the application refer to the versions that were current in the database as of the filing date of the application. ID: Q6L9W6; SEQ ID NO: 252); murine b4galnt3 (GenBank accession no. AB114826; UniProt ID: Q6L8S8); human B4GALNT4 (GenBank accession no. AB089939.1; UniProt ID: Q76KP1.1); and murine b4galnt4 (GenBank accession no. AB114827; UniProt ID: Q766D5.1).
[0071] Fucosyltransferases (FUTs) are glycosyltransferases involved in the synthesis of cellsurface antigens through catalyzing the transfer of fucose from GDP-fucose to acceptor sugars on biomolecules. The FUT family includes enzymes catalyzing al, 2-, al, 3/4-, al, 6- and protein O-FUT linkages. The al ,3/4-FUT group includes at least eight members: FUT3, FUT4, FUT5, FUT6, FUT7, FUT9, FUT10, and FUT11. FUT4 is a known to catalyze the alpha (l->3) linkage of beta-L-fucose to the GlcNAc of type 2 lactosmaines (LacNAc, Gal- beta (l->4) GlcNAc). See Lowe JB et al. “Molecular cloning of a human fucosyltransferase gene that determines expression of the Lewis x and VIM-2 epitopes but not ELAM-1- dependent cell adhesion”. J Biol Chem. 1991 Sep 15;266(26):17467-77. Examples of human FUTs include FUT3 (GenBank accession no. NM_000149; UniProt ID: P21217); FUT4 (SEQ ID NO: 253) (GenBank accession no. NM_002033; UniProt ID: P22083); FUT5 (GenBank accession no. NM_002034.2; UniProt ID: QI 1128); FUT6 (GenBank accession no. NM_000150.2; NM_001040701.1 ; UniProt ID: P51993.1); FUT7 (GenBank accession no. NM_004479; UniProt ID: QI 1130); FUT9 (GenBank accession no. NM_006581 ;
UniProt ID: Q9Y231); FUT10 (GenBank accession no. AJ582015; UniProt ID: Q6P4F1); and FUT11 (GenBank accession no. BC036037 UniProt ID: Q495W5). Examples of murine FUTs, include FUT4 (GenBank accession no. NM_010242; UniProt ID: B2RPT3); FUT7 (GenBank accession no. NM_013524; UniProt ID: QI 1131); FUT9 (GenBank accession no. NM_010243; UniProt ID: 088819); FUT10 (GenBank accession no. AJ880009; UniProt ID: Q5F2L2); and FUT11 (GenBank accession no. NM_028428.2; UniProt ID: Q8BHC9).
ANTI TUMOR ANTIBODIES
[0072] Anti-tumor antibody AB -006410 was discovered in antibody repertoires generated by Immune Repertoire Capture® (IRC®) technology from plasmablast B cells isolated from a melanoma patient who had undergone treatment with a pembrolizumab. The patient exhibited an active anti-tumor immune response evidenced by tumor-selective antibodies derived from their plasmablast B cells.
[0073] AB-006410, and other anti-tumor antibodies provided herein, bind to tumors and the binding is dependent on the expression of one or more specific glycosyltransferases in the tumor, that is, in the absence of expression of the one or more glycosyltransferases, the anti- tumor antibody will not show detectable binding to the tumor. In some embodiments, one of the one or more glycosyltransferases has N-acetyl-galactosaminyltransferase activity, such as the activity of B4GALNT3 or B4GALNT4. In some embodiments, the one of the one or more glycosyltransferases has fucosyltransferase activity, such as the activity of FUT3, FUT4, FUT5, FUT6, FUT7, FUT9, FUT10, and FUT11. In some embodiments, the one of the one or more glycosyltransferases has fucosyltransferase activity, such as the activity of FUT4, FUT5, FUT6, and FUT9. In some embodiments, the one of the one or more glycosyltransferases has N-acetyl-galactosaminyltransferase activity, such as the activity of B4GALNT3 or fucosyltransferase activity, such as the activity of FUT9. In some embodiments, the one of the one or more glycosyltransferases has N-acetyl- galactosaminyltransferase activity, such as the activity of B4GALNT3 and one of the one or more glycosyltransferases has fucosyltransferase activity, such as the activity of FUT9. [0074] In some embodiments, the binding of the anti-tumor antibody to the tumor is dependent on the expression one or more specific glycosyltransferases in the tumor, that is, in the absence of the expression of the one or more glycosyltransferases, the anti-tumor antibody will not show detectable binding to the tumor.
[0075] In some embodiments, each of the one or more glycosyltransferases is selected from the group consisting of B4GALNT3, B4GALNT4, FUT3, FUT4, FUT5, FUT6, FUT7, FUT9, FUT10, and FUT11. In some embodiments, the binding is dependent on the expression of the glycosyltransferase B4GALNT3 or the glycosyltransferase FUT9. In some embodiments, the anti-tumor antibody binds to a tumor cell that expresses a glycan comprising GalNAcbetal,4GlcNAc (LacdiNAc). In some embodiments, the anti-tumor antibody binds to a tumor cell that expresses GalNAcbetal,4GlcNAc (LacdiNAc). In some embodiments, the LacdiNAc is fucosylated.
[0076] In some embodiments, an anti-tumor antibody disclosed herein binds to an extracellular, tumor- ssociated glycan. The presence of the glycan (or display of the glycan on the tumor cell surface) is dependent on the expression of one or more specific glycosyltransferases in the tumor, that is, in the absence of expression of the one or more glycosyltransferases, the glycan will not be produced in the tumor cells or will not be displayed on the surface of tumor cells. In some embodiments, each of the one or more glycosyltransferases is selected from the group consisting of B4GALNT3, B4GALNT4, FUT3, FUT4, FUT5, FUT6, FUT7, FUT9, FUT10, and FUT11. In some embodiments, each of the one or more glycosyltransferases is selected from the group consisting of B4GALNT3, B4GALNT4, FUT4, FUT5, FUT6, and FUT9. In some embodiments, the glycan is dependent on the expression of the glycosyltransferase B4GALNT3 or the glycosyltransferase FUT9. In some embodiments, the glycan is dependent on the expression of B4GALNT3 and FUT9. In some embodiments, the anti-tumor antibody binds to a glycan comprising GalNAcbetal,4GlcNAc (LacdiNAc). In some embodiments, the anti-tumor antibody binds to GalNAcbetal,4GlcNAc (LacdiNAc). In some embodiments, the antitumor antibody binds to a tumor cell that expresses a glycan comprising GalNAcbetal,4GlcNAc (LacdiNAc). In some embodiments, the anti-tumor antibody binds to a tumor cell that expresses GalNAcbetal ,4GlcNAc (LacdiNAc). In some embodiments, the LacdiNAc is fucosylated.
STRUCTURES OF THE ANTI TUMOR ANTIBODIES
[0077] In some embodiments, an anti-tumor antibody comprises an HCDR1 having an amino acid sequence according to any one of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20 or a variant HCDR1 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence; an HCDR2 having an amino acid sequence according to any one of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-62, or a variant HCDR2 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence; and an HCDR3 having an amino acid sequence according to any one of SEQ ID NO: 63-85 or a variant HCDR3 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising: an LCDR1 having an amino acid sequence according to any one of SEQ ID NO: 86, 87, 88, 91-11 1, or a variant LCDR1 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence; an LCDR2 having an amino acid sequence according to any one of SEQ ID NO: 112-122, or variant LCDR2 in which 1, 2, or 3 amino acids are substituted relative to the sequence; and an LCDR3 having an amino acid sequence according to any one of SEQ ID NO: 123-126, or a variant LCDR3 in which 1, 2, 3, 4, or 5 amino acids are substituted relative to the sequence. [0078] In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 70% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 75% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 80% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 85% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 95% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 96% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 97% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 98% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid sequence at least 99% identical to any one of SEQ ID NO: 127-208. In some embodiments, an anti-tumor antibody comprises a heavy chain variable region comprising an amino acid identical to any one of SEQ ID NO: 127-208.
[0079] In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 70% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 75% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 80% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 85% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 95% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 96% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 97% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 98% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence at least 99% identical to any one of SEQ ID NO: 209-251. In some embodiments, an anti-tumor antibody comprises a light chain variable region comprising an amino acid sequence identical to any one of SEQ ID NOs: 209-251. [0080] In some embodiments an anti-tumor antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region. The VH region has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 127-208; and comprises an HCDR1 having an amino acid sequence according to any one of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, or the HCDR1 of any one of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20 in which 1 , 2, 3, 4, or 5 amino acids are substituted; an HCDR2 having an amino acid sequence according to any one of SEQ ID NO:
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-62, or the HCDR2 of any one of SEQ ID NO: 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31-62 in which 1, 2, 3, 4, or 5 amino acids are substituted; an HCDR3 having an amino acid sequence according to SEQ ID NO: 63-85 or the HCDR3 of SEQ ID NO: 63-85 in which 1, 2, 3, 4, or 5 amino acids are substituted. The VL region has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 209-251, and comprises an LCDR1 having an amino acid sequence according to SEQ ID NO: 86, 87, 88, 91-111 or the LCDR1 of SEQ ID NO: 86, 87, 88, 91-111 in which 1, 2, 3, 4, or 5 amino acids are substituted; an LCDR2 having an amino acid sequence according to SEQ ID NO: 112-122, or the LCDR2 of SEQ ID NO: 112-122 in which 1, 2, or 3 amino acids are substituted; an LCDR3 having an amino acid sequence according to SEQ ID NO: 123-126 or the LCDR3 of SEQ ID NO: 123-126 in which 1, 2, 3, 4, or 5 amino acids are substituted.
[0081] In some embodiments, an anti-tumor antibody comprises: a Vn region comprising amino acid sequence SEQ ID NO: 127-208 or 252 and a VL region comprising amino acid sequence SEQ ID NO: 209-251.
[0082] In some embodiments, an anti-tumor antibody of the present disclosure has one, two, or three CDRs of a VL sequence (LCDRs) having an amino acid sequence according to a sequence set forth in Table 2. In some embodiments, the anti-tumor antibody has at least one mutation and no more than 10, 20, 30, 40 or 50 mutations in the VL amino acid sequences compared to a VL sequence set forth in Table 3. In some embodiments, the anti-tumor antibody has a VL amino acid sequence set forth in Table 3. In some embodiments, the VL amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions compared to a VL sequence set forth in Table 3. In some embodiments, the VL amino acid sequence may comprise a deletion or insertion, e.g., a 1, 2, 3, 4, 5, 6, or 7 amino acid deletion or insertion, relative to an LCDR sequence shown in Table 2. In some embodiments, the VL region comprises an LCDR1 having 1 or 2 substitutions in relative to an LCDR1 sequence shown in Table 2. In some embodiments, an LCDR1 has 3, 4, or 5 substitutions relative to a CDR1 sequence shown in Table 2. In some embodiments, the VL region comprises an LCDR2 that has 1 or 2; or 1, 2, or 3; substitutions relative to the LCDR2 sequence shown in Table 2. In some embodiments, the VL region comprises an LCDR3 that has 1, 2, or 3; or 1, 2, 3, or 4; substitutions relative to an LCDR3 sequence shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1 , CDR2, and CDR3, each having at least 70% identity to an LCDR1 , LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 75% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 80% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 85% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 90% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 95% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having 100% identity to an LCDR1, LCDR2, and LCDR3 as shown in Table 2. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3 of an antibody designated as AB-011110, AB-011367, AB-011622, Ab-011788, AB-011861, AB-011263, AB-011628, AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213.
[0083] In some embodiments, an anti-tumor antibody of the present disclosure has one, two, or three CDRs of a VH sequence (HCDRs) having an amino acid sequence according to a sequence set forth in Table 1. In some embodiments, the anti-tumor antibody has at least one mutation and no more than 10, 20, 30, 40 or 50 mutations in the VH amino acid sequences compared to a VH sequence set forth in Table 3. In some embodiments, the anti-tumor antibody has a VH amino acid sequence set forth in Table 3. In some embodiments, the VH amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions compared to a VH sequence set forth in Table 3. In some embodiments, the VH amino acid sequence may comprise a deletion or insertion, e.g., a 1, 2, 3, 4, 5, 6, or 7 amino acid deletion or insertion, relative to an HCDR sequence shown in Table 1. In some embodiments, the VH region comprises an HCDR1 having 1 or 2 substitutions in relative to an HCDR1 sequence shown in Table 1. In some embodiments, an HCDR1 has 3, 4, or 5 substitutions relative to an HCDR1 sequence shown in Table 1. In some embodiments, the VH region comprises an HCDR2 that has 1 or 2; or 1, 2, or 3; substitutions relative to the HCDR2 sequence shown in Table 1. In some embodiments, the VH region comprises an HCDR3 that has 1, 2, or 3; or 1, 2, 3, or 4; substitutions relative to an HCDR3 sequence shown in Table 1. In some embodiments, an anti-tumor antibody of the present disclosure comprises an HCDR1, HCDR2, and HCDR3, each having at least 70% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 75% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 80% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 85% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having at least 90% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments, an anti-tumor antibody of the present disclosure comprises a CDR1 , CDR2, and CDR3, each having at least 95% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments an anti-tumor antibody of the present disclosure comprises a CDR1, CDR2, and CDR3, each having 100% identity to an HCDR1, HCDR2, and HCDR3 as shown in Table 1. In some embodiments, an anti-tumor antibody of the present disclosure comprises an HCDR1 , HCDR2, and HCDR3 of an antibody designated as AB-011110, AB- 011367, AB-011622, Ab-011788, AB-011861, AB-011263, AB-011628, AB-012179, AB- 012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB- 012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB- 012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB- 012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB- 012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB- 012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB- 012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB- 012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-
012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-
012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-
012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-
012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-
012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-
012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-
012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-
012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-
012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-
012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-
012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-
013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB- 013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB- 013209, AB-013210, AB-013211, AB-013212, or AB-013213.
[0084] The sequences of the anti-tumor antibodies described herein are shown in Tables 1-3.
Table 1. Heavy chain CDR (HCDR) sequences
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Table 2. Light chain CDR (LCDR) sequences
Figure imgf000043_0002
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Table 3. Heavy and light variable region sequences
Figure imgf000051_0002
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Variants
[0085] In some embodiments, variants of any of the anti-tumor antibodies disclosed herein can be generated by introducing mutations to the heavy chain and/or light chain sequences. In some embodiments, the mutation(s) are introduced into one or more of the CDRs of an anti-tumor antibody disclosed herein, e.g., AB-006410, AB-01110, AB-0011367, AB- 011788, AB-001 1628 or other antibodies disclosed in Table 1 or 2. In some embodiments, the mutation(s) are introduced in the framework regions. In some embodiments, a variant is engineered to be as much like self as possible to minimize immunogenicity. One approach to do so is to identify a close germline sequence and mutate one of the anti-tumor antibodies at as many mismatched positions (also known as “germline deviations”) to the germline residue type as possible.
[0086] For any the mutation disclosed in this application, the name indicates the antibody ID of the parental antibody (e.g., 11110 indicates parental antibody AB- 11110), whether the mutation is a heavy chain or light chain, the position in the heavy chain or light chain of the mutation (with the numbering based on the sequences as provided in Table 3 and not on Kabat or other amino acid sequence numbering conventions), the amino acid residue at the position before introduction of the mutation, and the amino acid at the position after introduction of the mutation. For example, L26NS, refers to that the asparagine (N) in the light chain position 26 is mutated to a serine (S). In some embodiments, the anti-tumor antibodies comprise amino acid residue mutation(s) of the heavy chain at position 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 111 , 1 12, 113, 114, 115, and/or 1 18. In some embodiments, the anti-tumor antibodies comprise an amino acid residue mutation of the heavy chain at position 107. In some embodiments, the anti-tumor antibodies comprise amino acid residue mutaiton(s) of the light chain at position 27, 28, 31, 57, and/or 97. In some embodiments, the anti-tumor antibodies comprise amino acid residue mutation(s) of the heavy chain at position 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 111, 112, 113, 114, 115, and/or 118 and amino acid residue mutation(s) of the light chain at position 27, 28, 31, 57, and/or 97. In some embodiments, the anti-tumor antibodies comprise amino acid residue mutation(s) selected from Table 6. In some embodiments, the mutation(s) are in a CDR region. In some embodiments, the mutation(s) are in a framework region. In some embodiments, one or more mutations are selected from the group consisting of L74ND, H54DK, H57HY, H31NV, H32AT, H33WY, H34VI, H35NH, H50RK, H52RH, H52RN, H53SV, H53SA, H54DN, H54DH, H54DR, H54DS, H56ES, H56ED, H56EN, H57GY, H57GK, H57GS, H58WY, H59TA, H59TW, H59TN, H61DN, H61DY, H61DS, H61DF, H62YW, H73SY, H74RA, H75EA, H76EA, H99NE, H101GA, H102YW, H103WY, H106SY, H106SA, H107AN, H107AS, H107AV, H108FY, H108FW, Hl 1 IND, Hl 1 INS, Hl 12RK, Hl BAE, Hl 14YW, Hl 14YP, H115AD, H118VQ, L28SN, L28SD, L29LN, L29LD, L29LF, L30QH, L31RT, L30QR, L35HY, L35HK, L36FY, L55GH, L55GY, L56FS, L56FY, L57RN, L57RY, L58RL, L97NR, H107VW, H107VY, L27EN, L28SA, L31RD, H107AW, H107AY, L97DN, L30RH, and L30RQ. In some embodiments, one or more mutations are selected from the group consisting of L74ND, H54DK, H57HY, H31NV, H32AT, H33WY, H34VI, H35NH, H50RK, H52RH, H52RN, H53SV, H53SA, H54DN, H54DH, H54DR, H54DS, H56ES, H56ED, H56EN, H57GY, H57GK, H57GS, H58WY, H59TA, H59TW, H59TN, H61DN, H61DY, H61DS, H61DF, H62YW, H73SY, H74RA, H75EA, H76EA, H99NE, H101 GA, H102YW, H103WY, H106SY, H106SA, H107AN, H107AS, H107AV, H108FY, H108FW, Hl 1 IND, Hl 1 INS, H112RK, Hl BAE, H114YW, H114YP, Hl BAD, H118VQ, L28SN, L28SD, L29LN, L29LD, L29LF, L30QH, L31RT, L30QR, L35HY, L35HK, L36FY, L55GH, L55GY, L56FS, L56FY, L57RN, L57RY, L58RL, L97NR, H107VW, H107VY, L27EN, L28SA, L31RD, H 107 AW, Hl 07 AY, L97DN, L30RH, and L30RQ as compared to parental antibodies AB-01 1110, AB-011622, AB-011788, AB-01 1861. In some embodiments entire CDR regions or entire framework regions of parental antibodies (e.g., those listed in Tables 1- 3) are replaced with an entire CDR region or entire framework region from a different antibody (e.g., those listed in Tables 1-3). In some embodiments, the one or more of the CDRs of the anti-tumor antibodies disclosed in Table 1 or 2 are mutated to generate variants with improved properties.
[0087] Methods of generating variants are further described in the section entitled “ENGINEERING VARIANTS” and Example 1 below.
Fc variants
[0088] In some embodiments, the anti-tumor antibodies disclosed herein comprise a modified Fc region (as further explained herein). Fc regions typically comprises one or more Fc chains. An IgG Fc chain typically contains two constant heavy domains (Cu2 and CH3) and a hinge region connected to the CH2 domain. Fc regions may typically comprise two Fc chains which dimerize with one another; however, an Fc region may have a single chain or more than two Fc chains, e.g., as may be present in some antibody formats.
[0089] In some embodiments, the anti-tumor antibodies comprise an IgGl Fc region (e.g., human IgGl Fc region), that is, except for having particular residue(s) at certain positions as noted herein, the Fc region has an amino acid sequence that is substantially similar to that of the Fc region within a wild type IgGl Fc. In some embodiments, the wild type IgGl Fc is a human IgGl. In some embodiments, the anti-tumor antibodies comprise an Fc region, each Fc chain of which has an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to that of an Fc chain within a wild-type IgGl Fc.
[0090] In some embodiments, the anti-tumor antibodies comprise an IgG2 Fc region (e.g., human IgG2 Fc region), that is, except for having particular residue(s) at certain positions as noted herein, the Fc region has an amino acid sequence that is substantially similar to that of the Fc region within a wild type IgG2 Fc. In some embodiments, the wild type IgG2 Fc is a human IgG2 Fc. In some embodiments, the anti-tumor antibodies comprise an Fc region, each Fc chain of which has an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to that of an Fc chain within a wild-type IgG2 Fc.
[0091] In some embodiments, the anti-tumor antibodies comprise an IgG4 Fc region (e.g., human IgG4 Fc region), that is, except for having particular residue(s) at certain positions as noted herein, the Fc region has an amino acid sequence that is substantially similar to that of the Fc region within a wild type IgG4 Fc. In some embodiments, the wild type IgG4 Fc is a human IgG4 Fc. In some embodiments, the anti-tumor antibodies comprise an Fc region, each Fc chain of which has an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to that of an Fc chain within a wild-type IgG4 Fc.
[0092] In certain embodiments, Fc regions are modified (e.g., substituted) at one more amino acid residues. In certain embodiments, such modifications alter the half-life of a molecule e.g., binding protein) which comprises the Fc region by altering (e.g., enhancing) binding to an Fc receptor such as the neonatal Fc receptor (FcRn.)
[0093] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
[0094] In some embodiments, one or more modifications in the modified Fc region is selected from the group consisting of: S298A, E333A, K334A, K326A, F243L, R292P, Y300L, V305I, P396L, F243L, R292P, Y300L, L235V, P396L, F243L, S239D, I332E, A330L, S267E, L328F, D265S, S239E, K326A, A327H, G237F, K326E, G236A, D270L, H268D, S324T, L234F, N325L, V266L, and S267D. In some embodiments, one or more modifications in the modified Fc region is selected from the group consisting of S228P, M252Y, S254T, T256E, T256D, T250Q, H285D, T307A, T307Q, T307R, T307W, L309D, Q411H, Q311V, A378V, E38OA, M428L, N434A, N434S, N297A, D265A, L234A, L235A, and N434W.
[0095] In some embodiments, the modified Fc region comprises a specific combination of amino acid substitutions selected from the group consisting of: L234A/L235A; V234A/G237A; L235A/G237A/E318A; S228P/L236E; H268Q/V309L/A330S/A331S; C220S/C226S/C229S/P238S; C226S/C229S/E3233P/L235V/L235A; L234F/L235E/P331S; C226S/P230S; L234A/G237A; L234A/L235A/G237A; Q311R/M428L;
L234A/L235A/P329G (LALAPG); and L234A/L235A/P329A (LALAPA). In some embodiments, the modified Fc region comprises a specific combination of amino acid substitutions consisting of L234A/L235 A/P329A (LALAPA).
[0096] In some embodiments, the modified Fc region comprises a specific combination of amino acid substitutions selected from the group consisting of M428L/N434S (LS); M252Y/S254T/T256E (YTE); T250Q/M428L; T307A/E380A/N434A; T256D/T307Q (DQ); T256D/T307W (DW); M252Y/T256D (YD); T307Q/Q311V/A378V (QVV);
T256D/H285D/T307R/Q311V/A378V (DDRVV); L309D/Q311H/N434S (DHS); S228P/L235E (SPLE); L234A/L235A (LALA); M428L/N434A (LA); L234A/G237A (LAGA); L234A/L235A/G237A (LALAGA); L234A/L235A/P329G (LALAPG); L234A/L235A/P329A (LALAPA); N297A/YTE; D265A/YTE; LALA/YTE; LAGA/YTE; LALAGA/YTE; LALAPG/YTE; N297A/LS; D265A/LS; LALA/LS; LAGA/LS;
LALAGA/LS; LALAPG/LS; N297A/DHS; D265A/DHS; LALA/DHS; LAGA/DHS; LALAGA/DHS; LALAPG/DHS; SP/YTE; SPLE/YTF; SP/LS; SPLE/LS; SP/DHS; SPLE/DHS; N297A/LA; D265A/LA; LALA/LA; LAGA/LA; LALAGA/LA; LALAPG/LA; N297A/N434A; D265A/N434A; LALA/N434A; LAGA/N434A; LALAGA/N434A;
LALAPG/N434A; N297A/N434W; D265A/N434W; LALA/N434W; LAGA/N434W;
LALAGA/N434W; LALAPG/N434W; N297A/DQ; D265A/DQ; LALA/DQ; LAGA/DQ; LALAGA/DQ; LALAPG/DQ; N297A/DW; D265A/DW; LALA/DW; LAGA/DW;
LALAGA/DW; LALAPG/DW; N297A/YD; D265A/YD; LALA/YD; LAGA/YD;
LALAGA/YD; LALAPG/YD; N297A/QVV; D265A/QVV; LALA/QVV; LAGA/QVV, LALAGA/QVV; LALAPG/QVV; N297A/DDRVV; D265A/DDRVV; LALA/DDRVV; LAGA/DDRVV; LALAGA/DDRVV; LALAPG/DDRVV; SP/Q311R/M428L;
SPLE/Q311R/M428L; N297A/Q311R/M428L; D265A/Q311R/M428L;
LALA/Q311R/M428L; LAGA/Q311R/M428L; LALAGA/Q311R/M428L; and LALAPG/Q311R/M428L.
[0097] In addition to the variants discussed above, there are several useful Fc amino acid modifications that can be made for a variety of reasons, including, but not limited to, altering binding to one or more FcyR receptors, altered binding to FcRn receptors and the like as discussed below. Accordingly, the antibodies provided herein (heterodimeric, as well as homodimeric) can include such amino acid modifications with or without the heterodimerization variants outlined herein (e.g., the pl variants and steric variants). Each set of variants can be independently and optionally included or excluded from any heterodimeric protein.
FcyR Variants
[0098] Accordingly, there are several useful Fc substitutions that can be made to alter binding to one or more of the FcyR receptors. In certain embodiments, the subject antibody includes modifications that alter the binding to one or more FcyR receptors (/.<?., “FcyR variants”). Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcyRIIIa generally results in increased ADCC (antibody dependent cell-mediated cytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell). Similarly, decreased binding to FcyRIIb (an inhibitory receptor) can be beneficial as well in some circumstances. Amino acid substitutions that find use in the antibodies described herein include those listed in US Patent Nos. 8,188,321 (particularly Figure 41) and 8,084,582, and US Publ. App. Nos.
20060235208 and 20070148170. Particular variants that find use include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D/332E/330L, 243A, 243L, 264 A, 264V and 299T.
[0099] In addition, there are additional Fc substitutions that find use in increased binding to the FcRn receptor and increased serum half-life, as specifically disclosed in USSN 12/341,769 including, but not limited to, 434S, 434 A, 428L, 308F, 2591, 428L/434S, 259U308F, 436U428L, 4361 or V/434S, 436V/428L and 259I/308F/428L. Such modification may be included in one or both Fc domains of the subject antibody. In certain embodiments, the anti-tumor antibodies comprising Fc substitutions that have increased binding to the FcRn receptor and increased serum half-life have an increased half-life that is about 10,000-fold, 1,000-fold, 500-fold, 100-fold, 50-fold, 20-fold, 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4.5-fold, 4-fold, 3.5-fold, 3-fold, 2.5-fold, 2-fold, 1.95-fold, 1.9-fold, 1.85-fold, 1.8-fold, 1.75-fold, 1.7-fold, 1.65-fold, 1.6-fold, 1.55-fold, 1.50-fold, 1.45-fold, 1.4-fold, 1.35-fold, 1.3-fold, 1.25-fold, 1.2-fold, 1.15-fold, 1.1-fold, or 1.05-fold longer compared to a binding protein comprising a wild-type Fc region.
[0100] In some embodiments, an anti-tumor antibody disclosed herein, including antibody fragments, of the present disclosure comprises an Fc region that has effector function, e.g., exhibits antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc region may be an Fc region engineered to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or ADCC. Accordingly, an Fc region can comprise additional mutations to increase or decrease effector functions, i.e., the ability to induce certain biological functions upon binding to an Fc receptor expressed on an immune cell. Immune cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans’ cells, natural killer (NK) cells, and cytotoxic T cells.
[0101] In some embodiments, an Fc region described herein can include additional modifications that modulate effector function. Examples of Fc region amino acid mutations that modulate an effector function include, but are not limited to, one or more substitutions at positions 228, 233, 234, 235, 236, 237, 238, 239, 243, 265, 269, 270, 297, 298, 318, 326, 327 , 329, 330, 331, 332, 333, and 334 (EU numbering scheme) of an Fc region.
[0102] Illustrative substitutions that decrease effector functions include the following: position 329 may have a mutation in which proline is substituted with a glycine or arginine or an amino acid residue large enough to destroy the Fc/Fcy receptor interface that is formed between proline 329 of the Fc and tryptophan residues Trp 87 and Trp 110 of FcyRIII. Additional illustrative substitutions that decrease effector functions include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may also be present, e.g., L234A and L235A of a human IgGl Fc region; L234A, L235A, and P329G of a human IgGl Fc region; S228P and L235E of a human IgG4 Fc region; L234A and G237A of a human IgGl Fc region; L234A, L235A, and G237A of a human IgGl Fc region; V234A and G237A of a human IgG2 Fc region; L235A, G237A, and E318A of a human IgG4 Fc region; and S228P and L236E of a human IgG4 Fc region, to decrease effectors functions. Examples of substitutions that increase effector functions include, e.g., E333A, K326W/E333S, S239D/I332E/G236A, S239D/A330L/I332E, G236A/S239D/A330L/I332E, F243L, G236A, and S298A/E333A/K334A. In some embodiments, the Fc mutations include P329G, L234A, L235A, or a combination thereof. Descriptions of amino acid mutations in an Fc region that can increase or decrease effector functions can be found in, e.g., Wang et al., Protein Cell. 9(1): 63-73, 2018; Saunders, Front Immunol. Jun 7, eCollection, 2019; Kellner et al., Transfus Med Hemother. 44(5): 327-336, 2017; and Lo et al., J Biol Chem. 292(9):3900- 3908, 2017. [0103] In some embodiments, an Fc region may have one or more amino acid substitutions that modulate ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region, according to the EU numbering scheme. Specifically, S298A, E333A, and K334A can be introduced to an Fc region to increase the affinity of the Fc region to FcyRIIIa and decrease the affinity of the Fc region to FcyRIIa and FcyRIIb.
[0104] An Fc region can also comprise additional mutations to increase serum half-life. Through enhanced binding to the neonatal Fc receptor (FcRn), such mutations in an Fc region can improve the pharmacokinetics of the antibody. Examples of substitutions in an Fc region that increase the serum half-life of an antibody include, e.g., M252Y/S254T/T256E, T250Q/M428L, N434A, N434H, T307A/E380A/N434A, M428L/N434S, M252Y/M428L, D259I/V308F, N434S, V308W, V308Y, and V3O8F. Descriptions of amino acid mutations in an Fc region that can increase the serum half-life of an antibody can be found in, e.g., Dumet et al., MAbs. 26:1-10, 2019; Booth et al., MAbs. 10(7): 1098-1110, 2018; and Dall’Acqua et al., J Biol Chem. 281(33):23514-24, 2006.
[0105] In some embodiments, an anti-tumor antibody described herein comprise an Fc region having altered glycosylation that increases the ability of the antibody to recruit NK cells and/or increase ADCC. In some embodiments, the Fc region comprises glycan containing no fucose (i.e. , the Fc region is afucosylated). Afucosylated antibodies can be produced using cell lines that express a heterologous enzyme that depletes the fucose pool inside the cell (e.g., GlymaxX® by ProBioGen AG, Berlin, Germany). Non-fucosylated antibodies can also be produced using a host cell line in which the endogenous a-1,6- fucosyltransferase (FUT8) gene is deleted. See Satoh, M. et al., “Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies,” Expert Opinion on Biological Therapy, 6:11, 1161-1173, DOI: 10.1517/14712598.6.11.1161.
[0106] Furthermore, in some embodiments, an antibody of the disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified, e.g., produced in cell lines and/or in cell culture conditions to alter its glycosylation (e.g., hypofucosylation, afucosylation, or increased sialylation), to alter one or more functional properties of the antibody. For example, the antibody can be linked to one of a variety of polymers, for example, polyethylene glycol. In some embodiments, an antibody may comprise mutations to facilitate linkage to a chemical moiety and/or to alter residues that are subject to post-translational modifications, e.g., glycosylation. ACTIVITY
Tumor-binding activity
[0107] The activity of the anti-tumor antibodies as described herein can be assessed for binding in binding assays. Nonlimiting examples of suitable assays include surface plasmon resonance analysis using a biosensor system such as a Biacore® system or a flow cytometry assay, which are further described in the EXAMPLES section.
[0108] In some embodiments, binding to glycans protein is assessed in a competitive assay format with a reference antibody AB-006410 or a reference antibody having the variable regions of AB-006410. In some embodiments, a variant anti-tumor antibody in accordance with the present disclosure may block binding of the reference antibody in a competition assay by about 50% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 80% or more, or about 90% or more.
[0109] In some embodiments, binding assays to assess variant activity are performed on tumor tissues or tumor cells ex vivo, e.g., on tumor cells that were grown as a tumor graft in a syngeneic (immune-matched) mouse in vivo then harvested and processed within 24-48 hrs. Binding can be assessed by any number of means including flow cytometry and immunohistochemistry or immunofluorescence-based assays.
[0110] In some embodiments the antibody is added to a cancer cell line and the binding is analyzed by flow cytometry. In one illustrative example, AB-006410 was shown to bind A549 cells and the binding of AB-006410 diminished in A549 cells in which one or more selected glycosyltransferase have been knocked out. In some embodiments, the glycosyltransferase is B4GALNT3 or FUT9. In some embodiments, the glycosyltransferase is B4GALNT3 and FUT9.
[0111] In some embodiments, the binding of the antibodies to bind to tumor cells are assessed by immunofluorescence methods, as described in the EXAMPLES. The anti-tumor antibodies preferentially bind to various tumors but not to normal human tissues. In one illustrative example, the AB-006410 showed preferential binding to ovarian, lung, pancreatic, and esophageal cancer tissues relative to the respective tumor adjacent tissues (TATs).
[0112] In some embodiments, the binding activity of the antibodies is assessed by determining ECso values, and in some embodiments additionally determining delta activity, i.e. , the difference in specific activity between lower and upper plateaus of the activation curve expressed as percent of activity of a selected antibody having known in vitro activity. In typical embodiments, ECso values are compared to a reference antibody. For purposes of this disclosure, an antibody comprising the VH and VL regions of an anti-tumor antibody disclosed herein and a mouse IgG2a Fc region when testing ex vivo binding using a mouse tumor model, is employed as a reference antibody and included in an assay to assess variant activity relative to the reference antibody. The fold over EC50 is calculated by dividing the EC 50 of the reference antibody by the EC50 of the test antibody. Based on the resulting values, the antibodies were assigned to groups and given a ranking from 0-4 as follows: 0= (>500 nM); 1 = <0.5 pM; 2 = 0.5 to 2 pM; 3 = 2 to 4 pM; 4 = >4 pM.
Fc effector function
[0113] In some embodiments, an anti-tumor antibody of the present disclosure comprises an Fc region that has effector function. Examples of effector functions include, but are not limited to, Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding (e.g., FcyR binding), ADCC, antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptor), and B-cell activation. Effector functions may vary with the antibody class. For example, native human IgGl and lgG3 antibodies can elicit ADCC and CDC activities upon binding to an appropriate Fc receptor present on an immune system cell; and native human IgGl, IgG2, IgG3, and IgG4 can elicit ADCP functions upon binding to the appropriate Fc receptor present on an immune cell.
[0114] In some embodiments, the Fc region of an anti-tumor antibody disclosed herein may be an Fc region engineered to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or ADCC. Accordingly, an Fc region can comprise additional mutations to increase or decrease effector functions, i.e., the ability to induce certain biological functions upon binding to an Fc receptor expressed on an immune cell. Immune cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans’ cells, natural killer (NK) cells, and cytotoxic T cells. In some embodiments, an antibody of the present disclosure has enhanced ADCC and/or serum stability compared to antibody AB-006410 when the antibody is assayed in a human IgGl isotype format.
[0115] The anti-tumor antibodies of the present disclosure may be evaluated in various assays for their ability to mediate FcR-dependent activity. In one assay, the binding activity of an anti-tumor antibody is evaluated in an Fc receptor engagement assay. For purposes of testing variants, “engagement” of an Fc receptor occurs when a variant antibody binds to both a target tumor cell via its Fv region and an FcyR present on an immune cell via the antibody Fc region in such as manner to transduce a signal. If the Fc region is kept constant among variants that differ in their Fv regions, then the assay allows an evaluation of tumor binding activity across such variants in the context of potential signal transduction through a particular Fc region binding a particular Fc receptor. In some embodiments, binding of the antibody Fc region can result in clustering and/or internalization of the FcR, resulting in a luminescence signal in cells harboring a NFAT-RE-Lucif erase reporter construct.
ADCC activity
[0116] In some embodiments, an anti-tumor antibody of the present disclosure has ADCC when the antibodies are assayed in a mouse IgG2a isotype format.
ADCP activity
[0117] In some embodiments, ADCP activity of an anti-tumor antibody is assessed using fluorescently labeled, in vitro cultured tumor cells and Raw264.7 murine macrophages. In certain embodiments, opsonization of the tumor cell by the antibody leads to phagocytosis detected by flow cytometry. Variations of this assay have been described and can include co-labeling of tumor and effector cells or assessment of phagocytosis through FcyRIIa engagement (e.g., FcyRIIa-H ADCP Reporter Bioassay from Promega).
ADC activity
[0118] An anti-tumor antibody is deemed to have ADC activity if, when the antibody is conjugated to a drug molecule (toxin) to form an antibody drug conjugate (ADC), said ADC can kill target cells. In some embodiments, the antibody is deemed to have ADC activity if the ECso of the assay measuring the cell killing activity of the ADC is less than 1 x 10'8. In one exemplary assay, the ADC activity of an anti-tumor antibody is evaluated using a drug- conjugated secondary antibody. The antibody-drug conjugate assay involves tumor target cells, primary antibodies of interest (the antibody to be tested), and a secondary antibody that is conjugated to a drug molecule, where the secondary antibody recognizes the primary antibody. Briefly, primary antibody dilutions are incubated with target cells at room temperature for a first period (for example, 10-30 minutes). The drug-conjugated secondary antibody is then added to the incubation mixture containing the target cells and the primary antibody. The mixture is then incubated for second period before measuring the extent of target cell lysis. In some embodiments, the assay generates a 100% cell lysis value by adding cell lysis buffer directly to target cell sample, which are not treated by the primary or drug-conjugated second antibody, and cell killing data from samples treated the antibody mixtures as disclosed above can be normalized to the value of 100% cell lysis. The results of the assay can be used to predict whether an ADC produced by conjugating a particular antibody and the drug molecule can kill target cells.
In vivo activity
[0119] In some embodiments, activity of an anti-tumor antibody is evaluated in vivo in a suitable animal tumor model. A reduction in tumor load of a subject treated with a test article relative to the tumor load of a subject treated with a control article reflects the antitumor function of an antibody. An anti-tumor antibody, or anti-tumor antibody immunoconjugate, disclosed herein can reduce tumor load of a subject by at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60%, or at least 70%, or greater relative to the tumor load of a control subject.
[0120] In some embodiments, a variant of an antibody as described herein has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or greater, of the antitumor activity of a reference antibody as shown in Tables 1-3 when evaluated under the same assay conditions to measure the anti-tumor activity in vivo. In some embodiments, an anti-tumor antibody exhibits improved activity, i.e., greater than 100% activity, compared to the reference antibody.
ANTIBODY FORMATS
[0121] In some embodiments, an anti-tumor antibody in accordance with the present disclosure is in a monovalent format. In some embodiments, the tumor-targeting antibody is in a fragment format, e.g., a Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment. In some embodiments, of the disclosure, an anti-tumor antibody in accordance with the disclosure may be an antibody fragment, e.g., a Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment. In another embodiment, the antibody is a substantially full-length antibody, e.g., an IgG antibody or other antibody class or isotype as defined herein. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.
[0122] In some embodiments, an anti-tumor antibody according to the present disclosure that is administered to a patient is an IgG of the IgGl subclass. In some embodiments, such an antibody is an IgG of the IgG2, IgG3, or IgG4 subclass. In some embodiments, such an antibody is an IgM. In some embodiments, such an antibody has a lambda light chain constant region. In some embodiments, such an antibody has a kappa light chain constant region.
[0123] In some embodiments, an anti-tumor antibody of the present disclosure is employed in a bispecific or multi- specific format, e.g., a tri-specific format. For example, in some embodiments, the antibody may be incorporated into a bispecific or multi-specific antibody that comprises a further binding domain that binds to the same or a different antigen.
[0124] There are a variety of possible formats that can be used in bispecific or multi -specific antibodies. The formats can vary elements such as the number of binding arms, the format of each binding arm (e.g., Fab, scFv, scFab, or Vu-only), the number of antigen binding domains present on the binding arms, the connectivity and geometry of each arm with respect to each other, the presence or absence of an Fc domain, the Ig class (e.g. , IgG or IgM), the Fc subclass (e.g., hlgGl, hIgG2, or hIgG4), and any mutations to the Fc (e.g., mutations to reduce or increase effector function or extend serum half-life).. Also see Speiss, et al. , Alternative Molecular Formats and Therapeutic Applications for Bispecific Antibodies, Mol Immunol, 67, 95-106 (2015), particularly FIG. 1, for examples of bispecific and multispecific formats.
[0125] In some embodiments of any of the above bispecific or multispecific antibody constructs, the tumor-targeting binding domain comprises all six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) sequences from the individual antibodies disclosed in Tables 1 and 2.
[0126] In some embodiments of any of the above bispecific or multispecific antibody constructs, the tumor-targeting binding domain comprises the VH and VL sequences from the individual antibodies disclosed in Tables 3.
[0127] In some embodiments of any of the above bispecific or multispecific antibody constructs, the tumor-targeting binding domain comprises all six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) sequences from any one of antibodies AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921 , AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213.
[0128] In some embodiments of any of the above bispecific or multispecific antibody constructs, the tumor-targeting binding domain comprises the Vu and VL sequences of AB- 012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB- 012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB- 012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB- 012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB- 012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB- 012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB- 012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB- 012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB- 012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB- 012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB- 012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB- 012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB- 012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB- 012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB- 012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB- 012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB- 012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB- 012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB- 012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB- 013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB- 013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB- 013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213.
[0129] In some embodiments, an anti-tumor antibody disclosed herein is constructed as a multivalent antibody. In some embodiments an anti-tumor antibody is constructed as a tetravalent molecule, comprising four glycan-binding arms per molecule. Such constructs exhibit increased ADCC activity, as well as increased binding to tumor cells a measured by flow cytometry.
[0130] In some embodiments, an anti-tumor antibody of the present disclosure is employed in a bispecific or multi-specific format, e.g., a tri-specific format. For example, in some embodiments, the antibody may be incorporated into a bispecific or multi-specific antibody that comprises a further binding domain that binds to the same or a different antigen.
[0131] There are a variety of possible formats that can be used in bispecific or multi-specific antibodies. The formats can vary elements such as the number of binding arms, the format of each binding arm (e.g., Fab, scFv, scFab, or Vn-only), the number of antigen binding domains present on the binding arms, the connectivity and geometry of each arm with respect to each other, the presence or absence of an Fc domain, the Ig class (e.g. , IgG or IgM), the Fc subclass (e.g., hlgGl, hIgG2, or hIgG4), and any mutations to the Fc (e.g., mutations to reduce or increase effector function or extend serum half-life).
[0132] Illustrative antigens that can be targeted by a further binding domain in a bispecific or multi- specific antibody that comprises an antigen binding domain of an anti-tumor antibody described herein, include, but are not limited to, antigens on T cells to enhance T cell engagement and/or activate T cells. Illustrative examples of such an antigen include, but are not limited to, CD3, CD2, CD4, CD5, CD6, CD8, CD28, CD40L, CD44, IL-15Ra, CD122, CD132, or CD25. In some embodiments, the antigen is CD3. In some embodiments, the antigen is in a T cell activating pathway, such as a 4-1BB/CD137, 4-1BBL/CD137L, 0X40, OX40L, GITRL, GITR, CD27, CD70, CD28, ICOS, HVEM, or LIGHT antigen.
Multi-specific formats that bind to CD3
[0133] In some embodiments, an anti-tumor antibody is incorporated into a bispecific or multi- specific antibody that comprises a binding domain that binds to a T-cell antigen. These bispecific antibodies or multi-specific antibodies can direct T cells to attach and lyse targeted tumor cells, i.e., the glycan expressing tumor cells. In some embodiments, the bispecific or multispecific antibody comprises a binding domain that binds to CD3. In some embodiments, the bispecific or multispecific antibody comprises a binding domain that binds to human CD3 comprising the anti-tumor antibodies described herein.
[0134] As will be appreciated by those in the art, any collection of anti-CD3 CDRs, anti- CD3 variable light and variable heavy domains, Fabs and scFvs as depicted in any of the Figures can be used. Similarly, any of the anti-glycans antigen binding domains can be used, whether CDRs, variable light and variable heavy domains, Fabs and scFvs, can be used, optionally and independently combined in any combination.
Multi-specific formats that bind to 4- IBB
[0135] In some embodiments, an anti-tumor antibody is incorporated into a multi- specific antibody that comprises a binding domain from an agonist antibody that binds to 4-1BB. In some embodiments, the 4- IBB agonist antibody is a bispecific antibody that is capable of binding to both glycans and 4- IBB. For purposes of this application, the term “4- IBB engager,” refers to the portion of a molecule (e.g. , a bispecific antibody capable of binding to both 4-1BB and glycans) that binds to 4-1 BB. In some embodiments, the 4-1BB engager is an antibody or an antibody fragment e.g. , scFv) that binds to 4- IBB. In some embodiments, the 4- IBB engager is a multimeric 4- IBB ligand (“4-1BBL”), for example, a 4-1BBL trimer. In some embodiments, as further described below, the bispecific antibody comprises one or more scFv fragments of an anti-4- IBB antibody and an anti-tumor antibody disclosed herein. In some embodiments, the 4- IBB agonist antibody is a trispecific antibody.
[0136] In some embodiments, the fusion molecule comprises a silenced human IgGl with three human 4- IBB ligand ectodomains attached via flexible linkers. In some embodiments, an anti-tumor antibody is incorporated into a fusion molecule comprising one or more 4- 1BB ligands (4-1 BBL). In some embodiments, a trimer of 4-1 BBL is fused to the C-terminal of either the light chain or heavy chain of an anti-tumor antibody. In some embodiments, one or more individual 4-1 BBL domains are connected via linkers, with one of the domains additionally fused to the anti-tumor antibodies via a linker. The 4-1 BBL domains comprise the entire ECD portion of the molecule or truncated forms that can still bind and activate 4- 1BB. GENERATION OF ANTIBODIES
[0137] The anti-tumor antibodies can be produced using vectors and recombinant methodology well known in the art. Reagents, cloning vectors, and kits for genetic manipulation are available from commercial vendors. Accordingly, in some embodiments of the disclosure, provided herein are isolated nucleic acids encoding a VH and/or VL region, or fragment thereof, of any of the tumor-targeting antibodies as described herein; vectors comprising such nucleic acids and host cells into which the nucleic acids are introduced that are used to replicate the antibody-encoding nucleic acids and/or to express the antibodies. Such nucleic acids may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of the tumor-targeting antibody (e.g., the light and/or heavy chains of the antibody). In some embodiments, the host cell contains (1) a vector containing a polynucleotide that encodes the VL amino acid sequence and a polynucleotide that encodes the VH amino acid sequence, or (2) a first vector containing a polynucleotide that encodes the VL amino acid sequence and a second vector containing a polynucleotide that encodes the VH amino acid sequence.
[0138] In some embodiments, the disclosure provides a method of making an anti-tumor antibody as described herein. In some embodiments, the method includes culturing a host cell as described in the preceding paragraph under conditions suitable for expression of the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).
[0139] Suitable vectors containing polynucleotides encoding antibodies of the present disclosure, or fragments thereof, include cloning vectors and expression vectors. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally can self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g. , pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl plasmids, pCRl, RP4, phage DNAs, and shuttle vectors. These and many other cloning vectors are available from commercial vendors.
[0140] Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure. The expression vector can be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids and viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and any other vector. [0141] Suitable host cells for expressing an anti-tumor antibody as described herein include both prokaryotic and eukaryotic cells. For example, an anti-tumor antibody may be produced in bacteria when glycosylation and Fc effector function are not needed. After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. Alternatively, the host cell may be a eukaryotic host cell, including eukaryotic microorganisms, such as filamentous fungi or yeast, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern, vertebrate, invertebrate, and plant cells. Examples of invertebrate cells include insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells. Plant cell cultures can also be utilized as host cells.
[0142] In some embodiments, vertebrate host cells are used for producing an anti-tumor antibody of the present disclosure. For example, mammalian cell lines such as a monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59,1977; baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251, 1980 monkey kidney cells (CV 1); African green monkey kidney cells (VERO- 76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g. , in Mather et al. , Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC 5 cells; and FS4 cells may be used to express an tumor-targeting antibodies. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); and myeloma cell lines such as Y0, NS0 and Sp2/0. Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268, 2003.
[0143] In some embodiments, an anti-tumor antibody of the present disclosure is produced by a CHO cell line, e.g., the CHO-K1 cell line. One or more expression plasmids can be introduced that encode heavy and light chain sequences. For example, an expression plasmid encoding a heavy chain disclosed herein, and an expression plasmid encoding a light chain disclosed herein are transfected into host cells. The expression plasmids can be introduced as linearized plasmids at a ratio of 1:1 in the CHO-K1 host cell line using reagents such as Freestyle Max reagent. Fluorescence- activated cell sorting (FACS) coupled with single cell imaging can be used as a cloning method to obtain a production cell line.
[0144] A host cell transfected with an expression vector encoding an anti-tumor antibody of the present disclosure, or fragment thereof, can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptides may be secreted and isolated from a mixture of cells and medium containing the polypeptides. Alternatively, the polypeptide may be retained in the cytoplasm or in a membrane fraction and the cells harvested, lysed, and the polypeptide isolated using a desired method.
[0145] In some embodiments, an anti-tumor antibody of the present disclosure can be produced by in vitro synthesis (see, e.g., Sutro Biopharma biochemical protein synthesis platform).
[0146] In some embodiments, provided herein is a method of generating variants of an antitumor antibody as disclosed herein. Thus, for example, a construct encoding a variant of a VH CDR3 as described herein can be modified and the VH region encoded by the modified construct can be tested for binding activity to LoVo cells and/or in vivo tumor-targeting activity in the context of a VH region as described herein, that is paired with a VL region or variant region as described herein. Similarly, a construct encoding a variant of a VL CDR3 as described herein can be modified and the VL region encoded by the modified construct can be tested for binding to CT26 cells, or other tumor cells, and/or in vivo tumor- targeting activity efficacy. Such an analysis can also be performed with other CDRs or framework regions and an antibody having the desired activity can then be selected.
TUMOR-TARGETING ANTIBODY CONJUGATES/ CO-STIMULATORY AGENTS
[0147] In some embodiments, an anti-tumor antibody disclosed herein may be conjugated or linked to therapeutic, imaging/detectable moieties, or enzymes. For example, the anti-tumor antibody may be conjugated to a detectable marker, a cytotoxic agent, an immunomodulating agent, an imaging agent, a therapeutic agent, an oligonucleotide, or an enzyme. Methods for conjugating or linking antibodies to a desired molecule are well known in the art. The moiety may be linked to the antibody covalently or by non-covalent linkages. [0148] In some embodiments, the antibody is conjugated, either directly or via a cleavable or non-cleavable linker or spacer, to a cytotoxic moiety or other moiety that exerts their effects on critical cellular processes required for survival (“payload”) to form an antibodydrug conjugate (“ADC”). [0149] In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, the linker is an enzymatic cleavable linker. In some embodiments, the linker is a pH-sensitive linker. In some embodiments, the linker is a reducible linker (e.g., sulfo-SPDB).
[0150] In some embodiments, the linker is cleaved in response to changes in pH or redox potential. In some embodiments, the linker is cleaved when contacted with lysosomal enzymes.
[0151] In some embodiments, the linker is selected from the group consisting of Maleimidocaproyl (MC), Maleimidocaproyl-Valine-Citrulline- p-amino-benzyloxy carbonyl (MC-VC-PAB), Maleimidomethyl cyclohexane- 1 -carboxylate (SMCC), N-succinimidyl-4- (2-pyridyldithio)butanoate (SPDB) and Lys-P AB-CO (Lysine- p-aminobenzyl -C=O). Each possibility represents a separate embodiment of the disclosure.
[0152] In some embodiments, the anti-tumor antibody is conjugated to an auristatin to form an ADC. In some embodiments, the ADC comprises an anti-tumor antibody conjugated to a ZymeLink™ Auristatin (ZLA) payload.
[0153] In some embodiments, an ADC of the present disclosure is conjugated to a microtubule inhibitor that induces apoptosis in cells undergoing mitosis by, for example, causing cell cycle arrest at G2/M. Nonlimiting examples of microtubule inhibitors that can be used include maytansine derivatives (DM1/DM4), or auristatins (monomethyl auristatin E (MMAE)/ monomethyl auristatin F (MMAF)) and variants thereof, such as monomethyl auristatin D, PF-06380101, duostatin5, AS269, Tapl8Hrl, AGD-0182, HPA-Auristatin F. In some embodiments, the payload is a tubulin-targeting agent, for example, hemiasterlin, tubulysin, or eribulin. In some embodiments, the payloads are DNA-damaging payloads, which include enediynes (calicheamicin), duocarmycin derivatives, pyrrolobenzodiazepine dimers (PBD dimers), and indolinobenzodiazepine pseudo-dimers.
[0154] In some embodiments, the antibody is conjugated to a cytotoxic agent including, but not limited to, e.g., ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, methotrexact, actinomycin, a diphtheria toxin, extotoxin A from Pseudomonas, Pseudomonas exotoxin 40, abrin, abrin A chain, modeccin A chain, alpha sarcin, gelonin, mitogellin, restrictocin, cobran venom factor, a ribonuclease, engineered Shiga toxin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolastatin, ccl065, or a cisplatin. In some embodiments, the antibody may be linked to an agent such as an enzyme inhibitor, a proliferation inhibitor, a lytic agent, a DNA or RNA synthesis inhibitors, a membrane permeability modifier, a DNA metabolite, a dichloroethylsulfide derivative, a protein production inhibitor, a ribosome inhibitor, or an inducer of apoptosis. In some embodiments, the antibody is conjugated to a drug such as a topoisomeriase Inhibitor, e.g., a topoisomeraise I inhibitor. Topoisomeraise I inhibitors include but are not limited to quinoline alkaloids (e.g., SN-38, DXd).
[0155] In some embodiments, the antibody is conjugated to one or more of the cytotoxic and/or anti-mitotic compounds.
[0156] In some embodiments, an anti-tumor antibody as described herein is joined to a molecule that facilitates transport of the antibody across a biological membrane, e.g. , by enhancing penetration of the membrane, facilitating protein translocation across membranes. Thus, for example, the antibody may be linked to a cell penetration agent, such as a cellpenetrating peptide. Examples of cell penetrating peptides include TAT, penetrating, polyarginine molecules, Kunitz domain-derived peptides, e.g., angiopep-2, SynB, buforin, transportan, amphiphathic peptides and others. In some embodiments, the antibody may be conjugated with a cationic molecule such as a polyamine. In some embodiments, the antibody may be conjugated to an agent that facilitates transport across the blood brain barrier, e.g., transcytosis. Thus, for example, the antibody may be conjugated to an agent that binds to endothelial cell receptors that are internalized, e.g., transferrin receptor, insulin receptor, insulin-like growth factor receptor, or a low-density lipoprotein receptor, and the like. In some embodiments, the antibody may be conjugated to a toxin facilitating entry of the antibody into the cytoplasm, e.g., Shiga toxin. In some embodiments, an anti-tumor antibody as described herein can be conjugated to an engineered toxin body (ETBs) to facilitate internalization of the antibody into a cell.
[0157] In some embodiments, an anti-tumor antibody described herein is conjugated or administered with a polypeptide immunomodulating agent, e.g., an adjuvant. Examples of immunomodulating agents include, but are not limited to, cytokines e.g., transforming growth factor- p (TGF0)), growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL- 15, an IL-15/IL-15Ra, e.g., sushi domain, complex, IL- 18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF), interferons (e.g., interferon-a, -0 or -y, erythropoietin and thrombopoietin, or a combination thereof. In some embodiments, the antibody is linked or administered with a compound that stimulates the innate immune system, such as an adjuvant, a Toll-like receptor (TLR) agonist, a C-type lectin receptor (CLR) agonist, a retinoic acid-inducible gene I-like receptor (RLR) agonist, a saponin, a polysaccharide such as chitin, chitosan, P-glucan, an ISCOM, QS-21, a stimulator of interferon genes (STING) agonist, or another immunopotentiating agent.
[0158] In some embodiments, an anti-tumor antibody described herein is conjugated to or administered with an IL- 15 receptor agonist, such as an IL- 15 fusion construct, an IL-15:IL- 15Ra fusion construct or a single-chain IL-15:IL-15Ra (sushi) fusion construct. In some embodiments, the tumor-targeting antibody conjugated to an IL- 15 receptor agonist is a bispecific or multispecific antibody. In some embodiments, the antibody is a bispecific or multispecific antibody comprising an antigen binding domain described herein that further comprises an IL- 15 receptor agonist.
[0159] In some embodiments, an anti-tumor antibody described herein is administered with a single-chain IL-15:IL-15Ra (sushi) fusion construct. In some embodiments, an anti-tumor antibody is administered with a polymer-conjugated IL-15 construct, such as NKTR-255.
[0160] The !L-15:lL-15Ra single chain constructs can be administered to a subject in a therapeutically effective dose, for example, a dose in a range from less than about 0.01 mg/kg body weight to about 25 mg/kg body weight, for example, 0.1 - 10 mg/kg body weight. In some embodiments, the dose is about 0.1 to about 25 mg/kg, about 1 to about 20 mg/kg, about 2 to about 15 mg/kg, about 3 to about 10 mg/kg, about 4 to about 5 mg/kg body weight, In some embodiments, the constructs can be administered in a dose of approximately 1 mg - 2 g per patient, or approximately 50 mg - 1000 mg per patient, or approximately 100 mg - 500 mg per patient.
[0161] In some embodiments, the single-chain IL- 15 fusion construct comprises IL- 15 joined to IL-15Ra (sushi) with a polypeptide linker. In some embodiments, the single-chain IL- 15 fusion construct is joined via a polypeptide linker to another protein, such as an Fc for long half-life. See, for example, FIG. 9B in W02018071919A1 (corresponding to U.S.
Patent No. 10550185). In some embodiments, the IL- 15 is joined or fused to the N-terminus of the heavy chain of an Fc, and IL- 15Ra( sushi) is joined or fused to the other Fc heavy chain N-terminus, using a heavy chain heterodimerization technology to form the desired hybrid Fc.
[0162] In some embodiments, the IL-15:IL-15Ra (sushi) single chain constructs are fused to the C-terminus of an antibody light chain, or the C-terminus of an antibody heavy chain, in both cases producing a molecule with two tumor-targeting binding sites (the Fab arms), and two IL15:IL15Ra units. In another embodiment, one copy of an IL 15 :IL 15 Rot fusion construct is fused to an anti-tumor antibody, thereby producing an antibody molecule comprising two tumor-targeting binding sites (the Fab arms) and only one ILI 5:ILI 5Rcx unit, for example using a knob-in-holes approach to heavy chain heterodimerization, or other heterodimerization technology.
[0163] In some embodiments, the IL-15:IL-15Ra (sushi) fusion constructs or the antibodies comprising the fusion constructs comprise a low affinity IL- 15 variant having improved pharmacokinetics (PK). In some embodiments, the IL-15:IL-15Ra (sushi) fusion constructs comprise a high affinity IL- 15 variant having increased agonist activity. In some embodiments, the high affinity IL- 15 variant has an N72D mutation. In some embodiments, the high affinity variant is fused to a dimeric IL-15Ra sushi domain-IgGl Fc fusion protein. In some embodiments, the IL-15:IL-15Ra (sushi) fusion construct is ALT- 803.
[0164] In some embodiments, antibodies comprising the IL15:IL15Ra fusion construct comprise one or more mutations in the Fc region described herein, for example E333A, K326W/E333S, S239D/I332E/G236A, S239D/A330L/I332E, G236A/S239D/A330L/I332E, F243L, G236A, and S298A/E333A/K334A. In some embodiments, antibodies comprising the IL15:IL15Ra fusion comprise one or more mutations in the Fc region that increase binding of the antibody to tumor cells, for example the mutations P329G, L234A, L235A, or a combination thereof.
[0165] In some embodiments, an anti-tumor antibody described herein is conjugated to or administered with an IL-2 receptor agonist. In some embodiments, the tumor-targeting antibody conjugated to an IL-2 receptor agonist is a bispecific or multispecific antibody. In some embodiments, the antibody is a bispecific or multispecific antibody comprising an antigen binding domain of an antibody described herein (e.g., AB-006410) that further comprises an IL-2 receptor agonist. In some embodiments, the IL-2 receptor agonist is pegylated IL-2.
[0166] In some embodiments, an anti-tumor antibody described herein is conjugated to or administered with a construct that can act as a trap for transforming growth factor-^ (TGFp). In some embodiments, the TGFp trap comprises the extracellular domain (ECD) of TGFp. In some embodiments, the TGFP trap comprises the extracellular domain (ECD) of TGFPRII. In some embodiments, the TGF trap is in the form of a bispecific antibody. The TGF RII ECD can preferably trap TGFP 1, and its low affinity to TGFp2 may mitigate potential cardiac toxicity.
[0167] In some embodiments, an anti-tumor antibody described herein comprises an extracellular domain (ECD) of the TGFP Receptor fused to the C-terminus of the heavy chain or to the C-terminus of the light chain. In some embodiments, the TGFP trap is a single trap construct. In some embodiments, the single TGFP trap is a bispecific tumortargeting TGF trap comprising a TGFp RII ECD fused to any one of the antibodies disclosed herein via a flexible linker to the C-terminus of the heavy chain or to the C- terminus of the light chain.
[0168] In some embodiments, the TGFP trap is a tandem trap construct. In some embodiments, the tandem TGFP trap comprises an IgG fused to two TGF RII ECDs. In some embodiments, the tandem TGFP trap comprises two TGFP2RII ECDs. In some embodiments, the two TGFP2RII ECDs are fused in series and are linked by a short linker (for example LIO or L25). In some embodiments, the two TGFP2RII ECDs are fused directly in series without a linker (L0). In some embodiments, the tandem TGFPRII ECDs are fused to the C-terminus of the heavy chain (HC-Cter), and the heavy chains were designed as an asymmetric pair such that the tandem-Trap is on only one heavy chain. In some embodiments, the asymmetric pair of heavy chains comprise knob-in-hole mutation that promote pairing of the heavy chains. For example, in some embodiments, one heavy chain comprises the amino acid substitutions T366S+L368A+Y407V (and optionally Y349C), and the other heavy chain comprises the amino acid substitution T336W (and optionally S354C). In some embodiments, the asymmetric single heavy chain C-ter fusion improves steric access of the Fc region to Fc gamma receptors and thereby improve function.
[0169] In some embodiments, the tandem TGFP trap is fused to the C-terminus of the light chain (LC-Cter), such that both light chains comprise two TGFPRII ECDs. In these embodiments, the net molecule exhibits twice the TGFP trapping capacity per molecule, and therefore may exhibit improved function.
[0170] In some embodiments, the bispecific TGFP trap construct comprises human variable regions. In some embodiments, the bispecific TGFP trap construct comprises a IgGl or IgG2 constant region. In some embodiments, the bispecific TGFP trap construct comprises a human IgGl constant region. In some embodiments, the bispecific TGFP trap construct comprises a mouse IgG2a constant region. In some embodiments, the variable regions of the TGFP trap construct are fused in frame to the IgG constant regions.
[0171] Binding of the TGFP trap construct can be determined using an ELISA assay, as described in the Examples. The ability of TGF trap constructs to bind to target tumor cells can be determined, for example, using flow-cytometry, as described in the Examples. The ability of TGFP trap constructs to engage and stimulate Fc-gamma Receptor in the presence of target tumor cells can be determined using a reporter bioassay, as described in the Examples. The ability of TGFP trap constructs to inhibit tumor growth can be determined, for example, in a syngeneic mouse model, as described in the Examples.
[0172] In some embodiments, the antibody may be linked to a radionuclide, an iron-related compound, a dye, a fluorescent agent, or an imaging agent. In some embodiments, an antibody may be linked to agents, such as, but not limited to, metals; metal chelators; lanthanides; lanthanide chelators; radiometals; radiometal chelators; positron-emitting nuclei; microbubbles (for ultrasound); liposomes; molecules microencapsulated in liposomes or nanosphere; monocrystalline iron oxide nanocompounds; magnetic resonance imaging contrast agents; light absorbing, reflecting and/or scattering agents; colloidal particles; fluorophores, such as near-infrared fluorophores.
TREATMENT OF CANCER
[0173] Provided herein are anti-tumor antibodies described herein, or a variant thereof, that can be used as a therapeutic agent to treat cancer. In some embodiments, the anti-tumor antibody comprises a modified Fc region comprising mutations described herein. For example, in some embodiments, the antibody comprises an Fc mutation that increases effector function selected from E333A, K326W/E333S, S239D/I332E/G236A, S239D/A330L/I332E, G236A/S239D/A330L/I332E, F243L, G236A, S298A/E333A/K334A, and P329G/L234A/L235A, or a combination thereof. In some embodiments, the antibody comprises a modified Fc region that is afucosylated. In some embodiments, the antibody is conjugated to or administered with an IL- 15 receptor agonist, a TGF trap, a TLR agonist, or an agonist anti-4- IBB antibody. In some embodiments, the antibody is a bispecific or multispecific antibody described herein.
[0174] In some embodiments, the disclosure additionally provides methods of identifying subjects who are candidates for treatment with an anti-tumor antibody having tumortargeting effects. Thus, in some embodiments, the disclosure provides a method of identifying a patient who can benefit from treatment with an anti-tumor antibody disclosed herein. In some embodiments, the patient has a tumor that expresses a tumor-associated glycan. In some embodiments, the tumor sample is from a primary tumor. In alternative embodiments, the tumor sample is a metastatic lesion. Binding of antibody to tumor cells through a binding interaction with the glycans can be measured using any assay, such as immunohistochemistry or flow cytometry. In some embodiments, binding of antibody to at least 0.2%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, or at least 50%, of the tumor cells in a sample may be used as a selection criterion for determining a patient to be treated with an anti-tumor antibody as described herein.
[0175] An anti-tumor antibody or an anti-tumor antibody immunoconjugate (e.g., an antibody- drug conjugate) disclosed herein can be used to treat several different cancers. In some embodiments, a cancer patient who can benefit from the treatment of the anti-tumor antibody or antibody immunoconjugate (e.g., an anti-tumor antibody-drug conjugate) has a cancer expressing a tumor-associated glycan. In some embodiments, the cancer is a carcinoma, a melanoma, or a sarcoma. In some embodiments, the cancer is colorectal, pancreatic, gastric, or uterine cancer. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is breast cancer, prostate cancer, testicular cancer, renal cell cancer, bladder cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, colorectal cancer, anal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, a brain cancer, e.g., glioblastoma, melanoma, or a bone or soft tissue sarcoma. In some embodiments, the cancer is acral melanoma. In some embodiments, the cancer is acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, astrocytoma, basal-cell carcinoma, bile duct cancer, bone tumor, brainstem glioma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, bronchial adenomas, Burkitt’s lymphoma, central nervous system lymphoma, cerebellar astrocytoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, epithelioid hemangioendothelioma (EHE), esophageal cancer, Ewing’s sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, a cancer of the eye, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, gastric carcinoma, hairy cell leukemia, hepatocellular carcinoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, childhood, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, leukemias, lip and oral cavity cancer, liposarcoma, liver cancer, non-small cell lung cancer, small-cell lung cancer, lymphomas, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma of bone, medulloblastoma, Merkel cell cancer, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndromes, myelogenous leukemia, myeloid leukemia, adult acute, myeloproliferative disorders, chronic, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma, supratentorial primitive neuroectodermal tumors, pituitary adenoma. Plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing sarcoma, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, melanoma,, small intestine cancer, squamous cell carcinoma, squamous neck cancer, stomach cancer, cutaneous T-Cell lymphoma, throat cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, gestational, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms tumor.
[0176] In some embodiments, the cancer is lung cancer, e.g., non-small cell lung adenocarcinoma or squamous cell carcinoma; breast cancer, e.g., Triple-, ER/PR+Her2-, ER/PR Her2+, or Triple-; colorectal cancer, e.g., adenocarcinoma, mucinous adenocarcinoma, or papillary adenocarcinoma; esophageal cancer; stomach cancer; kidney cancer, e.g., kidney clear cell cancer; ovarian cancer, e.g., ovarian endometrioid carcinoma, ovarian mucinous cystadenocarcinoma, or ovarian serous cystadenomcarcinoma; melanoma, e.g., acral melanoma, cutaneous melanoma, or mucosal melanoma; uterine or cervical cancer; liver cancer, e.g., hepatocellular carcinoma or bile duct carcinoma; bladder cancer, e.g., transitional or urothelial bladder cancer; or testicular cancer.
[0177] In some embodiments, the cancer is pancreatic adenocarcinoma, esophageal adenocarcinoma, NSCLC adenocarcinoma, or ovarian mucinous adenocarcinoma. [0178] In some embodiments, methods of the disclosure comprise administering an antitumor antibody disclosed herein, or a variant thereof, as a pharmaceutical composition to a cancer patient in a therapeutically effective amount using a dosing regimen suitable for treatment of the cancer. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the compositions for proper formulation.
[0179] The anti-tumor antibody is provided in a solution suitable for administration to the patient, such as a sterile isotonic aqueous solution for injection. The antibody is dissolved or suspended at a suitable concentration in an acceptable carrier. In some embodiments the carrier is aqueous, e.g., water, saline, phosphate buffered saline, and the like. The compositions may contain auxiliary pharmaceutical substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and the like.
ADMINISTRATION
[0180] The pharmaceutical compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the disease or symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” A therapeutically effective dose is determined by monitoring a patient’ s response to therapy. Typical benchmarks indicative of a therapeutically effective dose includes the amelioration of symptoms of the disease in the patient. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient’s health, including other factors such as age, weight, gender, administration route, and the like Single or multiple administrations of the antibody may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the methods provide a sufficient quantity of tumor-targeting antibody to effectively treat the patient. [0181] An anti-tumor antibody can be administered by any suitable means, including, for example, parenteral, intrapulmonary, and intranasal, administration, as well as local administration, such as intratumor administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibody may be administered by insufflation. In an illustrative embodiment, the antibody may be stored at 10 mg/ml in sterile isotonic aqueous saline solution for injection at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient. In some embodiments, the antibody is administered by intravenous infusion over the course of 1 hour at a dose of between 0.01 and 25 mg/kg. In some embodiments, the dose is about 0.1 to about 25 mg/kg, about 1 to about 20 mg/kg, about 2 to about 15 mg/kg, about 3 to about 10 mg/kg, about 4 to about 5 mg/kg body weight, In some embodiments, the constructs can be administered in a dose of approximately 1 mg - 2 g per patient, or approximately 50 mg - 1000 mg per patient, or approximately 100 mg - 500 mg per patient. In other embodiments, the antibody is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In some embodiments, the dose is administered over a period of about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 100 minutes, about 110 minutes, or about 120 minutes. In some embodiments the dose is administered over a period of about 0.5 hours, about 1 hour, about 1.5 hours or about 2 hours. In still other embodiments, the administration procedure is via sub-cutaneous bolus injection.
[0182] The dose of antibody is chosen to provide effective therapy for the patient and is in the range of less than 0.01 mg/kg body weight to about 25 mg/kg body weight or in the range 1 mg - 2 g per patient. Preferably the dose is in the range 0. 1 - 10 mg/kg or approximately 50 mg - 1000 mg / patient. The dose may be repeated at an appropriate frequency which may be in the range once per day to once every three months, or every six months, depending on the pharmacokinetics of the antibody (e.g., half-life of the antibody in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect of the antibody). In some embodiments, the in vivo half-life of the antibody is between about 7 and about 25 days and antibody dosing is repeated between once per week and once every 3 months or once every 6 months. In other embodiments, the antibody is administered approximately once per month.
[0183] In some embodiments, the antibody may be stored at 10 mg/ml or 20 mg/ml in a sterile isotonic aqueous solution. The solution can comprise agents such as buffering agents and stabilizing agents. For example, in some embodiments, a buffering agent such as histidine is included to maintain a formulation pH of about 5.5. Additional reagents such as sucrose or alternatives can be added to prevent aggregation and fragmentation in solution and during freezing and thawing. Agents such as polysorbate 80 or an alternative can be included to lower surface tension and stabilizes the antibody against agitation-induced denaturation and air- liquid and ice- liquid surface denaturation. In some embodiments, the solution for injection is stored at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient. COMBINATION THERAPY
[0184] An anti-tumor antibody may be administered with one or more additional therapeutic agents, e.g., radiation therapy, chemotherapeutic agents and/or immunotherapeutic agents. [0185] In some embodiments, an anti-tumor antibody can be administered in conjunction with an agent that targets an immune checkpoint antigen. In some embodiments, the agent is a biologic therapeutic or a small molecule. In some embodiments, the agent is a monoclonal antibody, a humanized antibody, a human antibody, a fusion protein, or a combination thereof. In certain embodiments, the agents inhibit, e.g., by blocking ligand binding to receptor, a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDO1, IDO2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, GITR, HAVCR2, LAG3, KIR, LAIR1 , LIGHT, MARCO, OX-40, SLAM, , 2B4, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137 (4-1BB), CD160, CD39, VISTA, TIGIT, a SIGLEC, CGEN-15049, 2B4, CHK1, CHK2, A2aR, B-7 family ligands or their receptors, or a combination thereof. In some embodiments, the agent targets PD-1, e.g., an antibody that blocks PD-L1 binding to PD-1 or otherwise inhibits PD-1. In some embodiments, the agent targets CTLA-4. In some embodiments, the agent targets LAG3. In some embodiments, the agent targets TIM3. In some embodiments, the agents target ICOS.
[0186] In some embodiments, an anti-tumor antibody can be administered in conjunction with a therapeutic antibody, such as an antibody that targets a tumor cell antigen. Examples of therapeutic antibodies include as rituximab, trastuzumab, tositumomab, ibritumomab, alemtuzumab, atezolizumab, avelumab, durvalumab, pidilizumab, AMP-224, AMP-514, PDR001, cemiplimab, BMS-936559, CK-301, epratuzumab, bevacizumab, elotuzumab, necitumumab, blinatumomab, brentuximab, cetuximab, daratumumab, denosumab, dinutuximab, gemtuzumab ibritumomab ipilimumab, nivolumab, obinutuzumab, ofatumumab, ado-trastuzumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, and ranibizumab. In some embodiments, an anti-tumor antibody can be administered in conjunction with a therapeutic antibody that binds an extracellular RNA-protein complex comprising polyadenylated RNA, such as the antibody designated ATRC-101.
[0187] In some embodiments, an anti-tumor antibody is administered with a chemotherapeutic agent. Examples of cancer chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5- fluorouracil; 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; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2’,2”-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; docetaxel, platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as bexarotene, alitretinoin; denileukin diftitox; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, mifepristone, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 1 17018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Further cancer therapeutic agents include sorafenib and other protein kinase inhibitors such as afatinib, axitinib, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, vandetanib, vemurafenib, and sunitinib; sirolimus (rapamycin), everolimus and other mTOR inhibitors. Examples of additional chemotherapeutic agents include topoisomerase I inhibitors (e.g. , irinotecan, topotecan, camptothecin and analogs or metabolites thereof, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, decarbazine, methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimetics (e.g., 5-fhiorouracil, capecitibine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea). Illustrative chemotherapeutic agents additionally include paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, and related analogs; thalidomide, lenalidomide, and related analogs (e.g., CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g. , imatinib mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-KB inhibitors, including inhibitors of IKB kinasei and other inhibitors of proteins or enzymes known to be upregulated, over-expressed or activated in cancers, the inhibition of which down regulates cell replication. Additional agents include asparaginase and a Bacillus Calmete- Guerin preparation.
[0188] In some embodiments, an anti-tumor antibody as described herein is administered after, or at the same time, as a therapeutic agent, e.g., a chemotherapeutic agent, such as doxorubicin, that induces stress granules (“SG-inducing agent”). Increasing the amount of stress granules in cancer cells can promote targeting the tumor cells by the tumor-targeting antibody. Other exemplary therapeutic agents that can induce stress granules include pyrimidine analogs (e.g., 5-FU, under trade names of Adrucil®, Carac®, Efudex®, Efudix®); protease inhibitors (e.g., Bortezomib, under the trade name of Velcade®); kinase inhibitors (e.g, Sorafenib and Imatinib, under the trade names of Nexavar® and Gleevec® , respectively); Arsenic compounds (e.g. , Arsenic trioxide, under the trade name of Trisenox®); Platinum-based compounds that induce DNA damage (e.g., Cisplatin and Oxaliplatin®, under the trade names of Platinol® and Eloxatin®, respectively); agents that disrupt microtubules (e.g., Vinblastin, under the trade name of Velban® or alkabban-AQ®; vincristin, under the trade name of Vincasar®, Marqibo®, or Oncovin®; Vinorelbin, under the trade name of Navelbin®); topoisomerase II inhibitor (e.g., Etoposide, under the trade name of Etopophos, Toposar®, VePesid®); and agents that induce DNA damage, e.g., irradiation. Several exemplary therapeutic agents that can induce stress granules formation are disclosed in Mahboubi et al., Biochimica et Biophysica Acta 1863 (2017) 884-895.
[0189] Various combinations with the anti-tumor antibody and the SG-inducing agent (or a combination of such agents) described herein may be employed to treat a cancer patient. By “combination therapy” or “in combination with”, it is not intended to imply that the therapeutic agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope described herein. The tumor- targeting antibody and the SG-inducing agent can be administered following the same or different dosing regimen. In some embodiments, the tumor-targeting antibody and the SG-inducing agent is administered sequentially in any order during the entire or portions of the treatment period. In some embodiments, the tumor-targeting antibody and the SG- inducing agent is administered simultaneously or approximately simultaneously (e.g., within about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or about 30 minutes of each other). In still other embodiments, the SG-inducing agent may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days before administration of the tumortargeting antibody. In some embodiments, the SG-inducing agent is administered from 1 to 4 weeks, 2 to 8 weeks, 3 to 12 weeks, 4 to 16 weeks or longer, before the tumor-targeting antibody is administered.
[0190] An anti-tumor antibody may also be administered to a cancer patient in conjunction with a cell-based therapy, such as natural killer (NK) cell therapy or a cancer vaccine. In some instances, a cancer vaccine is a peptide-based vaccine, a nucleic acid-based vaccine, a cell-based vaccine, a virus-based or viral fragment-based vaccine or an antigen presenting cell (APC) based vaccine e.g., dendritic cell-based vaccine). Cancer vaccines include Gardasil®, Cervarix®, sipuleucel-T (Provenge®), NeuVax™, HER-2 ICD peptide-based vaccine, HER-2/neu peptide vaccine, AdHER2/neu dendritic cell vaccine, HER-2 pulsed DC1 vaccine, Ad-sig-hMUC-l/ecdCD40L fusion protein vaccine, MVX-0NC0-1, hTERT/ survivin /CMV multipeptide vaccine, E39, J65, PlOs-PADRE, rV-CEA-Tricom, GVAX®, Lucanix®, HER2 VRP, AVX901, ONT-10, ISA1O1, ADXS1 1-001, VGX-3100, INO-9012, GSK1437173A, BPX-501, AGS-003, IDC-G3O5, HyperAcute®-Renal (HAR) immunotherapy, Prevenarl3, MAGER-3. Al, NA17.A2, DCVax-Direct, latent membrane protein-2 (LMP2)-loaded dendritic cell vaccine (NCT02115126), HS410-101 (NCT02010203, Heat Biologies), EAU RF 2010-01 (NCT01435356, GSK), 140036 (NCT02015104, Rutgers Cancer Institute of New Jersey), 130016 (NCTO 1730118, National Cancer Institute), MVX-201101 (NCT02193503, Maxivax SA), ITL-007-ATCR- MBC (NCT01741038, Immuno vative Therapies, Limited), CDR0000644921 (NCT00923143, Abramson cancer center of the University of Pennsylvania), SuMo-Sec-01 (NCT00108875, Julius Maximilians Universitaet Hospital), or MCC-15651 (NCT01176474, Medarex, Inc, BMS).
[0191] In some embodiments, the anti-tumor antibody can be administered with an agent, e.g., a corticosteroid, that mitigates side-effects resulting from stimulation of the immune system.
[0192] In the context of the present disclosure a therapeutic agent that is administered in conjunction with an anti-tumor antibody of the present disclosure can be administered prior to administrations of the tumor-targeting antibody or after administration of the tumortargeting antibody. In some embodiments, an anti-tumor antibody may be administered at the same time as the additional therapeutic agent. In some embodiments, an anti-tumor antibody and an additional therapeutic agent described above can be administered following the same or different dosing regimens. In some embodiments, the tumor-targeting antibody and the therapeutic agent are administered sequentially in any order during the entire treatment period or portions thereof. In some embodiments, the tumor-targeting antibody and the therapeutic agent are administered simultaneously or approximately simultaneously e.g., within about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or about 30 minutes of each other). In still other embodiments, the therapeutic agent may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days before the administration of the tumor-targeting antibody. In still other embodiments, the therapeutic agent may be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the administration of the tumortargeting antibody.
FUNCTIONAL ASSAYS
[0193] Also described herein are functional assays that can be used to determine the ability of the antibodies described herein to mediate FcR-dependent activity. In some embodiments, the assay measures antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), or complement-dependent cytoxicity (CDC). [0194] In some embodiments, binding and activation of FcyRIII by an antibody described herein is determined. In some embodiments, binding and activation of FcyRIIa by an antibody described herein is determined. In some embodiments, binding and activation of the FcR is determined using an ex vivo bioluminescent cell-based assay. In the bioluminescent cell-based assay, primary effector cells are replaced with a Jurkat cell line that stably expresses human a FcyR and a NFAT-induced luciferase. Following engagement with the Fc region by an antibody described herein bound to a target tumor cell, the effector cells expressing FcyRIIa or FcyRIII transduce intracellular signals resulting in NFAT- mediated luciferase activity that can be easily quantified.
IN VIVO ASSAYS
[0195] In some embodiments, the activity of the antibodies is evaluated in vivo in an animal model that is known for specific human tumors. One exemplary model is the CT26 mouse model. Tumor-targeting activity of these antibodies in vivo may be assessed by using several assays, including but not limited to using flow cytometry to analyze the immune profiling of the blood and tumor, monitoring tumor growth, and performing immunofluorescence to semi-quantitative estimate tumor infiltration. In some embodiments, the effect of the antibody can be assessed using Survival, a normalized area above the curve metric (NAAC), and a normalized growth rate metric (NGRM), where NAAC and NGRM were both developed at Atreca. An “in vivo active’- determination can be based on the in vivo activity was assessed by a p- value < 0.05 in at least one of the analyses of survival, NAAC, and NGRM, i.e., if an antibody exhibited a p-value of less than or equal to 0.05 for survival, NAAC, and/or NGRM (any one alone being sufficient), the antibody is considered “in vivo active”.
[0196] In some embodiments, provided herein are antibodies that exhibit inhibitory effects on tumors, including decreasing rate of tumor growth, size, tumor invasion and/or metastasis. Such antibodies exhibit tumor-targeting effects in vivo, e.g., when administered to subjects that has a tumor expressing a tumor-associated glycan.
ENGINEERING VARIANTS
[0197] In some embodiments, an antibody or variant thereof described herein is modified to have improved developability (i.e., reduced development liabilities), including but not limited to, decreased heterogeneity, increased yield, increased stability, improved net charges to improve pharmacokinetics, and or/reduced immunogenicity. In some embodiments, antibodies having improved developability can be obtained by introducing mutations to reduce or eliminate potential development liabilities. In some embodiments, antibodies having improved developability possess modifications as compared to a reference or control antibody in their amino acid sequence.
[0198] In some embodiments, the antibodies or variants thereof disclosed herein have improved developability while maintaining comparable or improved binding affinity to the target antigen as compared to a reference or control (unmodified) antibody. In some embodiments, the antibodies or variants thereof disclosed herein have improved developability while maintaining activities similar to a reference or control (unmodified) antibody.
[0199] In some embodiments, the antibodies or variants thereof have improved developability, e.g., as identified through various in vitro assays, such as aggregation assessment by HPLC or UPLC, hydrophobic interaction chromatography (HIC), polyspecificity assays (e.g., baculovirus particle binding), self-interaction nanoparticle spectroscopy (SINS), or mass spec analysis after incubation in an accelerated degradation condition such as high temperature, low pH, high pH, or oxidative H2O2. Mutations are successful if activity is maintained (or enhanced) while removing or reducing the severity of the liability.
[0200] Improved properties of antibodies or variants thereof as described herein include: (1) fits a standard platform (expression, purification, formulation); (2) high yield; (3) low heterogeneity (glycosylation, chemical modification, and the like); (4) consistent manufacturability (batch-to-batch, and small-to-large scale); (5) high stability (years in liquid formulation), e.g., minimal chemical degradation, fragmentation, and aggregation; and (6) long PK (in vivo half-life), e.g., no off-target binding, no impairment of FcRn recycling, and stable. Antibody liabilities are further described in Table 4.
Table 4. Description of potential development liabilities
Figure imgf000107_0001
Figure imgf000108_0001
proline.
2Sharma et al., Proc. Natl. Acad. Sci. USA 111 :18601-18606, 2014
3 This motif consists of a K or R, followed by a K or R. Stated differently, the motif can be KK, KR, RK, or RR.
4The dipeptide NG poses a medium risk of development liability. The dipeptides NA, NN, NS, and NT pose a low risk of development liability. N may also exhibit low risk of liability for other successor residues, e.g., D, H, or P. Stated differently, dipeptide ND, NH, or NP poses a low risk of development liability.
Similarly to the above, the dipeptide DG poses a medium risk of development liability. The dipeptides DA, DD, DS, and DT pose a low risk of development liability. D may also exhibit low risk of development liability for other successor residues, e.g., N, H, or P.
6“Free cysteine” refers to a cysteine that does not form a disulfide bond with another cysteine and thus is left “free” as thiols. The presence of free cysteines in the antibody can be a potential development liability. Typically, an odd net number of cysteines in the protein shows a likelihood there is a free cysteine.
[0201] In some embodiments, antibodies described herein comprise improved efficacy and/or reduced immunogenicity. In some embodiments, CDR3 is modified to improve efficacy and/or reduce immunogenicity.
[0202] Development liabilities can be removed or reduced by one or more mutations. Mutations are designed to preserve antibody structure and function while removing or reducing development liabilities and to improve function. In some embodiments, mutations to chemically similar residues can be identified that maintain size, shape, charge, and/or polarity. Illustrative mutations are described in Table 5.
Table 5. Preferred mutations to remove development liabilities
Figure imgf000108_0002
Figure imgf000109_0001
to C can be mutated to either A or S to remove development liabilities.
METHODS FOR ALTERING THE GLYCOSYLATION OF AN ANTIBODY
[0203] In some embodiments, the antibodies described herein comprise an Fc region having altered glycosylation that increase the ability of the antibody to recruit NK cells and/or increase ADCC. In some embodiments, the Fc region comprises glycan containing no fucose (i.e. the Fc region is afucosylated). Fucosylated antibodies can be produced using cell lines that express a heterologous enzyme that depletes the fucose pool inside the cell (e.g., GlymaxX® by ProBioGen AG, Berlin, Germany). Non-fucosylated antibodies can also be produced using a host cell line in which the endogenous a-l,6-fucosyltransferase (FUT8) gene is deleted.
EXAMPLES
EXAMPLE 1. GENERATING ANTIBODY VARIANTS
[0204] AB-006410, discovered in antibody repertoires generated by Immune Repertoire Capture® (IRC®) technology from plasmablast B cells isolated from a melanoma patient who had undergone treatment with a pembrolizumab, was previously mutated to generate antibodies with improved properties. AB -006410 was mutated to remove an N-linked glycosylation site in the light chain CDR1 to generate AB-011110 and AB-011111. AB- 011110 was then used as the basis for generation of additional variants designed to address other potential liabilities. One of these variants, AB-011622, contained the mutations R30Q in the light chain CDR1 and D97N in the light chain CDR3 designed to remove two liabilities. Additional variants were generated based on AB-011622, including AB-011788 which contains the mutation D54K in the heavy chain CDR2. AB-011367 was generated using a consensus of three sibling antibodies with a light chain CDR1 N-glycosylation site and a light chain CDR3 free cysteine removed. Additional variants were generated based on AB-011367, including AB-011628, which contains the mutation R57N in the light chain CDR2 and AB-011861 which contains the mutation D54N in the heavy chain HCDR2. The variants tested retained the thermostability of the parental antibodies. These variants were optimized further as shown in Table 6. The name of the mutation in Table 6 indicates the antibody ID of the parental antibody (e.g., 11110 indicates parental antibody AB-11110), whether the mutation is a heavy chain or light chain, the position in the heavy chain or light chain of the mutation (with the numbering based on the sequences as provided in Table 3 and not on Kabat or other amino acid sequence numbering conventions), the amino acid residue at the position before introduction of the mutation, and the amino acid at the position after introduction of the mutation. For example, L26NS, refers to that the asparagine (N) in the light chain position 26 is mutated to a serine (S). As shown in Examples 2 and 3, certain of the new variants showed improved properties over their parental antibody. Of note, those antibodies that contain the change in position 107 of the heavy chain to a tryptophan (H107VW and H107AW) showed more potent binding and reduced polyreactivity as compared to the parental antibody that did not contain that mutation.
Table 6. Antibody variants
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
EXAMPLE 2, IN VITRO STUDIES
[0205] The binding of antibodies to LoVo cell lysate was analyzed via ELISA. Results of the studies, shown in FIGs. 1A-1C and Table 7, indicate more potent binding of those variants that contained the H107VW or H107AW mutation. For example, AB-012934 exhibited improved binding over its parental antibody AB-11110 and AB-012909 exhibited improved binding over its parental antibody AB-011628. The variants that contained the Hl 07 VW mutation or Hl 07 AW also exhibited a reduction in binding to human serum, indicating a decrease in prospectivity as compared to the AB-0011628 and AB-0011861 parental antibodies (FIG. ID).
Table 7. ELISA results
Figure imgf000114_0001
[0206] Surface binding in tumor cells
[0207] Surface binding to colorectal cancer cell line LoVo was assessed by flow cytometry. Cells were staining in BSA-containing buffer with primary antibody for 30 minutes at 4°C with shaking. Cells were then washed and stained with secondary PE-labeled antibody for 30 minutes at 4°C with shaking. Before analysis, cells were counterstained with DAPI.
MedFI values from live, single cells was expressed as fold over isotype control.
[0208] Results of the assay indicated that all of the variants tested, AB-012909, AB-012918, AB-012921, AB-012926, AB-012929 and AB-012934, showed improved binding to the cell line as compared to the parental antibodies AB-01 110, AB-011628, and AB-01 1861 (FIG. 2).
[0209] Binding in tumor tissues
[0210] Immunohistochemistry staining was performed on formalin-fixed paraffin-embedded tissue sections of several human tumors. Standard dewaxing and antigen retrieval techniques were performed followed by standard immunostaining protocols. Briefly, fixed tissues were incubated with Dako serum free protein to block non-specific binding. Sections were incubated with a primary antibody in Dako background reducing diluent. Sections were incubated with a polymer HRP conjugated secondary antibody and stained using Hematoxylin. The primary antibodies were utilized with species-specific secondary antibodies and detected using standard methodology. AB-006410 variants showed robust and tumor-selective signal in >50% of tumor cells. All variants demonstrated increased binding on colorectal cancer tissue sections relative to the parental antibodies AB-011110, AB-01 1788 and AB-01 1628 (FIG. 5). Variant AB-012934 displayed the highest level of binding above all variants and parental antibodies.
[0211] Immunohistochemical staining of variants AB-012909 and AB-12934 and their respective parental antibodies AB-011628 and AB-011110 were performed on additional human cancer types. Variants AB-012934 and AB-012909 demonstrated increased reactivity on the majority of the cancer types relative to the parental antibodies. Six of these cancer types included: colorectal, lung, endometrial, breast, stomach and esophageal (FIG. 6 A and 6B).
[0212] ADC
[0213] The antibody-drug conjugate (ADC) activity of the anti-tumor antibodies were assessed on LoVo and NUGC4 cells. Briefly, the antibodies were tested for ADC activity using a secondary, toxin-conjugated antibody. Target cells were detached from the culture plate and cell concentration was adjusted to 31,250 cells/mL in assay media. 2,500 cells were added to each well of a 96 well plate and incubated with different concentrations of primary antibody for 15 min at room temperature. Following, secondary Fab anti-mouse IgG Fc conjugated to MMAF with a cleavable linker was added at a final concentration of 250 ng/mL. Cells were incubated for 72 h at 37 °C and 5% CO2. At the end of the incubation period, 100 pl CellTiter-Glo® was added to each well and allowed to incubate for 5-10 min at room temperature before reading luminescence. Data was then normalized to a maximum lysis control and plotted using graph pad prism.
[0214] Results of the assay showed that the tested variants exhibited improved ADC cytotoxicity in both LoVo (FIG. 3) and NUGC4 cells (FIG. 4) as compared to the parental antibodies AB-01110 and AB-011628. EC50 values, Area Under the Curve (AUC) and maximum % cytotoxicity values from the assays are shown in Table 8 (LoVo) and Table 9 (NUGC4).
Table 8. ADC data on LoVo cells
| | EC50 (nM) | Max. Cytox. (%) | AUC |
Figure imgf000116_0001
Table 9. ADC data on NUGC4 cells
Figure imgf000116_0002
[0215] ADC Activity
[0216] Antibody-drug conjugate activity can be tested on antibodies directly conjugated with a toxic payload, such as Exatecan. LoVo target cells are detached from the culture plate and cell concentration is adjusted to 31,250 cells/mL in assay media. 2,500 cells are added to each well of a 96 well plate and incubated with different concentrations of directly conjugated primary antibody for 15 min at room temperature. Cells are than incubated for 72 h at 37 °C and 5% CO2. At the end of the incubation period, 100 pl CellTiter-Glo® are added to each well and allowed to incubate for 5-10 min at room temperature before reading luminescence. Data is then normalized to a maximum lysis control and plotted using GraphPad Prism.
EXAMPLE 3, IN VIVO STUDIES
[0217] Antibodies in the ADC conjugate construct format can be tested in mice carrying tumors from LoVo tumor cells. In brief, 1 x 107 LoVo tumor cells are injected subcutaneously into female BALB/c nude mice. Tumors are allowed to establish and randomized at around 150 mm3. Dosing is performed at day of randomization.
[0218] While the disclosure has been described with reference to the specific examples and illustrations, changes can be made and equivalents can be substituted to adapt to a particular context or intended use as a matter of routine development and optimization and within the purview of one of ordinary skill in the art, thereby achieving benefits of the disclosure without departing from the scope of what is claimed and their equivalents.

Claims

1. An antibody that binds to a tumor or a fragment thereof, comprising: a) a heavy chain variable region (VH) comprising (i) a complementarity determining region (HCDR) 1 having an amino acid sequence according to any one of SEQ ID NOs: 16, 17, 18, 19, and 20; (ii) a HCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 25, 26, 27, 28, 29, 30, and 31-62; and (iii) a HCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 65-85; and b) a light chain variable region (VL) comprising (i) a LCDR1 having an amino acid sequence according to any one of SEQ ID NOs: 86, 87, 88, and 91-111 ; (ii) a LCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 112-122; and (iii) a LCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 123-126.
2. The antibody of claim 1, wherein the VL sequence comprises (i) a LCDR1 having an amino acid sequence according to any one of SEQ ID NOs: 89 and 91-111 ; (ii) a LCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 116-122; and (iii) a LCDR3 having an amino acid sequence according to SEQ ID NO: 126.
3. The antibody of any one of claims 1-2, wherein the VH sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 131-208 and 252.
4. The antibody of any one of claims 1-3, wherein the VL sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 214-251.
5. The antibody of any one of claims 1 -4, wherein the VH sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 131-208 and 252, and the VL sequence comprises an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 214-251.
6. An antibody that binds to a tumor or a fragment thereof, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 127-208 and 252 and at least one modification in a position selected from the group consisting of: 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 111, 112, 113, 114, 115, and 118; and/or b) a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to the sequence as set forth in any one of SEQ ID NOs: 209-251 and at least one modification in a position selected from the group consisting of: 27, 28, 31, 57, and 97.
7. The antibody of claim 6, wherein the modification is selected from a modification listed in Table 6.
8. An antibody that binds to a tumor or a fragment thereof, comprising: a) a heavy chain variable region (VH) comprising (i) a complementarity determining region (HCDR) 1 having an amino acid sequence according to any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, and 20; (ii) a HCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31-62; and (iii) a HCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 63-85; and at least one modification in a position selected from the group consisting of: 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 111, 112, 113, 114, 115, and 118; and/or b) a light chain variable region (VL) comprising (i) a LCDR1 having an amino acid sequence according to any one of SEQ ID NOs: 86, 87, 88, and 91-111 ; (ii) a LCDR2 having an amino acid sequence according to any one of SEQ ID NOs: 112-122; and (iii) a LCDR3 having an amino acid sequence according to any one of SEQ ID NOs: 123-126; and at least one modification in a position selected from the group consisting of: 27, 28, 31, 57, and 97.
9. The antibody of claim 8, wherein the modification is selected from a modification listed in Table 6.
10. An immunoconjugate, comprising the antibody of any one of claims 1-9, and a cytotoxic agent conjugated to the antibody using a linker.
11. The immunoconjugate of claim 10, wherein the cytotoxic agent is selected from the group consisting of: ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, methotrexact, actinomycin, a diphtheria toxin, extotoxin A from Pseudomonas, Pseudomonas exotoxin 40, abrin, abrin A chain, modeccin A chain, alpha sarcin, gelonin, mitogellin, restrictocin, cobran venom factor, a ribonuclease, engineered Shiga toxin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolastatin, ccl065, and a cisplatin.
12. An antibody that binds to a tumor, wherein the antibody comprises: a) a heavy chain variable region comprising: an HCDR1, HCDR2, and/or HCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB- 012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB- 012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB- 012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB- 012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB- 012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB- 012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB-012265, AB- 012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB- 012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB- 012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB- 012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB- 013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, , AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213 listed in Table 1, or variants of the HCDR1, HCDR2, and/or HCDR3 amino acid sequence in which 1, 2, 3, 4, 5, or more amino acids are substituted; and at least one modification in a position selected from the group consisting of: 31, 32, 33, 34, 35, 50, 52, 53, 54, 56, 57, 58, 59, 61, 62, 73, 74, 75, 76, 99, 101, 102, 103, 106, 107, 108, 111, 112, 113, 1 14, 115, and 118; and/or b) a light chain variable region comprising: an LCDR1, LCDR2, and/or LCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB- 012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB- 012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB- 012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB- 012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB- 012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB- 012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB- 012260, AB-012261, AB-012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB- 012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB- 012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB- 012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB- 013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB- 013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB- 013213, or variants of the LCDR1, LCDR2, and/or LCDR3 amino acid sequence in which 1, 2, 3, 4, 5, or more amino acid are substituted; and at least one modification in a position selected from the group consisting of: 27, 28, 31, 57, and 97.
13. The antibody of claim 12, wherein the heavy chain variable region of the antibody comprises an HCDR3 variant of an HCDR3 of any one of AB-012179, AB-012180, AB- 012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB- 012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB- 012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB- 012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB- 012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB- 012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB- 012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB- 012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB- 012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB- 012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB- 012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB- 012258, AB-012259, AB-012260, AB-012261 , AB-012262, AB-012263, AB-012264, AB- 012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB- 012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB- 012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB- 012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB- 012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB- 012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB- 012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB- 013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB- 013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB- 013210, AB-013211, AB-013212, or AB-013213, in which the amino acid residue at position 107 has been substituted with a tryptophan.
14. The antibody of claim 12, wherein the heavy chain variable region of the antibody comprises an HCDR3 variant comprising SEQ ID NO: 84.
15. The antibody of claim 12, wherein the antibody comprises all six CDRs of an antibody selected from the group consisting of designated as AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB- 012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB- 012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB- 012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB- 012226, AB-012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB- 012239, AB-012240, AB-012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB- 012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB- 012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB- 012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB- 012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB- 012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB- 013199, AB-013200, AB-013201 , AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB- 013212, and AB-013213.
16. The antibody of any one of claims 12-15, wherein the antibody comprises a VH region comprising a VH amino acid sequence in Table 3 or an amino sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the VH amino acid sequence in Table 3, and/or wherein the antibody comprises a VL region comprising a VL amino acid sequence in Table 3; and an amino sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the VL amino acid sequence in Table 3.
17. The antibody of any one of claims 12-15, wherein the antibody comprises: a VH region having an amino acid sequence at least 80% identical to the amino acid sequence according to any one of SEQ ID NOs: 127-208 or 252; and/or a VL region having an amino acid sequence at least 80% identical to the amino acid sequence according to any one of SEQ ID NOs: 209-251.
18. The antibody of any one of claims 12-17, wherein the antibody comprises both the VH and VL of an antibody selected from the group consisting of designated as AB-012179, AB- 012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB- 012187, AB-012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB- 012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB- 012201, AB-012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB- 012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB- 012215, AB-012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB- 012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB- 012229, AB-012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB- 012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB- 012243, AB-012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB- 012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB- 012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB- 012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB- 012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB- 012915, AB-012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB- 012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB- 012929, AB-012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB- 012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB- 012943, AB-012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB- 013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB- 013202, AB-013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB- 013209, AB-013210, AB-01321 1, AB-013212, AB-013213, and a variant thereof.
19. The antibody of any one of claims 12-18, wherein the the antibody comprises both the VH and VL of an antibody selected from the group consisting of designated as AB- 012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB- 012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB- 012205, AB-012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB-012213, AB-012214, AB-012215, AB-012216, AB-012217, AB- 012218, AB-012219, AB-012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB-012230, AB- 012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB- 012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB- 012257, AB-012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB-012269, AB- 012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB- 012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB- 012933, AB-012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB-012944, AB-012945, AB- 012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB-013203, AB- 013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, and AB-013213.
20. The antibody of any one of claims 1-9 or 12-19 or the immunoconjugate of any one of claims 10-11 , wherein binding of the antibody to the tumor is dependent on expression of one or more glycosyltransferases in the tumor.
21. The antibody or immunoconjugate of claim 20, wherein one of the one or more glycosyltransferases has N-acetyl-galactosaminyltransferase activity.
22. The antibody or immunoconjugate of claim 20 or 21 , wherein one of the one or more glycosyltransferases has fucosyltransferase activity.
23. The antibody or immunoconjugate of any one of claims 20-22, wherein the glycosyltransferase that has N-acetyl-galactosaminyltransferase activity is selected from the group consisting of B4GALNT3 and B4GALNT4.
24. The antibody or immunoconjugate of claim 23, wherein the glycosyltransferase is B4GALNT3.
25. The antibody or immunoconjugate of any one of claims 20-24, wherein one of the one or more glycosyltransferases that has fucosyltransferase activity is selected from the group consisting of FUT4, FUT5, FUT6, and FUT9.
26. The antibody or immunoconjugate of claim 25, wherein the glycosyltransferase is FUT9.
27. The antibody or immunoconjugate of any one of claims 1-26, wherein the tumor expresses a tumor-associated glycan.
28. The antibody or immunoconjugate of claim 27, wherein the tumor-associated glycan is an extracellular glycan.
29. The antibody or immunoconjugate of claim 27, wherein the presence of the tumor- associated glycan is dependent on the expression of B4GALNT3 and FUT9 in the tumor.
30. The antibody of any one of claims 1-9 or 12-29 or the immunoconjugate of any one of claims 10-11, wherein the antibody or immunoconjugate preferentially binds to a tumor tissue relative to a normal tissue.
31. The antibody of any one of claims 1-9 or 12-30 or the immunoconjugate of any one of claims 10-11, wherein the antibody or immunoconjugate is internalized by the tumor cells upon contacting the tumor.
32. A polynucleotide encoding a polypeptide comprising a VH sequence of the antibody of any one of claims 1-9 or 12-19 or the immunoconjugate of any one of claims 10- 11, and/or a VL sequence of the antibody of any one of claims 1-9 or 12-31 or the immunoconjugate of any one of claims 10-11.
33. An expression vector comprising a polynucleotide encoding the VH region and/or the VL region of the antibody of any one of claims 1-9 or 12-31 or the immunoconjugate of any one of claims 10-11.
34. A host cell that comprises an expression vector of claim 33.
35. A pharmaceutical composition comprising the antibody of any one of claims 1-9 or 12-31 or the immunoconjugate of any one of claims 10-11 and a pharmaceutically acceptable carrier.
36. A method of treating a cancer patient, the method comprising administering the antibody of any one of claims 1-9 or 12-31 or the immunoconjugate of any one of claims 10- 11 to the patient.
37. The method of claim 36, wherein the cancer is selected from the group consisting of colorectal, lung, endometrial, breast, stomach and esophageal cancer.
38. Use of antibody of any one of claims 1-9 or 12-31 or the immunoconjugate of any one of claims 10-11 for a method of treating cancer.
39. The use of claim 38, wherein the cancer is selected from the group consisting of colorectal, lung, endometrial, breast, stomach and esophageal cancer.
40. A polypeptide comprising a heavy chain variable region, wherein the heavy chain variable region comprises an HCDR1, HCDR2, and/or HCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB- 012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB- 012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB- 012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB- 012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB- 012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB- 012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB- 012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB- 012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB- 012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB- 012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB- 012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB- 012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB- 012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB- 012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB- 012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB- 012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB- 012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB- 012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB- 013192, AB-013193, AB-013195, AB-013196, AB-013197, AB-013198, AB-013199, AB- 013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB- 013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213 listed in Table 1, or variants of the HCDR1, HCDR2, and/or HCDR3 amino acid sequence in which 1, 2, 3, 4, 5, or more amino acids are substituted.
41. The polypeptide of claim 40, wherein the heavy chain variable region of the antibody comprises an HCDR3 variant of an HCDR3 of any one of AB-012179, AB-012180, AB- 012181, AB-012182, AB-012183, AB-012184, AB-012185, AB-012186, AB-012187, AB- 012188, AB-012189, AB-012190, AB-012191, AB-012192, AB-012193, AB-012194, AB- 012195, AB-012196, AB-012197, AB-012198, AB-012199, AB-012200, AB-012201, AB- 012202, AB-012203, AB-012204, AB-012205, AB-012206, AB-012207, AB-012208, AB- 012209, AB-012210, AB-01221 1, AB-012212, AB-012213, AB-012214, AB-012215, AB- 012216, AB-012217, AB-012218, AB-012219, AB-012220, AB-012221, AB-012222, AB- 012223, AB-012224, AB-012225, AB-012226, AB-012227, AB-012228, AB-012229, AB- 012230, AB-012231, AB-012232, AB-012233, AB-012234, AB-012235, AB-012236, AB- 012237, AB-012238, AB-012239, AB-012240, AB-012241, AB-012242, AB-012243, AB- 012244, AB-012245, AB-012246, AB-012247, AB-012248, AB-012249, AB-012250, AB- 012251, AB-012252, AB-012253, AB-012254, AB-012255, AB-012256, AB-012257, AB- 012258, AB-012259, AB-012260, AB-012261, AB-012262, AB-012263, AB-012264, AB- 012265, AB-012266, AB-012267, AB-012268, AB-012269, AB-012270, AB-012271, AB- 012909, AB-012910, AB-012911, AB-012912, AB-012913, AB-012914, AB-012915, AB- 012916, AB-012917, AB-012918, AB-012919, AB-012920, AB-012921, AB-012922, AB- 012923, AB-012924, AB-012925, AB-012926, AB-012927, AB-012928, AB-012929, AB-
012930, AB-012931, AB-012932, AB-012933, AB-012934, AB-012935, AB-012936, AB- 012937, AB-012938, AB-012939, AB-012940, AB-012941, AB-012942, AB-012943, AB- 012944, AB-012945, AB-012946, AB-012947, AB-013192, AB-013193, AB-013195, AB-
013196, AB-013197, AB-013198, AB-013199, AB-013200, AB-013201, AB-013202, AB- 013203, AB-013204, AB-013205, AB-013206, AB-013207, AB-013208, AB-013209, AB- 013210, AB-013211, AB-013212, or AB-013213, in which the amino acid residue at position 107 has been substituted with a tryptophan.
42. The polypeptide of claim 40, wherein the heavy chain variable region of the antibody comprises an HCDR3 variant comprising SEQ ID NO: 84.
43. A polypeptide comprising a light chain variable region, wherein the light chain variable region comprises an LCDR1, LCDR2, and/or LCDR3 amino acid sequence of any one of AB-012179, AB-012180, AB-012181, AB-012182, AB-012183, AB-012184, AB-
012185, AB-012186, AB-012187, AB-012188, AB-012189, AB-012190, AB-012191, AB- 012192, AB-012193, AB-012194, AB-012195, AB-012196, AB-012197, AB-012198, AB- 012199, AB-012200, AB-012201, AB-012202, AB-012203, AB-012204, AB-012205, AB- 012206, AB-012207, AB-012208, AB-012209, AB-012210, AB-012211, AB-012212, AB- 012213, AB-012214, AB-012215, AB-012216, AB-012217, AB-012218, AB-012219, AB- 012220, AB-012221, AB-012222, AB-012223, AB-012224, AB-012225, AB-012226, AB- 012227, AB-012228, AB-012229, AB-012230, AB-012231, AB-012232, AB-012233, AB- 012234, AB-012235, AB-012236, AB-012237, AB-012238, AB-012239, AB-012240, AB- 012241, AB-012242, AB-012243, AB-012244, AB-012245, AB-012246, AB-012247, AB- 012248, AB-012249, AB-012250, AB-012251, AB-012252, AB-012253, AB-012254, AB- 012255, AB-012256, AB-012257, AB-012258, AB-012259, AB-012260, AB-012261, AB- 012262, AB-012263, AB-012264, AB-012265, AB-012266, AB-012267, AB-012268, AB- 012269, AB-012270, AB-012271, AB-012909, AB-012910, AB-012911, AB-012912, AB- 012913, AB-012914, AB-012915, AB-012916, AB-012917, AB-012918, AB-012919, AB- 012920, AB-012921, AB-012922, AB-012923, AB-012924, AB-012925, AB-012926, AB- 012927, AB-012928, AB-012929, AB-012930, AB-012931, AB-012932, AB-012933, AB- 012934, AB-012935, AB-012936, AB-012937, AB-012938, AB-012939, AB-012940, AB- 012941, AB-012942, AB-012943, AB-012944, AB-012945, AB-012946, AB-012947, AB- 013192, AB-013193, AB-013195, AB-013196 , AB-013197, AB-013198, AB-013199, AB- 013200, AB-013201, AB-013202, AB-013203, AB-013204, AB-013205, AB-013206, AB- 013207, AB-013208, AB-013209, AB-013210, AB-013211, AB-013212, or AB-013213 listed in Table 1, or variants of the HCDR1, HCDR2, and/or HCDR3 amino acid sequence in which 1, 2, 3, 4, 5, or more amino acids are substituted.
44. A polypeptide comprising a VH sequence of the antibody of any one of claims 1-9 or 12-31 or the immunoconjugate of any one of claims 10-11, and/or a VL sequence of the antibody of any one of claims 1-9 or 12-31 or the immunoconjugate of any one of claims 10- 11.
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