Anti-FAP antibodies and uses thereof

Abstract

The present application relates to particular anti-FAP antibodies, including multispecific antibodies, as well as nucleic acids encoding the antibodies, vectors and host cells comprising the nucleic acids, and methods of making and using the antibodies.

Description

ANTI-FAP ANTIBODIES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional application No. 63 / 656,763, filed June 6, 2024, entitled “ANTI-FAP ANTIBODIES AND USES THEREOF”, the contents of which are incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The contents of the Sequence Listing (217372000740SEQLIST.xml; Size: 19,111 bytes; and Date of Creation: May 30, 2025) are herein incorporated by reference in its entirety.FIELD

[0003] The present application relates to anti-FAP antibodies and antigen binding fragments thereof, nucleic acids encoding the antibodies and antigen binding fragments thereof, vectors and host cells comprising the nucleic acids, and methods of making and using the antibodies and antigen binding fragments thereof.BACKGROUND

[0004] There exists a need for safe and effective agents and methods for treating, preventing and managing cancer, including for cancers that are refractory to standard treatments, while reducing or avoiding the toxicities and / or side effects associated with some existing therapies.

[0005] There also exists a need for safe and effective agents and methods for treating, preventing and managing fibrosis.

[0006] The present disclosure provides such agents, including anti-FAP antibodies and antigen binding fragments thereof.SUMMARY

[0007] The present disclosure relates to anti-FAP antibodies and antigen binding fragments thereof, nucleic acids encoding the antibodies and antigen binding fragments thereof, vectors and host cells comprising the nucleic acids, and methods of both making and using the antibodies and antigen binding fragments thereof. For example, embodiments of the disclosure include the following:Embodiment 1. An antibody or antigen binding fragment thereof that binds fibroblast activation protein alpha (FAP), wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1 or 7, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 8, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 9; and a light chain variable region comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 10, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 or GAS (SEQ ID NO: 11), and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or 12.Embodiment 2. The antibody or antigen binding fragment thereof of embodiment 1, wherein the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.Embodiment 3. The antibody or antigen binding fragment thereof of embodiment 1, wherein the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a LCDR2 comprising the amino acid sequence of GAS (SEQ ID NO: 11), and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.Embodiment 4. The antibody or antigen binding fragment thereof of any one of embodiments 1-3, wherein the heavy chain variable region comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15; and the light chain variable region comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.Embodiment 5. The antibody or antigen binding fragment thereof of any one of embodiments 1-3, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.Embodiment 6. The antibody or antigen binding fragment thereof of any one of embodiments 1-5, wherein the heavy chain variable region comprises the amino acid sequenceof SEQ ID NO: 13 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14, or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16.Embodiment 7. The antibody or antigen binding fragment thereof of any one of embodiments 1-6, wherein the antibody or antigen binding fragment thereof is an antibody fragment.Embodiment 8. The antibody or antigen binding fragment thereof of embodiment 7, wherein the antibody fragment is selected from a Fab, Fab’, F(ab)2, and scFv.Embodiment 9. The antibody or antigen binding fragment thereof of embodiment 8, which is an scFv.Embodiment 10. The antibody or antigen binding fragment thereof of embodiment 9, wherein the antibody or antigen binding fragment thereof comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 17. Embodiment 11. The antibody or antigen binding fragment thereof of embodiment 9 or embodiment 10, wherein the antibody or antigen binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 17.Embodiment 12. A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of embodiments 1-11 and a pharmaceutically acceptable carrier.Embodiment 13. An isolated nucleic acid that encodes the antibody or antigen binding fragment thereof of any one of embodiments 1-11.Embodiment 14. The isolated nucleic acid of embodiment 13, which is an expression vector.Embodiment 15. A host cell that expresses the antibody or antigen binding fragment thereof of any one of embodiments 1-11.Embodiment 16. A host cell comprising the nucleic acid of embodiment 13 or embodiment 14.Embodiment 17. A method of producing an antibody or antigen binding fragment thereof comprising culturing the host cell of embodiment 15 or embodiment 16 under conditions suitable for expressing the antibody or antigen binding fragment thereof.Embodiment 18. The method of embodiment 17, comprising isolating the antibody or antigen binding fragment thereof.Embodiment 19. A multispecific antibody or antigen binding fragment thereof comprising a first antigen-binding domain that binds FAP and at least one second antigen-binding domainthat binds a second target antigen, wherein the first antigen binding domain comprises a first heavy chain variable region and a first light chain variable region, wherein: a) the first heavy chain variable region comprises a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1 or 7, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 8, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 9; and b) the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 10, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 or GAS (SEQ ID NO: 11), and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or 12.Embodiment 20. The multispecific antibody or antigen binding fragment thereof of embodiment 19, wherein the first heavy chain variable region comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.Embodiment 21. The multispecific antibody or antigen binding fragment thereof of embodiment 19, wherein the first heavy chain variable region comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a LCDR2 comprising the amino acid sequence of GAS (SEQ ID NO: 11), and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.Embodiment 22. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-21, wherein the first heavy chain variable region comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15; and the first light chain variable region comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.Embodiment 23. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-22, wherein the first antigen binding domain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15, and / or a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.Embodiment 24. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-23, wherein the first antigen binding domain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, or wherein the first antigen binding domain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.Embodiment 25. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-24, wherein the first antigen binding domain is a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv).Embodiment 26. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-25, wherein the first antigen binding domain is a scFv or sdFv.Embodiment 27. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-26, wherein the first antigen binding domain comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 17.Embodiment 28. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-27, wherein the first antigen binding domain comprises the amino acid sequence of SEQ ID NO: 17.Embodiment 29. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-28, wherein the second antigen binding domain comprises a second heavy chain variable region and a second light chain variable region.Embodiment 30. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-29, wherein the second antigen binding domain is a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv).Embodiment 31. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-30, wherein the second target antigen is not CD40.Embodiment 32. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-31, wherein the multispecific antibody or antigen binding fragment thereof comprises a third antigen binding domain that binds to a third target antigen, wherein the second target antigen and the third target antigen are the same or different.Embodiment 33. The multispecific antibody or antigen binding fragment thereof of embodiment 32, wherein the third antigen binding domain is a Fab, Fab', F(ab')2, Fd, Fv, singlechain Fv (scFv) or disulfide-linked Fv (sdFv).Embodiment 34. The multispecific antibody of antigen binding fragment thereof of embodiment 32 or embodiment 33, wherein the third target antigen is not CD40.Embodiment 35. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 32-34, wherein the second antigen binding domain and the third antigen binding domain are the same or different.Embodiment 36. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 32-35, wherein: a) the second antigen binding domain comprises a second heavy chain variable region and a second light chain variable region; and b) the third antigen binding domain comprises a third heavy chain variable region and a third light chain variable region.Embodiment 37. The multispecific antibody or antigen binding fragment thereof of embodiment 36, wherein: c) the second heavy chain variable region is fused to a first heavy chain constant region and the second light chain variable region is fused to a first light chain constant region; and d) the third heavy chain variable region is fused to a second heavy chain constant region and the third light chain variable region is fused to a second light chain constant region.Embodiment 38. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-37, wherein the first antigen binding domain that binds to FAP is a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv), and wherein the first antigen binding domain is fused to the C-terminus of the first or second heavy chain constant region.Embodiment 39. The multispecific antibody or antigen binding fragment thereof of embodiment 37 or embodiment 38, wherein the first heavy chain constant region and the second heavy chain constant region are different.Embodiment 40. The multispecific antibody or antigen binding fragment thereof of embodiment 39, wherein the first heavy chain constant region and the second heavy chain constant region form a heterodimer.Embodiment 41. The multispecific antibody or antigen binding fragment thereof of embodiment 39 or embodiment 40, wherein the first heavy chain constant region comprises at least one first heterodimerization mutation and the second heavy chain constant region comprises at least one second heterodimerization mutation.Embodiment 42. The multispecific antibody or antigen binding fragment thereof of embodiment 41, wherein at least one first heterodimerization mutation comprises T366W and at least one second heterodimerization mutation comprises one or more of T366S, L368A, and / or Y407V, or wherein at least one first heterodimerization mutation comprises one or more of T366S, L368A, and / or Y407V and at least one second heterodimerization mutation comprises T366W, wherein mutation position is according to Kabat numbering.Embodiment 43. The multispecific antibody or antigen binding fragment thereof of embodiment 41, wherein at least one first heterodimerization mutation comprises T366W and at least one second heterodimerization mutation comprises T366S, L368A, andY407V, or wherein at least one first heterodimerization mutation comprises T366S, L368A, and Y407V and at least one second heterodimerization mutation comprises T366W, wherein mutation position is according to Kabat numbering.Embodiment 44. The multispecific antibody or antigen binding fragment thereof of embodiment 41, wherein at least one first heterodimerization mutation comprises one or more of T350V, L351Y, S400E, F405A, and / or Y407V and at least one second heterodimerization mutation comprises one or more of T350V, T366L, N390R, K392M, K392L, and / or T394W, or wherein at least one first heterodimerization mutation comprises one or more of T350V, T366L, N390R, K392M, K392L, and / or T394W and at least one second heterodimerization mutation comprises one or more of T350V, L351Y, S400E, F405A, and / or Y407V, wherein mutation position is according to Kabat numbering.Embodiment 45. The multispecific antibody or antigen binding fragment thereof of embodiment 41, wherein at least one first heterodimerization mutation comprises T350V, L351Y, F405A, and Y407V and at least one second heterodimerization mutation comprises T350V, T366L, K392L, and T394W, or wherein at least one first heterodimerization mutation comprises one or more of T350V, T366L, K392L, and T394W and at least one second heterodimerization mutation comprises one or more of T350V, L351Y, F405A, and Y407V, wherein mutation position is according to Kabat numbering.Embodiment 46. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-45, wherein the second target antigen is an immune cell antigen.Embodiment 47. The multispecific antibody or antigen binding fragment thereof of embodiment 46, wherein the immune cell antigen is expressed on the surface of a T cell, a natural killer (NK) cell, or a dendritic cell.Embodiment 48. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-45, wherein the second target antigen is expressed on fibroblasts.Embodiment 49. The multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-48, wherein the second target antigen is selected from CD3 and TGFRpiI. Embodiment 50. A pharmaceutical composition comprising the multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-49 and a pharmaceutically acceptable carrier.Embodiment 51. An isolated nucleic acid that encodes the multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-49.Embodiment 52. The isolated nucleic acid of embodiment 51, which is an expression vector.Embodiment 53. A host cell that expresses the multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-49.Embodiment 54. A host cell comprising the nucleic acid of embodiment 51 or embodiment 52.Embodiment 55. A method of producing a multispecific antibody or antigen binding fragment thereof comprising culturing the host cell of embodiment 53 or embodiment 54 under conditions suitable for expressing the multispecific antibody or antigen binding fragment thereof.Embodiment 56. The method of embodiment 55, comprising isolating the multispecific antibody or antigen binding fragment thereof.Embodiment 57. A method of treating cancer comprising administering to a subject in need thereof the antibody or antigen binding fragment thereof of any one of embodiments 1-11, the multispecific antibody or antigen binding fragment thereof of any one of embodiments 19-49, or the pharmaceutical composition of embodiment 12 or embodiment 50.Embodiment 58. The method of embodiment 57, wherein the cancer is a solid cancer. Embodiment 59. A method of treating fibrosis comprising administering to a subject in need thereof the antibody or antigen binding fragment thereof of any one of embodiments 1-11, the multispecific antibody or antigen binding fragment thereof of any one of embodiments 19- 49, or the pharmaceutical composition of embodiment 12 or embodiment 50.

[0008] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims. All references cited herein are incorporated in their entirety by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGs. 1A-1B show activation of human dendritic cells (DCs) (FIG. 1A) and macrophages (FIG. IB) with a multispecific antibody that binds FAP and CD40. FIG. 1Ashows IL12p40 secretion in response to increasing amounts of CD40xFAP.LP62 in a co-culture assay of DCs with HFF1 or HFF1 FAP KO cells (n=8). FIG. IB shows TNFa secretion in response to increasing amounts of CD40xFAP.LP62 in a co-culture assay of macrophages with HFF1 or HFF1 FAP KO cells (representative data from n=8 donors across 4 independent experiments).

[0010] FIGs. 2A-2B show CCL22 (FIG. 2A) and IL12p40 (FIG. 2B) secretion in response to increasing amounts of mCD40xFAP.LP62 in a co-culture assay of bone marrow-derived macrophages (BMDMs) with MC38-FAP or MC38-FAP KO cells (representative data from 4 pooled mice across 2 independent experiments).

[0011] FIG. 3 shows cumulative KPCY tumor growth curves upon treatment (QDxl) with 3 or 10 mg / kg mCD40xFAP.LP62 compared to mCD40xHEL (hen egg lysozyme) isotype control. Data shown are representative of 10 mice per group across 2 independent experiments.Statistical significance was determined by 2-way Anova (repeat measure). ** P<0.01, *** P<0.001; **** P<0.0001.

[0012] FIGs. 4A-4E shows immune profiling of KPCY tumor-bearing mice dosed with 10 mg / kg mCD40xFAP.LP62 or isotype control. CD86 expression (FIG. 4A), % migratory crosspresenting dendritic cells (cDCl) (FIG. 4B), and percent of activated cDCl cells that migrated to tumor-draining lymph nodes (TDLN) (FIG. 4C) are all increased at 48 hours. cDCl cells in TDLNs showed only modest (p<0.01), if any, increase, in CD86 activation (FIG. 4D). The ratio of CD8+ T cells to Tregs (CD8 T:Treg) was increased at 240 hours (FIG. 4E). Error bars indicate mean ± SD. Statistical analyses were performed by using unpaired parametric Student’s t-test with Welch’s correction. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Data shown are representative of 10 mice per group across 2 independent experiments.

[0013] FIG. 5 shows cytokine levels (IFNy, IL-ip, IP10, TNFa, and MIP-la) in KPCY tumors 240 hours post-treatment with mCD40xFAP.LP62 or mCD40xHEL isotype control (10 mg / kg, QDxl). Statistical significance was determined by Student’s T test (nonparametric, Mann- Whitney). ** P<0.01, **** P<0.0001. Data are representative of 10 mice per group across 2 independent experiments for the isotype control and 3 and 10 mice per group for mCD40xFAP.LP62 across two independent experiments.

[0014] FIG. 6 shows pSMAD inhibition (measured as % control) after treatment with varying concentrations of three different anti-FAP / TGFpR2 bispecific antibodies or control anti- HEL / TGFPR2 antibody.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTSI. Definitions

[0015] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0016] In this application, the use of “or” means “and / or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0017] Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are described, e.g., in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), among other places.

[0018] As used herein, the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., + / -5- 10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as “at least” and “about” precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

[0019] The term “polypeptide” refers to a polymer of amino acid residues, and is not limited to a minimum length. A “protein” may comprise one or more polypeptides. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide” or “protein” refers to a polypeptide or protein, respectively, which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification. A protein may comprise two or more polypeptides.

[0020] ‘ ‘FAP” or “fibroblast activating protein alpha” as used herein, refers to human FAP (UniProt ID: Q12884.1; NP_004451.2), unless expressly noted otherwise (i.e., murine FAP, cynomolgus FAP, or the like). An exemplary mature human FAP amino acid sequences is shown in SEQ ID NO: 18. FAP is expressed in some tumor tissues, for example, in the tumor microenvironment (TME), including during development of malignant tumors. For example, cancer-associated fibroblasts (CAFs) in the tumor stroma can have high levels of FAP expression, where it plays a role in promoting tumor growth, invasion, metastasis, and immunosuppression.

[0021] The term “antibody” herein refers to a molecule comprising at least complementaritydetermining region (CDR) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to antigen. The term is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, etc.), full length antibodies, single-chain antibodies, antibody conjugates, and antibody fragments, so long as they exhibit the desired FAP- specific binding activity.

[0022] An “isolated” antibody is one that has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0023] An “antigen” refers to the target of an antibody, i.e., the molecule to which the antibody specifically binds. The term “epitope” denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes on a protein can be formed both from contiguous amino acid stretches (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g., coming in spatial proximity due to the folding of the antigen, i.e., by the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by an antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents.

[0024] An “anti-FAP antibody” or “anti-FAP antibody or antigen binding fragment thereof’ or a “FAP-antibody” “FAP-antibody or antigen binding fragment thereof’ or an “antibody thatspecifically binds to FAP” and similar phrases refer to an antibody or antigen binding fragment thereof that specifically binds to FAP as defined herein.

[0025] The term “heavy chain” refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term “full-length heavy chain” refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0026] The term “light chain” refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term “full-length light chain” refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0027] The term “complementarity determining regions” (“CDRs”) as used herein refers to each of the regions of an antibody variable region which are hypervariable in sequence and which determine antigen binding specificity. Generally, antibodies comprise six CDRs: three in the VH (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Unless otherwise indicated, the CDRs are determined according to the sequence table herein. In some embodiments, CDRs are determined according to Kabat definitions. In some embodiments, CDRs are determined according to IMGT.

[0028] ‘ ‘Framework,” “framework region,” or “FR” refers to the residues of the variable region residues that are not part of the complementary determining regions (CDRs). The FR of a variable region generally consists of four FRs: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR- H1(CDR-L1)-FR2- CDR-H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4.

[0029] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). See, e.g., Kindt et al. Kuby Immunology, 6thed., W.H. Freeman and Co., page 91 (2007). A variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and / or FR4; and light chain (LC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and / or FR4. That is, a variable domain may lack a portion of FR1 and / or FR4 so long as it retains antigenbinding activity. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH orVL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150 :880-887 (1993) ; Clarkson et al., Nature 352 :624-628 (1991).

[0030] The light chain and heavy chain “constant regions” of an antibody refer to additional sequence portions outside of the FRs and CDRs and variable regions. Certain antibody fragments may lack all or some of the constant regions. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.

[0031] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain at Gly446 and Lys447 (EU numbering). Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine and lysine, respectively. Therefore, the C-terminal lysine, or the C-terminal glycine and lysine, of the Fc region may or may not be present. Thus, a “full-length heavy chain constant region” or a “full length antibody” for example, which is a human IgGl antibody, includes an IgGl with both a C-terminal glycine and lysine, without the C-terminal lysine, or without both the C-terminal glycine and lysine. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0032] ‘ ‘Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0033] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called ex, 8, e, y, and p., respectively. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda ( ), based on the amino acid sequence of its constant domain.

[0034] An “antibody fragment” or “antigen-binding fragment” or “antigen binding fragment” or “antigen-binding portion” refers to a fragment or portion of an antibody other than an intact antibody that binds the antigen (e.g., FAP) to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0035] The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or, in the case of an IgG antibody, having heavy chains that contain an Fc region as defined herein above.

[0036] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0037] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0038] A “human antibody” as used herein refers to antibodies produced from human immunoglobulin sequences, such as antibodies produced in non-human animals that comprise human immunoglobulin genes (such as XenoMouse® and Veloclmmune® mice), and antibodies selected using in vitro methods, such as phage display, wherein the antibody repertoire is based on a human immunoglobulin sequence.

[0039] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprisingthe population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0040] A “multispecific” antibody is one that binds specifically to more than one target antigen, while a “bispecific” antibody is one that binds specifically to two antigens. An “antibody conjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a therapeutic agent or a label.

[0041] Antibodies may be modified as part of the production process in certain host cells or through metabolism in vivo. An antibody or antibody region amino acid sequence herein is intended to encompass not only the specific amino acid sequence, but also that sequence as post- translationally modified, for instance, including side chain modifications and cleavages. Such a post-translational modification can occur, for instance, as a result of production of the antibody in a host cell and / or as a result of post-translational modification in vivo in an animal (e.g., a human).

[0042] In some embodiments, an antibody disclosed herein comprises a post-translational modification (e.g., one or more post-translational modifications). Post-translational modifications can include, e.g., ubiquitination, phosphorylation, acetylation, hydroxylation, methylation, glycyosylation, AMPylation, prenylation, deamidation, elimylation, citrullination, and carbamoylation. In some embodiments, the antibody is not post-translationally modified.

[0043] As noted above, antibodies can undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain, often a Gly-Lys. This cleavage can occur, for instance, as a result of the process of production of the antibody in a host cell. An antibody produced by expression of a specific nucleic acid molecule encoding a full-length heavy chain can include the full-length heavy chain, or it can include a cleaved variant of the full-length heavy chain, such as a heavy chain lacking a C-terminal Lys or a C- terminal Gly-Lys.

[0044] Other types of post-translational modifications can occur during production of antibodies, or otherwise in vivo, such as the modification of an amino acid side chain. For instance, an N-terminal Glu or Gin residue on an antibody chain can be post-translationallymodified to an N-terminal pyroglutamate (also known as pyrrolidine carboxylate; abbreviated pE).

[0045] ‘ ‘Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0046] The term “signal sequence” or “leader sequence” refers to a sequence of amino acid residues located at the N terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell. A leader sequence may be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached. Nonlimiting exemplary leader sequences also include leader sequences from heterologous proteins. In some embodiments, an antibody lacks a leader sequence. In some embodiments, an antibody comprises at least one leader sequence, which may be selected from native antibody leader sequences and heterologous leader sequences.

[0047] The term “nucleic acid molecule” or “polynucleotide” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine I, guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugarsor phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA, circular RNA) vectors, can be unmodified or modified.

[0048] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0049] ‘ ‘Isolated nucleic acid encoding an anti-FAP antibody” refers to one or more nucleic acid molecules encoding anti-FAP antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0050] ‘ ‘Isolated nucleic acid that encodes a multispecific antibody” refers to one or more nucleic acid molecules encoding multispecific antibodies that bind FAP in one or more polynucleotides, including such nucleic acid molecule(s) in a single vector or as separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0051] The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

[0052] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.

[0053] In this disclosure, “binds” or “binding” or “specific binding” and similar terms, when referring to a protein and its ligand or an antibody and its antigen target for example, or some other binding pair, means that the binding affinity between the members of the binding pair is sufficiently strong that the interaction cannot be due to random molecular associations (i.e. “nonspecific binding”). Such binding typically requires a dissociation constant (KD) of IpM or less, and may often involve a KD of 100 nM or less.

[0054] “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 affinitywhich reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can generally be represented by the dissociation constant (KD). Affinity of an antibody for an antigen can be measured by common methods known in the art, such as biolayer interferometry or surface plasmon resonance (SPR), for instance.

[0055] The terms “reduce” or “inhibit” more generally refer to a decrease or cessation of any event (such as protein ligand binding) or to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and / or amount as compared to a reference. It is not necessary that the inhibition or reduction be complete. For example, in certain embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater. In another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.

[0056] ‘ ‘Treatment” or “treating” as used herein, covers any administration or application of a therapeutic for disease in a human, and includes inhibiting the disease or progression of the disease or one or more disease symptoms, inhibiting or slowing the disease or its progression or one or more of its symptoms, arresting its development, partially or fully relieving the disease or one or more of its symptoms, or preventing a recurrence of one or more symptoms of the disease.

[0057] The terms “subject” and “patient” are used interchangeably herein to refer to a human unless expressly indicated otherwise (i.e., a murine subject or the like).

[0058] The term “cancer” is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. A cancer may be benign (also referred to as a benign tumor), pre-malignant, or malignant. Cancer cells may be solid cancer cells or leukemic cancer cells.

[0059] As used herein, “solid cancer” or “solid tumor” refers to abnormally high levels of proliferation and growth in one or more solid organ and / or tissue, including but not limited to the stomach, colon, pancreas, lungs, breast, and mesothelium. The solid tumor has an associated “tumor microenvironment” (or “TME”), which may comprise, for example, cells (e.g., immune cells and stromal cells), molecules and metabolites, blood vessels, and an extracellular matrix (ECM) that surrounds the solid tumor and supports cancer cell survival, local invasion, and / or metastatic dissemination. In some instances, an antibody binds an antigen expressed in the tumor microenvironment. In some instances, an antibody binds FAP expressed in the tumor microenvironment.

[0060] Nonlimiting exemplary solid cancers include squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer, testis cancer, cholangiocarcinoma, gallbladder carcinoma, gastric cancer, melanoma, and various types of head and neck cancer (including squamous cell carcinoma of the head and neck).

[0061] As used herein, the term “tumor,” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. “Neoplastic,” as used herein, refers to any form of dysregulated or unregulated cell growth, whether malignant or benign, resulting in abnormal tissue growth. Thus, “neoplastic cells” include malignant and benign cells having dysregulated or unregulated cell growth.

[0062] The term “effective amount” or “therapeutically effective amount” refers to an amount of a drug effective for treatment of a disease or disorder in a subject, such as to partially or fully relieve one or more symptoms. In some embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0063] A “biological sample” as used herein refers to a sample taken from a subject or from an animal. Examples of biological samples include tissue samples and liquid biological samples, such as whole blood, serum, plasma, blood supernatant, or synovial fluid. A biological sample may be taken directly from a subject or may be first chemically or physically modified in some fashion prior to use, for example, in order to assist in analysis of the sample.

[0064] A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction.II. Exemplary Anti-FAP Antibodies

[0065] In some embodiments, antibodies that comprising at least one antigen-binding domain specifically binds FAP, such as human FAP, are provided. Such antibodies include, but are not limited to, monoclonal antibodies, multispecific antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and / or light chain CDRs discussed herein. In some embodiments, an isolated antibody comprising at least one antigen-binding domain that binds to FAP is provided. In some embodiments, a monoclonal antibody comprising at least one antigen-binding domain that binds to FAP is provided. In some embodiments, the antibody binds to human FAP. In some embodiments, the antibody binds to human FAP comprising the amino acid sequence of SEQ ID NO: 18.

[0066] In some embodiments, an anti-FAP antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, an anti-FAP antibody comprises at least one heavy chain comprising a heavy chain variable region, and at least one light chain comprising a light chain variable region. In some embodiments, an antigen binding domain that binds FAP is a single-chain Fv (scFv). As used herein, a single-chain Fv (scFv), or any other antibody that comprises, for example, a single polypeptide chain comprising all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain is the region of the anti-FAP antibody that comprises the three heavy chain CDRs. In some embodiments, the light chain is the region of the anti-FAP antibody that comprises the three light chain CDRs.

[0067] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof comprises at least one, two, three, four, five, or six CDRs selected from a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 1 or 7; a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 2 or 8; a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 3 or 9; a light chain complementarity determining region 1 (LCDR1) comprising the amino acid of SEQ ID NO: 4 or 10; a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 5 or GAS (SEQ ID NO: 11); and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 6 or 12.

[0068] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises six CDRs including a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 7; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 8; a HCDR3comprising the amino acid sequence of SEQ ID NO: 3 or 9; a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 10; a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 or GAS (SEQ ID NO: 11); and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or 12.

[0069] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof comprises at least one, two, three, four, five, or six CDRs selected from a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0070] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof comprises at least one, two, three, four, five, or six CDRs selected from a HCDR1 comprising the amino acid sequence of SEQ ID NO: 7; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 8; a HCDR3 comprising the amino acid sequence of SEQ ID NO: 9; a LCDR1 comprising the amino acid sequence of SEQ ID NO: 10; a LCDR2 comprising the amino acid sequence of GAS (SEQ ID NO: 11); and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.

[0071] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises six CDRs including a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0072] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises six CDRs including a HCDR1 comprising the amino acid sequence of SEQ ID NO: 7; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 8; a HCDR3 comprising the amino acid sequence of SEQ ID NO: 9; a LCDR1 comprising the amino acid sequence of SEQ ID NO: 10; a LCDR2 comprising the amino acid sequence of GAS (SEQ ID NO: 11); and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.

[0073] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises at least one, at least two, or all three VH CDR sequences selected from (i) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 1 or 7; (ii) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 2 or 8; and (iii) a heavy chaincomplementarity determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 3 or 9.

[0074] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises at least one, at least two, or all three VH CDR sequences selected from (i) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3.

[0075] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises at least one, at least two, or all three VH CDR sequences selected from (i) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 7; (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 8; and (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0076] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises at least one, at least two, or all three VL CDR sequences selected from (i) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 10; (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 or GAS (SEQ ID NO: 11); and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or 12.

[0077] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises at least one, at least two, or all three VL CDR sequences selected from (i) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0078] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises at least one, at least two, or all three VL CDR sequences selected from (i) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 10; (ii) an LCDR2 comprising the amino acid sequence of GAS (SEQ ID NO: 11); and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.

[0079] In some embodiments, any of the six anti-FAP CDRs as defined by Kabat can be combined as subparts with any of the anti-FAP CDRs as defined by IM GT, for a total of six CDRs in a construct. Thus, in some embodiments, two CDRs defined by Kabat (for example, HCDR1 and HCDR2) can be combined with four CDRs as defined by IM GT (HCDR3, LCDR1, LCDR2, and LCDR3). In some embodiments, two or fewer residues in one or more of the CDRs can be replaced to obtain a variant thereof. In some embodiments, two or fewer residues can be replaced in 1, 2, 3, 4, 5, or 6 of the CDRs.

[0080] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises (I) a heavy chain variable region comprising at least one, at least two, or all three heavy chain variable region CDR sequences selected from (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 7; (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 8; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 9; and (II) a light chain variable region comprising at least one, at least two, or all three light chain variable region CDR sequences selected from (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 10; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 or GAS (SEQ ID NO: 11); and (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or 12; wherein the heavy chain variable region comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13 or 15, and the light chain variable region comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14 or 16.

[0081] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13 or 15. In some embodiments, a heavy chain variable region sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FAP antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind FAP. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 13 or 15. In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (that is, in the framework regions (FRs)). In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, including post- translational modifications of that sequence. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-FAP antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, including post-translational modifications of that sequence.

[0082] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof is provided, wherein the antibody comprises a light chain variable region (VL) having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14 or 16. In some embodiments, a light chain variable region sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FAP antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to FAP. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 14 or 16. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (that is, in the FRs). In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, including post- translational modifications of that sequence. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-FAP antibody or antigenbinding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, including post-translational modifications of that sequence.

[0083] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13 or 15 and a light chain variable domain (VL) having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14 or 16. In some embodiments, a VH sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions relative to the reference sequence, and a VL sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-FAP antibody or antigenbinding fragment thereof comprising that sequence retains the ability to bind to FAP. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 13 or 15. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and / or deleted in SEQ ID NO: 14 or 16. In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (that is, in the FRs). In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, including post- translational modifications of one or both sequences. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, including post-translational modifications of one or both sequences.

[0084] In some embodiments, the anti-FAP antibody is an antibody fragment. In some embodiments, the antibody fragment is a Fab, Fab', F(ab)2, Fd, Fv, single-chain Fv (scFv), or disulfide-linked Fv (sdFv). In some embodiments, the antibody fragment is a scFv. In some embodiments, the anti-FAP antibody comprises one or more substitutions selected from VH- G44C and VE-Q100C. In some embodiments, the antibody comprises a G44C substitution in the VH and a Q100C substitution in the VE, wherein substitution positions are according to Kabat numbering. In some embodiments, the antibody comprising cysteine substitutions at these two amino acid positions produces a disulfide bond between the VH and VE domains. In some embodiments, the scFv comprises as amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO: 17.

[0085] In various embodiments, the antibody is a monoclonal antibody. In various embodiments, the antibody is a multispecific antibody.

[0086] In various embodiments, an anti-FAP antibody or antigen-binding fragment thereof may comprise any of the following properties, singly or in combination. In some embodiments, the antibody binds to human FAP with an affinity (KD) between 0.5 and 10 nM, or 0.5 and 7 nM, or 1 and 5 nM, as determined by surface plasmon resonance. In some embodiments, the antibodybinds to cynomolgus monkey FAP with a KD between 0.5 and 10 nM, or 0.5 and 7 nM, or 1 and 5 nM, as determined by surface plasmon resonance. In some embodiments, the antibody binds human FAP expressed on the surface of cells with an EC50 of between 0.5 and 10 nM, or 0.5 and 7 nM, or 1 and 5 nM.

[0087] In some embodiments, the antibody binds human FAP. In some embodiments, the human FAP comprises the amino acid sequence of SEQ ID NO: 18.III. Exemplary Multispecific Antibodies

[0088] In certain embodiments, an antibody provided herein is a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In some embodiments, the multispecific antibody comprises a first antigen-binding domain that binds FAP and at least one second antigen-binding domain that binds a second target antigen. In some embodiments, bispecific antibodies may bind to two different epitopes of FAP. Multispecific antibodies may also be used to localize drugs such as other antibodies or antigen binding domains or cytotoxic agents or to localize detection labels to cells that express FAP. In some embodiments, FAP is overexpressed in cancer cells. In some embodiments, FAP is overexpressed in fibrotic cells. In some embodiments, FAP is highly expressed in the tumor microenvironment. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0089] In some embodiments, a multispecific antibody provided herein comprises a first antigen-binding domain that binds FAP and at least one second antigen-binding domain that binds a second target antigen. The antigen-binding domain that binds to FAP may be any anti- FAP antibody or antigen-binding fragment thereof described herein.

[0090] In some embodiments, the multispecific antibody comprises a first antigen-binding domain that binds FAP and at least one second antigen-binding domain that binds a second target antigen, wherein the first antigen-binding domain is an antibody fragment. In some embodiments, the first antigen-binding domain is a Fab, Fab', F(ab’)2, Fd, Fv, single-chain Fv (scFv), or disulfide-linked Fv (sdFv). In some embodiments, the first antigen-binding domain comprises as amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the first antigen binding domain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the first antigen binding domain consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments, the second antigen binding domain is a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv).

[0091] In some embodiments, the multispecific antibody comprising a first antigen-binding domain that binds FAP and at least one second antigen-binding domain that binds a second target antigen comprises a first antigen binding domain comprising a scFv. In some embodiments, the multispecific antibody comprising a first antigen-binding domain that binds FAP and at least one second antigen-binding domain that binds a second target antigen comprises at least a first heavy chain constant region. In some embodiments, the scFv is fused to the C-terminus of the first heavy chain constant region. In some embodiments, the scFv is fused directly to the C-terminus of the first heavy chain constant region. In some embodiments, the scFv is fused indirectly to the C-terminus of the first heavy chain constant region via a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker comprises glycine and / or serine residues.

[0092] In some embodiments, the multispecific antibody provided herein comprises a first antigen-binding domain that is an scFv antigen binding domain that binds to FAP, and an Fc domain. In particular embodiments, the Fc is a heterodimeric Fc comprising a first and second Fc polypeptide chain each comprising a hinge-CH2-CH3, in which the interface of one of the CH3 domains is modified or altered to promote the formation of the multispecific antibody. In some embodiments, the scFv that binds to FAP is linked with its N-terminus to the C-terminus of the CH3 of one of the Fc polypeptide chains. In some embodiments, the linkage is via a peptide linker. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of (GGGGS)n. In some such embodiments, the spacer has between 5 and 30 amino acids. In some embodiments, n is between 1 and 6 (inclusive). In some embodiments, n=4 and the linker is set forth as GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 19). In some embodiments, the peptide linker is set forth as GGSGGGGSGGGGSGGGGS (SEQ ID NO:20).

[0093] In some embodiments, the multispecific antibody comprises a post-translational modification. In some embodiments, the post-translational modifications may include amino acid modifications as a result of oxidation and / or glycation. Certain amino acids of a protein can be modified post-transcriptionally and the amino acid sequences provided herein include amino acids that contain a post-translational modification, e.g., glycation and / or oxidation. In some embodiments, the post-translational modification includes oxidation. In some embodiments, the oxidation is of methionine (Met) residues to form methionine sulfoxide or methionine sulfone. In some embodiments, the post-translational modification includes glycation of a lysine residue. In some embodiments, provided are compositions comprising proteins in which the presence of a glycated lysine represents less than 20%, less than 17%, less than 15%, less than 13% or less than 10% of lysine residues of proteins in the composition. 1

[0094] In some embodiments, the multispecific antibody comprises a first antigen-binding domain that binds FAP and a second antigen-binding domain that binds a second antigen and a third antigen binding domain that binds to a third antigen. In some embodiments, the second antigen and the third antigen are the same. In some embodiments, the second antigen and the third antigen are different. In some embodiments, the second antigen binding domain and the third antigen binding domain are the same. In some embodiments, the second antigen binding domain and the third antigen binding domain are different. In some embodiments, the second antigen binding domain comprises a second heavy chain variable region and a second light chain variable region; and the third antigen binding domain comprises a third heavy chain variable region and a third light chain variable region. In some embodiments, the second antigen binding domain is a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv). In some embodiments, the third antigen binding domain is a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv). In some embodiments, the second antigen binding domain and the third antigen binding domain are each independently a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv). In some embodiments, the second heavy chain variable region is fused to a first heavy chain constant region and the second light chain variable region is fused to a first light chain constant region; and the third heavy chain variable region is fused to a second heavy chain constant region and the third light chain variable region is fused to a second light chain constant region. In some embodiments, the first antigen binding domain that binds to FAP is a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide- linked Fv (sdFv), and the first antigen binding domain is fused to the C-terminus of the first or second heavy chain constant region.

[0095] In some embodiments, the first heavy chain constant region and the second heavy chain constant region are different. In some embodiments, the first heavy chain constant region and the second heavy chain constant region form a heterodimer. Methods and variants for heterodimerization also include those described in published international PCT App. WO2014 / 145806, including “knobs and holes” mutations (also called “skew” variants), mutations that relate to “electrostatic steering” or “charge pairs,” and pl variants. Heterodimeric variants also include any as described in U.S. published Appl. No. US2012 / 0149876 or US2018 / 011883.

[0096] In some embodiments, modifications include introduction of a protuberance (knob) into a first heavy chain constant region and a cavity (hole) into a second heavy chain constant region such that the protuberance is positionable in the cavity to promote complexing of the first and second heavy chain constant regions. Amino acids targeted for replacement and / or modificationto create protuberances or cavities in a polypeptide are typically interface amino acids that interact or contact with one or more amino acids in the interface of a second polypeptide.

[0097] In some embodiments, the first heavy chain constant region is modified to contain protuberance (hole) amino acids include replacement of a native or original amino acid with an amino acid that has at least one side chain which projects from the interface of the first heavy chain constant region and is therefore positionable in a compensatory cavity (hole) in an adjacent interface of a second heavy chain constant region. Most often, the replacement amino acid is one which has a larger side chain volume than the original amino acid residue. One of skill in the art knows how to determine and / or assess the properties of amino acid residues to identify those that are ideal replacement amino acids to create a protuberance. In some embodiments, the replacement residues for the formation of a protuberance are naturally occurring amino acid residues and include, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the original residue identified for replacement is an amino acid residue that has a small side chain such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0098] In some embodiments, the second heavy chain constant region is modified to contain a cavity (hole) is one that includes replacement of a native or original amino acid with an amino acid that has at least one side chain that is recessed from the interface of the second heavy chain constant region and thus is able to accommodate a corresponding protuberance from the interface of the first heavy chain constant region. Most often, the replacement amino acid is one which has a smaller side chain volume than the original amino acid residue. One of skill in the art knows how to determine and / or assess the properties of amino acid residues to identify those that are ideal replacement residues for the formation of a cavity. Generally, the replacement residues for the formation of a cavity are naturally occurring amino acids and include, for example, alanine (A), serine (S), threonine (T) and valine (V). In some examples, the original amino acid identified for replacement is an amino acid that has a large side chain such as, for example, tyrosine, arginine, phenylalanine, or tryptophan.

[0099] The CH3 interface of human IgGl, for example, involves sixteen residues on each domain located on four anti-parallel P-strands which buries 1090 A2 from each surface (see e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgway et al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168).Modifications of a CH3 domain to create protuberances or cavities are described, for example, in U.S. Pat. No. 5,731,168; International Patent Applications WO98 / 50431 and WO 2005 / 063816; and Ridgway et al., (1996) Prot. Engin., 9: 617-621. In some examples, modifications of a CH3 domain to create protuberances or cavities are typically targeted toresidues located on the two central anti-parallel P-strands. The aim is to minimize the risk that the protuberances which are created can be accommodated by protruding into the surrounding solvent rather than being accommodated by a compensatory cavity in the partner CH3 domain.

[0100] In some embodiments, to promote heterodimerization of both polypeptides of the Fc and / or constant region, the heterodimer contain paired or complementary amino acid modifications. Exemplary paired amino acid modification of polypeptides of an Fc fusion are set forth in Table 1.

[0101] In some embodiments, the first heavy chain constant region and / or the second heavy chain constant region comprise one or more heterodimerization mutations. In some embodiments, the heterodimerization mutation comprises one or more of T350V, L351Y, T366W, T366S, T366L, L368A, N390R, K392M, K392L, T394W, S400E, F405A, and / or Y407V, wherein mutation positions are according to Kabat numbering.

[0102] In some embodiments, the first heavy chain constant region comprises at least one first heterodimerization mutation and the second heavy chain constant region comprises at least one second heterodimerization mutation. In some embodiments, at least one first heterodimerization mutation comprises T366W and at least one second heterodimerization mutation comprises one or more of T366S, L368A, and / or Y407V, wherein mutation positions are according to Kabat numbering. In some embodiments, at least one first heterodimerization mutation comprises one or more of T366S, L368A, and / or Y407V and at least one second heterodimerization mutation comprises T366W, wherein mutation positions are according to Kabat numbering. In some embodiments, at least one first heterodimerization mutation comprises T366W and at least one second heterodimerization mutation comprises T366S, L368A, andY407V, wherein mutation positions are according to Kabat numbering. In some embodiments, at least one first heterodimerization mutation comprises T366S, L368A, and Y407V and at least one secondheterodimerization mutation comprises T366W, wherein mutation positions are according to Kabat numbering. In some embodiments, at least one first heterodimerization mutation comprises one or more of T350V, L351Y, S400E, F405A, and / or Y407V and at least one second heterodimerization mutation comprises one or more of T350V, T366L, N390R, K392M, K392L, and / or T394W, wherein mutation positions are according to Kabat numbering. In some embodiments, at least one first heterodimerization mutation comprises one or more of T350V, T366L, N390R, K392M, K392L, and / or T394W and at least one second heterodimerization mutation comprises one or more of T350V, L351Y, S400E, F405A, and / or Y407V, wherein mutation positions are according to Kabat numbering. In some embodiments, at least one first heterodimerization mutation comprises T350V, L351Y, F405A, and Y407V and at least one second heterodimerization mutation comprises T350V, T366L, K392L, and T394W, wherein mutation positions are according to Kabat numbering. In some embodiments, at least one first heterodimerization mutation comprises one or more of T350V, T366L, K392L, and T394W and at least one second heterodimerization mutation comprises one or more of T350V, L351 Y, F405A, and Y407V, wherein mutation positions are according to Kabat numbering. See, e.g., Escobar-Cabrera et al., Antibodies (Basel), 6(2):7 (2017), and WO 2013 / 166594.

[0103] In some embodiments, the first heavy chain constant region and the second heavy chain constant region are IgGl, IgG2, IgG3, or IgG4 constant regions. In some embodiments, the first heavy chain constant region and / or the second heavy chain constant region are IgGl constant regions. In some embodiments, the first heavy chain constant region and the second heavy chain constant region are IgGl constant regions.

[0104] In some embodiments, the first heavy chain constant region and the second heavy chain constant region comprise one or more mutation that reduced binding to FcyR. In some embodiments, the first heavy chain constant region and the second heavy chain constant region do not bind FcyR or have reduced binding to FcyR compared to wild-type constant regions of the same isotype. In some embodiments, the first heavy chain constant region and the second heavy chain constant region do not bind FcyR or have reduced binding to FcyR compared to wild-type IgG constant regions. In some embodiments, the first heavy chain constant region and the second heavy chain constant region comprise one or more mutation selected from E234A, E235A, and D265S, wherein mutation positions are according to Kabat. In some embodiments, the first heavy chain constant region and the second heavy chain constant region comprise two or more mutation selected from E234A, E235A, and D265S, wherein mutation positions are according to Kabat. In some embodiments, the first heavy chain constant region and the second heavy chain constant region comprise E234A, E235A, and D265S mutations, wherein mutation positions are according to Kabat.

[0105] In some embodiments, the multispecific antibody comprises a first antigen-binding domain that binds FAP and at least one second antigen-binding domain that binds a second target antigen, wherein the second target antigen is an immune cell antigen. In some embodiments, the second target antigen is a fibroblast antigen. In some embodiments, the second target antigen is expressed on the same cell as FAP. In some embodiments, the second target antigen is expressed on a different cell from FAP, such as on an immune cell. In some embodiments, the immune cell antigen is expressed on the surface of a T cell, a natural killer (NK) cell, and / or a dendritic cell. In some embodiments, the second target antigen is selected from CD3 and TGFRpiI. In some embodiments, the second target antigen is not CD40.

[0106] In some embodiments, the multispecific antibody further comprises a third antigen binding domain that binds a third target antigen. In some embodiments, the third target antigen is an immune cell antigen. In some embodiments, the third target antigen is a fibroblast antigen. In some embodiments, the third target antigen is expressed on the same cell as FAP. In some embodiments, the third target antigen is expressed on a different cell from FAP, such as on an immune cell. In some embodiments, the immune cell antigen is expressed on the surface of a T cell, a natural killer (NK) cell, and / or a dendritic cell. In some embodiments, the third target antigen is selected from CD3 and TGFRpiI. In some embodiments, the third target antigen is not CD40.IV. Exemplary Antibody Variants, Fragments, and Constant Regions

[0107] In many embodiments, an antibody specifically binding to FAP (e.g., a multispecific antibody) may further incorporate any of the features, singly or in combination, as described in the sections that follow.A. Antibody Fragments

[0108] In certain embodiments, an antibody or antigen-binding fragment thereof provided herein is an antibody fragment. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody fragment selected from an Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv), and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,187,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0109] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0110] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0111] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.B. Human Antibodies

[0112] In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be made by any suitable method. Nonlimiting exemplary methods include making human antibodies in transgenic mice that comprise human immunoglobulin loci. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al., Nature 362: 255-8 (1993); Lonberg et al., Nature 368: 856-9 (1994); and U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.

[0113] Nonlimiting exemplary methods also include selecting human antibodies from phage display libraries. See, e.g., Hoogenboom et al., J. Mol. Biol. 227: 381-8 (1992); Marks et al., J. Mol. Biol. 222: 581-97 (1991); and PCT Publication No. WO 99 / 10494.C. Chimeric and Humanized Antibodies

[0114] In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0115] In certain embodiments, a chimeric antibody is a humanized antibody. In some embodiments, an antibody provided herein is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0116] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’lAcad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43- 60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

[0117] Human framework regions that may be used for humanization include but are not limited to framework regions selected using the “best- fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carte r et al. Pro c. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0118] In some embodiments, the humanized antibodies may comprise a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant region.D. Glycosylation and Pegylation Variants

[0119] In certain embodiments, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation can increase the affinity of theantibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0120] Glycosylation of the constant region on N297 can be prevented by mutating the N297 residue to another residue, e.g., N297A, and / or by mutating an adjacent amino acid, e.g., 298 to thereby reduce glycosylation on N297.

[0121] Additionally, or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies described herein to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases {e.g., beta(l,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).

[0122] Another modification of the antibodies described herein is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. In some embodiments, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI-CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be appliedto the antibodies described herein. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.E. Constant Regions

[0123] In some embodiments, an antibody is a full-length antibody. In some embodiments, a multispecific antibody comprises a full-length antibody. In some embodiments, an antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from K and . In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG antibody lacking a C-terminal lysine in the heavy chain constant region. In some embodiments, an antibody described herein comprises a human IgG constant region, such as an IgGl, IgG2, IgG3, or IgG4. In some embodiments, the antibody is an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, an antibody described herein comprises a human IgGl heavy chain constant region. In some embodiments, an antibody described herein comprises a human IgGl constant region and a human K light chain.

[0124] In some embodiments, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain at Gly446 and Lys447 (EU numbering). Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain and / or a cleaved variant of the full-length heavy chain. In some embodiments, the C-terminal lysine, or the C-terminal glycine and lysine, of the Fc region may or may not be present. Thus, a “full-length heavy chain constant region” or a “full length antibody” for example, which is a human IgGl antibody, includes an IgGl with both a C-terminal glycine and lysine, without the C-terminal lysine, or without both the C-terminal glycine and lysine.

[0125] The choice of heavy chain constant region can determine whether or not an antibody will have effector function in vivo. Such effector function, in some embodiments, includes antibodydependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), and can result in killing of the cell to which the antibody is bound. In some methods of treatment, including methods of treating some cancers, cell killing may be desirable, for example, when the antibody binds to a cell that supports the maintenance or growth of the tumor. Exemplary cells that may support the maintenance or growth of a tumor include, but are not limited to, tumor cells themselves, cells that aid in the recruitment of vasculature to the tumor, and cells that provide ligands, growth factors, or counter-receptors that support orpromote tumor growth or tumor survival. In some embodiments, when effector function is desirable, an antibody comprising a human IgGl heavy chain or a human IgG3 heavy chain is selected.

[0126] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0127] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibodies with certain improved properties. For example, an antibody may be afucosylated, for example, by mutating residues such as Asn297 that are normally glycosylated with fucose-containing glycosylations, or through other means.

[0128] Antibodies are also provided with amino-terminal leader extensions. For example, one or more amino acid residues of the amino-terminal leader sequence are present at the aminoterminus of any one or more heavy or light chains of an antibody. An exemplary amino-terminal leader extension comprises or consists of three amino acid residues, VHS, present on one or both light chains of an antibody.

[0129] The in vivo or serum half-life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice, in humans, or in non-human primates to which the polypeptides with a variant Fc region are administered. See also, e.g., Petkova et al. International Immunology 18(12): 1759- 1769 (2006).

[0130] In certain embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue 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, 265, 297, 318, 320, 322, 330, and / or 331 (EU numbering) 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 Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0131] In some examples, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

[0132] In some embodiments, a multispecific antibody comprises one or more mutations in the heavy chain constant region that reduce or eliminate binding to FcyR. In some embodiments, the multispecific antibody does not bind FcyR or has reduced binding to FcyR compared to a multispecific antibody having the same antigen binding domains but a wild-type constant region of the same isotype. In some examples, the Fc region can be modified to decrease antibody dependent cellular cytotoxicity (ADCC) and / or to decrease the affinity for an Fey receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241 , 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305,307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337,338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437,438 or 439 (EU numbering). Exemplary substitutions include 234A, 235A, 236A, 239D, 239E, 265S, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 234A / 235A / 265S, 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T (EU numbering). Other Fc modifications that can be made to Fes are those for reducing or ablating binding to FcyR and / or complement proteins, thereby reducing or ablating Fc-mediated effector functions such as ADCC, ADCP, and CDC.Exemplary modifications include but are not limited substitutions, insertions, and deletions at positions 234, 235, 236, 237, 265, 267, 269, 325, 328, 330, and / or 331 (e.g., 330 and 331), wherein numbering is according to the EU index. Exemplary substitutions include but are not limited to 234A, 235A, 235E, 236R, 237A, 265S, 267R, 269R, 325L, 328R, 330S, and 33 IS (e.g., 330S, and 33 IS), wherein numbering is according to the EU index. An Fc variant can comprise 234A / 235A / 265S. Other modifications for reducing FcyR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of the glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691. For example, a human IgGl constant region may comprise L234A, L235E, and G237A substitutions. In some embodiments, a human IgGl constant region may comprise a P238K substitution. In some embodiments, a human IgGlconstant region may comprise L234A, L235E, G237A, A330S, and P331S substitutions. Insome embodiments, a human IgGlconstant region may comprise L234A, L235A, and D265S substitutions. (All numbering under the EU index.)

[0133] Fc variants that enhance affinity for an inhibitory receptor FcyRIIb can also be used. Such variants can provide an Fc fusion protein with immunomodulatory activities related to FcyRIIb cells, including for example, B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcyRIIb relative to one or more activating receptors. Modifications for altering binding to FcyRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331, and 332, according to the EU index. Exemplary substitutions for enhancing FcyRIIb affinity include but are not limited to 234A, 234D, 234E, 234F, 234W, 235D, 235E, 235F, 235R, 235Y, 236D, 236N, 237A, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, 330S, 33 IS, and 332E. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to FcyRIIb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F. (All numbering under the EU index.)

[0134] Other modifications for enhancing FcyR and complement interactions include but are not limited to substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243E, 292P, 300E, 396E, 3051, and 396E. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691. Fc modifications that increase binding to an Fey receptor include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285,298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330,335, 337, 338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437,438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Patent Publication No. WO 00 / 42072.

[0135] Optionally, the Fc region can comprise a non-naturally occurring amino acid residue at additional and / or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCX Patent Publications WO 00 / 42072; WO 01 / 58957; WO 02 / 06919; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752; WO 04 / 074455; WO 04 / 099249; WO 04 / 063351; WO 05 / 070963; WO 05 / 040217, WO 05 / 092925 and WO 06 / 0201 14).

[0136] The affinities and binding properties of an Fc region for its ligand can be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art including but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (EEISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE analysis), and othermethods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods can utilize a label on one or more of the components being examined and / or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0137] In certain embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region for FcRn, For example, one or more of more of following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375. Specific exemplary substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including for example 2591, 308F, 428L, 428M, 434S, 4341 1. 434F, 434Y, and 434X1. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428 L, 428F, 250Q / 428L (Hinton et al. 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 31 1A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 31 1 S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S (Dall’Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dal’Acqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0138] In certain embodiments, hybrid IgG isotypes with particular biological characteristics can be used. For example, an IgGl / IgG3 hybrid variant can be constructed by substituting IgGl positions in the CH2 and / or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody can be constructed that comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In some embodiments described herein, an IgGl / IgG2 hybrid variant can be constructed by substituting IgG2 positions in the CH2 and / or CH3 region with amino acids from IgGl at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody can beconstructed that comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, +236G (referring to an insertion of a glycine at position 236), and 327A.

[0139] Moreover, the binding sites on human IgGl for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.F. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcyRIII. Additionally, the following combination mutants were shown to improve FcyRIII binding: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A, which has been shown to exhibit enhanced FcyRIIIa binding and ADCC activity (Shields et al., 2001). Other IgGl variants with strongly enhanced binding to FcyRIIIa have been identified, including variants with S239D / I332E and S239D / I332E / A330E mutations which showed the greatest increase in affinity for FcyRIIIa, a decrease in FcyRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Eazar et al., 2006). Introduction of the triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu- specific), rituximab (CD20- specific), and cetuximab (EGFR- specific) translated into greatly enhanced ADCC activity in vitro, and the S239D / I332E variant showed an enhanced capacity to deplete B cells in monkeys (Eazar et al., 2006). In addition, IgGl mutants containing E235V, F243E, R292P, Y300E and P396E mutations which exhibited enhanced binding to FcyRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcyRIIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that can be used include: S298A / E333A / E334A, S239D / I332E, S239D / I332E / A330E, E235V / F243E / R292P / Y300E / P396E, and M428E / N434S.

[0140] In certain embodiments, an Fc is chosen that has essentially no effector function, i.e., it has reduced binding to FcyRs and reduced complement fixation. An exemplary Fc, e.g., IgGl Fc, that is effectorless comprises the following three mutations: E234A, E235A, and D265S.

[0141] When using an IgG4 constant domain, it can include the substitution S228P, which mimics the hinge sequence in IgGl and thereby stabilizes IgG4 molecules. Fc modifications described in WO 2017 / 087678 or WO2016081746 may also be used.

[0142] In certain embodiments, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation of the constant region on N297 can be prevented by mutating the N297 residue to another residue, e.g., N297A, and / or by mutating an adjacent amino acid, e.g., 298 to thereby reduce glycosylation on N297.V. Pharmaceutical Compositions

[0143] Pharmaceutical compositions comprising an antibody described herein are provided. In some embodiments, the pharmaceutical composition comprises the antibody and a pharmaceutically acceptable carrier.

[0144] In various embodiments, compositions comprising a antibody provided herein are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, which include vehicles, adjuvants, and diluents, are available. Moreover, various pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are also available.

[0145] In some embodiments, the pharmaceutically acceptable carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.VI. Nucleic Acid Molecules Encoding Anti-FAP Antibodies

[0146] Nucleic acid molecules comprising polynucleotides that encode one or more chains of anti-FAP antibodies described herein are provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an anti-FAP antibody. In some embodiments, provided is an isolated nucleic acid encoding any one of the anti-FAP antibodies provided herein. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an anti-FAP antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

[0147] In some such embodiments, the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when an antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.

[0148] In some embodiments, a polynucleotide encoding a heavy chain or light chain of an anti- FAP antibody comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain. The leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.

[0149] Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.

[0150] In some embodiments, the isolated nucleic acid is an expression vector. Also provided herein are host cells that express the anti-FAP antibodies provided herein. In some embodiments, the host cells comprise isolated nucleic acids encoding the anti-FAP antibodies or expression vectors comprising nucleic acids encoding the anti-FAP antibodies. Also provided herein are methods of producing the anti-FAP antibodies provided herein, comprising culturing the host cells under conditions suitable for expressing the anti-FAP antibodies. In some embodiments, the methods comprise isolating the anti-FAP antibodies.VII. Anti-FAP Antibody Expression and ProductionA. Vectors

[0151] Vectors comprising polynucleotides that encode anti-FAP heavy chains and / or anti-FAP light chains are provided. In some embodiments, provided herein is a vector comprising a nucleic acid encoding any one of the anti-FAP antibodies described herein. Vectors comprising polynucleotides that encode anti-FAP heavy chains and / or anti-FAP light chains are also provided. Such vectors include, but are not limited to, DNA vectors, RNA vectors (e.g, mRNA and circular RNA, self-amplifying RNA vectors, etc.), phage vectors, viral vectors (e.g., pox virus vectors, vaccinia virus vectors, adenovirus vectors, modified vaccinia virus Ankara (MVA) vectors, etc.), retroviral vectors, etc. In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.

[0152] In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a mole- ormass-ratio of between 5: 1 and 1:5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1:1 and 1:5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1:2 for the vector encoding the heavy chain and the vector encoding the light chain is used.

[0153] In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0154] In some embodiments, a vector is chosen for in vivo expression anti-FAP heavy chains and / or anti-FAP light chains in animals, including humans. In some such embodiments, expression of the polypeptide is under the control of a promoter that functions in a tissuespecific manner. For example, liver- specific promoters are described, e.g., in PCT Publication No. WO 2006 / 076288.B. Host Cells

[0155] In various embodiments, anti-FAP heavy chains and / or anti-FAP light chains may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. In some embodiments, provided herein are host cells comprising an isolated nucleic acid encoding any one of the anti-FAP antibodies described herein. In some embodiments, provided herein are host cells comprising a vector comprising a nucleic acid encoding any one of the anti-FAP antibodies described herein. In some embodiments, provided herein is a host cell that produces any one of the anti-FAP antibodies described herein. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, anti-FAP heavy chains and / or anti-FAP light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006 / 0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the anti-FAP heavy chains and / or anti-FAP light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0156] Introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Nonlimiting exemplary methods are described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rded. Cold Spring Harbor Eaboratory Press (2001). Nucleicacids may be transiently or stably transfected in the desired host cells, according to any suitable method.

[0157] In some embodiments, one or more polypeptides may be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method.

[0158] In some embodiments, provided herein is a method for making an anti-FAP antibody described herein, comprising culturing a host cell described herein under conditions suitable for expression of the antibody. In some embodiments, the method further comprises recovering the antibody produced by the host cell.C. Purification of Anti-FAP Antibodies

[0159] Anti-FAP antibodies may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography or size exclusion chromatography.D. Cell-free Production of Anti-FAP Antibodies

[0160] In some embodiments, an anti-FAP antibody is produced in a cell-free system. Nonlimiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).VIII. Therapeutic Compositions and MethodsA. Methods of Treating Diseases

[0161] In one embodiment, provided herein is a method of treating and / or preventing cancer, which comprises administering to a subject in need thereof an antibody that binds FAP, a multispecific antibody comprising a first antigen-binding domain that binds FAP, or a pharmaceutical composition thereof provided herein. The antibodies provided herein are useful for targeting an antigen overexpressed in cancer (e.g., FAP) and / or fibrosis.

[0162] In another embodiment, provided herein is method of managing cancer, which comprises administering to a patient an antibody that binds FAP, a multispecific antibody comprising a first antigen-binding domain that binds FAP, or a pharmaceutical composition thereof provided herein.

[0163] Also provided herein are methods of treating patients who have been previously treated for cancer but are non-responsive to standard therapies, as well as those who have not previously been treated. Also encompassed are methods of treating patients regardless of patient's age, although some diseases or disorders are more common in certain age groups. Further encompassed are methods of treating patients who have undergone surgery in an attempt to treatthe disease or condition at issue, as well as those who have not. Because patients with cancer have heterogeneous clinical manifestations and varying clinical outcomes, the treatment given to a patient may vary, depending on his / her prognosis. The skilled clinician will be able to readily determine without undue experimentation specific secondary agents, types of surgery, and types of non-drug based standard therapy that can be effectively used to treat an individual patient with cancer.

[0164] In certain embodiments, the cancer is a solid cancer. In certain embodiments, the solid cancer is metastatic. In certain embodiments, the solid cancer is gastric cancer, pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), or squamous cell carcinoma of head and neck (SCCHN).

[0165] In certain embodiments, the patient to be treated with one of the methods provided herein has not been treated with anticancer therapy prior to the administration of an antibody provided herein. In certain embodiments, the patient to be treated with one of the methods provided herein has been treated with anticancer therapy prior to the administration of an antibody provided herein. In certain embodiments, the patient to be treated with one of the methods provided herein has developed drug resistance to the anticancer therapy.

[0166] In some embodiments, provided herein is a method of treating fibrosis comprising administering to a subject in need thereof an antibody that binds FAP, a multispecific antibody comprising a first antigen-binding domain that binds FAP, or a pharmaceutical composition thereof provided herein. In some embodiments, the fibrosis is idiopathic pulmonary fibrosis (IPF).

[0167] The methods provided herein encompass treating a patient regardless of patient's age, although some diseases or disorders are more common in certain age groups.B. Routes of Administration and Carriers

[0168] Provided herein are compositions (e.g., pharmaceutical compositions) comprising an antibody, including a multispecific antibody provided herein and one or more pharmaceutically acceptable carriers.

[0169] In various embodiments, an antibody, including a multispecific antibody provided herein may be administered in vivo by various routes, including, but not limited to, oral, intra-arterial, parenteral (including intravenous and subcutaneous), intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The subject compositions may be formulated into preparations, such as liquid formulations or formulations suitable for injections, inhalations, and the like. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid orcarrier, for example, sterile water. The appropriate formulation and route of administration may be selected according to the intended application.IX. EXAMPLESExample 1: Generation of Anti-FAP Antibodies

[0170] Screening and discovery of fully human immunoglobulin G (IgG) antibodies against human FAP was performed by phage selection using the SuperHuman 2.0 synthetic naive scFv library and panning against recombinant human and mouse extracellular domains and HEK-293 human FAP overexpression cell lines. Positive clones were sequenced and scFvs were produced from bacterial periplasmic extracts. Purified scFvs were screened for cross -reactive binding to human, cyno, and mouse FAP by ELISA, while displaying no binding to the related proline selective peptidase dipeptidyl peptidase 4 (DPP4). Eighty-two human, cyno, and mouse cross- reactive FAP positive clones were reformatted and produced as recombinant fully human IgGl antibodies. Purified FAP antibodies were used to determine relative affinities using single point Mirror Ball measurements, followed by dose titrations for affinity measurement by FACS.Three clones demonstrated significant human, cyno, and mouse cross -reactivity and were selected for affinity maturation by phage display using a CDR focused library. SPR affinity measurements identified three clones with 10-fold greater affinities relative to each of the three parental antibodies for human and cyno FAP. These clones were selected for reformatting as CD40 x FAP bispecific antibodies and were screened to identify the most potent FAP driven clustering dependent CD40 agonism. The anti-FAP clone LP62 was found to drive the greatest CD40 agonism, while possessing favorable biophysical traits.Example 2: Generation of Trivalent Bispecific Anti-CD40 / FAP Antibody

[0171] CD40.9H3xFAP.LP62 is a humanized immunoglobulin G1 (IgGl) bispecific antibody comprised of a bivalent arm that recognizes CD40 and a single chain variable fragment (scFv) that recognizes fibroblast activating protein (FAP). The scFv that binds FAP comprises the amino acid sequence of SEQ ID NO: 17.Example 3: Binding Characterization of CD40xFAP.LP62

[0172] CD40xFAP.LP62 binding to human and cynomolgus (cyno) FAP was evaluated by surface plasmon resonance (SPR) using human and cyno FAP proteins. CD40xFAP.LP62 bound to human FAP in a dose-dependent manner with a mean (± SD) equilibrium dissociation constant (KD) of 9.2 ± 0.6 x 10'8M and 1.96 ± 0.07 x 10'9M, respectively. The rates ofassociation and dissociation of CD40xFAP.LP62 to human and cyno FAP are summarized in Table El.

[0173] SPR studies also demonstrated that CD40xFAP.LP62 does not block binding of CD40 to its physiologic ligand, CD40L (data not shown).Table El: Binding Kinetics of CD40xFAP.LP62 to Human and Cyno FAP

[0174] Binding of CD40xFAP.LP62 was also evaluated across several human and cyno FAP expressing cells compared to isotype controls. Dose-dependent binding was observed on human macrophages and human FAP-expressing human embryonic kidney (HEK) cells. Mean (± SD) half maximal effective concentrations (EC50) for binding to cells are summarized in Table E2. Table E2: CD40xFAP.LP62 Binds to Human and Cyno CD40- and FAP-Expressing CellsExample 4: CD40xFAP.LP62 Activates Human Dendritic Cells and Macrophages in a FAP-Dependent Manner

[0175] The activity of CD40xFAP.LP62 was evaluated for the ability to induce IL12p40 secretion in a human dendritic cell (DC) and HFF1 co-culture activity assay. DCs were cocultured with either HFF1 or HFF1 FAP KO cells. HFF1 cells express FAP receptors on their cell surface with a mean (± SD) of 50617 ± 19408 that is comparable to levels observed in cancer-associated fibroblasts from human tumors.

[0176] The results are shown in FIG. 1A. Dose-dependent IL12p40 secretion was observed with a mean (± SD) EC50 concentration of 0.079 ± 0.043 nM (n=8 donors across 4 independent experiments). No IL12p40 secretion was observed when DCs were co-cultured with HFF1 FAP KO cells (absence of FAP) or anti-HELxFAP isotype control antibody. Similar FAP dependent and dose-dependent responses were observed from CD40-positive macrophages co-cultured with HFF1 cells in the presence of CD40xFAP.LP62 with a mean (± SD) EC50 concentration for TNFa release of 0.12 ± 0.05 nM (n=6 donors across 3 independent experiments) (FIG. IB).

[0177] Macrophages can activate CD4+ T cells, so the ability of CD40xFAP.LP62-activated macrophages to induce CD4+ T cell function was also evaluated. In a tri-culture assay of macrophages, HFF1 cells, and CD4+ T cells, CD40xFAP.LP62 induced secretion of the T cell-specific cytokine IL2 in a dose-dependent manner, while no IL2 was observed with the anti- HELxFAP isotype control. (n=3 donors across 2 independent experiments, data not shown).Example 5: Mouse Surrogate mCD40xFAP.LP62 Characterization

[0178] CD40xFAP.LP62 does not bind to mouse CD40. Therefore, a 2+1 anti-mouse CD40xFAP bispecific antibody (mCD40xFAP.LP62) having a similar structure as CD40xFAP.LP62, with the same LP62 anti-FAP scFv, was developed as a surrogate for use in mouse tumor efficacy studies. mCD40xFAP.LP62 was comprehensively assessed through binding and in vitro functional assays and found to be comparable to CD40xFAP.LP62. mCD40xFAP.LP62 contains three amino acid substitutions (L234A, L235A, P329G) in the mouse IgG2a Fc backbone that attenuate binding to Fc gamma receptors (FcyR). Thus, similar to CD40xFAP.LP62, CD40 agonism is mediated by FAP-dependent clustering of multiple mCD40xFAP.LP62 antibodies.

[0179] mCD40xFAP.LP62 binding to mouse CD40 and FAP were evaluated by SPR using mouse CD40 and FAP proteins. mCD40xFAP.LP62 bound to mouse CD40 and FAP, similar to CD40xFAP.LP62, in a dose-dependent manner with a mean (+ SD) equilibrium dissociation constant (KD) of 4.58 + 0.09 x 10'8M and 3.4 + 0.2 x 10'9M, respectively. Binding of mCD40xFAP.LP62 was also evaluated on different mouse CD40- and FAP-expressing cells compared to isotype controls and commercially available comparator anti-CD40 and anti-FAP antibodies. Comparable to CD40xFAP.LP62, dose-dependent binding was observed on murine macrophages and mouse FAP-expressing HEK cells (data not shown). Mean (+ SD) half maximal effective concentrations (EC50) for binding to cells are summarized in Table E3.Table E3: mCD40xFAP.LP62 Binds to Mouse CD40- and FAP-Expressing CellsExample 6: mCD40xFAP.LP62 Activates CD40 in a FAP-Dependent Manner

[0180] To confirm comparable functional activity of mCD40xFAP.LP62 to CD40xFAP.LP62, increasing concentrations of mCD40xFAP.LP62 were added to primary murine bone marrow- derived macrophages (BMDM) co-cultured with MC38 cells that express FAP (MC38-FAP) or MC38-FAP KO cells.

[0181] The FAP expressed on the surface of MC38-FAP cells [mean (+ SD) of 27943 + 9551 FAP receptors (data not shown)] enables mCD40xFAP.LP62 clustering to drive CD40activation and is comparable to levels found in patient tumors [mean (+ SD) of 35024 ± 25653 FAP receptors (data not shown)]. The CCL22 and IL12p40 cytokines downstream of CD40 activation were induced in a mCD40xFAP.LP62 dose-dependent manner with mean (+ SD) EC50 concentrations of 1.02 ± 0.13 nM and 1.43 ± 0.56 nM, respectively (n= 4 pooled mice across 2 independent experiments). See FIGs. 2A-2B. mCD40xFAP.LP62 activity was dependent on FAP as no CCL22 and IL12p40 were detected upon co-culture with MC38 cells that did not express FAP, nor with mCD40xHEL, an anti-mCD40 / anti-hen egg lysozyme isotype-matched bispecific antibody. Collectively, these data support the use of mCD40xFAP.LP62 as a relevant in vivo surrogate for CD40xFAP.LP62 in mice.Example 7: Antitumor Efficacy and Pharmacodynamic Activity of mCD40xFAP.LP62 in KPCY Syngeneic Mouse Tumor Model

[0182] The antitumor effects of mCD40xFAP.LP62 were evaluated in the KPCY tumor-bearing mouse model, which harbors tumor stromal cells expressing FAP levels comparable to human tumors (data not shown). Tumor volumes following a single dose of mCD40xFAP.LP62 as monotherapy was monitored along with immune cell phenotyping and cytokine changes.

[0183] C57 / BL6 mice were implanted with 5xl05KPCY cells subcutaneously, randomized into treatment groups of 10 mice each when average tumor volumes reached 75 mm3to 150 mm3, and dosed via the intraperitoneal (IP) route with either mCD40xFAP.LP62 or an anti-hen egg lysozyme isotype bispecific antibody control, mCD40xHEL. Because this isotype antibody contains the same anti-CD40 antibody arms as mCD40xFAP.LP62, this control enabled the evaluation of FAP binding-dependent CD40 agonist activity in vivo.

[0184] The results are shown in FIG. 3. Significant single agent activity of mCD40xFAP.LP62 was observed at the 10 mg / kg dose, with 5 of 10 tumor-free mice as well as at a lower dose of 3 mg / kg (n=10 mice per group across 2 independent experiments). Mean tumor volumes are summarized in Table E4.Table E4. mCD40xFAP.LP62 Drives Single Agent Efficacy in KPCY Tumor Models

[0185] Immune profiling of KPCY tumor-bearing mice dosed with 10 mg / kg mCD40xFAP.LP62 was subsequently performed. KPCY mice were treated with mCD40xFAP.LP62 or anti-KLH isotype control (10 mg / kg, QDxl). Tumors and tumor-draininglymph nodes (TDLNs) were profiled by flow cytometry at 48 hours and 240 hours posttreatment (n=10 mice per group across 2 independent experiments). Cell populations for which there were <100 cells were excluded from analyses. This experiment demonstrated that tumoral cross-presenting dendritic cells (cDCl) upregulated expression of CD86, a costimulatory receptor and marker of cDCl activation, as soon as 48 hours post-treatment compared to isotype control-treated mice (FIG. 4A). Additionally, the percent of activated cDCl cells migrating to TDLNs was increased as compared to anti-KLH isotype (FIGS. 4B and 4C), consistent with published reports of systemically active anti-CD40 agonists. See, e.g., Lin et al., J Exp Med. 2020 Aug 3;217(8):e20190673. Resident cDCl cells in TDLNs showed only modest (p<0.01), if any, increases, in CD86 activation (FIG. 4D), indicating that mCD40xFAP.LP62 activity was restricted to the FAP-expressing tumor. Finally, these changes were associated with increases in the ratio of CD8 T cells to Tregs (CD8 T:Treg) compared to anti-KLH isotype control 240 hours post-treatment (FIG. 4E).

[0186] Thl cytokine levels in KPCY tumors at 240 hours post-treatment with mCD40xFAP.LP62 or mCD40xHEL isotype control (10 mg / kg, QDxl) was also assessed. As shown in FIG. 5, mCD40xFAP.LP62 induced several Thl cytokines in the tumor, including IFN-y, IL-ip, IP10, TNF-a, and MIP-la 240 hours post-treatment as compared to mCD40xHEL isotype control-treated mice (n=10 mice per group across 2 independent experiments for the isotype control and 3 and 10 mice per group for mCD40xFAP.LP62 across two independent experiments).

[0187] No significant cytokine induction was observed in the periphery compared to control- treated mice, thus supporting the modality and mechanism of action for localized tumoral activation while sparing systemic CD40 activation (data not shown).

[0188] Taken together, these data demonstrate that mCD40xFAP.LP62 activates CD40- mediated innate immune responses in the tumor that engage adaptive immunity leading to efficacy.Example 8: Binding Characterization of Bispecific TGF|JRIIxFAP.LP62 Antibodies

[0189] A bispecific TGFPR2 / FAP antibody comprising one TGFPR2 antigen binding domain and one FAP antigen binding domain was constructed. The construct was designed with an effectorless IgGl Fc for prolonged PK and reduced or eliminated cell killing. The FAP.LP62 antigen binding domain was tested with a TGFPR2 binding domain, and with a non-targeting HEL binding domain. The following bispecific FAP antibodies were constructed and tested for binding kinetics using surface plasmon resonance (SPR): TGFpR2.LP120xFAP.LP62 and aHELxFAP.LP62.

[0190] Affinity for FAP was measured in two orientations by surface plasmon resonance. In the first orientation for measuring SPR for FAP, an antibody capture method was used with a bivalent model fit. Antibodies that capture the bispecific antibodies were anchored to a dextran matrix and captured the bispecific antibodies. The captured bispecific antibodies then captured FAP and measurements were taken. In the second orientation for measuring SPR for FAP, a FAP immobilization method was used with a bispecific model fit. HuFAP was anchored to a dextran matrix, and the bispecific antibodies bound to the anchored huFAP and measurements were taken. It was observed that the second orientation for measuring FAP, the FAP immobilization method, had a faster off-rate and lower affinity to bispecific antibodies. The FAP immobilized off-rate was much faster than when in solution, suggesting this orientation gives an affinity-based measurement. The rates of association and dissociation of TGFpR2.LP120xFAP.LP62 and aHELxFAP.LP62 to human FAP are summarized in Table E5 and Table E6.Table E5. Binding Kinetics of Bispecific FAP.LP62 Antibodies to huFAP As Measured by SPR, Antibody Capture MethodTable E6. Binding Kinetics of Bispecific FAP.LP62 Antibodies to huFAP As Measured by SPR, FAP Immobilized Method

[0191] For affinity for TGFpRII, an antibody capture method was used. Antibodies that capture the bispecific antibodies were anchored to a dextran matrix and captured the bispecific antibodies. The captured bispecific antibodies then captured human TGFpRII and measurements were taken. The rates of association and dissociation of TGFpR2.LP120xFAP.LP62 and TGFpR2.LP120xHEL to human TGFpRII are summarized in Table E7.Table E7. Binding Kinetics of Bispecific TGFPRIFFAP.LP62 Antibodies to huTGFpRII As Measured by SPR, Antibody Capture MethodExample 9: TGFpR2.LP120xFAP.LP62 Inhibits TGF Signaling in a FAP-Dependent Manner

[0192] The activity of TGFpR2.LP120xFAP.LP62 was evaluated for the ability to inhibit TGFP signaling, as measured by inhibition of pSMAD3 signaling (a type of TGFP signaling activated when TGFP binds both receptors TGFpRII and TGFpRI), in target cells, U138MG (FAP+TGFbRII+), and non-target cells, HepG2 (TGFbRII+). Target and non-target cells were pretreated with TGFpR2.LP120xFAP.LP62 for 45 minutes. Pre-treated cells were then incubated for 1 hour with TGFP prior to measuring pSMAD3 by ELISA. Target U138MG cells exhibited a FAP / TGFbRII ratio of 15. The results are shown in Table E8.Table E8. TGFpR2.LP120xFAP.LP62 Inhibition of pSMAD3 Signaling in U138MG Cells and HepG2 Cells

[0193] These data show that recruitment of a TGFbR2 inhibitor to the surface of a target cell via a bispecific anti-TGFpR2 / FAP provides increased potency over the non-targeted comparator.

[0194] The activity of TGFpR2.LP120xFAP.LP62 was evaluated in another experiment for the ability to inhibit TGFP signaling, as measured by inhibition of pSmad3 signaling in a target cell U138MG (FAP+TGFbRII+). Target cells were pretreated with TGFpR2.LP120xFAP.LP62 for 45 minutes. Pre-treated cells were then incubated for 1 hour with TGFP prior to measuring pSMAD3 by ELISA. The results are shown in Table E9 and FIG. 6. Table E9 also displays FAP Kd (nM) and maximum cell binding MFI at 200 nM for TGFpR2.LP120xFAP.LP62.Table E9. TGFpR2.LP120xFAP.LP62 Inhibition of pSMAD3 Signaling in U138MG Cells and HepG2 Cells

[0195] These data show that a bispecific anti-TGFpR2 / FAP antibody inhibits pSMAD3 signalling in fibroblasts with an IC50 almost 10-fold lower than non-targeted anti-TGFRpR2 antibody.X TABLE OF CERTAIN SEQUENCES

Claims

CLAIMS1. An antibody or antigen binding fragment thereof that binds fibroblast activation protein alpha (FAP), wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1 or 7, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 8, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 9; and a light chain variable region comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 10, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 or GAS (SEQ ID NO: 11), and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or 12.

2. The antibody or antigen binding fragment thereof of claim 1, wherein the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

3. The antibody or antigen binding fragment thereof of claim 1, wherein the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a LCDR2 comprising the amino acid sequence of GAS (SEQ ID NO: 11), and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.

4. The antibody or antigen binding fragment thereof of any one of claims 1-3, wherein the heavy chain variable region comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15; and the light chain variable region comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.

5. The antibody or antigen binding fragment thereof of any one of claims 1-3, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.

6. The antibody or antigen binding fragment thereof of any one of claims 1-5, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 13 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14, or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16.

7. The antibody or antigen binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen binding fragment thereof is an antibody fragment.

8. The antibody or antigen binding fragment thereof of claim 7, wherein the antibody fragment is selected from a Fab, Fab’, F(ab)2, and scFv.

9. The antibody or antigen binding fragment thereof of claim 8, which is an scFv.

10. The antibody or antigen binding fragment thereof of claim 9, wherein the antibody or antigen binding fragment thereof comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 17.

11. The antibody or antigen binding fragment thereof of claim 9 or claim 10, wherein the antibody or antigen binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 17.

12. A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of claims 1-11 and a pharmaceutically acceptable carrier.

13. An isolated nucleic acid that encodes the antibody or antigen binding fragment thereof of any one of claims 1-11.

14. The isolated nucleic acid of claim 13, which is an expression vector.

15. A host cell that expresses the antibody or antigen binding fragment thereof of any one of claims 1-11.

16. A host cell comprising the nucleic acid of claim 13 or claim 14.

17. A method of producing an antibody or antigen binding fragment thereof comprising culturing the host cell of claim 15 or claim 16 under conditions suitable for expressing the antibody or antigen binding fragment thereof.

18. The method of claim 17, comprising isolating the antibody or antigen binding fragment thereof.

19. A multispecific antibody or antigen binding fragment thereof comprising a first antigen-binding domain that binds FAP and at least one second antigen-binding domain that binds a second target antigen, wherein the first antigen binding domain comprises a first heavy chain variable region and a first light chain variable region, wherein: a) the first heavy chain variable region comprises a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1 or 7, aHCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 8, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 9; and b) the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 10, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 or GAS (SEQ ID NO: 11), and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or 12.

20. The multispecific antibody or antigen binding fragment thereof of claim 19, wherein the first heavy chain variable region comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

21. The multispecific antibody or antigen binding fragment thereof of claim 19, wherein the first heavy chain variable region comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the first light chain variable region comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a LCDR2 comprising the amino acid sequence of GAS (SEQ ID NO: 11), and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.

22. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-21, wherein the first heavy chain variable region comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15; and the first light chain variable region comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.

23. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-22, wherein the first antigen binding domain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15, and / or a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.

24. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-23, wherein the first antigen binding domain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, or wherein the first antigenbinding domain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

25. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-24, wherein the first antigen binding domain is a Fab, Fab', F(ab')2, Fd, Fv, singlechain Fv (scFv) or disulfide-linked Fv (sdFv).

26. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-25, wherein the first antigen binding domain is a scFv or sdFv.

27. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-26, wherein the first antigen binding domain comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 17.

28. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-27, wherein the first antigen binding domain comprises the amino acid sequence of SEQ ID NO: 17.

29. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-28, wherein the second antigen binding domain comprises a second heavy chain variable region and a second light chain variable region.

30. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-29, wherein the second antigen binding domain is a Fab, Fab', F(ab')2, Fd, Fv, singlechain Fv (scFv) or disulfide-linked Fv (sdFv).

31. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-30, wherein the second target antigen is not CD40.

32. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-31, wherein the multispecific antibody or antigen binding fragment thereof comprises a third antigen binding domain that binds to a third target antigen, wherein the second target antigen and the third target antigen are the same or different.

33. The multispecific antibody or antigen binding fragment thereof of claim 32, wherein the second antigen binding domain and the third antigen binding domain are each independently a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv).

34. The multispecific antibody of antigen binding fragment thereof of claim 32 or claim 33, wherein the second target antigen and the third target antigen are each not CD40.

35. The multispecific antibody or antigen binding fragment thereof of any one of claims 32-34, wherein the second antigen binding domain and the third antigen binding domain are the same or different.

36. The multispecific antibody or antigen binding fragment thereof of any one of claims 32-35, wherein: a) the second antigen binding domain comprises a second heavy chain variable region and a second light chain variable region; and b) the third antigen binding domain comprises a third heavy chain variable region and a third light chain variable region.

37. The multispecific antibody or antigen binding fragment thereof of claim 36, wherein: a) the second heavy chain variable region is fused to a first heavy chain constant region and the second light chain variable region is fused to a first light chain constant region; and b) the third heavy chain variable region is fused to a second heavy chain constant region and the third light chain variable region is fused to a second light chain constant region.

38. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-37, wherein the first antigen binding domain that binds to FAP is a Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv (scFv) or disulfide-linked Fv (sdFv), and wherein the first antigen binding domain is fused to the C-terminus of the first or second heavy chain constant region.

39. The multispecific antibody or antigen binding fragment thereof of claim 37 or claim 38, wherein the first heavy chain constant region and the second heavy chain constant region are different.

40. The multispecific antibody or antigen binding fragment thereof of claim 39, wherein the first heavy chain constant region and the second heavy chain constant region form a heterodimer.

41. The multispecific antibody or antigen binding fragment thereof of claim 39 or claim 40, wherein the first heavy chain constant region comprises at least one first heterodimerization mutation and the second heavy chain constant region comprises at least one second heterodimerization mutation.

42. The multispecific antibody or antigen binding fragment thereof of claim 41, wherein at least one first heterodimerization mutation comprises T366W and at least one second heterodimerization mutation comprises one or more of T366S, L368A, and / or Y407V, or wherein at least one first heterodimerization mutation comprises one or more of T366S, L368A, and / or Y407V and at least one second heterodimerization mutation comprises T366W, wherein mutation position is according to Kabat numbering.

43. The multispecific antibody or antigen binding fragment thereof of claim 41, wherein at least one first heterodimerization mutation comprises T366W and at least one secondheterodimerization mutation comprises T366S, L368A, andY407V, or wherein at least one first heterodimerization mutation comprises T366S, L368A, and Y407V and at least one second heterodimerization mutation comprises T366W, wherein mutation position is according to Kabat numbering.

44. The multispecific antibody or antigen binding fragment thereof of claim 41, wherein at least one first heterodimerization mutation comprises one or more of T350V, L351Y, S400E, F405A, and / or Y407V and at least one second heterodimerization mutation comprises one or more of T350V, T366L, N390R, K392M, K392L, and / or T394W, or wherein at least one first heterodimerization mutation comprises one or more of T350V, T366L, N390R, K392M, K392L, and / or T394W and at least one second heterodimerization mutation comprises one or more of T350V, L351Y, S400E, F405A, and / or Y407V, wherein mutation position is according to Kabat numbering.

45. The multispecific antibody or antigen binding fragment thereof of claim 41, wherein at least one first heterodimerization mutation comprises T350V, L351Y, F405A, and Y407V and at least one second heterodimerization mutation comprises T350V, T366L, K392L, and T394W, or wherein at least one first heterodimerization mutation comprises one or more of T350V, T366L, K392L, and T394W and at least one second heterodimerization mutation comprises one or more of T350V, L351Y, F405A, and Y407V, wherein mutation position is according to Kabat numbering.

46. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-45, wherein the second target antigen is an immune cell antigen.

47. The multispecific antibody or antigen binding fragment thereof of claim 46, wherein the immune cell antigen is expressed on the surface of a T cell, a natural killer (NK) cell, or a dendritic cell.

48. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-45, wherein the second target antigen is expressed on fibroblasts.

49. The multispecific antibody or antigen binding fragment thereof of any one of claims 19-48, wherein the second target antigen is selected from CD3 and TGFRpiI.

50. A pharmaceutical composition comprising the multispecific antibody or antigen binding fragment thereof of any one of claims 19-49 and a pharmaceutically acceptable carrier.

51. An isolated nucleic acid that encodes the multispecific antibody or antigen binding fragment thereof of any one of claims 19-49.

52. The isolated nucleic acid of claim 51, which is an expression vector.

53. A host cell that expresses the multispecific antibody or antigen binding fragment thereof of any one of claims 19-49.

54. A host cell comprising the nucleic acid of claim 51 or claim 52.

55. A method of producing a multispecific antibody or antigen binding fragment thereof comprising culturing the host cell of claim 53 or claim 54 under conditions suitable for expressing the multispecific antibody or antigen binding fragment thereof.

56. The method of claim 55, comprising isolating the multispecific antibody or antigen binding fragment thereof.

57. A method of treating cancer comprising administering to a subject in need thereof the antibody or antigen binding fragment thereof of any one of claims 1-11, the multispecific antibody or antigen binding fragment thereof of any one of claims 19-49, or the pharmaceutical composition of claim 12 or claim 50.

58. The method of claim 57, wherein the cancer is a solid cancer.

59. A method of treating fibrosis comprising administering to a subject in need thereof the antibody or antigen binding fragment thereof of any one of claims 1-11, the multispecific antibody or antigen binding fragment thereof of any one of claims 19-49, or the pharmaceutical composition of claim 12 or claim 50.

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