Anti-FAP antibodies and methods of use thereof

Abstract

Provided are antibodies that specifically bind to FAP (e.g., human FAP), and such anti-FAP antibodies further comprising a TGFβ-binding moiety. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

Description

ANTI-FAP ANTIBODIES AND METHODS OF USE THEREOF CROSS REFERENCE

[0001] This application claims the priority of U.S. Provisional Application No. 63 / 632,889, filed April 11, 2024, and U.S. Provisional Application No. 63 / 587,885, filed October 4, 2023, each of which is hereby incorporated by reference in their entirety. REFERENCE TO SEQUENCE LISTING

[0002] The content of the electronically submitted sequence listing in .XML file (Name: 212698_Seqlisting_ST26.XML; Size: 272,268 bytes; and Date Created: October 2, 2024), filed with the application, is incorporated herein by reference in its entirety. FIELD

[0003] The present disclosure relates to antibodies that specifically bind to fibroblast activation protein (FAP), including fusions thereof with a transforming growth factor beta BACKGROUND

[0004] FAP is a type II membrane-bound serine protease belonging to the dipeptidyl peptidase 4 family, which consists of 760 amino acids with residues 1-4 constituting the intracellular domain, 5–25 composing the transmembrane domain, and 26–760 forming a large extracellular domain. This unique serine protease exhibits enzymatic activity as a homodimer, cleaving the peptide bond at a post-proline dipeptidyl peptidase and endopeptidase activities. FAP is expressed on the surface of most cancer-associated fibroblasts (CAFs), with no presence in benign tumors and minimal presence in normal tissues such as fetal cells, stromal fibroblasts, or injured tissues. CAFs have emerged as key architects of a pro-tumorigenic and immunosuppressive tumor microenvironment (TME) to promote cancer growth and immune escape. FAP-positive CAFs (FAP+CAFs) have been implicated in resistance to immunotherapy.

[0005] cell proliferation, differentiation, apoptosis, angiogenesis, fibrosis, and immune activation. BUSINESS.32102391.1determining the specificity of intracellular signals elicited by the cytokines of the TGF-beta the tumor-stromal crosstalk and a key factor in inducing the transdifferentiation of fibroblasts chemotherapy, targeted therapy, and immunotherapy. Thus, there is a need for therapies + CAFs.

[0006] Given the complementary roles of FAP and TGF in modulating tumorigenicity and immune response to tumors, therapeutic antibodies targeting FAP and TGF hold great promise for cancer therapy. SUMMARY

[0007] The instant disclosure provides antibodies that specifically bind to FAP (e.g., provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies. The antibodies disclosed herein are particularly strong binders to FAP, and thus, are especially useful for increasing T cell infiltration of tumors, overcoming resistance to immunotherapy, and treating cancer in a subject.

[0008] Accordingly, in one aspect, the present disclosure provides an antibody that specifically binds to human FAP, the antibody comprising a VH comprising the CDRH1, CDRH2, and CDRH3 sequences of the VH amino acid sequence set forth in SEQ ID NO: 76, and a VL comprising the CDRL1, CDRL2, and CDRL3 of the VL amino acid sequence set forth in SEQ ID NO: 92.

[0009] In certain embodiments, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequence, respectively, set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and BUSINESS.32102391.183; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87.

[0010] In certain embodiments, the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 11, 38, and 39. In certain embodiments, the antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 42, 46, and 49. In certain embodiments, the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, and 39; 1, 13, and 39; 1, 14, and 39; 1, 15, and 39; 2, 16, and 39; 3, 17, and 39; 1, 18, and 39; 4, 19, and 39; 5, 20, and 39; 6, 21, and 39; 1, 22, and 39; 4, 23, and 39; 7, 24, and 39; 8, 25, and 39; 1, 26, and 39; 1, 27, and 39; 1, 28, and 39; 9, 29, and 39; 1, 30, and 39; 1, 31, and 39; 1, 32, and 39; 1, 33, and 39; 1, 34, and 39; 10, 35, and 39; 1, 36, and 39; or 1, 37, and 39. In certain embodiments, the antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 40, 43, and 47; 40, 44, and 47; 40, 43, and 48; 41, 45, and 47; or 41, 43, and 47. In certain embodiments, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, 39, 40, 43, and 47; 1, 12, 39, 40, 44, and 47; 1, 13, 39, 40, 43, and 47; 1, 14, 39, 40, 43, and 47; 1, 15, 39, 40, 43, and 47; 2, 16, 39, 40, 43, and 47; 3, 17, 39, 40, 43, and 48; 1, 18, 39, 40, 43, and 47; 4, 19, 39, 40, 43, and 47; 5, 20, 39, 41, 45, and 47; 6, 21, 39, 40, 43, and 47; 1, 22, 39, 40, 43, and 47; 4, 23, 39, 40, 43, and 47; 7, 24, 39, 40, 43, and 47; 8, 25, 39, 40, 43, and 47; 1, 26, 39, 40, 43, and 47; 1, 27, 39, 40, 43, and 47; 1, 28, 39, 40, 43, and 47; 9, 29, 39, 40, 43, and 47; 1, 30, 39, 40, 43, and 47; 1, 31, 39, 40, 43, and 47; 1, 32, 39, 40, 43, and 47; 1, 33, 39, 40, 43, and 47; 1, 34, 39, 41, 43, and 47; 10, 35, 39, 40, 43, and 47; 1, 36, 39, 40, 43, and 47; or 1, 37, 39, 40, 43, and 47. In certain embodiments, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 14, 39, 40, 43, and 47.

[0011] In certain embodiments, the antibody comprises the VH amino acid sequence of SEQ ID NO: 76. In certain embodiments, the antibody comprises the VH amino acid sequence of any one of SEQ ID NOs: 50-75.

[0012] In certain embodiments, the antibody comprises a heavy chain constant region, optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, and IgM. In certain embodiments, the heavy chain constant region is a human IgG1 heavy chain constant region comprising a D at position 239, an L at position 330, and an E at position 332, numbered according to the EU numbering system. In certain embodiments, the antibody BUSINESS.32102391.1comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 134, 135, or 136.

[0013] In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 93-118.

[0014] In certain embodiments, the antibody comprises the VL amino acid sequence of SEQ ID NO: 92. In certain embodiments, the antibody comprises the VL amino acid sequence of any one of SEQ ID NOs: 77-91.

[0015] In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119-133.

[0016] In certain embodiments, the VH and VL comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87. In certain embodiments, the VH and VL comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 52 and 80.

[0017] In certain embodiments, the antibody comprises a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 93 and 119; 93 and 120; 94 and 121; 95 and 122; 96 and 123; 97 and 121; 98 and 124; 99 and 119; 100 and 125; 101 and 126; 102 and 127; 103 and 128; 104 and 121; 105 and 129; 106 and 130; 107 and 121; 108 and 121; 109 and 131; 110 and 131; 111 and 119; 112 and 122; 113 and 125; 114 and 123; 115 and 132; 116 and 133; 117 and 130; or 118 and 129. In certain embodiments, the antibody comprises a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 95 and 122.

[0018] In certain embodiments, the antibody is an scFv, a Fab, or chimeric antigen receptor.

[0019] BUSINESS.32102391.1covalently linked to the C-terminal residue of a heavy chain of the antibody. In certain C-terminal residue of the heavy chain via a peptide linker. In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: at least 75% (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 137 or 138. In certain embodiments, the

[0020] In certain embodiments, the heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 149-157.

[0021] In one aspect, the present disclosure provides an antibody that specifically binds human FAP, the antibody comprising a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 157 and 122; 150 and 122; 156 and 120; 149 and 121; 154 and 124; 151 and 119; 152 and 121; 153 and 130; or 155 and 122. In certain embodiments, the heavy chain and the light chain comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 157 and 122.

[0022] In one aspect, the present disclosure provides a polypeptide comprising a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the polypeptide comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in any one of SEQ ID NOs: 50-75.

[0023] In certain embodiments, the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 11, 38, and 39. In certain embodiments, the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, and 39; 1, 13, and 39; 1, 14, and 39; 1, 15, and 39; 2, 16, and 39; 3, 17, and 39; 1, 18, and 39; 4, 19, and 39; 5, 20, and 39; 6, 21, and 39; 1, 22, and 39; 4, 23, and 39; 7, 24, and 39; 8, 25, and 39; 1, 26, and 39; 1, 27, and 39; 1, 28, and 39; 9, 29, and 39; 1, 30, and 39; 1, 31, and 39; 1, 32, and 39; 1, 33, and 39; 1, 34, and 39; 10, 35, and 39; 1, 36, and 39; or 1, 37, and 39. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 76. In certain embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 50-75. BUSINESS.32102391.1

[0024] In certain embodiments, the polypeptide comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 93-118 or 149-157.

[0025] In one aspect, the present disclosure provides a polypeptide comprising a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 92. In certain embodiments, the polypeptide comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in any one of SEQ ID NOs: 77-91.

[0026] In certain embodiments, the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 42, 46, and 49. In certain embodiments, the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 40, 43, and 47; 40, 44, and 47; 40, 43, and 48; 41, 45, and 47; or 41, 43, and 47. In certain embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 92. In certain embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NOs: 77-91.

[0027] In certain embodiments, the polypeptide comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119-133.

[0028] In certain embodiments, a polypeptide disclosed herein is a chimeric antigen receptor.

[0029] In certain embodiments, an antibody or polypeptide disclosed herein is conjugated to a cytotoxic agent, cytostatic agent, toxin, radionuclide, or detectable label.

[0030] In one aspect, the present disclosure provides a polynucleotide encoding: a VH, a VL, a heavy chain, and / or a light chain of an antibody disclosed herein; or a polypeptide disclosed herein.

[0031] In one aspect, the present disclosure provides a vector comprising a polynucleotide disclosed herein.

[0032] In one aspect, the present disclosure provides a recombinant host cell comprising: (a) a polynucleotide disclosed herein; (b) a vector disclosed herein; (c) a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody disclosed herein and a second polynucleotide encoding a light chain variable region or a light chain of an antibody disclosed herein; or (d) a first vector comprising a first polynucleotide encoding a heavy chain variable BUSINESS.32102391.1region or a heavy chain of an antibody disclosed herein and a second vector comprising a second polynucleotide encoding a light chain variable region or a light chain of an antibody disclosed herein.

[0033] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a host cell disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0034] In one aspect, the present disclosure provides a method of producing an antibody, the method comprising culturing a host cell disclosed herein under suitable conditions so that the polynucleotide is expressed and the antibody is produced.

[0035] In one aspect, the present disclosure provides a method of increasing T cell infiltration of a tumor in a subject, the method comprising administering to the subject an effective amount of an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0036] In one aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0037] In one aspect, the present disclosure provides use of an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein, for the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

[0038] In one aspect, the present disclosure provides an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein, for use in medicine.

[0039] In one aspect, the present disclosure provides an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein, for use in the treatment of cancer in a subject in need thereof.

[0040] In one aspect, the present disclosure provides an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a BUSINESS.32102391.1host cell disclosed herein, or a pharmaceutical composition disclosed herein, for use in increasing T cell infiltration of a tumor in a subject.

[0041] In one aspect, the present disclosure provides a multispecific antibody binding moiety. In certain embodiments, the antigen binding region is a scFv, VH, VL, or Fab, that specifically binds to human FAP. In certain embodiments, the multispecific antibody comprises a full-length antibody (e.g., a heterotetrameric antibody or a heavy chain antibody) comprises a full-length heterotetrameric antibody (comprising two heavy chains and two light binding moiety is covalently linked to the C-terminal residue of a heavy chain of the antibody. linked to the C-terminal residue of the heavy chain, optionally via a peptide linker. In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group comprises an amino acid sequence at least 75% (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 137 or SEQ ID NO: 137 or 138. In certain embodiments, the multispecific antibody comprises two heavy chains, each comprising from N-terminus to C-terminus: an antigen binding region that specifically binds to human FAP; a CH2 domain; a CH3 domain; a peptide linker of SEQ ID multispecific antibody comprises a full length heterotetrameric antibody (comprising two heavy chains and two light chains), wherein the C-terminus of each heavy chain is linked to NO: 144, such that each heavy chain comprises from N-terminus to C-terminus: a VH; a CH1 binding moiety of SEQ ID NO: 144. BUSINESS.32102391.1

[0042] The instant disclosure also provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a first antibody that specifically binds to human FAP and a second antibody that specifically binds to human CTLA-4, PD-1, or PD-L1.

[0043] In certain embodiments, the method further comprises administering to the subject a third antibody that specifically binds to human CTLA-4, PD-1, or PD-L1.

[0044] In certain embodiments, the second antibody specifically binds to human CTLA-4 and the third antibody specifically binds to human PD-1 or PD-L1. In certain embodiments, the second antibody specifically binds to CTLA-4 and the third antibody specifically binds to PD-1. In certain embodiments, the second antibody specifically binds to CTLA-4 and the third antibody specifically binds to PD-L1.

[0045] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds comprises a human IgG1 heavy chain constant region comprising S239D / A330L / I332E mutations, numbered according to the EU numbering system.

[0046] In certain embodiments, the antibody that specifically binds to human CTLA-4 competes for binding with or binds to the same epitope as botensilimab, ipilimumab, or tremelimumab.

[0047] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and a CDRL3 with an amino acid sequence that is identical to the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively, of botensilimab, ipilimumab, or tremelimumab. In certain embodiments, the antibody that specifically binds to human CTLA-4 is botensilimab.

[0048] In certain embodiments, the antibody that specifically binds to human PD-1 or PD-L1 is selected from the group consisting of adebrelimab, atezolizumab, avelumab, balstilimab, camrelizumab, cemiplimab, cosibelimab, dostarlimab, durvalumab, enlonstobart, envafolimab, nivolumab, pembrolizumab, penpulimab, pidilizumab, prolgolimab, pucotenlimab, retifanlimab, serplulimab, sintilimab, socazolimab, sugemalimab, tagitanlimab, tislelizumab, toripalimab, and zimberelimab. In certain embodiments, the antibody that specifically binds to human PD-1 is balstilimab. BUSINESS.32102391.1

[0049] In certain embodiments, the second antibody is botensilimab and the third antibody is balstilimab, nivolumab, or pembrolizumab.

[0050] In certain embodiments, the first antibody that specifically binds to human FAP comprises a VH comprising the CDRH1, CDRH2, and CDRH3 sequences of the VH amino acid sequence set forth in SEQ ID NO: 76, and a VL comprising the CDRL1, CDRL2, and CDRL3 of the VL amino acid sequence set forth in SEQ ID NO: 92.

[0051] In certain embodiments, the first antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequence, respectively, set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87.

[0052] In certain embodiments, the first antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 11, 38, and 39.

[0053] In certain embodiments, the first antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 42, 46, and 49.

[0054] In certain embodiments, the first antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, and 39; 1, 13, and 39; 1, 14, and 39; 1, 15, and 39; 2, 16, and 39; 3, 17, and 39; 1, 18, and 39; 4, 19, and 39; 5, 20, and 39; 6, 21, and 39; 1, 22, and 39; 4, 23, and 39; 7, 24, and 39; 8, 25, and 39; 1, 26, and 39; 1, 27, and 39; 1, 28, and 39; 9, 29, and 39; 1, 30, and 39; 1, 31, and 39; 1, 32, and 39; 1, 33, and 39; 1, 34, and 39; 10, 35, and 39; 1, 36, and 39; or 1, 37, and 39.

[0055] In certain embodiments, the first antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 40, 43, and 47; 40, 44, and 47; 40, 43, and 48; 41, 45, and 47; or 41, 43, and 47.

[0056] In certain embodiments, the first antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, 39, 40, 43, and 47; 1, 12, 39, 40, 44, and 47; 1, 13, 39, 40, 43, and 47; 1, 14, 39, 40, 43, and 47; 1, 15, 39, 40, 43, and 47; 2, 16, 39, 40, 43, and 47; 3, 17, 39, 40, 43, and 48; 1, 18, 39, 40, 43, and 47; 4, 19, 39, 40, 43, and 47; 5, 20, 39, 41, 45, and 47; 6, 21, 39, 40, 43, and 47; 1, 22, 39, 40, 43, and 47; 4, 23, 39, 40, 43, and 47; 7, 24, 39, 40, 43, and 47; 8, 25, 39, 40, 43, and 47; 1, 26, 39, 40, 43, and 47; 1, 27, 39, 40, 43, and 47; 1, 28, 39, 40, 43, and 47; 9, 29, 39, 40, 43, and 47; 1, 30, 39, 40, 43, and 47; 1, 31, 39, 40, 43, and 47; 1, 32, 39, 40, 43, and BUSINESS.32102391.147; 1, 33, 39, 40, 43, and 47; 1, 34, 39, 41, 43, and 47; 10, 35, 39, 40, 43, and 47; 1, 36, 39, 40, 43, and 47; or 1, 37, 39, 40, 43, and 47.

[0057] In certain embodiments, the first antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 14, 39, 40, 43, and 47.

[0058] In certain embodiments, the first antibody comprises the VH amino acid sequence of SEQ ID NO: 76.

[0059] In certain embodiments, the first antibody comprises the VH amino acid sequence of any one of SEQ ID NOs: 50-75.

[0060] In certain embodiments, the first antibody comprises a heavy chain constant region, optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, and IgM. In certain embodiments, the heavy chain constant region of the first antibody is a human IgG1 heavy chain constant region comprising a D at position 239, an L at position 330, and an E at position 332, numbered according to the EU numbering system.

[0061] In certain embodiments, the first antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy comprises the amino acid sequence of SEQ ID NO: 134, 135, or 136.

[0062] In certain embodiments, the first antibody comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 93-118.

[0063] In certain embodiments, the first antibody comprises the VL amino acid sequence of SEQ ID NO: 92.

[0064] In certain embodiments, the first antibody comprises the VL amino acid sequence of any one of SEQ ID NOs: 77-91.

[0065] In certain embodiments, the first antibody comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119-133.

[0066] In certain embodiments, the VH and VL of the first antibody comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87.

[0067] In certain embodiments, the VH and VL of the first antibody comprise the BUSINESS.32102391.1amino acid sequences, respectively, set forth in SEQ ID NOs: 52 and 80.

[0068] In certain embodiments, the first antibody comprises a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 93 and 119; 93 and 120; 94 and 121; 95 and 122; 96 and 123; 97 and 121; 98 and 124; 99 and 119; 100 and 125; 101 and 126; 102 and 127; 103 and 128; 104 and 121; 105 and 129; 106 and 130; 107 and 121; 108 and 121; 109 and 131; 110 and 131; 111 and 119; 112 and 122; 113 and 125; 114 and 123; 115 and 132; 116 and 133; 117 and 130; or 118 and 129.

[0069] In certain embodiments, the first antibody comprises a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 95 and 122.

[0070] covalently linked to the C-terminal residue of a heavy chain of the antibody.

[0071] covalently linked to the C-terminal residue of the heavy chain via a peptide linker. In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 139-148.

[0072] sequence at least 75% (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 137 or 138.

[0073] sequence of SEQ ID NO: 137 or 138.

[0074] In certain embodiments, the heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 149-157.

[0075] In certain embodiments, the first antibody that specifically binds to human FAP is an scFv, a Fab, or chimeric antigen receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] FIG. 1 is a graph showing the binding of anti-FAP antibodies, reference anti- FAP antibodies 28H1 and ESC11, or an isotype control antibody, to Chinese hamster ovary BUSINESS.32102391.1(CHO) cells expressing human FAP protein, as measured by flow cytometry. Binding is shown by the geometric median fluorescence intensity (MFI) values (y-axis) plotted against the respective antibody (x-axis) at a concentration of 20 µg / mL.

[0077] FIG. 2A-FIG.2B are graphs showing the binding of a dose series of anti-FAP antibodies, reference antibodies 28H1 and ESC11, and isotype control antibody, to CHO cells expressing human FAP, as measured by flow cytometry. Binding is shown by the MFI values (y-axis) relative to antibody concentrations from 20 to 0.0012 µg / mL (x-axis).

[0078] FIG. 3A-FIG.3B are graphs showing the binding of a dose series of anti-FAP antibodies, reference antibodies 28H1 and ESC11, and isotype control antibody, to CHO cells expressing cynomolgus monkey FAP, as measured by flow cytometry. Binding is shown by the geometric MFI values (y-axis) relative to serial dilutions of the antibody concentrations (x- axis).

[0079] FIG. 4A-FIG.4B are graphs showing the binding of a dose series of anti-FAP antibodies, reference antibodies 28H1 and ESC11, and isotype control antibody, to CHO cells expressing mouse FAP, as measured by flow cytometry. Binding is shown by the geometric MFI values (y-axis) relative to serial dilutions of the antibody concentrations (x-axis).

[0080] FIG. 5A-FIG.5B are graphs showing the binding of a dose series of anti-FAP antibodies, reference antibodies 28H1 and ESC11, and isotype control antibody, to glioblastoma cell line U138MG that endogenously expresses human FAP, as measured by flow cytometry. Binding is shown by the geometric MFI values (y-axis) relative to serial dilutions of the antibody concentrations (x-axis).

[0081] FIG. 6A-FIG. 6B are graphs showing the ability of anti-FAP antibodies, reference antibodies 28H1 and ESC11, and isotype control antibody, to activate Fc receptors (Fc R) on Jurkat reporter cells expressing Fc RIIIA (V158) and an NFAT–Luciferase reporter gene construct, in the presence of FAP-expressing human embryonic kidney (HEK) cells. Fc R activation is shown by Luminescence Units (LUM) counts per seconds (cps) (y-axis) relative to serial dilutions of the antibody concentrations (x-axis).

[0082] FIG. 7A-FIG. 7B are graphs showing the ability of anti-FAP antibodies, reference antibodies 28H1 and ESC11, and isotype control antibody, to mediate antibody dependent cellular cytotoxicity (ADCC) in FAP-expressing U138MG cells (FIG. 7A) and human embryonic kidney (HEK) cells (FIG.7B) by primary NK cells. Cellular cytotoxicity is plotted as percentage cell death relative to the no-treatment control (y-axis), versus serial dilutions of the antibody concentrations (x-axis). BUSINESS.32102391.1

[0083] FIG.8 distinct binding events that are consistent with the antibody binding simultaneously to human

[0084] FIG.9A-FIG.9B are graphs showing the binding of a dose series of anti-FAP- antibody, to CHO cells expressing human FAP, as measured by flow cytometry. Binding is shown by the gMFI values (y-axis) relative to serial dilutions of the antibody concentrations (x-axis).

[0085] FIG. 10A-FIG. 10D expressing human FAP (FIG. 10A), cynomolgus monkey FAP (FIG. 10B), or mouse FAP (FIG. 10C), or wild-type CHO cells as a control (FIG. 10D), as measured by flow cytometry. Binding is shown by the gMFI values (y-axis) relative to serial dilutions of the antibody concentrations (x-axis).

[0086] FIG. 11A-FIG. 11B driven luciferase reporter gene. The TGF -induced reporter gene activation is shown by Relative Luminescence Units (RLU) (y-axis) relative to serial dilutions of the antibody concentrations (x-axis)

[0087] FIG. 12A-FIG. 12C are graphs showing the ability of different Fc variants of expressing a SMAD-driven luciferase reporter gene. The TGF -induced reporter gene dilutions of the antibody concentrations (x-axis).

[0088] FIG. 13A-FIG. 13C expressing human Fc RIIB (FIG.13A), human Fc RIIIA F158 (low affinity allele) (FIG.13B), or human Fc RIIIA V158 (high affinity allele) (FIG. 13C), as measured by flow cytometry. Binding is shown by the gMFI values (y-axis) plotted against the respective antibody concentrations (µg / mL) (x-axis).

[0089] FIG.14A-FIG.14D are graphs showing the ability of Fc variants of anti-FAP- R receptors on Jurkat BUSINESS.32102391.1reporter cells expressing Fc RIIIA V158 (FIGs.14A and 14C) or human Fc RIIIA F158 (FIGs. 14B and 14D) and an NFAT-Luciferase reporter gene construct, in the presence of FAP- expressing HEK cells (FIGs. 14A and 14B) or U138MG cells (FIGs. 14C and 14D). Fc R activation is shown by the luminescence levels (y-axis) relative to serial dilutions of the antibody concentrations (x-axis).

[0090] FIG. 15A-FIG. 15E are graphs showing the ability Fc variants of anti-FAP- cellular cytotoxicity (ADCC) on FAP-expressing human embryonic kidney (HEK) cells (FIG. 15A) and U138MG cells (FIGs. 15B-15E), via NK92 cell line (FIGs. 15A-15B) or primary PBMC-derived NK cells (FIGs. 15C-15E). Cellular cytotoxicity is plotted as percentage cell death relative to the no-treatment control (y-axis) versus serial dilutions of the antibody concentrations (x-axis).

[0091] FIG.16A-FIG.16B are graphs showing the expression levels of red fluorescent protein (RFP), which correlate with TGF 1-induced expression of Vimentin protein in A549- as measured by flow cytometry. The RFP values are plotted as % Vimentin (y-axis) normalized to no treatment, in the presence of serial dilutions of the antibody concentrations (x-axis).

[0092] FIG. 17A-FIG.17B are graphs showing in vivo inhibition of tumor growth by anti-FAP-TGF RII bispecific antibodies in combination with anti-PD-1 treatment. FIG. 17A shows tumor growth curves of 10 mice / group implanted with MC38 tumor cells in the mammary fat pad and treated with the indicated antibodies at the indicated schedules. FIG.17B is a survival plot of the same groups.

[0093] FIG. 18A-FIG. 18D are graphs showing the in vivo pharmacodynamic effects of treatment with anti-FAP-TGF RII bispecific murine surrogate antibody PE22267 on tumor stroma and T cell infiltration of the tumor microenvironment. FIG.18A shows the % reduction control. FIG.18B is a histogram showing the motility of CD8+ T cells into the tumor core with anti-FAP-TGF RII bispecific antibody treatment, versus the tumor periphery. FIG.18C shows the increase in % of CD8+ T cell proliferation with anti-FAP-TGF RII bispecific antibody treatment, as measured using a Ki67 marker. FIG.18D shows TGF and 3) plasma levels in mice, as measured by Elisa (Luminex), compared to vehicle control.

[0094] FIG. 19A-FIG.19B are graphs showing in vivo inhibition of tumor growth by anti-FAP-TGF RII bispecific murine surrogate antibodies in combination with anti-CTLA-4 BUSINESS.32102391.1and anti-PD-1 antibodies. Tumor growth curves of 12 mice / group implanted with 4T1 tumor cells in the mammary fat pad and treated with either a combination of anti-CTLA-4 and anti- PD-1 antibodies, anti-FAP-TGF RII bispecific antibody alone, a combination of anti-FAP- TGF RII bispecific antibody with anti-CTLA-4 and anti-PD-1 antibodies, or isotype control at the indicated schedules are shown over 33 days.

[0095] FIG. 20A-FIG. 20R are a series of pictures and graphs showing the in vivo pharmacodynamic effects of treatment with anti-FAP-TGF RII bispecific murine surrogate antibody PE21208-1 and combination therapy with anti-CTLA-4 and anti-PD-1 antibodies on tumor stroma and T cell infiltration of the tumor microenvironment. FIG. 20A-FIG. 20D are representative pictures showing tumor stromal markers of ECM (collagen) in tumor tissue from mice treated with anti-FAP-TGF RII bispecific antibody alone or in combination with anti- CTLA-4 and anti-PD-1 antibodies, as compared to isotype control and anti-CTLA-4 + anti- PD-1 control. FIG.20E is a histogram quantifying the % reduction in collagen. FIG.20F-FIG. 20Q are representative pictures showing enhancement of CD3+ T cell infiltration in 4T1 tumors with the combination of anti-FAP-TGF RII bispecific antibody, anti-PD-1, and anti-CTLA-4 treatment. FIG. 20R shows the quantitative analysis of image data, revealing the increased frequency of CD3+ T cells (n=4 mice per group). Error bars represent mean±SEM; statistical significance denoted by *p<0.05, determined via unpaired T-test with Welch’s correction.

[0096] FIG. 21A-FIG.21E are graphs showing in vivo inhibition of tumor growth by anti-FAP-TGF RII bispecific murine surrogate antibody PE21208-1 in combination with anti- CTLA-4. FIG. 21A is a graph showing tumor growth over time in 13 mice / group implanted with MC38 tumor cells and treated with the indicated antibodies at the indicated schedules. FIG. 21B-FIG. 21E are graphs showing the individual spaghetti plots of each group. CR=complete response; PR=partial response. DETAILED DESCRIPTION

[0097] The present disclosure provides antibodies that specifically bind to FAP (e.g., provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies. The antibodies disclosed herein are particularly potent binders to FAP, and thus, are especially useful for increasing T cell infiltration of tumors, overcoming resistance to immunotherapy, and treating cancer in a subject. BUSINESS.32102391.1

[0098] The present disclosure also relates to methods of administering to subjects in need antibodies that specifically bind to fibroblast activation protein (FAP), including fusions immune checkpoint inhibitors, including antibodies that specifically bind to CTLA-4, PD-1, and / or PD-L1.

[0099] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of 5% to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., up to 5% to 10% below) the value or range remain within the intended meaning of the recited value or range.

[0100] As used herein, the term “FAP,” refers to fibroblast activation protein alpha. As used herein, the term “human FAP” refers to a FAP protein encoded by a wild-type human FAP gene, e.g., RefSeq accession number NG_027991.1. Exemplary immature wild-type human FAP amino acid sequences are provided as in RefSeq accession numbers NP_004451.2 and NP_001278736.1.

[0101] TGFB1, TGFB2, and TGFB3 by a human TGFB1 gene (e.g., a wild-type human TGFB1 gene). An exemplary wild-type TGFB2 gene (e.g., a wild-type human TGFB2 provided as in RefSeq accession numbers NP_001129071.1 and NP_003229.1. As used herein, TGFB3 gene (e.g., a wild-type human TGFB3 in RefSeq accession numbers NP_003230.1, NP_001316868.1, and NP_001316867.1.

[0102] As used herein, the terms “transforming growth factor beta receptor” and by the TGFBR1, TGFBR2, and TGFBR3 TGFBR1 gene (e.g., a wild-type human TGFBR1 by GenBank accession numbers NP_004603.1, NP_001124388.1, and NP_001293139.1. As encoded by a human TGFB2 gene (e.g., a wild-type human TGFB2 gene). Exemplary wild- BUSINESS.32102391.1TGFB3 gene (e.g., a wild-type human TGFB3 GenBank accession numbers NP_003230.1, NP_001316868.1, and NP_001316867.1.

[0103] As used herein, the terms “antibody” and “antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, without limitation, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above, and conjugates or fusion proteins comprising any of the above. In certain embodiments, antibodies described herein refer to polyclonal antibody populations. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1or IgA2), or any subclass (e.g., IgG2aor IgG2b) of immunoglobulin molecule. In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody. In certain embodiments, an antibody, as described herein, comprises a full-length antibody (e.g., a full-length antibody that specifically binds to FAP), or an antigen-binding fragment thereof, linked to a ligand binding moiety that specifically binds to a molecule other than FAP (e.g., a ligand binding e.g., a ligand binding moiety comprising one or more receptor comprising a VH and / or a VL disclosed herein linked to a transmembrane domain and one or more intracellular domains that are capable of providing signals for T-cell activation. BUSINESS.32102391.1

[0104] “Multispecific antibodies” are antibodies (e.g., bispecific antibodies) that specifically bind to two or more different antigens or two or more different regions of the same antigen. Multispecific antibodies include bispecific antibodies that contain two different antigen-binding sites (exclusive of the Fc region). Multispecific antibodies can include, for example, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, heteroconjugate antibodies, linked single-chain antibodies or linked-single-chain Fvs (scFv), camelized antibodies, affybodies, linked Fab fragments, F(ab’)2 fragments, chemically-linked Fvs, and disulfide-linked Fvs (sdFv). Multispecific antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, multispecific antibodies described herein are IgG antibodies, or a class (e.g., human IgG1, IgG2, or IgG4) or subclass thereof.

[0105] As used herein, the term “CDR” or “complementarity determining region” means the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are herein incorporated by reference in their entireties, where the definitions include overlapping or subsets of amino acid residues when compared against each other. In certain embodiments, the term “CDR” is a CDR as defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term “CDR” is a CDR as defined by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In certain embodiments, heavy chain CDRs and light chain CDRs of an antibody are defined using different conventions. In certain embodiments, heavy chain CDRs and / or light chain CDRs are defined by performing structural analysis of an antibody and identifying residues in the variable region(s) predicted to make contact with an epitope region of a target molecule (e.g., human and / or cynomolgus FAP). CDRH1, CDRH2, and CDRH3 denote the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 denote the light chain CDRs. BUSINESS.32102391.1

[0106] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0107] As used herein, the terms “VH” and “VL” refer to antibody heavy and light chain variable regions, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is herein incorporated by reference in its entirety.

[0108] As used herein, the terms “constant region” and “constant domain” are interchangeable and are common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with an Fc receptor (e.g., Fc gamma receptor).

[0109] As used herein, the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g. based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0110] As used herein, the term “light chain” when used in reference to an antibody can refer to any distinct type, e.g. BUSINESS.32102391.1of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.

[0111] As used herein, the terms “specifically binds,” “specifically recognizes,” “immunospecifically binds,” and “immunospecifically recognizes” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope or immune complex) as such binding is understood by one skilled in the art. For example, a molecule that specifically binds to an antigen can bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to the antigen with a KA that is at least 2 logs (e.g., factors of 10), 2.5 logs, 3 logs, 4 logs, or greater than the KA when the molecules bind non- specifically to another antigen.

[0112] As used herein, the term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0113] As used herein, the term “EU numbering system” refers to the EU numbering convention for the constant regions of an antibody, as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al, Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, each of which is herein incorporated by reference in its entirety.

[0114] As used herein, the term “binding moiety” refers to a moiety that has the ability to specifically bind to a target molecule or complex. The binding moiety can comprise a small molecule, peptide, modified peptide (e.g., peptides having non-natural amino acid residues, and / or stapled peptides), polypeptide, protein, antibody, antibody fragment, scFv, Fc- containing polypeptide, fusion antibody, ligand, aptamer, nucleic acid, variants thereof, or any combination thereof. In certain embodiments, the binding moiety comprises a domain of a natural protein or a variant thereof, wherein the natural protein binds to the target molecule or e.g. BUSINESS.32102391.1(e.g.

[0115] As used herein, the term “linked to” refers to covalent or noncovalent binding between two molecules or moieties. The skilled worker will appreciate that when a first molecule or moiety is linked to a second molecule or moiety, the linkage need not be direct, but instead, can be via an intervening molecule or moiety. For example, when a heavy chain variable region of a full-length antibody is linked to a ligand-binding moiety, the ligand- binding moiety can bind a constant region (e.g., a heavy chain constant region) of the full- length antibody (e.g., via a peptide bond), rather than bind directly to the heavy chain variable region.

[0116] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays (e.g., constraining peptides using CLIPS (Chemical Linkage of Peptides onto Scaffolds) to map discontinuous or conformational epitopes), and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, each of which is herein incorporated by reference in its entirety). Antibody:antigen crystals may be studied using well known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; U.S.2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), each of which is herein incorporated by reference in its entirety. Mutagenesis mapping studies may be accomplished using any method known to one of skill in BUSINESS.32102391.1the art. See, e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085, each of which is herein incorporated by reference in its entirety, for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0117] As used herein, the term “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration of an antibody to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0118] As used herein, the term “effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.

[0119] As used herein, the term “subject” includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In one embodiment, the subject is a human.

[0120] As used herein with respect to an antibody or polynucleotide, the term “isolated” refers to an antibody or polynucleotide that is separated from one or more contaminants (e.g., polypeptides, polynucleotides, lipids, or carbohydrates, etc.) which are present in a natural source of the antibody or polynucleotide. All instances of “isolated antibodies” described herein are additionally contemplated as antibodies that may be, but need not be, isolated. All instances of “isolated polynucleotides” described herein are additionally contemplated as polynucleotides that may be, but need not be, isolated. All instances of “antibodies” described herein are additionally contemplated as antibodies that may be, but need not be, isolated. All instances of “polynucleotides” described herein are additionally contemplated as polynucleotides that may be, but need not be, isolated.

[0121] The determination of “percent identity” between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is BUSINESS.32102391.1herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0122] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted. Anti-FAP antibodies

[0123] In one aspect, the instant disclosure provides antibodies that specifically bind to FAP (e.g., human FAP, cynomolgus FAP, or mouse FAP). The amino acid sequences of exemplary antibodies are set forth in Table 1. Table 1. Amino acid sequences of exemplary anti-FAP antibodies.BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1

[0124] In certain embodiments, the instant disclosure provides an isolated antibody that specifically binds to FAP (e.g., human FAP), the antibody comprising a VH domain comprising one, two, or all three of the CDRs of a VH domain set forth in Table 1. In certain embodiments, the antibody comprises the CDRH1 of a VH domain set forth in Table 1. In certain embodiments, the antibody comprises the CDRH2 of a VH domain set forth in Table 1. In certain embodiments, the antibody comprises the CDRH3 of a VH domain set forth in Table 1.

[0125] In certain embodiments, the instant disclosure provides an isolated antibody that specifically binds to FAP (e.g., human FAP), the antibody comprising a VL domain comprising one, two, or all three of the CDRs of a VL domain disclosed in Table 1 herein. In certain embodiments, the antibody comprises the CDRL1 of a VL domain set forth in Table 1. In certain embodiments, the antibody comprises the CDRL2 of a VL domain set forth in Table 1. In certain embodiments, the antibody comprises the CDRL3 of a VL domain set forth in Table 1. BUSINESS.32102391.1

[0126] The individual CDRs of an antibody disclosed herein can be determined according to any CDR numbering scheme known in the art.

[0127] In certain embodiments, one or more of the CDRs of an antibody disclosed herein can be determined according to Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest (1991), each of which is herein incorporated by reference in its entirety.

[0128] In certain embodiments, the instant disclosure provides antibodies that specifically bind to FAP (e.g., human FAP) and comprise CDRs of an antibody disclosed in Table 1 herein as determined by the Kabat numbering scheme.

[0129] In certain embodiments, one or more of the CDRs of an antibody disclosed herein can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No.7,709,226, all of which are herein incorporated by reference in their entireties).

[0130] In certain embodiments, the instant disclosure provides antibodies that specifically bind to FAP (e.g., human FAP) and comprise CDRs of an antibody disclosed in Table 1 herein, as determined by the Chothia numbering system.

[0131] In certain embodiments, one or more of the CDRs of an antibody disclosed herein can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745, herein incorporated by reference in its entirety. See also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), herein incorporated by reference in its entirety.

[0132] In certain embodiments, the instant disclosure provides antibodies that specifically bind to FAP (e.g., human FAP) and comprise CDRs of an antibody disclosed in Table 1 herein, as determined by the MacCallum numbering system.

[0133] In certain embodiments, the CDRs of an antibody disclosed herein can be determined according to the IMGT numbering system as described in: Lefranc M-P, (1999) The Immunologist 7: 132-136; Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212, each of which is herein incorporated by reference in its entirety; and Lefranc M-P et al., (2009) Nucleic Acids Res 37: D1006-D1012. BUSINESS.32102391.1

[0134] In certain embodiments, the instant disclosure provides antibodies that specifically bind to FAP (e.g., human FAP) and comprise CDRs of an antibody disclosed in Table 1 herein, as determined by the IMGT numbering system.

[0135] In certain embodiments, the CDRs of an antibody disclosed herein can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (Oxford Molecular Group, Inc.), herein incorporated by reference in its entirety.

[0136] In certain embodiments, the instant disclosure provides antibodies that specifically bind to FAP (e.g., human FAP) and comprise CDRs of an antibody disclosed in Table 1 herein as determined by the AbM numbering scheme.

[0137] In certain embodiments, the CDRs of an antibody disclosed herein can be determined according to the AHo numbering system, as described in Honegger and Plückthun, A., J. Mol. Biol.309:657-670 (2001), herein incorporated by reference in its entirety.

[0138] In certain embodiments, the instant disclosure provides antibodies that specifically bind to FAP (e.g., human FAP) and comprise CDRs of an antibody disclosed in Table 1 herein, as determined by the AHo numbering system.

[0139] In certain embodiments, the individual CDRs of an antibody disclosed herein are each independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, wherein the structural analysis identifies residues in the variable region(s) predicted to make contact with an epitope region of FAP.

[0140] In certain embodiments, the instant disclosure provides an antibody that specifically bind FAP (e.g., human FAP), wherein the antibody comprises a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 76, and a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 92, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, wherein the structural analysis identifies residues in the variable region(s) predicted to make contact with an epitope region of FAP (e.g., human FAP).

[0141] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), wherein the antibody comprises the CDRH1, 37 BUSINESS.32102391.1CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequence, respectively, set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, wherein the structural analysis identifies residues in the variable region(s) predicted to make contact with an epitope region of FAP (e.g., human FAP).

[0142] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), wherein the antibody comprises a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 11, 38, and 39.

[0143] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), wherein the antibody comprises a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 42, 46, and 49.

[0144] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), wherein the antibody comprises a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions, respectively, comprise the amino acid sequences set forth in SEQ ID NOs: 11, 38, 39, 42, 46, and 49.

[0145] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), wherein the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, and 39; 1, 13, and 39; 1, 14, and 39; 1, 15, and 39; 2, 16, and 39; 3, 17, and 39; 1, 18, and 39; 4, 19, and 39; 5, 20, and 39; 6, 21, and 39; 1, 22, and 39; 4, 23, and 39; 7, 24, and 39; 8, 25, and 39; 1, 26, and 39; 1, 27, and 39; 1, 28, and 39; 9, 29, and 39; 1, 30, and 39; 1, 31, and 39; 1, 32, and 39; 1, 33, and 39; 1, 34, and 39; 10, 35, and 39; 1, 36, and 39; or 1, 37, and 39.

[0146] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), wherein the antibody comprises the CDRL1, BUSINESS.32102391.1CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 40, 43, and 47; 40, 44, and 47; 40, 43, and 48; 41, 45, and 47; or 41, 43, and 47.

[0147] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, 39, 40, 43, and 47; 1, 12, 39, 40, 44, and 47; 1, 13, 39, 40, 43, and 47; 1, 14, 39, 40, 43, and 47; 1, 15, 39, 40, 43, and 47; 2, 16, 39, 40, 43, and 47; 3, 17, 39, 40, 43, and 48; 1, 18, 39, 40, 43, and 47; 4, 19, 39, 40, 43, and 47; 5, 20, 39, 41, 45, and 47; 6, 21, 39, 40, 43, and 47; 1, 22, 39, 40, 43, and 47; 4, 23, 39, 40, 43, and 47; 7, 24, 39, 40, 43, and 47; 8, 25, 39, 40, 43, and 47; 1, 26, 39, 40, 43, and 47; 1, 27, 39, 40, 43, and 47; 1, 28, 39, 40, 43, and 47; 9, 29, 39, 40, 43, and 47; 1, 30, 39, 40, 43, and 47; 1, 31, 39, 40, 43, and 47; 1, 32, 39, 40, 43, and 47; 1, 33, 39, 40, 43, and 47; 1, 34, 39, 41, 43, and 47; 10, 35, 39, 40, 43, and 47; 1, 36, 39, 40, 43, and 47; or 1, 37, 39, 40, 43, and 47. In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 14, 39, 40, 43, and 47.

[0148] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP) comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 76.

[0149] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP) comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence of any one of SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VH comprising the amino acid sequence of any one of SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, the amino acid sequence BUSINESS.32102391.1of the VH consists of the amino acid sequence of any one of SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75.

[0150] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 92. In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VL comprising an amino acid sequence set forth in SEQ ID NO: 92. In certain embodiments, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 92.

[0151] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence of any one of SEQ ID NOs: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VL comprising the amino acid sequence of any one of SEQ ID NOs: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In certain embodiments, the amino acid sequence of the VL consists of the amino acid sequence of any one of SEQ ID NOs: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91.

[0152] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 76, and a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 92. In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VH comprising the amino acid sequence of SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 92. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 76; and the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 92. BUSINESS.32102391.1

[0153] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in any one of SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75, and a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in any one of SEQ ID NOs: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising a VH comprising an amino acid sequence of any one of SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75, and a VL comprising an amino acid sequence of any one of SEQ ID NOs: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in any one of SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75; and the amino acid sequence of the VL consists of the amino acid sequence set forth in any one of SEQ ID NOs: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91.

[0154] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 76 and 92, respectively. In certain embodiments, the amino acid sequences of VH and VL consist of the amino acid sequences set forth in SEQ ID NOs: 76 and 92, respectively.

[0155] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87, respectively. In certain embodiments, the amino acid sequences of VH and VL consist of the amino acid sequences set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and BUSINESS.32102391.191; 74 and 88; or 75 and 87, respectively. In certain embodiments, the VH and VL comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 52 and 80. In certain embodiments, the VH and VL consist of the amino acid sequences, respectively, set forth in SEQ ID NOs: 52 and 80.

[0156] In certain embodiments, the instant disclosure provides an antibody that cross- competes for binding to FAP (e.g., human FAP) with an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 76 and 92, respectively. In certain embodiments, the instant disclosure provides an antibody that cross-competes for binding to FAP (e.g., human FAP) with an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87, respectively.

[0157] In certain embodiments, the instant disclosure provides an antibody that binds to the same or an overlapping epitope of FAP (e.g., an epitope of human FAP) as an antibody described herein, e.g., an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 76 and 92, respectively. In certain embodiments, the instant disclosure provides an antibody that binds to the same or an overlapping epitope of FAP (e.g., an epitope of human FAP) as an antibody described herein, e.g., an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87, respectively.

[0158] In certain embodiments, the epitope of an antibody can be determined by, e.g., NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are herein incorporated by reference in their entireties). Antibody:antigen crystals may be studied using well known X- BUSINESS.32102391.1ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.; U.S. Patent Application No.2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323, all of which are herein incorporated by reference in their entireties). Mutagenesis mapping studies may be accomplished using any method known to one of skill in the art. See, e.g., Champe M et al., (1995) supra and Cunningham BC & Wells JA (1989) supra for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques. In a specific embodiment, the epitope of an antibody is determined using alanine scanning mutagenesis studies. In addition, or antibodies that recognize and bind to the same or overlapping epitopes of FAP (e.g., human FAP) can be identified using routine techniques such as an immunoassay, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competition binding assays also can be used to determine whether two antibodies have similar binding specificity for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as FAP (e.g., human FAP). Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253); solid phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137: 3614-9); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1): 7-15); solid phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176: 546-52); and direct labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32: 77-82), all of which are herein incorporated by reference in their entireties. Typically, such an assay involves the use of purified antigen (e.g., FAP, such as human FAP) bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually, the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it will inhibit BUSINESS.32102391.1specific binding of a reference or antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. A competition binding assay can be configured in a large number of different formats using either labeled antigen or labeled antibody. In a common version of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block the binding of labeled antibodies to the antigen is then measured using radioactive or enzyme labels. For further details see, for example, Wagener C et al., (1983) J Immunol 130: 2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest 21: 523- 538; Kuroki M et al., (1992) Hybridoma 11: 391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, pp. 386-389, all of which are herein incorporated by reference in their entireties.

[0159] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), the antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118.

[0160] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), the antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133. In certain embodiments, the amino acid sequence of the light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133.

[0161] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), comprising the heavy chain and light chain, wherein the heavy chain and light chain comprise the amino acid sequences of SEQ ID NOs: 93 and 119; 93 and 120; 94 and 121; 95 and 122; 96 and 123; 97 and 121; 98 and 124; 99 and 119; 100 and 125; 101 and 126; 102 and 127; 103 and 128; 104 and 121; 105 and 129; 106 and 130; 107 and 121; 108 and 121; 109 and 131; 110 and 131; 111 and 119; 112 and 122; 113 and 125; 114 and 123; 115 and 132; 116 and 133; 117 and 130; or 118 and 129, respectively.

[0162] In certain embodiments, the amino acid sequences of the heavy chain and light chain consist of amino acid sequences selected from the groups consisting of SEQ ID NOs: 93 BUSINESS.32102391.1and 119; 93 and 120; 94 and 121; 95 and 122; 96 and 123; 97 and 121; 98 and 124; 99 and 119; 100 and 125; 101 and 126; 102 and 127; 103 and 128; 104 and 121; 105 and 129; 106 and 130; 107 and 121; 108 and 121; 109 and 131; 110 and 131; 111 and 119; 112 and 122; 113 and 125; 114 and 123; 115 and 132; 116 and 133; 117 and 130; or 118 and 129, respectively.

[0163] The anti-FAP antigen-binding molecules of the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bispecific or a multispecific antibody with a second or additional binding specificity.

[0164] In certain embodiments, the antibody disclosed herein is conjugated to a cytotoxic agent, cytostatic agent, toxin, radionuclide, or detectable label. In certain embodiments, the cytotoxic agent is able to induce death or destruction of a cell in contact therewith. In certain embodiments, the cytostatic agent is able to prevent or substantially reduce proliferation and / or inhibits the activity or function of a cell in contact therewith. In certain embodiments, the cytotoxic agent or cytostatic agent is a chemotherapeutic agent. In certain embodiments, the radionuclide is selected from the group consisting of the isotopes3H,14C,32P,35S,36Cl,51Cr,57Co,58Co,59Fe,67Cu,90Y,99Tc,111In,117Lu,121I,124I,125I,131I,198Au,211At,213Bi,225Ac, and186Re. In certain embodiments, the detectable label comprises a fluorescent moiety or a click chemistry handle.

[0165] Any immunoglobulin (Ig) constant region can be used in the antibodies disclosed herein. In certain embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.

[0166] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), the antibody comprising a heavy chain constant region, optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, and IgM.

[0167] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), the antibody comprising a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy BUSINESS.32102391.1

[0168] In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), the antibody comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 134, 135, or 136. In certain embodiments, the instant disclosure provides an antibody that specifically binds to FAP (e.g., human FAP), the antibody comprising a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 134, 135, or 136.

[0169] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into an Fc region (e.g., a CH2 domain (residues 231-340 of human IgG1)) and / or a CH3 domain (residues 341-447 of human IgG1, numbered according to the EU numbering system) and / or a hinge region (residues 216-230, numbered according to the EU numbering system) of an antibody described herein, to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0170] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of an antibody described herein, such that the number of cysteine residues in the hinge region is altered (e.g., increased or decreased) as described in, e.g., U.S. Patent No. 5,677,425, herein incorporated by reference in its entirety. The number of cysteine residues in the hinge region may be altered to, e.g., facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody.

[0171] In a specific embodiment, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant region, or FcRn- binding fragment thereof (preferably an Fc or hinge-Fc fragment) to alter (e.g., decrease or increase) half-life of the antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are herein incorporated by reference in their entireties, for examples of mutations that will alter (e.g., decrease or increase) the half-life of an antibody in vivo. In certain embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant region, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc fragment) to decrease the half-life of the antibody in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant region, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc fragment) to increase the half-life of the antibody in vivo. In a specific embodiment, the antibodies may have one or more amino acid mutations (e.g., BUSINESS.32102391.1substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU numbering system. In a specific embodiment, the constant region of the IgG1 of antibody described herein comprises a methionine (M) to tyrosine (Y) substitution in position 252, a serine (S) to threonine (T) substitution in position 254, and a threonine (T) to glutamic acid (E) substitution in position 256, numbered according to the EU numbering system. See U.S. Patent No. 7,658,921, which is herein incorporated by reference in its entirety. This type of mutant IgG, referred to as “YTE mutant” has been shown to display fourfold increased half- life as compared to wild-type versions of the same antibody (see Dall’Acqua WF et al., (2006) J Biol Chem 281: 23514-24, which is herein incorporated by reference in its entirety). In certain embodiments, an antibody comprises an IgG constant region comprising one, two, three, or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0172] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into an Fc region (e.g., a CH2 domain (residues 231-340 of human IgG1)) and / or a CH3 domain (residues 341-447 of human IgG1, numbered according to the EU numbering system) and / or a hinge region (residues 216-230, numbered according to the EU numbering system) of an antibody described herein, to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into the Fc receptor or fragment thereof are known to one of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody for an Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, all of which are herein incorporated by reference in their entireties.

[0173] In certain embodiments, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain variant human heavy chain constant region, e.g., a variant human IgG1, a variant human IgG2, or a variant human IgG4heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region comprises one or more of the following amino acid BUSINESS.32102391.1mutations, according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In certain embodiments, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F, according to the EU numbering system. In certain embodiments, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0174] In a further embodiment, one, two, or more amino acid substitutions are introduced into an IgG constant region Fc region to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered according to the EU numbering system, can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen- binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each of which is herein incorporated by reference in its entirety. In certain embodiments, the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating antibody thereby increasing tumor localization. See, e.g., U.S. Patent Nos.5,585,097 and 8,591,886, each of which is herein incorporated by reference in its entirety, for a description of mutations that delete or inactivate the constant region and thereby increase tumor localization. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276: 6591-604, which is herein incorporated by reference in its entirety). In various embodiments, one or more of the following mutations in the constant region of an antibody described herein may be made: an N297A substitution; an N297Q substitution; an L234A substitution; an L234F substitution; an L235A substitution; an L235F substitution; an L235V substitution; an L237A substitution; an S239D substitution; an E233P substitution; an L234V substitution; a C236 deletion; a P238A substitution; an F243L substitution; a D265A substitution; an S267E substitution; an L328F substitution; an R292P substitution; a Y300L BUSINESS.32102391.1substitution; an A327Q substitution; a P329A substitution; an A330L substitution; an I332E substitution; or a P396L substitution, numbered according to the EU numbering system.

[0175] In certain embodiments, a mutation selected from the group consisting of D265A, P329A, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235A, L237A, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of S239D, I332E, optionally A330L, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein.

[0176] In a specific embodiment, an antibody described herein comprises the constant region of an IgG1with an N297Q or N297A amino acid substitution, numbered according to the EU numbering system. In certain embodiments, an antibody described herein comprises the constant region of an IgG1 with a mutation selected from the group consisting of D265A, P329A, and a combination thereof, numbered according to the EU numbering system. In another embodiment, an antibody described herein comprises the constant region of an IgG1 with a mutation selected from the group consisting of L234A, L235A, and a combination thereof, numbered according to the EU numbering system. In another embodiment, an antibody described herein comprises the constant region of an IgG1 with a mutation selected from the group consisting of L234F, L235F, N297A, and a combination thereof, numbered according to the EU numbering system. In certain embodiments, amino acid residues in the constant region of an antibody described herein in the positions corresponding to positions L234, L235, and D265 in a human IgG1heavy chain, numbered according to the EU numbering system, are not L, L, and D, respectively. This approach is described in detail in International Publication No. BUSINESS.32102391.1WO 14 / 108483, which is herein incorporated by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively, numbered according to the EU numbering system.

[0177] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 in the constant region of an antibody described herein, numbered according to the EU numbering system, can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.), which is herein incorporated by reference in its entirety. In certain embodiments, one or more amino acid residues within amino acid positions 231 to 238 in the N-terminal region of the CH2 domain of an antibody described herein are altered to thereby alter the ability of the antibody to fix complement, numbered according to the EU numbering system. This approach is described further in International Publication No. WO 94 / 29351, which is herein incorporated by reference in its entirety. In certain embodiments, the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fc receptor by mutating one or more amino acids (e.g., introducing amino acid substitutions) at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, numbered according to the EU numbering system. This approach is described further in International Publication No. WO 00 / 42072, which is herein incorporated by reference in its entirety.

[0178] In certain embodiments, an antibody described herein comprises a modified constant region of an IgG1, wherein the modification increases the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC). In certain embodiments, 0.1, 1, or 10 µg / mL of the antibody is capable of inducing cell death of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of FAP-expressing cells within 1, 2, or 3 hours, as assessed by methods described herein and / or known to a person of skill in the art. In certain embodiments, the modified constant region of an IgG1comprises S239D and I332E substitutions, numbered according to the EU numbering system. In certain embodiments, the modified constant region BUSINESS.32102391.1of an IgG1 comprises S239D, A330L, and I332E substitutions, numbered according to the EU numbering system. In certain embodiments, the modified constant region of an IgG1 comprises L235V, F243L, R292P, Y300L, and P396L substitutions, numbered according to the EU numbering system. In certain embodiments, the antibody is capable of inducing cell death in effector T cells and Tregs, wherein the percentage of Tregs that undergo cell death is higher than the percentage of effector T cells that undergo cell death by at least 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, or 5 fold.

[0179] In certain embodiments, an antibody described herein comprises the constant region of an IgG2 antibody and the cysteine at amino acid residue 127 of the heavy chain, numbered according to the EU numbering system, is substituted for serine.

[0180] In certain embodiments, an antibody described herein comprises the constant region of an IgG4 antibody and the serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is substituted for proline.

[0181] In certain embodiments, an antibody described herein comprises the constant region of an IgG1 antibody comprising a D at position 239, an L at position 330, and an E at position 332, numbered according to the EU numbering system.

[0182] In certain embodiments, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody described herein having two heavy chain constant regions. Anti-FAP antibodies comprising ligand binding moieties

[0183] In one aspect, the instant disclosure provides antibodies that specifically bind to FAP (e.g., human FAP, cynomolgus FAP, or mouse FAP) and comprise a ligand binding moiety (e.g.

[0184] Table 2 herein.BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1BUSINESS.32102391.1

[0185] A ligand binding moiety (e.g. portion of an immunoglobulin chain of an anti-FAP antibody, including the N- or C-terminal amino acid residue. The ligand binding moiety (e.g. (covalently or non-covalently) to the immunoglobulin chain directly or via a linker (e.g., a peptide linker). Covalent linkage can be a chemical linkage or a genetic linkage (i.e., to form a fusion protein). In certain embodiments, the ligand binding moiety (e.g. moiety) is linked (e.g., covalently) to the C-terminal amino acid residue of the immunoglobulin chain. In certain embodiments, the ligand binding moiety (e.g. linked to the C-terminal amino acid residue of an immunoglobulin chain without a linker (e.g., via a peptide bond). In certain embodiments, the ligand binding moiety (e.g. BUSINESS.32102391.1moiety) is linked (e.g., covalently) to the C-terminal amino acid residue of the immunoglobulin chain via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker has the general formula (G4S)n, where n is an integer. In certain embodiments, the peptide linker comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 139, 140, 141, 142, 143, 144, 145, 146, 147, or 148. In certain embodiments, the peptide linker consists of an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 139, 140, 141, 142, 143, 144, 145, 146, 147, or 148. In certain embodiments, the peptide linker is linked to the immunoglobulin chain via a peptide bond.

[0186] In certain embodiments, the ligand binding moiety (e.g. moiety) is linked (e.g., covalently) to a heavy chain of the anti-FAP antibody. The ligand binding moiety (e.g. e.g., covalently) to any portion of the heavy chain. In certain embodiments, the ligand binding moiety (e.g. moiety) is linked (e.g., covalently) to the C-terminal amino acid residue of the heavy chain. In certain embodiments, the ligand binding moiety (e.g. C-terminal amino acid residue of the heavy chain without a linker (e.g., via a peptide bond). In certain embodiments, the ligand binding moiety (e.g. e.g., covalently) to the C-terminal amino acid residue of the heavy chain via linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 139, 140, 141, 142, 143, 144, 145, 146, 147, or 148. In certain embodiments, the peptide linker consists of an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 139, 140, 141, 142, 143, 144, 145, 146, 147, or 148. In certain embodiments, the peptide linker is linked to the heavy chain via a peptide bond.

[0187] In certain embodiments, the anti-FAP antibody comprises a binding moiety the anti-FAP antibody comprises a binding moiety capable of antagonizing and / or inhibiting BUSINESS.32102391.1e.g. e.g. e.g. e.g. e.g., human e.g. e.g. e.g. e.g. e.g. e.g. et al. (2008) Development, Humana Press; Zwaagstra et al. (2012) Mol Cancer Ther. 11(7):1477-87; Ravi et al. (2018) Nat. Commun. 9:741; EP0975771B1, US7786261B2, US8993524B2, and US20150225483A1, each of which is incorporated herein by reference in its entirety.

[0188] e.g. e.g. (e.g. e.g., a e.g. e.g. e.g., human e.g. moiety comprises an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in binding moiety consists of an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth amino acid sequence set forth in SEQ ID NO: 137 or 138.

[0189] moiety comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, BUSINESS.32102391.191%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in binding moiety comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth binding moiety comprising the amino acid sequence set forth in SEQ ID NO: 137 or 138. In an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 137 or 138. of the amino acid sequence set forth in SEQ ID NO: 137 or 138.

[0190] moieties comprise the amino acid sequences set forth in SEQ ID NOs: 137 and 138; 137 and 137; or 138 and 138, respectively. In certain embodiments, the anti-FAP antibody comprises a 138; 137 and 137; or 138 and 138, respectively. In certain embodiments, the anti-FAP antibody amino acid sequence set forth in SEQ ID NO: 137. In certain embodiments, the anti-FAP comprises the amino acid sequence set forth in SEQ ID NO: 138.

[0191] e.g. e.g. (e.g. antibodies include fresolimumab and metelimumab, and are described in US6492497B1, US7151169B2, US7723486B2, which are incorporated herein by reference in their entirety.

[0192] In certain embodiments, the antibody that specifically binds to FAP (e.g., amino acid sequence set forth in any one of SEQ ID NO: 149, 150, 151, 152, 153, 154, 155, 156, or 157. In certain embodiments, the antibody that specifically binds to FAP (e.g., human BUSINESS.32102391.1acid sequence set forth in any one of SEQ ID NO: 149, 150, 151, 152, 153, 154, 155, 156, or 157.

[0193] In certain embodiments, the antibody that specifically binds to FAP (e.g., light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 157 and 122; 150 and 122; 156 and 120; 149 and 121; 154 and 124; 151 and 119; 152 and 121; 153 and 130; or 155 and 122.

[0194] In certain embodiments, the antibody that specifically binds to FAP (e.g., light chain consisting of the amino acid sequences, respectively, set forth in SEQ ID NOs: 157 and 122; 150 and 122; 156 and 120; 149 and 121; 154 and 124; 151 and 119; 152 and 121; 153 and 130; or 155 and 122.

[0195] In certain embodiments, the antibody that specifically binds to FAP (e.g., light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 157 and 122. In certain embodiments, the antibody that specifically binds to FAP (e.g., human consisting of the amino acid sequences, respectively, set forth in SEQ ID NOs: 157 and 122. Pharmaceutical compositions

[0196] Provided herein are compositions comprising an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody disclosed herein having the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as BUSINESS.32102391.1sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN , PLURONIC , or polyethylene glycol (PEG).

[0197] In a specific embodiment, pharmaceutical compositions comprise an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody disclosed herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In a specific embodiment, pharmaceutical compositions comprise an effective amount of an antibody described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In certain embodiments, the antibody is the only active ingredient included in the pharmaceutical composition. Pharmaceutical compositions described herein can be useful in decreasing or inhibiting FAP (e.g., human, mouse, or cynomolgus FAP) activity and treating a condition, such as cancer. In one embodiment, the present invention relates to a pharmaceutical composition of the present invention comprising an anti-FAP antibody of the present invention for use as a medicament. In another embodiment, the present invention relates to a pharmaceutical composition of the present invention for use in a method for the treatment of cancer.

[0198] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include Sodium Chloride Injection, Ringer’s Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringer’s Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN®80). A sequestering or chelating agent of metal ions includes EDTA. Pharmaceutical carriers also BUSINESS.32102391.1include ethyl alcohol, polyethylene glycol, and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0199] A pharmaceutical composition may be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral. Parenteral administration, characterized by either subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0200] Preparations for parenteral administration of an antibody include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.

[0201] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0202] Topical mixtures comprising antibody proteins are prepared as described for the local and systemic administration. The resulting mixture can be a solution, suspension, emulsions, or the like and can be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches, or any other formulations suitable for topical administration.

[0203] An anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody disclosed herein can be formulated as an aerosol for topical application, such as by inhalation (see, e.g., U.S. Patent Nos.4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma and are herein BUSINESS.32102391.1incorporated by reference in their entireties). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflations, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will, in one embodiment, have diameters of less than 50 microns, in one embodiment less than 10 microns.

[0204] An anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody disclosed herein can be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the antibody alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[0205] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art, and can be used to administer an antibody. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are herein incorporated by reference in their entireties.

[0206] In certain embodiments, a pharmaceutical composition comprising an antibody described herein is a lyophilized powder, which can be reconstituted for administration as solutions, emulsions, and other mixtures. It may also be reconstituted and formulated as solids or gels. The lyophilized powder is prepared by dissolving an antibody described herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In certain embodiments, the lyophilized powder is sterile. The solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. Reconstitution of this lyophilized powder BUSINESS.32102391.1with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends upon the selected compound. Such amount can be empirically determined.

[0207] The anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies disclosed herein and other compositions provided herein can also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are herein incorporated by reference in their entireties. In a specific embodiment, an antibody described herein is targeted to a tumor.

[0208] The compositions to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes. Methods of use and uses

[0209] In another aspect, the instant disclosure provides a method of treating a subject using the anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies disclosed herein. Any disease or disorder in a subject that would benefit from decrease of FAP (e.g., human, mouse, FAP (e.g., human, mouse or cynomolgus FAP) antibodies disclosed herein. The anti-FAP (e.g., human FAP) antibodies disclosed herein are particularly useful for increasing T cell infiltration of tumors, and accordingly can be used as an immunotherapy for subjects with cancer. For example, in certain embodiments, the instant disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody, polynucleotide, vector, or pharmaceutical composition, as disclosed herein.

[0210] In another aspect, the instant disclosure provides a method of increasing T cell infiltration of a tumor in a subject using the anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies disclosed herein. The antigen binding molecules can also be used to induce mesenchymal transition (EMT) in tumor cells, debulk the tumor stroma, and / or deplete immunosuppressive FAP+cancer-associated fibroblasts (CAFs). BUSINESS.32102391.1

[0211] The anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies provided herein can be used for treating diseases characterized by FAP expression, particularly by abnormal expression (e.g., overexpression, or expression in a different pattern in the cell) of FAP compared to normal tissue of the same cell type.

[0212] The anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies disclosed herein can be used to inhibit tumor growth or kill tumor cells. For example, the anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies can bind to FAP that is on the membrane or cell surface of cancerous cells (tumor cells or cells of the tumor stroma) and elicit, e.g., ADCC or other effector-mediated killing of the cancerous cells.

[0213] The anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies can alternatively be used in order to block the function of FAP, for example by physically interfering with its binding of another compound. For example, the antigen binding molecules can be used to block the enzymatic activity of FAP (e.g., serine peptidase, gelatinase, collagenase activity), FAP mediated ECM degradation, and / or FAP mediated cell invasion or migration.

[0214] In one aspect, an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody for use as a medicament is provided. In further aspects, an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody for use in treating a disease characterized by expression of FAP is provided. In certain embodiments, an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody for use in a method of treating a subject having a disease characterized by expression of FAP, comprising administering to the subject an effective amount of the anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is an anti-cancer agent, e.g., a chemotherapeutic agent, an inhibitor of tumor cell proliferation, or an activator of tumor cell apoptosis.

[0215] In a further aspect, the use of an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody in the manufacture or preparation of a medicament is provided. In one embodiment, the medicament is for treatment of a disease characterized by expression of FAP. In a further embodiment, the medicament is for use in a method of treating a disease characterized by expression of FAP comprising administering to a subject having a disease BUSINESS.32102391.1characterized by expression of FAP an effective amount of the medicament. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is an anti-cancer agent, e.g., a chemotherapeutic agent, an inhibitor of tumor cell proliferation, or an activator of tumor cell apoptosis.

[0216] For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the antibody, and the discretion of the attending physician.

[0217] In one embodiment, the present invention relates to an antibody and / or pharmaceutical composition of the present invention for use in a method of the present invention, wherein the method further comprises administering an additional therapeutic agent to the subject. In one embodiment, the present invention relates to (a) an antibody and / or pharmaceutical composition of the present invention and (b) an additional therapeutic agent for use as a medicament. In one embodiment, the present invention relates to (a) an antibody and / or pharmaceutical composition of the present invention and (b) an additional therapeutic agent for use in a method for the treatment of cancer. In a further embodiment, the present invention relates to a pharmaceutical composition, kit or kit-of-parts comprising (a) an antibody and / or pharmaceutical composition of the present invention and (b) an additional therapeutic agent. In one embodiment, the additional therapeutic agent is a chemotherapeutic, a radiotherapeutic, or a checkpoint targeting agent.

[0218] In certain embodiments, an anti-PD-1 antibody is used in methods disclosed herein. In certain embodiments, the anti-PD-1 antibody is nivolumab, also known as BMS- 936558 or MDX1106, developed by Bristol-Myers Squibb. In certain embodiments, the anti- PD-1 antibody is pembrolizumab, also known as lambrolizumab or MK-3475, developed by Merck & Co. In certain embodiments, the anti-PD-1 antibody is pidilizumab, also known as CT-011, developed by CureTech. In certain embodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514, developed by MedImmune. In certain embodiments, the anti-PD-1 antibody is PDR001 developed by Novartis Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is REGN2810 developed by Regeneron Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is PF-06801591 developed BUSINESS.32102391.1by Pfizer. In certain embodiments, the anti-PD-1 antibody is BGB-A317 developed by BeiGene. In certain embodiments, the anti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. In certain embodiments, the anti-PD-1 antibody is SHR-1210 developed by Hengrui.

[0219] Further non-limiting examples of anti-PD-1 antibodies that may be used in treatment methods disclosed herein are disclosed in the following patents and patent applications, all of which are herein incorporated by reference in their entireties for all purposes: U.S. Patent No. 6,808,710; U.S. Patent No. 7,332,582; U.S. Patent No. 7,488,802; U.S. Patent No.8,008,449; U.S. Patent No.8,114,845; U.S. Patent No.8,168,757; U.S. Patent No. 8,354,509; U.S. Patent No. 8,686,119; U.S. Patent No. 8,735,553; U.S. Patent No. 8,747,847; U.S. Patent No. 8,779,105; U.S. Patent No. 8,927,697; U.S. Patent No. 8,993,731; U.S. Patent No.9,102,727; U.S. Patent No.9,205,148; U.S. Publication No. US 2013 / 0202623 A1; U.S. Publication No. US 2013 / 0291136 A1; U.S. Publication No. US 2014 / 0044738 A1; U.S. Publication No. US 2014 / 0356363 A1; U.S. Publication No. US 2016 / 0075783 A1; and PCT Publication No. WO 2013 / 033091 A1; PCT Publication No. WO 2015 / 036394 A1; PCT Publication No. WO 2014 / 179664 A2; PCT Publication No. WO 2014 / 209804 A1; PCT Publication No. WO 2014 / 206107 A1; PCT Publication No. WO 2015 / 058573 A1; PCT Publication No. WO 2015 / 085847 A1; PCT Publication No. WO 2015 / 200119 A1; PCT Publication No. WO 2016 / 015685 A1; and PCT Publication No. WO 2016 / 020856 A1.

[0220] An antibody or pharmaceutical composition described herein may be delivered to a subject by a variety of routes. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intra-arterial, and subcutaneous routes. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray. In certain embodiments, the antibody or pharmaceutical composition described herein is delivered subcutaneously or intravenously. In certain embodiments, the antibody or pharmaceutical composition described herein is delivered intra-arterially. In certain embodiments, the antibody or pharmaceutical composition described herein is delivered intratumorally. In certain embodiments, the antibody or pharmaceutical composition described herein is delivered into a tumor draining lymph node.

[0221] The amount of an antibody or composition which will be effective in the treatment and / or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques. BUSINESS.32102391.1

[0222] The precise dose to be employed in a composition will also depend on the route of administration, and the seriousness of the infection or disease caused by it, and should be decided according to the judgment of the practitioner and each subject’s circumstances. For example, effective doses may also vary depending upon means of administration, target site, physiological state of the patient (including age, body weight, and health), whether the patient is human or an animal, other medications administered, or whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0223] An anti-FAP (e.g., human. mouse, or cynomolgus FAP) antibody described herein can also be used to assay FAP (e.g., human, mouse, or cynomolgus FAP) protein levels in a biological sample using classical immunohistological methods known to those of skill in the art, including immunoassays, such as the enzyme linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I,121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Such labels can be used to label an antibody described herein. Alternatively, a second antibody that recognizes an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein can be labeled and used in combination with an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody to detect FAP (e.g., human, mouse, or cynomolgus FAP) protein levels. Therefore, in one embodiment, the present invention relates to the use of an antibody of the present invention for in vitro detection of FAP (e.g., human, mouse, or cynomolgus FAP) protein in a biological sample. In a further embodiment, the present invention relates to the use of an anti-FAP antibody of the invention, for assaying and / or detecting FAP (e.g., human, mouse, or cynomolgus FAP) protein levels in a biological sample in vitro, optionally wherein the anti-FAP antibody is conjugated to a radionuclide or detectable label, and / or carries a label described herein, and / or wherein an immunohistological method is used.

[0224] Assaying for the expression level of FAP (e.g., human, mouse, or cynomolgus FAP) protein is intended to include qualitatively or quantitatively measuring or estimating the level of FAP (e.g., human, mouse, or cynomolgus FAP) protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level) or relatively BUSINESS.32102391.1(e.g., by comparing to the disease associated protein level in a second biological sample). FAP (e.g., human, mouse, or cynomolgus FAP) polypeptide expression level in the first biological sample can be measured or estimated and compared to a standard FAP (e.g., human, mouse, or cynomolgus FAP) protein level, the standard being taken, for example, from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once the “standard” FAP (e.g., human, mouse, or cynomolgus FAP) polypeptide level is known, it can be used repeatedly as a standard for comparison. Therefore, in a further embodiment, the present invention relates to an in vitro method for assaying and / or detecting FAP protein levels, for example human FAP protein levels, in a biological sample, comprising qualitatively or quantitatively measuring or estimating the level of FAP protein, for example of human FAP protein, in a biological sample, by an immunohistological method.

[0225] As used herein, the term “biological sample” refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing FAP (e.g., human, mouse, or cynomolgus FAP). Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Biological samples include peripheral mononuclear blood cells.

[0226] An anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description. Prognostic, diagnostic, monitoring and screening assays and kits for in vitro assessment and evaluation of immune system status and / or immune response may be utilized to predict, diagnose, and monitor to evaluate patient samples, including those known to have or suspected of having an immune system-dysfunction or with regard to an anticipated or desired immune system response, antigen response or vaccine response. The assessment and evaluation of immune system status and / or immune response is also useful in determining the suitability of a patient for a clinical trial of a drug or for the administration of a particular chemotherapeutic agent, a radiotherapeutic agent, or an antibody, including combinations thereof, versus a different agent or antibody. This type of prognostic and diagnostic monitoring and assessment is already in practice utilizing antibodies against the HER2 protein in breast cancer (HercepTestTM, Dako) where the assay is also used to evaluate patients for antibody therapy using Herceptin®. In vivo applications include directed cell therapy and immune system BUSINESS.32102391.1modulation and radio imaging of immune responses. Therefore, in one embodiment, the present invention relates to an anti-FAP antibody and / or pharmaceutical composition of the present invention for use as a diagnostic. In one embodiment, the present invention relates to an anti- FAP antibody and / or pharmaceutical composition of the present invention for use in a method for the prediction, diagnosis and / or monitoring of a subject having or suspected to have an immune system-dysfunction and / or with regard to an anticipated or desired immune system response, antigen response or vaccine response. In another embodiment, the present invention relates to the use of anti-FAP antibody of the invention, for predicting, diagnosing, and / or monitoring of a subject having or suspected to have an immune system-dysfunction and / or with regard to an anticipated or desired immune system response, antigen response, or vaccine response by assaying and / or detecting human FAP protein levels in a biological sample of the subject in vitro.

[0227] In one embodiment, an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody can be used in immunohistochemistry of biopsy samples. In one embodiment, the method is an in vitro method. In another embodiment, an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody can be used to detect levels of FAP (e.g., human, mouse, or cynomolgus FAP), or levels of cells which contain FAP (e.g., human, mouse, or cynomolgus FAP) on their membrane surface, the levels of which can then be linked to certain disease symptoms. Anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies described herein may carry a detectable or functional label, and / or may be conjugated to a radionuclide or detectable label. When fluorescence labels are used, currently available microscopy and fluorescence-activated cell sorter analysis (FACS) or combination of both methods procedures known in the art may be utilized to identify and to quantitate the specific binding members. Anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies described herein may carry or may be conjugated to a fluorescence label. Exemplary fluorescence labels include, for example, reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes. An anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody may carry or may be conjugated to a radioactive label or radionuclide, such as the isotopes3H,14C,32P,35S,36Cl,51Cr,57Co,58Co,59Fe,67Cu,90Y,99Tc,111In,117Lu,121I,124I,125I,131I,198Au,211At,213Bi,225Ac, and186Re. When radioactive labels are used, currently available counting procedures known in the art may be utilized to identify and quantitate the specific binding of anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody to FAP (e.g., human, mouse, or cynomolgus FAP). In the instance where the label is an enzyme, detection may be BUSINESS.32102391.1accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques as known in the art. This can be achieved by contacting a sample or a control sample with an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody under conditions that allow for the formation of a complex between the antibody and FAP (e.g., human, mouse, or cynomolgus FAP). Any complexes formed between the antibody and FAP (e.g., human, mouse, or cynomolgus FAP) are detected and compared in the sample and the control. In light of the specific binding of the antibodies described herein for FAP (e.g., human, mouse, or cynomolgus FAP), the antibodies can be used to specifically detect FAP (e.g., human, mouse, or cynomolgus FAP) expression on the surface of cells. The antibodies described herein can also be used to purify FAP (e.g., human, mouse, or cynomolgus FAP) via immunoaffinity purification. Also included herein is an assay system which may be prepared in the form of a test kit, kit, or kit-of-parts for the quantitative analysis of the extent of the presence of, for instance, FAP (e.g., human, mouse, or cynomolgus FAP) or FAP (e.g., human, mouse, or cynomolgus FAP) / FAP (e.g., human, mouse, or cynomolgus FAP) ligand complexes. The system, test kit, kit, or kit-of-parts may comprise a labeled component, e.g., a labeled antibody, and one or more additional immunochemical reagents.

[0228] In certain embodiments, the antibodies and pharmaceutical compositions described herein may be used or combined with one or more of a chemotherapeutic agent, an anti-cancer agent, an anti-angiogenic agent, an anti-fibrotic agent, an immunotherapeutic agent, a therapeutic antibody, a bispecific antibody, and “antibody-like” therapeutic protein (such as DARTs®, Duobodies®, Bites®, XmAbs®, TandAbs ®, Fab derivatives), an antibody-drug conjugate (ADC), a radiotherapeutic agent, an anti-neoplastic agent, an anti-proliferation agent, an oncolytic virus, a gene modifier or editor (such as CRISPR / Cas9, zinc finger nucleases or synthetic nucleases, TALENs), a CAR (chimeric antigen receptor) T-cell immunotherapeutic agent, an engineered T cell receptor (TCR-T), or any combination thereof. These therapeutic agents may be in the forms of compounds, antibodies, polypeptides, or polynucleotides. In certain embodiments, the application provides a product comprising a compound described herein and an additional therapeutic agent as a combined preparation for simultaneous, separate, or sequential use in therapy, e.g., a method of treating a disease, disorder, or condition BUSINESS.32102391.1PD-1 Inhibitors

[0229] PD-1 inhibitors that are useful in the methods and uses described herein include but are not limited to those described below. In certain embodiments, the PD-1 inhibitor is a human PD-1 inhibitor.

[0230] In certain embodiments, the human PD-1 inhibitor is an antibody that specifically binds to human PD-1 or human PD-L1. In certain embodiments, the antibody that specifically binds to human PD-1 comprises: a heavy chain variable region (VH) comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 167. In certain embodiments, the antibody that specifically binds to human PD-1 comprises: a light chain variable region (VL) comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 168. In certain embodiments, the antibody that specifically binds to human PD-1 comprises a heavy chain variable region (VH) comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 167 and a light chain variable region (VL) comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 168.

[0231] In certain embodiments, the antibody that specifically binds to human PD-1 comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 161, 162, and 163, respectively. In certain embodiments, the antibody that specifically binds to human PD-1 comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 164, 165, and 166, respectively. In certain embodiments, the antibody that specifically binds to human PD-1 comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 161, 162, 163, 164, 165, and 166, respectively.

[0232] In certain embodiments, the antibody that specifically binds to human PD-1 comprises a VH comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 167. In certain embodiments, the antibody that specifically binds to human PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 167.

[0233] In certain embodiments, the antibody that specifically binds to human PD-1 comprises a VL comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 168. In BUSINESS.32102391.1certain embodiments, the antibody that specifically binds to human PD-1 comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 168.

[0234] In certain embodiments, the antibody that specifically binds to human PD-1 comprises: a VH comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 167; and a VL comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 168. In certain embodiments, the antibody that specifically binds to human PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 167; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 168.

[0235] In certain embodiments, the antibody that specifically binds to human PD-1 comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the heavy chain constant region is IgG1. In certain embodiments, the heavy chain constant region is IgG2. In certain embodiments, the antibody comprises a light chain constant region selected from the group consisting of a human kappa light chain constant region and a human lambda light chain constant region.

[0236] In certain embodiments, the antibody that specifically binds to human PD-1 comprises an IgG4heavy chain constant region. In certain embodiments, the heavy chain constant region is a human IgG4heavy chain constant region comprising a P at position 228, numbered according to the EU numbering system.

[0237] In certain embodiments, the antibody that specifically binds to human PD-1 comprises a heavy chain comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 169. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 169.

[0238] In certain embodiments, the antibody that specifically binds to human PD-1 comprises a light chain comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 170. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 170.

[0239] In certain embodiments, the antibody that specifically binds to human PD-1 comprises a heavy chain comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 169; BUSINESS.32102391.1and a light chain comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 170. In certain embodiments, the antibody that specifically binds to human PD-1 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 169 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 170. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 169 and the amino acid sequence of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 170.

[0240] In certain embodiments the antibody that specifically binds to human PD-1 is balstilimab.

[0241] The amino acid sequences of exemplary anti-PD-1 antibodies are provided in Table 3 herein. Table 3. Amino acid sequences of exemplary anti-PD-1 antibodies.BUSINESS.32102391.1

[0242] In certain embodiments the antibody that specifically binds to human PD-1 or human PD-L1 is adebrelimab, atezolizumab, avelumab, camrelizumab, cemiplimab, cosibelimab, dostarlimab, durvalumab, enlonstobart, envafolimab, nivolumab, pembrolizumab, penpulimab, pidilizumab, prolgolimab, pucotenlimab, retifanlimab, serplulimab, sintilimab, socazolimab, sugemalimab, tagitanlimab, tislelizumab, toripalimab, and zimberelimab.

[0243] Further non-limiting examples of anti-PD-1 antibodies that may be used in treatment methods described herein are disclosed in the following patents and patent applications, which are incorporated herein by reference in their entireties for all purposes: U.S. Patent No.6,808,710; U.S. Patent No. 7,332,582; U.S. Patent No.7,488,802; U.S. Patent No. 8,008,449; U.S. Patent No. 8,114,845; U.S. Patent No. 8,168,757; U.S. Patent No. 8,354,509; U.S. Patent No.8,686,119; U.S. Patent No.8,735,553; U.S. Patent No.8,747,847; U.S. Patent No. 8,779,105; U.S. Patent No. 8,927,697; U.S. Patent No. 8,993,731; U.S. Patent No. 9,102,727; U.S. Patent No. 9,205,148; U.S. Publication No. US 2013 / 0202623 A1; U.S. Publication No. US 2013 / 0291136 A1; U.S. Publication No. US 2014 / 0044738 A1; U.S. Publication No. US 2014 / 0356363 A1; U.S. Publication No. US 2016 / 0075783 A1; and PCT Publication No. WO 2013 / 033091 A1; PCT Publication No. WO 2015 / 036394 A1; PCT Publication No. WO 2014 / 179664 A2; PCT Publication No. WO 2014 / 209804 A1; PCT Publication No. WO 2014 / 206107 A1; PCT Publication No. WO 2015 / 058573 A1; PCT Publication No. WO 2015 / 085847 A1; PCT Publication No. WO 2015 / 200119 A1; PCT Publication No. WO 2016 / 015685 A1; and PCT Publication No. WO 2016 / 020856 A1.

[0244] Further examples of anti-PD-L1 antibodies that may be used in treatment methods described herein are disclosed in the following patents and patent applications, which are incorporated herein by reference in their entireties for all purposes: U.S. Patent No. 7,943,743; U.S. Patent No. 8,168,179; U.S. Patent No. 8,217,149; U.S. Patent No. 8,552,154; U.S. Patent No. 8,779,108; U.S. Patent No. 8,981,063; U.S. Patent No. 9,175,082; U.S. Publication No. US 2010 / 0203056 A1; U.S. Publication No. US 2003 / 0232323 A1; U.S. Publication No. US 2013 / 0323249 A1; U.S. Publication No. US 2014 / 0341917 A1; U.S. Publication No. US 2014 / 0044738 A1; U.S. Publication No. US 2015 / 0203580 A1; U.S. Publication No. US 2015 / 0225483 A1; U.S. Publication No. US 2015 / 0346208 A1; U.S. Publication No. US 2015 / 0355184 A1; and PCT Publication No. WO 2014 / 100079 A1; PCT Publication No. WO 2014 / 022758 A1; PCT Publication No. WO 2014 / 055897 A2; PCT Publication No. WO 2015 / 061668 A1; PCT Publication No. WO 2015 / 109124 A1; PCT BUSINESS.32102391.1Publication No. WO 2015 / 195163 A1; PCT Publication No. WO 2016 / 000619 A1; and PCT Publication No. WO 2016 / 030350 A1.

[0245] In certain embodiments, the human PD-1 inhibitor is pidilizumab. CTLA-4 Inhibitors

[0246] Human CTLA-4 inhibitors that are useful in the methods and uses described herein include, but are not limited to, those described below.

[0247] In certain embodiments, the human CTLA-4 inhibitor is an antibody that specifically binds to human CTLA-4. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises: a heavy chain variable region (VH) comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 177. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises: a light chain variable region (VL) comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 178. In certain embodiments, the antibody comprises a heavy chain variable region (VH) comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 177 and a light chain variable region (VL) comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 178.

[0248] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 171, 172, and 173, respectively. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 174, 175, and 176, respectively. In certain embodiments, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 171, 172, 173, 174, 175, and 176, respectively.

[0249] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a VH comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 177. In certain embodiments, the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 177.

[0250] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a VL comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 178. In BUSINESS.32102391.1certain embodiments, the antibody comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 178.

[0251] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises: a VH comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 177; and a VL comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 178. In certain embodiments, the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 177 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 178.

[0252] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the heavy chain constant region is IgG1. In certain embodiments, the heavy chain constant region is IgG2. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises an IgG1 heavy chain constant region.

[0253] In certain embodiments, the antibody comprises a light chain constant region selected from the group consisting of a human kappa light chain constant region and a human lambda light chain constant region.

[0254] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds

[0255] In certain embodiments, the IgG region of the antibody that specifically binds with a wild-type Fc region, e.g., an IgG1 Fc. Sequence alterations that result in increased Methods 65: 105-113 (2014), Lazar et al., Proc Natl Acad Sci 103: 4005-4010 (2006), Shields et al., J Biol Chem.276(9):6591-6604 (2001), each of which is herein incorporated by reference in its entirety.

[0256] In certain embodiments, the antibody that specifically binds to human CTLA-4 is afucosylated. BUSINESS.32102391.1

[0257] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising a mutation selected from the group consisting of: L235V, G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L, and combinations thereof, numbered according to the EU numbering system.

[0258] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1constant region, or fragment thereof comprising S239D, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising T256A, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising K290A, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising S298A, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1constant region, or fragment thereof comprising I332E, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising E333A, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising K334A, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising A339T, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising S239D and I332E, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises an IgG1heavy chain constant region that comprises S239D / I332E mutations, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1constant region, or BUSINESS.32102391.1fragment thereof comprising S239D, A330L, and I332E, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises an IgG1 heavy chain constant region that comprises S239D / A330L / I332E mutations, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1constant region, or fragment thereof comprising S298A, E333A, and K334A, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1constant region, or fragment thereof comprising G236A, S239D, and I332E, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising F243L, R292P, Y300L, V305I, and P396L, numbered according to the EU numbering system. In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises an IgG1 heavy chain constant region that comprises L235V / F243L / R292P / Y300L / P396L mutations, numbered according to the EU numbering system. In certain embodiments, the heavy chain constant region is a human IgG1 heavy chain constant region comprising a D at position 239, a L at position 330, and an E at position 332, numbered according to the EU numbering system.

[0259] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 179. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 179.

[0260] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a light chain comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 180. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 180.

[0261] In certain embodiments, the antibody that specifically binds to human CTLA-4 comprises a heavy chain comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 179; and a light chain comprising an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 180. In BUSINESS.32102391.1certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 179 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 180. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 179 and the amino acid sequence of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 180.

[0262] In certain embodiments the antibody that specifically binds to human CTLA-4 is selected from the group consisting of botensilimab, ipilimumab, and tremelimumab. In certain embodiments the antibody that specifically binds to human CTLA-4 is botensilimab.

[0263] The amino acid sequences of exemplary anti-CTLA-4 antibodies are provided in Table 4 herein. Table 4. Amino acid sequences of exemplary anti-CTLA-4 antibodies.BUSINESS.32102391.1Polynucleotides, vectors, and methods of producing anti-FAP antibodies

[0264] In another aspect, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody described herein or a fragment thereof (e.g., a VH and / or VL) that specifically binds to a FAP (e.g., human, mouse, or cynomolgus FAP) antigen, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising nucleotide sequences encoding a polypeptide (e.g., a heavy and / or light chain) of any of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells.

[0265] As used herein, an “isolated” polynucleotide or nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source (e.g., in a mouse or a human) of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the language “substantially free” includes preparations of polynucleotide or nucleic acid molecule having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular less than about 10%) of other material, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors and / or other chemicals. In a specific embodiment, a nucleic acid molecule(s) encoding an antibody described herein is isolated or purified.

[0266] In particular aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies, which specifically bind to a FAP (e.g., human, mouse, or cynomolgus FAP) polypeptide and comprises an amino acid sequence as described herein, as well as antibodies which compete with such antibodies for binding to a FAP (e.g., human, mouse, or cynomolgus FAP) polypeptide (e.g., in a dose-dependent manner), or which binds to the same epitope as that of such antibodies.

[0267] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding a polypeptide (e.g., the light chain or heavy chain) of an antibody described herein. The polynucleotides can comprise nucleotide sequences encoding a light chain comprising the VL FRs and CDRs of antibodies described herein (see, e.g., Table 1) or nucleotide sequences encoding a heavy chain comprising the VH FRs and CDRs of antibodies described herein (see, e.g., Table 1).

[0268] Also provided herein are polynucleotides encoding an anti-FAP (e.g., human, BUSINESS.32102391.1mouse, or cynomolgus FAP) antibody that are optimized, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods to generate optimized nucleic acids encoding an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody or a fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain, optionally linked to a ligand binding moiety) for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, accordingly, all of which are herein incorporated by reference in their entireties. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression. The alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid. In certain embodiments, it can be desirable to alter one or more codons to encode a conservative mutation, e.g., a similar amino acid with similar chemical structure and properties and / or function as the original amino acid. Such methods can increase expression of an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody or fragment thereof by at least 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or more relative to the expression of an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody encoded by polynucleotides that have not been optimized.

[0269] In certain embodiments, an optimized polynucleotide sequence encoding an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein or a fragment thereof (e.g., VL domain and / or VH domain) can hybridize to an antisense (e.g., complementary) polynucleotide of an unoptimized polynucleotide sequence encoding an anti- FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein or a fragment thereof (e.g., VL domain and / or VH domain). In specific embodiments, an optimized nucleotide sequence encoding an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein or a fragment hybridizes under high stringency conditions to antisense polynucleotide of an unoptimized polynucleotide sequence encoding an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein or a fragment thereof. In a specific embodiment, an optimized nucleotide sequence encoding an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein or a fragment thereof hybridizes under high stringency, intermediate, or lower stringency hybridization conditions to an antisense polynucleotide of an BUSINESS.32102391.1unoptimized nucleotide sequence encoding an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein or a fragment thereof. Information regarding hybridization conditions has been described, see, e.g., U.S. Patent Application Publication No. US 2005 / 0048549 (e.g., paragraphs 72-73), which is herein incorporated by reference in its entirety.

[0270] The polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Nucleotide sequences encoding antibodies described herein, e.g., antibodies described in Table 1, and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody. Such a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6, herein incorporated by reference in its entirety), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing, and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0271] Alternatively, a polynucleotide encoding an antibody described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the light chain and / or heavy chain of an antibody. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the variable light chain region and / or the variable heavy chain region of an antibody. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies.

[0272] If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody described herein) by PCR amplification using synthetic primers BUSINESS.32102391.1hybridizable to the 3’ and 5’ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0273] DNA encoding anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies in the recombinant host cells.

[0274] To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a heavy chain constant region, e.g., the human gamma 1 or human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a light chain constant region, e.g., human kappa or lambda constant regions. In certain embodiments, the vectors for site for the variable region, constant domains, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co- transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.

[0275] The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the murine sequences, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0276] Also provided are polynucleotides that hybridize under high stringency, intermediate, or lower stringency hybridization conditions to polynucleotides that encode an BUSINESS.32102391.1antibody described herein. In specific embodiments, polynucleotides described herein hybridize under high stringency, intermediate, or lower stringency hybridization conditions to polynucleotides encoding a VH domain and / or VL domain provided herein.

[0277] Hybridization conditions have been described in the art and are known to one of skill in the art. For example, hybridization under stringent conditions can involve hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65°C; hybridization under highly stringent conditions can involve hybridization to filter-bound nucleic acid in 6xSSC at about 45°C followed by one or more washes in 0.1xSSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions are known to those of skill in the art and have been described, see, for example, Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3, which is herein incorporated by reference in its entirety.

[0278] In certain aspects, provided herein are cells (e.g., host cells) expressing (e.g., recombinantly) antibodies described herein which specifically bind to FAP (e.g., human, mouse, or cynomolgus FAP) and related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies or a fragment for recombinant expression in host cells, preferably in mammalian cells (e.g., CHO cells). Also provided herein are host cells comprising such vectors for recombinantly expressing anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies described herein (e.g., human or humanized antibody). In a particular aspect, provided herein are methods for producing an antibody described herein, comprising expressing such antibody from a host cell.

[0279] Recombinant expression of an antibody described herein (e.g., a full-length antibody, heavy and / or light chain of an antibody, or a single chain antibody optionally comprising a ligand binding moiety described herein) that specifically binds to FAP (e.g., human, mouse, or cynomolgus FAP) generally involves construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody, a polypeptide (e.g., heavy and / or light chain) of an antibody, or a fragment thereof (e.g., heavy and / or light chain variable regions) described herein has been obtained, the vector for the production of the antibody can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a BUSINESS.32102391.1polynucleotide containing an antibody or antibody fragment (e.g., light chain or heavy chain) encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (e.g., light chain or heavy chain) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody described herein, a polypeptide (e.g., heavy or light chain) of an antibody, a heavy or light chain variable region of an antibody or a fragment thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors can, for example, include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No.5,122,464, which are herein incorporated by reference in their entireties) and variable regions of the antibody can be cloned into such a vector for expression of an entire polypeptide (e.g., the entire heavy chain or the entire light chain), or both the entire heavy and light chains.

[0280] In certain embodiments, a vector comprises a polynucleotide encoding a VH, VL, heavy chain, and / or light chain of an antibody described herein. In another embodiment, a vector comprises a polynucleotide encoding the VH and the VL of an antibody described herein. In another embodiment, a vector comprises a polynucleotide encoding the heavy chain and the light chain of an antibody described herein.

[0281] An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody described herein or a fragment thereof. Thus, provided herein are host cells containing a polynucleotide encoding an antibody described herein or fragments thereof, or a heavy or light chain thereof, or fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of such sequences in the host cell.

[0282] In certain embodiments, for the expression of double-chained antibodies, vectors encoding both the heavy and light chains, individually, can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below. In certain embodiments, a host cell contains a vector comprising a polynucleotide encoding both the heavy chain and light chain of an antibody described herein, or a fragment thereof. In specific embodiments, a host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain or a VH of an antibody described herein, or a fragment BUSINESS.32102391.1thereof, and a second vector comprising a polynucleotide encoding a light chain or a VL of an antibody described herein, or a fragment thereof. In other embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or a VH of an antibody described herein, or a fragment thereof, and a second host cell comprises a second vector comprising a polynucleotide encoding a light chain or a VL of an antibody described herein. In specific embodiments, a heavy chain / VH expressed by a first cell associated with a light chain / VL of a second cell to form an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein. In certain embodiments, provided herein is a population of host cells comprising such first host cell and such second host cell.

[0283] In a particular embodiment, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding a light chain / VL of an anti- FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein, and a second vector comprising a polynucleotide encoding a heavy chain / VH of an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein.

[0284] A variety of host-expression vector systems can be utilized to express antibody described herein (see, e.g., U.S. Patent No. 5,807,715, which is herein incorporated by reference in its entirety). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with, e.g., recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with, e.g., recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with, e.g., recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with, e.g., recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with, e.g., recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK-293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, NIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, and BMT10 cells) harboring, e.g., recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein BUSINESS.32102391.1promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In a specific embodiment, cells for expressing antibodies described herein are Chinese hamster ovary (CHO) cells, for example CHO cells from the CHO GS System (Lonza). In certain embodiments, a polypeptide (e.g., the heavy chain and / or light chain) of an antibody produced by a CHO cell may have an N-terminal glutamine or glutamate residue replaced by pyroglutamate. In a particular embodiment, cells for expressing antibodies described herein are human cells, e.g., human cell lines. In a specific embodiment, a mammalian expression vector is pOptiVEC or pcDNA3.3. In a particular embodiment, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), especially for the expression of whole recombinant antibody, are used for the expression of a recombinant antibody. For example, mammalian cells such as CHO cells, in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101- 5; and Cockett MI et al., (1990) Biotechnology 8(7): 662-7, each of which is herein incorporated by reference in its entirety). In certain embodiments, antibodies described herein are produced by CHO cells or NS0 cells. In a specific embodiment, the expression of nucleotide sequences encoding antibodies described herein which specifically bind to FAP (e.g., human, mouse, or cynomolgus FAP) is regulated by a constitutive promoter, inducible promoter, or tissue specific promoter.

[0285] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions of an antibody, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the antibody coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509); and the like, all of which are herein incorporated by reference in their entireties. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The BUSINESS.32102391.1pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0286] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[0287] In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12): 3655-9, which is herein incorporated by reference in its entirety). Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol.153:516-544, which is herein incorporated by reference in its entirety).

[0288] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK-293, NIH 3T3, BUSINESS.32102391.1W138, BT483, Hs578T, HTB2, BT2O, T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibodies described herein are produced in mammalian cells, such as CHO cells.

[0289] In a specific embodiment, the antibodies described herein have reduced fucose content or no fucose content. Such antibodies can be produced using techniques known to one skilled in the art. For example, the antibodies can be expressed in cells deficient or lacking the fucosyltransferase can be used to produce antibodies with reduced fucose content. The Potelligent®system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.

[0290] For long-term, high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines which stably express an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein can be engineered. In specific embodiments, a cell provided herein stably expresses a light chain / light chain variable region and a heavy chain / heavy chain variable region which associate to form an antibody described herein, optionally wherein at least one of the light chain / light chain variable region and the heavy chain / heavy chain variable region is linked to a ligand binding moiety.

[0291] In certain aspects, rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA / polynucleotide, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express an anti- FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein or a fragment thereof. Such engineered cell lines can be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule. BUSINESS.32102391.1

[0292] A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-32); hypoxanthineguanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034); and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23) genes in tk-, hgprt- or aprt-cells, respectively, all of which are herein incorporated by reference in their entireties. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6): 3567-70; O’Hare K et al., (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191- 217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-56), all of which are herein incorporated by reference in their entireties. Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone and such methods are described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) J Mol Biol 150: 1-14, all of which are herein incorporated by reference in their entireties.

[0293] The expression levels of an antibody can be increased by vector amplification (for a review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987), which is herein incorporated by reference in its entirety). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66, which is herein incorporated by reference in its entirety).

[0294] The host cell can be co-transfected with two or more expression vectors described herein, the first vector encoding a heavy chain derived polypeptide and the second BUSINESS.32102391.1vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers which enable equal expression of heavy and light chain derived polypeptides. The host cells can be co-transfected with different amounts of the two or more expression vectors. For example, host cells can be transfected with any one of the following ratios of a first expression vector and a second expression vector: about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0295] Alternatively, a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and Köhler G (1980) PNAS 77: 2197-2199, each of which is herein incorporated by reference in its entirety). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. The expression vector can be monocistronic or multicistronic. A multicistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or in the range of 2-5, 5-10, or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can comprise, in the following order, a promoter, a first gene (e.g., heavy chain of an antibody optionally linked to a ligand binding moiety as described herein), and a second gene and (e.g., light chain of an antibody optionally linked to a ligand binding moiety as described herein). In such an expression vector, the transcription of both genes can be driven by the promoter, whereas the translation of the mRNA from the first gene can be by a cap-dependent scanning mechanism and the translation of the mRNA from the second gene can be by a cap-independent mechanism, e.g., by an IRES.

[0296] Once an antibody described herein has been produced by recombinant expression, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0297] In specific embodiments, an antibody described herein is isolated or purified. Generally, an isolated antibody is one that is substantially free of other antibodies with different antigenic specificities than the isolated antibody. For example, in a particular embodiment, a preparation of an antibody described herein is substantially free of cellular material and / or BUSINESS.32102391.1chemical precursors. The language “substantially free of cellular material” includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”) and / or variants of an antibody, for example, different post-translational modified forms of an antibody or other different versions of an antibody (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a specific embodiment, antibodies described herein are isolated or purified.

[0298] Antibodies or fragments thereof that specifically bind to FAP (e.g., human, mouse, or cynomolgus FAP) can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of BUSINESS.32102391.1which are herein incorporated by reference in their entireties.

[0299] In a specific embodiment, an antibody described herein is an antibody (e.g., recombinant antibody) prepared, expressed, created, or isolated by any means that involves creation, e.g., via synthesis, genetic engineering of DNA sequences. In certain embodiments, such an antibody comprises sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.

[0300] In one aspect, provided herein is a method of making an antibody which specifically binds to FAP (e.g., human, mouse, or cynomolgus FAP) comprising culturing a cell or host cell described herein. In one embodiment, the method is performed in vitro. In a certain aspect, provided herein is a method of making an antibody which specifically binds to FAP (e.g., human, mouse, or cynomolgus FAP) comprising expressing (e.g., recombinantly expressing) the antibody using a cell or host cell described herein (e.g., a cell or a host cell comprising polynucleotides encoding an antibody described herein). In a particular embodiment, the cell is an isolated cell. In a particular embodiment, the exogenous polynucleotides have been introduced into the cell. In a particular embodiment, the method further comprises the step of purifying the antibody obtained from the cell or host cell.

[0301] Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York, which is herein incorporated by reference in its entirety).

[0302] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which is herein incorporated by reference in its entirety. The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing a polypeptide of an antibody described herein or a fragment thereof, for example, light chain and / or heavy chain of such antibody. BUSINESS.32102391.1

[0303] In specific embodiments, a “monoclonal antibody,” as used herein, is an antibody produced by a single cell (e.g., hybridoma or host cell producing a recombinant antibody), wherein the antibody specifically binds to FAP (e.g., human, mouse, or cynomolgus FAP) as determined, e.g., by ELISA or other antigen-binding or competitive binding assay known in the art or in the examples provided herein. In particular embodiments, a monoclonal antibody can be a chimeric antibody or a humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody. In particular embodiments, a monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody). Monoclonal antibodies described herein can, for example, be made by the hybridoma method as described in Kohler G & Milstein C (1975) Nature 256: 495, which is herein incorporated by reference in its entirety, or can, e.g., be isolated from phage libraries using the techniques as described herein, for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0304] As used herein, an antibody binds to an antigen multivalently (e.g., bivalently) when the antibody comprises at least two (e.g., two or more) monovalent binding domains, each monovalent binding domain capable of binding to an epitope on the antigen. Each monovalent binding domain can bind to the same or different epitopes on the antigen.

[0305] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. For example, in the hybridoma method, a mouse or other appropriate host animal, such as a sheep, goat, rabbit, rat, hamster, or macaque monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein (e.g., FAP (e.g., human, mouse, or cynomolgus FAP)) used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), herein incorporated by reference in its entirety). Additionally, a RIMMS (repetitive immunization multiple sites) technique can be used to immunize an animal (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, herein incorporated by reference in its entirety).

[0306] In certain embodiments, mice (or other animals, such as rats, monkeys, donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen (e.g., FAP (e.g., human, BUSINESS.32102391.1mouse, or cynomolgus FAP)) and once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example, cells from cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limited dilution. In certain embodiments, lymph nodes of the immunized mice are harvested and fused with NS0 myeloma cells.

[0307] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0308] Specific embodiments employ myeloma cells that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cell lines are murine myeloma lines, such as the NS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), each of which is herein incorporated by reference in its entirety).

[0309] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against FAP (e.g., human, mouse, or cynomolgus FAP). The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, for example, immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

[0310] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and / or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, BUSINESS.32102391.1D-MEM or RPMI 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

[0311] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0312] Antibodies described herein include, e.g., antibody fragments which recognize a specific FAP (e.g., human, mouse, or cynomolgus FAP), and which can be generated by any technique known to those of skill in the art. For example, Fab and F(ab’)2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab’)2 fragments). A Fab fragment corresponds to one of the two identical arms of an antibody and contains the complete light chain paired with the VH and CH1 domains of the heavy chain. A F(ab’)2 fragment contains the two antigen-binding arms of an antibody molecule linked by disulfide bonds in the hinge region.

[0313] Further, the antibodies described herein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of affected tissues). The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13, and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT Application No. PCT / GB91 / 001134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and BUSINESS.32102391.1WO 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, all of which are herein incorporated by reference in their entireties.

[0314] In addition, the antibodies described herein can also be generated using specific immunization methods, alone or in a combination with phage display technologies as known in the art. For example, DNA immunization has been recently reported as an efficient strategy for vaccination. See Bolhassani and Yazdi (2009) Avicenna J Med Biotechnol.1:71-88; Liu et al. (2016) Emerg Microbes Infect. 5:e33; and van der Woning et al. (2016) MAbs. 8:1126- 1135, each of which is herein incorporated by reference in its entirety. Immunizing animals with DNA encoding a protein of interest can be performed using the similar methods as traditional animal immunization using the protein itself, and can include similar initial immunization and boosting steps. After DNA immunization, hybridoma cells can be prepared. Alternatively, or additionally, nucleic acid molecules (e.g., RNAs) can be isolated from tissues (e.g., spleen) of the immunized animal to generate a phase display library for antibody panning and screening.

[0315] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce antibody fragments such as Fab, Fab’, and F(ab’)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO 92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; and Better M et al., (1988) Science 240: 1041-1043, all of which are herein incorporated by reference in their entireties.

[0316] In certain embodiments, to generate whole antibodies, PCR primers, including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences from a template, e.g., scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell BUSINESS.32102391.1lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.

[0317] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain a variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, all of which are herein incorporated by reference in their entireties.

[0318] A humanized antibody is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a murine immunoglobulin). In particular embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The antibody also can include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5): 489-498; Studnicka GM et al., (1994) Prot Engineering 7(6): 805-814; and Roguska MA et al., (1994) PNAS 91: 969-973), chain shuffling (U.S. Patent No. 5,565,332), and techniques disclosed in, e.g., U.S. Pat. No.6,407,213, U.S. Pat. No.5,766,886, International Publication No. WO 93 / 17105; Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng.13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267- 79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10): 895904; Couto JR et al., (1995) Cancer Res.55 (23 Supp): 5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10; and Pedersen JT et al., (1994) J Mol Biol 235(3): 959-73, all of which are herein incorporated by reference in their entireties. See also U.S. Application Publication No. US 2005 / 0042664 A1 (Feb. 24, 2005), which is herein incorporated by reference in its entirety. BUSINESS.32102391.1

[0319] Methods for making multispecific (e.g., bispecific antibodies) have been described, see, for example, U.S. Patent Nos. 7,951,917, 7,183,076, 8,227,577, 5,837,242, 5,989,830, 5,869,620, 6,132,992, and 8,586,713, all of which are herein incorporated by reference in their entireties.

[0320] Single domain antibodies, for example, antibodies lacking the light chains, can be produced by methods well known in the art. See Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) J Biotechnol 74(4): 277-302; U.S. Patent No. 6,005,079; and International Publication Nos. WO 94 / 04678, WO 94 / 25591, and WO 01 / 44301, all of which are herein incorporated by reference in their entireties.

[0321] Further, antibodies that specifically bind to a FAP (e.g., human, mouse, or cynomolgus FAP) antigen can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” an antigen using techniques well known to those skilled in the art. See, e.g., Greenspan NS & Bona CA (1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438, each of which is herein incorporated by reference in its entirety.

[0322] In particular embodiments, an antibody described herein, which binds to the same epitope of FAP (e.g., human, mouse, or cynomolgus FAP) as an anti-FAP (e.g., human, mouse, or cynomolgus FAP) antibody described herein, is a human antibody. In particular embodiments, an antibody described herein, which competitively blocks (e.g., in a dose- dependent manner) any one of the antibodies described herein, from binding to FAP (e.g., human, mouse, or cynomolgus FAP), is a human antibody. Human antibodies can be produced using any method known in the art. For example, transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes, can be used. In particular, the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human BUSINESS.32102391.1antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of an antigen (e.g., FAP (e.g., human, mouse, or cynomolgus FAP)). Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93, herein incorporated by reference in its entirety. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patent Nos.5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are herein incorporated by reference in their entireties. Examples of mice capable of producing human antibodies include the XenoMouseTM(Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), the HuAb-MouseTM(Mederex, Inc. / Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569, 825), the Trans Chromo MouseTM(Kirin), and the KM MouseTM(Medarex / Kirin), all of which are herein incorporated by reference in their entireties.

[0323] Human antibodies that specifically bind to FAP (e.g., human, mouse, or cynomolgus FAP) can be made by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, all of which are herein incorporated by reference in their entireties.

[0324] In certain embodiments, human antibodies can be produced using mouse– human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse–human hybridomas secreting human monoclonal antibodies, and these mouse–human hybridomas can be screened to determine ones which secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., FAP (e.g., human, mouse, or cynomolgus FAP)). Such methods are known and are described in the art, see, e.g., Shinmoto H et al., (2004) Cytotechnology 46: BUSINESS.32102391.119-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31, each of which is herein incorporated by reference in its entirety. Kits

[0325] Also provided are kits comprising one or more antibodies described herein, or pharmaceutical compositions or conjugates thereof. In a specific embodiment, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more antibodies provided herein. In certain embodiments, the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein. In certain embodiments, the kits may contain a T cell mitogen, such as, e.g., phytohaemagglutinin (PHA) and / or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.

[0326] Also provided, are kits that can be used in the above methods. In one embodiment, a kit comprises an antibody described herein, preferably a purified antibody, in one or more containers. In a specific embodiment, kits described herein contain a substantially isolated FAP (e.g., human, mouse, or cynomolgus FAP) antigen as a control. In another specific embodiment, the kits described herein further comprise a control antibody which does not react with a FAP (e.g., human, mouse, or cynomolgus FAP) antigen. In another specific embodiment, kits described herein contain one or more elements for detecting the binding of an antibody to a FAP (e.g., human, mouse, or cynomolgus FAP) antigen (e.g., the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound, or a second antibody which recognizes the first antibody can be conjugated to a detectable substrate). In specific embodiments, a kit provided herein can include a recombinantly produced or chemically synthesized FAP (e.g., human, mouse, or cynomolgus FAP) antigen. The FAP (e.g., human, mouse, or cynomolgus FAP) antigen provided in the kit can also be attached to a solid support. In a more specific embodiment, the detecting means of the above-described kit includes a solid support to which a FAP (e.g., human, mouse, or cynomolgus FAP) antigen is attached. Such a kit can also include a non-attached reporter-labeled anti-human antibody or anti-mouse / rat antibody. In this BUSINESS.32102391.1embodiment, binding of the antibody to the FAP (e.g., human, mouse, or cynomolgus FAP) antigen can be detected by binding of the said reporter-labeled antibody. In one embodiment, the present invention relates to the use of a kit of the present invention for in vitro assaying and / or detecting FAP antigen (e.g., human, mouse, or cynomolgus FAP) in a biological sample. EXAMPLES

[0327] The following examples are offered by way of illustration and not by way of limitation. Example 1: Characterization of anti-FAP antibodies

[0328] This example describes the characterization of antibodies that specifically bind to human, cynomolgus, and mouse FAP proteins. The antibodies were isolated / discovered, and affinity matured by phage display. The amino acid sequences of these antibodies are set forth herein in Table 1.

[0329] The binding affinities of anti-FAP antibodies with IgG1 constant regions, to His-tagged extracellular domains (ECDs) of human FAP, cynomolgus FAP, mouse FAP, and related family member protein human CD26, were assessed by surface plasmon resonance (SPR). Reference anti-FAP clones 28H1 and ESC11 were used as a benchmark, and an irrelevant antibody was used as a negative isotype control.

[0330] Briefly, SPR experiments were performed using a Biacore 4000 instrument, and the association rate (Ka), dissociation rate (Kd), and dissociation constant (KD) were calculated from each experiment using a 1:1 binding model with Biacore 4000 Evaluation Software. Capture of antibodies from the Human Fc Capture kit type 2 (29234600, Cytiva) to the Series S CM5 sensor chip (29149603, Cytiva) was performed using the immobilization wizard for amine coupling from the Biacore control panel, and following the manufacturer’s recommendations included with the kit.

[0331] Approximately 2 µg / mL of anti-FAP antibodies and controls diluted in a running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Surfactant P20) were captured in individual flow cells of previously prepared anti-human Fc CM5 chip, keeping a single flow cell as a reference. Antibodies were captured using a 30 sec injection at a flow rate of 10 µL / min to reach about 150 response units (RU). Human FAP, cynomolgus FAP, mouse FAP, and human CD26 (diluted in the running buffer at the concentration of 100, 10, 1 and 0 nM) were flowed over the chip surface at a flow rate of 30 µL / min with a 2-minute association phase and a 5-minute dissociation phase. The sensor chip was regenerated between cycles with BUSINESS.32102391.1a 30 sec injection of 3M MgCl2 at a flow rate of 30 µL / minute followed by stabilization period of 60 sec. Sensorgrams were evaluated and fit to a simple Langmuir 1:1 interaction model using the BiacoreTM4000 evaluation software. Data quality was verified by visually inspecting deviations and curve fitting, and by evaluating the parameters of Rmax, Chi2, and Tc.

[0332] The binding kinetics of anti-FAP antibodies to FAP proteins are shown in Table 5. No binding was detected to human CD26 protein (data not shown). Table 5: Kinetic parameters of anti-FAP antibodies binding to ECD-His-tagged human, cynomolgus, and mouse FAP.BUSINESS.32102391.1control NBD = no binding detected Example 2: Binding of anti-FAP antibodies to FAP expressed on cells

[0333] The capacity of anti-FAP antibodies to bind to cells expressing human, cynomolgus, and / or mouse FAP was tested in a variety of cell types. Binding to CHO cells expressing human, cynomolgus monkey, or mouse FAP

[0334] Anti-FAP antibodies were assessed for their ability to bind to human FAP expressed on the surface of CHO cells. Briefly, CHO cells were transfected with a vector encoding human FAP protein domains. A clone stably expressing a relatively high level of FAP was cultured in Roswell Park Memorial Institute (RPMI)-1640 medium with glutamax +10% Heat inactivated FBS + 1% Pen / Strep, 10 µg / ml Blasticidin.

[0335] For the antibody binding assay, the cells were seeded in a 96-well U-bottom tissue culture plate at a density of 4×105cells per well in 50 L of PBS supplemented with 0.5% BSA, 1 mM EDTA and 0.05% sodium azide (FACS Buffer). The cells were incubated for 45 minutes at 4°C with 50 L of anti-FAP antibodies, VRC01 IgG1 isotype control, or reference antibodies 28H1 or ESC11, at a final concentration of 20 µg / mL in FACS Buffer.

[0336] For antibody staining, the cells were washed twice with cold FACS Buffer and resuspended in 100 L of FACS Buffer containing an R-Phycoerythrin goat anti-human IgG antibody (Jackson ImmunoResearch / 109-116-098) at a final dilution of 1 / 800. After a 45- minute incubation in the dark at 4°C, the cells were washed twice with cold FACS Buffer, and the cells were analyzed by flow cytometry (BD LSR Fortessa Flow Cytometer). CHO cells were identified using a plot of forward scatter-area (FSC-A) versus side scatter area (SSC-A) and another plot of SSC-A versus forward scatter-height (SSC-H) for selection of single cells. Samples were analyzed by sequentially gating on the single cell population and plotting histograms representing the fluorescence in the PE channel. For each sample, mean fluorescence intensity (MFI) was calculated, and the data were plotted by GraphPad Prism software.

[0337] As shown in FIG. 1, anti-FAP antibodies demonstrated binding to CHO cells over-expressing human FAP.

[0338] A panel of these anti-FAP antibodies was further tested for binding to CHO cells over-expressing human FAP using a dilution series with concentrations ranging from 20 to 0.0012 µg / mL. As shown in FIG. 2A, all anti-FAP antibodies tested bound to CHO cells BUSINESS.32102391.1expressing high levels of human FAP in a dose-dependent manner. Reference antibodies ESC11 and 28H1 also bound to the cells, but isotype control did not (FIG.2B). The calculated area under the curve (AUC) and 50% effective concentration (EC50) values are shown in Table 6. Table 6: Anti-FAP antibodies binding to human FAP-expressing CHO cells.

[0339] In similar experiments, anti-FAP antibodies bound to CHO cells over- expressing cynomolgus monkey FAP or mouse FAP. FIG. 3A and FIG. 3B show the binding of anti-FAP antibodies and controls, respectively, to CHO cells expressing cynomolgus monkey FAP. FIG. 4A and FIG. 4B show the binding of anti-FAP antibodies and controls, respectively, to CHO cells expressing mouse FAP. The calculated AUC and EC50 values are shown in Table 7 for each cell line. Table 7: Anti-FAP antibodies binding to cynomolgus monkey FAP-expressing and mouse FAP-expressing CHO cells.BUSINESS.32102391.1Binding to U138MG cells endogenously expressing human FAP

[0340] Anti-FAP antibodies were further tested for binding to U138MG cells, a human glioblastoma cell line that endogenously expresses human FAP. As shown in FIG. 5A, anti- FAP antibodies bound dose-dependently to U138MG cells. Reference antibodies ESC11 and 28H1 also bound to U138MG cells, while isotype control showed little or no binding (FIG. 5B). The calculated AUC and EC50 values are shown in Table 8. Table 8: Anti-FAP antibodies binding to U138MG cells.Example 3: Induction of Fc RIIIA signaling by anti-FAP antibodies

[0341] This example describes the capacity of anti-FAP antibodies to activate a reporter lines.

[0342] expressing FAP. Effector cells comprised engineered Jurkat reporter cells stably expressing the wherein the antigen is located on the surface of the target cells. This binding then triggers the promoter / reporter construct of the effector cells, leading to luciferase gene transcription and the subsequent generation of a luminescent signal.

[0343] human FAP were plated overnight in 96-well cell culture plates to adhere, then incubated with BUSINESS.32102391.1increasing concentrations (7-point dose titration, 100 to 0.006 nM) of anti-FAP antibodies, expressing NFAT reporter cells were added to the antibody-target cell mixture at a 1:2.5 ratio (6×104cells) in RPMI media, supplemented with 4% heat-inactivated low-IgG FBS, at 37°C and 5% CO2. After a 6-hour incubation, Bio-Glo Luciferase Assay Substrate (Promega, G720A) was added to each sample (1:1 v / v). Luminescence was measured using the EnVision®Multilabel Plate Reader (PerkinElmer). Data were plotted with GraphPad Prism software.

[0344] Consistent with the observation that anti-FAP antibodies showed potent binding (FIGs. 6A and 6B). Table 9 shows the AUC and EC50 values of the anti-FAP antibodies inExample 4: Induction of ADCC by anti-FAP antibodies

[0345] Antibody mediated cellular cytotoxicity (ADCC) is a cell-mediated mechanism adopted by host immune defense whereby an effector cell lyses a target cell. The nonspecific cytotoxic cells recognize specific antibodies bound to membrane-surface antigens on a target

[0346] The capacity of anti-FAP antibodies to mediate ADCC was tested using an assay system consisting of a co-culture of target cells engineered to express FAP (HEK-FAP), or endogenously expressing FAP (U138MG), labeled with a viability dye (Calcein AM), and BUSINESS.32102391.1antibodies. Cellular cytotoxicity was evaluated by measuring the fluorescence captured due to the release of the dye.

[0347] Specifically, NK cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and activated overnight in a cocktail of IL2 and IL15. The target cells were labeled with Calcein AM dye and plated at a density of 5000 cells / well. The activated NK cells were then plated at 2X density to the target cells. The anti-FAP antibodies and control antibodies were added to the cell-coculture in a titrated manner to assess their impact. After a 3-hour incubation, cell lysis was evaluated by measuring the fluorescence emitted by the release of the Calcein AM dye on a plate reader (PerkinElmer). Data was analyzed and plotted using Microsoft Excel and GraphPad Prism.

[0348] Consistent with the observation that anti-FAP antibodies showed potent demonstrated a strong ADCC potential towards FAP-expressing HEK cells and U138MG cells (FIGs. 7A-7B). Table 10 shows the target cell toxicity (%) and EC50 values of the anti-FAP antibodies. Table 10: EC50 values and target cell cytotoxicity % of FAP-expressing target cells via ADCC mediated by anti-FAP antibodies.BUSINESS.32102391.1Example 5: Binding kinetics of anti-FAP-TGF RII bispecific antibodies

[0349] The binding affinities of anti-FAP antibodies with IgG1 constant regions fused

[0350] Briefly, SPR experiments were performed using a Biacore T200 instrument and the association rate (Ka), dissociation rate (kd), and dissociation constant (KD) were calculated from each experiment using a 1:1 binding model with Biacore 200 Evaluation Software. To test human FAP-His binding, the test antibodies were captured using a Series S Protein A sensor chip (29127556, Cytiva).

[0351] a running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Surfactant P20) were captured in individual flow cells of the protein A chip, keeping a single flow cell as a reference. Antibodies were captured using a 30 sec injection at a flow rate of 10 µL / min to reach about 150 RU. Human FAP (diluted in the running buffer at the concentration of 100, 10, 1, and 0 nM) was flowed over the chip surface at a flow rate of 30 µL / min with a 3-minute association phase and a 10-minute dissociation phase. The sensor chip was regenerated between cycles with a 30 sec injection of 1 M Glycine-HCl pH 1.5 at a flow rate of 30 µL / min followed by stabilization period of 60 sec. Sensorgrams were evaluated and fit to a simple Langmuir 1:1 interaction model using the BiacoreTMEvaluation 3.1 software. Data quality was verified by visually inspecting deviations and curve fitting, and by evaluating the parameters of Rmax, Chi2, and Tc. The binding kinetics are shown in Table 11. BUSINESS.32102391.1tagged human FAP protein.variant TGF RII ECD

[0352] To enhance effector functions associated with the binding of the antibody Fc

[0353] Briefly, SPR experiments were performed using a Biacore T200 instrument. The association rate (Ka), dissociation rate (kd), and dissociation constant (KD) were calculated from each experiment using a bivalent analyte model using the Biacore Evaluation 3.0 software.

[0354] was captured using a Series S Sensor Chip CM7 (28953828, Cytiva) immobilized with anti- human IgG1 (Fc) capture antibody (29234600, Cytiva).

[0355] To test the binding to human FAP, cynomolgus FAP, and mouse FAP, in a running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) captured in individual flow cells of the anti-Human Fc antibody immobilized CM7 chip, keeping a single flow cell as a reference. The antibodies were captured using a 30-second injection at a flow rate of 10 µL / min to reach appropriate RU (~ 60 RU of capture for human FAP binding). Human FAP, cynomolgus FAP, and mouse FAP (diluted in the running buffer to a concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.09,8 and 0 nM) BUSINESS.32102391.1were flowed over the chip surface at a flow rate of 30 µL / min with a 3-minute association phase and a 20-minute dissociation phase.

[0356] controls diluted in a running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Surfactant P20) captured in individual flow cells of the anti-Human Fc antibody immobilized CM7 chip, keeping a single flow cell as a reference. The antibodies were captured using a 30- second injection at a flow rate of 10 µL / min to reach appropriate RU (~120 RU of capture for (diluted in the running buffer to a concentration of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0312, and 0 nM), were flowed over the chip surface at a flow rate of 30 µL / min with a 3- minute association phase and a 20-minute dissociation phase. The sensor chip was regenerated between cycles with two 30-second injections of 3.0 M MgCl2 at 30 µL / min followed by a stabilization period of 60 sec. Sensorgrams were evaluated and fit to a bivalent analyte model using the Biacore Insight Evaluation 3.1 software. Data quality was verified by visually inspecting deviations and curve fitting, and by evaluating the parameters of Rmax, Chi2, and Tc.

[0357]

[0358] In another experiment, the simultaneous binding of 23-B08-DLE-

[0359] Briefly, SPR experiments were performed using a BiacoreTM8K+ instrument. The antibodies were captured using a Series S Sensor Chip CM3 (BR-1005-36, Cytiva) immobilized with anti-human IgG1 (Fc) capture antibody (29234600, Cytiva). Approximately 111 BUSINESS.32102391.1running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Surfactant P20) and captured in individual flow cells of the anti-human Fc antibody immobilized CM3 chip, keeping a single flow cell as a reference. The antibodies were captured using a 60-second injection at a flow rate of 10 µL / min to reach approximately 150 RU. Using a dual injection combinations using running buffer as a control (Table 13). Each dual injection lasted for 180 sec at a flow rate of 30 µL / min, followed by a dissociation phase of 300 sec. Table 13: Combinations of injections.

[0360] The sensor chip was regenerated between cycles with 45-second injections of 10 mM glycine at pH 1.5 at 30 µL / min followed by a stabilization period of 60 sec. Data analysis was performed using BiacoreTMInsight Evaluation Software version 4.0.8.20368 for kinetic determination (GE Healthcare Life Sciences). The sensorgrams were evaluated manually using the visualization tools within the software to analyze the data and associated binding RU are reported (FIG.8).

[0361] to bind to cells expressing human FAP.

[0362] assessed by flow cytometry for their ability to bind CHO cells over-expressing human FAP, as described in Example 2. The concentration of the molecules ranged from 30 µg / mL to 0.0002 µg / mL.

[0363] demonstrated dose-dependent binding to FAP expressed on cells. FAP binding was also BUSINESS.32102391.1

[0364] g / mL) to FAP was determined by flow cytometry using CHO cells over-expressing human, cynomolgus, or mouse FAP, as described in Example 2.

[0365] cynomolgus monkey, and mouse FAP expressed on CHO cells, in a dose-dependent manner and with EC50values (±SD, N) of 0.20 g / mL (± 0.05 g / mL, 2), 0.18 g / mL (± 0.01 g / mL, 2), and 0.21 g / mL (± 0.03 g / mL, 2), respectively.

[0366] As expected, no binding was observed with isotype controls (FIGs. 10A-10C) or when CHO cells were not transfected with human FAP (FIG. 10D), demonstrating the Example 7: Inhibition of TGF signaling by anti-FAP-TGF RII bispecific antibodies

[0367] expressed a firefly luciferase reporter gene under the control of SMAD-responsive elements (HEK 293-SMAD luciferase cells, BPS Bioscience / 60653). The TGF neutralizing activity was compared to reference anti-FAP antibodies ESC11 and 28H1, or isotype control, all fused to wild-type TGF RII ECD. An irrelevant antibody was used as a negative isotype control.

[0368] HEK 293-SMAD luciferase cells were expanded with Minimal Essential Medium (MEM) with Earle’s Balanced Salt Solution (EBSS) and L-Glutamine, supplemented with 10% heat-inactivated FBS, Sodium Pyruvate (1X), 400 µg / mL Geneticin, and 1% penicillin-streptomycin. At the log-growth phase, the cells were trypsinized, counted, and seeded into a white 96-well flat-bottom assay plate to have a final concentration of 4x105cells / mL, and were incubated overnight in a humidified chamber at 5% CO2 and 37°C. Media was changed to MEM with EBSS and L-Glutamine, supplemented with 0.5% heat inactivated FBS, Sodium Pyruvate (1X), 400 µg / mL Geneticin, and 1% penicillin-streptomycin, by discarding the supernatant gently. Cells were then incubated in a humidified chamber for 4.5 hrs. After incubation, the media was gently removed and replaced by 50 µL of anti-FAP- TGF RII bispecific antibodies or control antibodies at a dose titration (20 to 0.0003 nM), and BUSINESS.32102391.1overnight. To measure luciferase activity, 100 µL of Bio-Glo luciferase assay system (Promega / G7940) were added to each well. The luminescence signal (RLU) was measured using a Tecan Infinite M1000-Pro plate reader. RLU values were plotted against anti-FAP- TGF RII bispecific antibody concentrations using GraphPad Prism software and 50% inhibitory concentration (IC50) values were calculated. The IC50 values are shown in Table 14.

[0369] As shown in FIGs. 11A and 11B, all anti-FAP-TGF RII bispecific antibodies activity. Table 14: Inhibition of TGF 1 signaling by anti-FAP-TGF RII bispecific antibodies.RII ECD

[0370] luciferase reporter cells as described above (dose range 100 to 0.00005 nM).

[0371] 50values (±SD, N) of 0.045 g / mL (±0.006 g / mL, 3) and 0.012 g / mL (±0.003 g / mL, 3), respectively. The calculated IC50values for TGF 1 and TGF are shown in Table 15. Consistent with its lower binding BUSINESS.32102391.1(Lopez-Casillas et al demonstrated.

[0372] signaling (FIGs.12A-12C). Table 15: IC50 values for inhibition of TGF 1 signaling.N / D: Not determined. Example 8: Binding to Fc R of anti-FAP-TGF RII bispecific antibodies

[0373] The capacity of anti-FAP-TGF RII bispecific antibody variants 23-B08-

[0374] the V / V or F / F polymorphism at position 158. Clones stably expressing a relatively high level 1640 medium (glutamax + 10% FBS + 1% NEAA + 1% G418) or Roswell Park Memorial Institute (RPMI)-1640 medium (glutamax + 10% FBS + 1% NEAA + 1% G418 + 0.25% Zeocin).

[0375] For the antibody binding assay, the cells were treated with PBS containing eBioscience Fixable Viability Dye Fluor 780 (1:1000) and seeded in a 96-well U-bottom tissue culture plate at a density of 4×105cells per well, and incubated at 37oC with 5% CO2 for 30 min. The cells were washed twice with cold FACS Buffer (PBS supplemented with 0.5% BSA, 1mM EDTA, and 0.05% sodium azide) and resuspended in 50 µL of FACS Buffer. The antibodies or control antibodies, at a final dose range of 0.003 to 50 µg / mL in FACS Buffer.

[0376] For detection of binding, the cells were washed twice with cold FACS Buffer BUSINESS.32102391.1IgG antibody (Jackson ImmunoResearch / 109-116-098) at a final dilution of 1 / 800. After a 45- minute incubation in the dark at 4°C, the cells were washed twice with cold FACS Buffer and analyzed by flow cytometry (BD LSR Fortessa Flow Cytometer). CHO cells were identified using a plot of forward scatter-area (FSC-A) versus side scatter area (SSC-A) and another plot of SSC-A versus forward scatter-height (SSC-H) for selection of single cells. Dead cells were identified by plotting SSC-A vs APC-CY7-A and gating out cells positive for the viability dye Fluor 780. Anti-FAP-TGF RII bispecific antibodies and control antibodies binding to cells were identified by plotting SSC-A vs PE-A. Samples were analyzed by sequentially gating on the single cell population, live / dead cells, and plotting histograms representing the fluorescence in the PE channel. For each sample, mean fluorescence intensity (MFI) was calculated, and the data were plotted by GraphPad Prism software.

[0377] As shown in FIG. 13A, fusion of the anti-FAP antibody 23-B08 to the variant TGF RII ECD decreased the binding to the inhibitory Fc RIIB receptor compared to the parental anti-FAP antibody. Decreased Fc RIIB binding was also observed when anti-FAP- TGF RII bispecific variants were made on an IgG1 backbone, including the DLE or the N297A substitutions.

[0378] Dose-dependent binding of anti-FAP-TGF RII bispecific variants to Fc RIIIA- expressing cell lines with V / V (high affinity for IgG1) and F / F (low affinity for IgG1) polymorphisms was observed, but fusion of the anti-FAP antibody to TGF RII ECD was associated with decreased binding (FIGs. 13B-13C). Compared to the wild-type IgG1 backbone, the largest decrease in binding was observed with the N297A variant, which substitutions in the IgG1 backbone increased the binding to Fc RIIIA (V / V) and (F / F). Variant RIIIA similarly as to 23-B08 and Isotype Control- RII, 23-B08-DLE showed the best binding to Fc RIIIA.

[0379] The calculated AUC and EC50 values are shown in Tables 16, 17, and 18 for Fc RIIB, Fc RIIIA (F / F), and Fc RIIIA (V / V), respectively. expressing CHO cells. BUSINESS.32102391.1(F / F)-expressing CHO cells.(V / V)-expressing CHO cells.N / D: Not determined. Example 9: Induction of Fc RIIIA signaling by anti-FAP-TGF RII bispecific antibodies

[0380] expressing cell lines, as described in Example 3.

[0381] BUSINESS.32102391.1(HEK cells or U138MG cells expressing human FAP) were plated overnight in 96-well cell culture plates to adhere, then incubated with increasing concentrations (7-point dose titration, expressing NFAT reporter cells were added to the antibody-target cell mixture at a 1:2.5 ratio (6×104cells) in RPMI media, supplemented with 4% heat-inactivated low-IgG FBS, at 37°C and 5% CO2. After a 6-hour incubation, Bio-Glo Luciferase Assay Substrate (Promega, G720A) was added to each sample (1:1 v / v). Luminescence was measured using the EnVision®Multilabel Plate Reader (PerkinElmer). Data were plotted with GraphPad Prism software.

[0382] affinity (V158) and the low affinity (F158) Jurkat effector cell lines, showing alleviation of the Example 10: Induction of ADCC by anti-FAP-TGF RII bispecific antibodies

[0383] tested as in Example 4.

[0384] Specifically, NK cells were isolated either from healthy donor peripheral blood mononuclear cells (PBMCs) and activated overnight in a cocktail of IL2 and IL15, or an NK92 with Calcein AM dye and plated at a density of 5000 cells / well. The activated NKs were then antibodies were added to the cell co-culture in a titrated manner (100, 10, 1, 0.1, 0.01, and 0.001 nM) to assess their impact. After a 3-hour incubation, cell lysis was evaluated by measuring the fluorescence emitted by release of the Calcein AM dye on a plate reader (PerkinElmer). Data was analyzed and plotted using Microsoft Excel and GraphPad Prism.

[0385] BUSINESS.32102391.1bispecific antibodies with DLE substitutions in the Fc region, known to enhance binding to

[0386] The calculated EC50and AUC values are shown in Table 19. Table 19: EC50 antibodies.anti-FAP-TGF RII bispecific antibodies

[0387] control.

[0388] Briefly, a red fluorescent protein (RFP) coding sequence was inserted at the 3’ end of the endogenous open reading frame (ORF) of vimentin gene in the lung cancer cell line A549 (ATCC CCL-185 EMT). The A549-Vimentin-RFP cell reporter line allowed the detection of vimentin protein, which is a marker of EMT, by measuring the fluorescence from RFP fused to vimentin.

[0389] temperature. A549-Vimentin-RFP cells were grown in F12-K media supplemented with 10% FBS at 37oC and 5% CO2 and were plated at a density of 20,000 cells per well in a 96-well flat BUSINESS.32102391.1incubated for 48 hours. The cells were washed with FACS buffer (PBS, 2mM EDTA, 5% BSA, pH 7.2) and analyzed using the LSRFortessa (BD Biosciences). Flow data were analyzed using FlowJo software and were plotted with GraphPad Prism software.

[0390] expression. Example 12: In vivo activity of anti-FAP-TGF RII bispecific antibodies a syngeneic model of colon cancer.

[0391] to reduce tumors and increase survival in a model of colon cancer.

[0392] Female C57 / BL6 mice (6-8 weeks old) were injected with 100,000 MC38 colon cancer cells in the mammary fat pad on day 0. Mice were treated with 10, 30, or 300 µg of 2) in combination with 200 µg of an anti-PD-1 antibody (clone RMP1-14, BioXcell), anti-PD- 1 antibody alone, or vehicle, administered intraperitonially beginning on day 7, when the tumors reached a volume range of 50-75 mm3. Tumor growth was monitored biweekly using a digital caliper.

[0393] synergized with anti-PD1 therapy to improve curative responses (FIG.17A) and survival (FIG. 17B), compared to the anti-PD-1 monotherapy, in all the doses tested. The combination therapy increased the survival of MC38 tumor-bearing mice up to 90%, as compared to 60% in the anti-PD-1 treated arm. rich stroma and increase T-cell infiltration of the tumor microenvironment.

[0394] One of the leading causes of anti-cancer therapy failure is the focus on solely targeting the cancer or malignant cells, while ignoring the tumor stroma or the tumor microenvironment (TME). The tumor stroma is a complex structure consisting of many cell types (epithelial, endothelial, immune, fibroblasts, mesenchymal, pericytes, adipocytes, and many more), along with acellular components such as extracellular matrix (ECM). Tumor stroma provides structure to the tumor and has critical roles in tumor initiation, progression, BUSINESS.32102391.1the tumor stroma by targeted depletion of the immunosuppressive FAP+CAF subset via differentiation, reversing EMT, and alleviating immunosuppression.

[0395] To elucidate the mechanism of action of the efficacy by which the anti-FAP- pharmacodynamic study was conducted in MC38 tumor-bearing mice. Specifically, morphological changes (necrosis and fibrosis) in the TME were assessed by histology. Stromal reorganization was characterized by measuring the CAF content and collagen content. T cell content (CD3, CD4, CD8), spatial organization with respect to CAFs, and motility of CD8+ T cells were also tested.

[0396] Six to 8-week-old female C57 / BL6 mice were injected with 100,000 MC38 cells in the mammary fat pad on day 0 and treated intraperitonially with 240 µg of PE22267 or vehicle on day 8, after randomization, when the tumors reached a range volume of 50-75 mm3. On day 11, the animals were sacrificed to isolate the tumor, and processed via cryosectioning for immunofluorescence and histology experiments. Immunofluorescence was used to evaluate the tumor stroma for T cell markers (CD3, CD4, and CD8), collagen (ECM protein), and CAF

[0397] As shown in FIG. 18A, treatment with PE22267 significantly reduced the collagen and CAF content, hence debulking the tumor and potentially enabling better penetration of therapeutics (antibodies, chemotherapeutics), and immune cells, such as T cells, into the TME. FIG. 18B demonstrates dramatic infiltration of CD8+ T cells in the tumor nest cells, whereas in the vehicle group, CD8+ T cells were sequestered in the periphery. FIG.18C shows increased proliferation of CD8+ T cells with PE22267 treatment, as demonstrated by (1-3) with PE22267 treatment.

[0398] bispecific antibodies can remodel the TME, making the tumor permissive to immune cells, activation / proliferation of infiltrated CD8+T cells to provide anti-tumor efficacy. This mechanism of action is complementary to immunotherapy, such as anti-PD-1, as demonstrated BUSINESS.32102391.1in Example 12. combination therapy reduce tumors in a syngeneic model of breast cancer

[0399] antibody that could contribute to the efficacy of treatment with immune checkpoint blockade (ICB) with anti-CTLA-4 and anti-PD-1 antibodies in a breast carcinoma syngeneic model, a pharmacodynamic study was conducted in 4T1 tumor-bearing mice. Morphological changes (necrosis, fibrosis) in the TME were assessed by histology. ECM density was characterized by measuring the collagen content. T cell infiltration (CD3), into the periphery and core of the tumor stroma was also tested.

[0400] Six to 8-week-old female Balb / c mice were injected with 20,0004T1 cells in bispecific murine surrogate antibodies (PE21208-1, a mouse surrogate of human clone 23-B08 (Clone 9D9.3M, Agenus) and anti-PD-1 antibody (clone RMP1-14, BioXCell) on day 7, after randomization, when the tumors reached a range volume of approximately 50 mm3. On day 19, 4 animals per group were sacrificed to isolate the tumor and processed via cryosectioning for immunofluorescence and histology experiments. Multiplex Immunofluorescence (CODEX platform, Akoya Biosciences) was used to evaluate the tumor stroma for T cell markers (CD3, CD4 and CD8), and Picrosirius red (PSR) staining was used to evaluate collagen (ECM protein).

[0401] synergized with anti-CTLA-4 + anti-PD-1 antibody therapy to improve anti-tumor responses, compared to the isotype control or individual therapies, in a difficult-to-treat orthotopic model of 4T1 mammary carcinoma.

[0402] As shown in FIG. 20A-FIG. 20E, treatment with PE21208-1 and the combination with anti-PD-1 and anti-CTLA-4 antibodies significantly reduced the collagen content, hence debulking the tumor. Moreover, along with the reduction in density of collagen, the collagen was also reorganized (from being organized, i.e., a bad prognosis, to disorganized, i.e., a good prognosis) in the PE21208-1 and PE21208-1 / anti-PD-1 / anti-CTLA-4 combination- treated group, potentially enabling better penetration of therapeutics (antibodies, BUSINESS.32102391.1chemotherapeutics) and immune cells such as T cells. A confirmation of this hypothesis can be seen in FIG.20F-FIG.20R which show enhancement of CD3+ T cell infiltration in 4T1 tumors treated with PE21208-1 in combination with anti-PD-1 / CTLA-4 treatment (FIG. 20P). Quantitative analysis of image data revealed increased frequency of CD3+ T cells in tumor tissue, as shown in FIG.20R. in a syngeneic model of colorectal cancer

[0403] antibodies in combination with ICB, another murine solid cancer indication was used. Six to 8-week-old female C57 / BL6 mice were injected with 100,000 MC38 colorectal carcinoma cells in the mammary fat pad on day 0, and treated intraperitonially with 240 µg of mouse surrogate bispecific PE21208-1, a murine anti-CTLA-4 antibody (clone 9D9, Agenus), or both, on day 8, after randomization, when the tumors reached a range volume of 50-75 mm3.

[0404] As shown in FIG. 21A, PE21208-1 synergized with anti-CTLA-4 therapy to improve anti-tumor responses, compared to the isotype control or the individual therapies. The benefit was observed at the dose of 240 µg of PE21208-1, administered twice weekly along with treatment of 100 µg anti-CTLA-4 once weekly. The combination arm demonstrated one curative response and 3 partial responses, as compared to no responses in any of the other groups (FIG.21A-FIG.21E).

[0405] ICB such as anti-PD-1 and anti-CTLA-4 antibodies. Further, the data illustrate that treatment permissive to immune cells, especially CD3+ T cells. This activation and proliferation of infiltrated CD3+ T cells provides increased anti-tumor efficacy. The remodeling of the TME interstitial pressure of the ECM, thereby making the tumor stroma less stiff, and leading to infiltration of small and large therapeutics such as chemotherapeutics. The combination regimens shown herein have complimentary functions and are particularly useful for modulating tumor stroma and immune response to tumors, and hence, are useful for treating cancer in a subject.

[0406] with ICB antibodies as well as first-line chemotherapy, to reduce tumorigenesis and provide clinical benefit to human patients. BUSINESS.32102391.1* * *

[0407] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0408] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0409] Other embodiments are within the following claims. BUSINESS.32102391.1

Claims

WHAT IS CLAIMED:

1. An antibody that specifically binds to human FAP, the antibody comprising a VH comprising the CDRH1, CDRH2, and CDRH3 sequences of the VH amino acid sequence set forth in SEQ ID NO: 76, and a VL comprising the CDRL1, CDRL2, and CDRL3 of the VL amino acid sequence set forth in SEQ ID NO:

92.

2. The antibody of claim 1, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequence, respectively, set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87.

3. The antibody of claim 1 or 2, wherein the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 11, 38, and 39.

4. The antibody of any one of the preceding claims, wherein the antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 42, 46, and 49.

5. The antibody of any one of the preceding claims, wherein the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, and 39; 1, 13, and 39; 1, 14, and 39; 1, 15, and 39; 2, 16, and 39; 3, 17, and 39; 1, 18, and 39; 4, 19, and 39; 5, 20, and 39; 6, 21, and 39; 1, 22, and 39; 4, 23, and 39; 7, 24, and 39; 8, 25, and 39; 1, 26, and 39; 1, 27, and 39; 1, 28, and 39; 9, 29, and 39; 1, 30, and 39; 1, 31, and 39; 1, 32, and 39; 1, 33, and 39; 1, 34, and 39; 10, 35, and 39; 1, 36, and 39; or 1, 37, and 39.

6. The antibody of any one of the preceding claims, wherein the antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 40, 43, and 47; 40, 44, and 47; 40, 43, and 48; 41, 45, and 47; or 41, 43, and 47. BUSINESS.32102391.

17. The antibody of any one of the preceding claims, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, 39, 40, 43, and 47; 1, 12, 39, 40, 44, and 47; 1, 13, 39, 40, 43, and 47; 1, 14, 39, 40, 43, and 47; 1, 15, 39, 40, 43, and 47; 2, 16, 39, 40, 43, and 47; 3, 17, 39, 40, 43, and 48; 1, 18, 39, 40, 43, and 47; 4, 19, 39, 40, 43, and 47; 5, 20, 39, 41, 45, and 47; 6, 21, 39, 40, 43, and 47; 1, 22, 39, 40, 43, and 47; 4, 23, 39, 40, 43, and 47; 7, 24, 39, 40, 43, and 47; 8, 25, 39, 40, 43, and 47; 1, 26, 39, 40, 43, and 47; 1, 27, 39, 40, 43, and 47; 1, 28, 39, 40, 43, and 47; 9, 29, 39, 40, 43, and 47; 1, 30, 39, 40, 43, and 47; 1, 31, 39, 40, 43, and 47; 1, 32, 39, 40, 43, and 47; 1, 33, 39, 40, 43, and 47; 1, 34, 39, 41, 43, and 47; 10, 35, 39, 40, 43, and 47; 1, 36, 39, 40, 43, and 47; or 1, 37, 39, 40, 43, and 47.

8. The antibody of any one of the preceding claims, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 14, 39, 40, 43, and 47.

9. The antibody of any one of the preceding claims, wherein the antibody comprises the VH amino acid sequence of SEQ ID NO:

76.

10. The antibody of any one of the preceding claims, wherein the antibody comprises the VH amino acid sequence of any one of SEQ ID NOs: 50-75.

11. The antibody of any one of the preceding claims, wherein the antibody comprises a heavy chain constant region, optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, and IgM.

12. The antibody of claim 11, wherein the heavy chain constant region is a human IgG1heavy chain constant region comprising a D at position 239, an L at position 330, and an E at position 332, numbered according to the EU numbering system. BUSINESS.32102391.

113. The antibody of any one of the preceding claims, wherein the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the 14. The antibody of any one of the preceding claims, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 134, 135, or 136.

15. The antibody of any one of the preceding claims, wherein the antibody comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 93-118.

16. The antibody of any one of the preceding claims, wherein the antibody comprises the VL amino acid sequence of SEQ ID NO:

92.

17. The antibody of any one of the preceding claims, wherein the antibody comprises the VL amino acid sequence of any one of SEQ ID NOs: 77-91.

18. The antibody of any one of the preceding claims, wherein the antibody comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119-133.

19. The antibody of any one of the preceding claims, wherein the VH and VL comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87.

20. The antibody of any one of the preceding claims, wherein the VH and VL comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 52 and 80.

21. The antibody of any one of the preceding claims, wherein the antibody comprises a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID BUSINESS.32102391.1NOs: 93 and 119; 93 and 120; 94 and 121; 95 and 122; 96 and 123; 97 and 121; 98 and 124; 99 and 119; 100 and 125; 101 and 126; 102 and 127; 103 and 128; 104 and 121; 105 and 129; 106 and 130; 107 and 121; 108 and 121; 109 and 131; 110 and 131; 111 and 119; 112 and 122; 113 and 125; 114 and 123; 115 and 132; 116 and 133; 117 and 130; or 118 and 129.

22. The antibody of any one of the preceding claims, wherein the antibody comprises a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 95 and 122.

23. The antibody of any one of claims 1-10, 16, 17, 19, or 20, wherein the antibody is an scFv, a Fab, or chimeric antigen receptor.

24. moiety.

25.

26.

27.

28.

29. linked to the heavy chain.

30. linked to the C-terminal residue of a heavy chain of the antibody. BUSINESS.32102391.

131. covalently linked to the C-terminal residue of the heavy chain via a peptide linker.

32. The antibody of claim 31, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 139-148.

33. amino acid sequence at least 75% (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 137 or 138.

34. amino acid sequence of SEQ ID NO: 137 or 138.

35. The antibody of any one of claims 30-34, wherein the heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 149-157.

36. An antibody that specifically binds human FAP, the antibody comprising a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 157 and 122; 150 and 122; 156 and 120; 149 and 121; 154 and 124; 151 and 119; 152 and 121; 153 and 130; or 155 and 122.

37. The antibody of claim 36, wherein the heavy chain and the light chain comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 157 and 122.

38. A polypeptide comprising a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO:

76.

39. The polypeptide of claim 38, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in any one of SEQ ID NOs: 50-75. BUSINESS.32102391.

140. The polypeptide of claim 38, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 11, 38, and 39.

41. The polypeptide of claim 38, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, and 39; 1, 13, and 39; 1, 14, and 39; 1, 15, and 39; 2, 16, and 39; 3, 17, and 39; 1, 18, and 39; 4, 19, and 39; 5, 20, and 39; 6, 21, and 39; 1, 22, and 39; 4, 23, and 39; 7, 24, and 39; 8, 25, and 39; 1, 26, and 39; 1, 27, and 39; 1, 28, and 39; 9, 29, and 39; 1, 30, and 39; 1, 31, and 39; 1, 32, and 39; 1, 33, and 39; 1, 34, and 39; 10, 35, and 39; 1, 36, and 39; or 1, 37, and 39.

42. The polypeptide of claim 38, wherein the VH comprises the amino acid sequence of SEQ ID NO:

76.

43. The polypeptide of claim 38, wherein the VH comprises the amino acid sequence of any one of SEQ ID NOs: 50-75.

44. The polypeptide of claim 38, wherein the polypeptide comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 93-118 or 149-157.

45. A polypeptide comprising a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO:

92.

46. The polypeptide of claim 45, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in any one of SEQ ID NOs: 77-91.

47. The polypeptide of claim 45, wherein the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 42, 46, and 49.

48. The polypeptide of claim 45, wherein the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 40, 43, and 47; 40, 44, and 47; 40, 43, and 48; 41, 45, and 47; or 41, 43, and 47. BUSINESS.32102391.

149. The polypeptide of claim 45, wherein the VL comprises the amino acid sequence of SEQ ID NO:

92.

50. The polypeptide of claim 45, wherein the VL comprises the amino acid sequence of any one of SEQ ID NOs: 77-91.

51. The polypeptide of any one of claims 38-43 and 45-50, wherein the polypeptide is a chimeric antigen receptor.

52. The polypeptide of claim 45, wherein the polypeptide comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119-133.

53. The antibody or polypeptide of any one of the preceding claims, wherein the antibody or polypeptide is conjugated to a cytotoxic agent, cytostatic agent, toxin, radionuclide, or detectable label.

54. A polynucleotide encoding: a VH, a VL, a heavy chain, and / or a light chain of an antibody of any one of claims 1-37; or a polypeptide of any one of claims 38-52.

55. A vector comprising the polynucleotide of claim 54.

56. A recombinant host cell comprising: (a) the polynucleotide of claim 54; (b) the vector of claim 55; (c) a first polynucleotide encoding a VH or a heavy chain of the antibody of any one of claims 1-37 and a second polynucleotide encoding a VL or a light chain of the antibody of any one of claims 1-37; or (d) a first vector comprising a first polynucleotide encoding a VL or a heavy chain of the antibody of any one of claims 1-37 and a second vector comprising a second BUSINESS.32102391.1polynucleotide encoding a VL or a light chain of the antibody of any one of claims 1- 37.

57. A pharmaceutical composition comprising the antibody of any one of claims 1-37 or 53, the polypeptide of any one of claims 38-53, the polynucleotide of claim 54, the vector of claim 55, or the host cell of claim 56, and a pharmaceutically acceptable carrier or excipient.

58. A method of producing an antibody, the method comprising culturing the host cell of claim 56 under suitable conditions so that the polynucleotide is expressed and the antibody is produced.

59. A method of increasing T cell infiltration of a tumor in a subject, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1-37 or 53, the polypeptide of any one of claims 38-53, the polynucleotide of claim 54, the vector of claim 55, the host cell of claim 56, or the pharmaceutical composition of claim 57.

60. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1-37 or 53, the polypeptide of any one of claims 38-53, the polynucleotide of claim 54, the vector of claim 55, the host cell of claim 56, or the pharmaceutical composition of claim 57.

61. Use of the antibody of any one of claims 1-37 or 53, the polypeptide of any one of claims 38-53, the polynucleotide of claim 54, the vector of claim 55, the host cell of claim 56, or the pharmaceutical composition of claim 57, for the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

62. The antibody of any one of claims 1-37 or 53, the polypeptide of any one of claims 38-53, the polynucleotide of claim 54, the vector of claim 55, the host cell of claim 56, or the pharmaceutical composition of claim 57, for use in medicine.

63. The antibody of any one of claims 1-37 or 53, the polypeptide of any one of claims 38-53, the polynucleotide of claim 54, the vector of claim 55, the host cell of claim 56, or the BUSINESS.32102391.1pharmaceutical composition of claim 57, for use in the treatment of cancer in a subject in need thereof.

64. The antibody of any one of claims 1-37 or 53, the polypeptide of any one of claims 38-53, the polynucleotide of claim 54, the vector of claim 55, the host cell of claim 56, or the pharmaceutical composition of claim 57, for use in increasing T cell infiltration of a tumor in a subject.

65. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a first antibody that specifically binds to human FAP and a second antibody that specifically binds to human CTLA-4, PD-1, or PD-L1.

66. The method of claim 1, further comprising administering to the subject a third antibody that specifically binds to human CTLA-4, PD-1, or PD-L1.

67. The method of claim 65 or 66, wherein the second antibody specifically binds to human CTLA-4 and the third antibody specifically binds to human PD-1 or PD-L1.

68. The method of any one of claims 65-67, wherein the second antibody specifically binds to CTLA-4 and the third antibody specifically binds to PD-1.

69. The method of any one of claims 65-67, wherein the second antibody specifically binds to CTLA-4 and the third antibody specifically binds to PD-L1.

70. The method of any one of claims 65-69, wherein the antibody that specifically binds to human CTLA-4 comprises a human IgG heavy chain constant region that is a variant of a wild- type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant BUSINESS.32102391.

171. The method of any one of claims 65-70, wherein the antibody that binds to human CTLA- 4 comprises a human IgG1 heavy chain constant region comprising S239D / A330L / I332E mutations, numbered according to the EU numbering system.

72. The method of any one of claims 65-71, wherein the antibody that specifically binds to human CTLA-4 competes for binding with or binds to the same epitope as botensilimab, ipilimumab, or tremelimumab.

73. The method of any one of claims 65-72, wherein the antibody that specifically binds to human CTLA-4 comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and a CDRL3 with an amino acid sequence that is identical to the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively, of botensilimab, ipilimumab, or tremelimumab.

74. The method of any one of claims 65-73, wherein the antibody that specifically binds to human CTLA-4 is botensilimab.

75. The method of any one of claims 65-74, wherein the antibody that specifically binds to human PD-1 or PD-L1 is selected from the group consisting of adebrelimab, atezolizumab, avelumab, balstilimab, camrelizumab, cemiplimab, cosibelimab, dostarlimab, durvalumab, enlonstobart, envafolimab, nivolumab, pembrolizumab, penpulimab, pidilizumab, prolgolimab, pucotenlimab, retifanlimab, serplulimab, sintilimab, socazolimab, sugemalimab, tagitanlimab, tislelizumab, toripalimab, and zimberelimab.

76. The method of any one of claims 65-75, wherein the antibody that specifically binds to human PD-1 is balstilimab.

77. The method of any one of claims 65-76, wherein the second antibody is botensilimab and the third antibody is balstilimab, nivolumab, or pembrolizumab.

78. The method of any one of claims 65-77, wherein the first antibody that specifically binds to human FAP comprises a VH comprising the CDRH1, CDRH2, and CDRH3 sequences of the BUSINESS.32102391.1VH amino acid sequence set forth in SEQ ID NO: 76, and a VL comprising the CDRL1, CDRL2, and CDRL3 of the VL amino acid sequence set forth in SEQ ID NO:

92.

79. The method of any one of claims 65-78, wherein the first antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequence, respectively, set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87.

80. The method of any one of claims 65-79, wherein the first antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 11, 38, and 39.

81. The method of any one of claims 65-80, wherein the first antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 42, 46, and 49.

82. The method of any one of claims 65-81, wherein the first antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 12, and 39; 1, 13, and 39; 1, 14, and 39; 1, 15, and 39; 2, 16, and 39; 3, 17, and 39; 1, 18, and 39; 4, 19, and 39; 5, 20, and 39; 6, 21, and 39; 1, 22, and 39; 4, 23, and 39; 7, 24, and 39; 8, 25, and 39; 1, 26, and 39; 1, 27, and 39; 1, 28, and 39; 9, 29, and 39; 1, 30, and 39; 1, 31, and 39; 1, 32, and 39; 1, 33, and 39; 1, 34, and 39; 10, 35, and 39; 1, 36, and 39; or 1, 37, and 39.

83. The method of any one of claims 65-82, wherein the first antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 40, 43, and 47; 40, 44, and 47; 40, 43, and 48; 41, 45, and 47; or 41, 43, and 47.

84. The method of any one of claims 65-83, wherein the first antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in BUSINESS.32102391.1SEQ ID NOs: 1, 12, 39, 40, 43, and 47; 1, 12, 39, 40, 44, and 47; 1, 13, 39, 40, 43, and 47; 1, 14, 39, 40, 43, and 47; 1, 15, 39, 40, 43, and 47; 2, 16, 39, 40, 43, and 47; 3, 17, 39, 40, 43, and 48; 1, 18, 39, 40, 43, and 47; 4, 19, 39, 40, 43, and 47; 5, 20, 39, 41, 45, and 47; 6, 21, 39, 40, 43, and 47; 1, 22, 39, 40, 43, and 47; 4, 23, 39, 40, 43, and 47; 7, 24, 39, 40, 43, and 47; 8, 25, 39, 40, 43, and 47; 1, 26, 39, 40, 43, and 47; 1, 27, 39, 40, 43, and 47; 1, 28, 39, 40, 43, and 47; 9, 29, 39, 40, 43, and 47; 1, 30, 39, 40, 43, and 47; 1, 31, 39, 40, 43, and 47; 1, 32, 39, 40, 43, and 47; 1, 33, 39, 40, 43, and 47; 1, 34, 39, 41, 43, and 47; 10, 35, 39, 40, 43, and 47; 1, 36, 39, 40, 43, and 47; or 1, 37, 39, 40, 43, and 47.

85. The method of any one of claims 65-84, wherein the first antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 1, 14, 39, 40, 43, and 47.

86. The method of any one of claims 65-85, wherein the first antibody comprises the VH amino acid sequence of SEQ ID NO:

76.

87. The method of any one of claims 65-86, wherein the first antibody comprises the VH amino acid sequence of any one of SEQ ID NOs: 50-75.

88. The method of any one of claims 65-87, wherein the first antibody comprises a heavy chain constant region, optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, and IgM.

89. The method of claim 88, wherein the heavy chain constant region of the first antibody is a human IgG1 heavy chain constant region comprising a D at position 239, an L at position 330, and an E at position 332, numbered according to the EU numbering system.

90. The method of any one of claims 65-89, wherein the first antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant BUSINESS.32102391.

191. The method of any one of claims 65-90, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 134, 135, or 136.

92. The method of any one of claims 65-91, wherein the first antibody comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 93-118.

93. The method of any one of claims 65-92, wherein the first antibody comprises the VL amino acid sequence of SEQ ID NO:

92.

94. The method of any one of claims 65-93, wherein the first antibody comprises the VL amino acid sequence of any one of SEQ ID NOs: 77-91.

95. The method of any one of claims 65-94, wherein the first antibody comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119-133.

96. The method of any one of claims 65-95, wherein the VH and VL of the first antibody comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 50 and 77; 50 and 78; 51 and 79; 52 and 80; 53 and 81; 54 and 79; 55 and 82; 56 and 77; 57 and 83; 58 and 84; 59 and 85; 60 and 86; 61 and 79; 62 and 87; 63 and 88; 64 and 79; 65 and 79; 66 and 89; 67 and 89; 68 and 77; 69 and 80; 70 and 83; 71 and 81; 72 and 90; 73 and 91; 74 and 88; or 75 and 87.

97. The method of any one of claims 65-96, wherein the VH and VL of the first antibody comprise the amino acid sequences, respectively, set forth in SEQ ID NOs: 52 and 80.

98. The method of any one of claims 65-97, wherein the first antibody comprises a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 93 and 119; 93 and 120; 94 and 121; 95 and 122; 96 and 123; 97 and 121; 98 and 124; 99 and 119; 100 and 125; 101 and 126; 102 and 127; 103 and 128; 104 and 121; 105 and 129; 106 and 130; 107 and 121; 108 and 121; 109 and 131; 110 and 131; 111 and 119; 112 and 122; 113 and 125; 114 and 123; 115 and 132; 116 and 133; 117 and 130; or 118 and 129. BUSINESS.32102391.

199. The method of any one of claims 65-98, wherein the first antibody comprises a heavy chain and a light chain comprising the amino acid sequences, respectively, set forth in SEQ ID NOs: 95 and 122. binding moiety. linked to the heavy chain. linked to the C-terminal residue of a heavy chain of the antibody. covalently linked to the C-terminal residue of the heavy chain via a peptide linker.

108. The method of claim 107, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 139-148. BUSINESS.32102391.1an amino acid sequence at least 75% (e.g., at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 137 or 138. the amino acid sequence of SEQ ID NO: 137 or 138.

111. The method of any one of claims 106-110, wherein the heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 149-157.

112. The method of any one of claims 65-111, wherein the first antibody that specifically binds to human FAP is an scFv, a Fab, or chimeric antigen receptor. BUSINESS.32102391.1

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