Fibroblast activation protein (FAP) CAR invariant natural killer T cells and uses thereof

Abstract

This disclosure is based, at least in part, on the discovery of novel anti-fibroblast activation protein (FAP) antibodies or antigen-binding fragments thereof, and demonstrates that genetically engineered cells (e.g., iNKT cells) expressing chimeric antigen receptors comprising anti-FAP antibodies or antigen-binding fragments thereof have improved properties, including increased binding to FAP, killing of FAP-expressing cancer cells in vitro and in vivo, and improved persistence in subjects receiving therapy. In some embodiments, the anti-FAP CAR iNKT cells are engineered to express an arming molecule (e.g., soluble IL-15).

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of the filing date of U.S. Provisional Application No. 63 / 413,236, filed October 4, 2022, the entire contents of which are incorporated herein by reference.

[0002] Reference to the electronic sequence listing The contents of the electronic sequence listing (A132770004WO00-SEQ-LGE.xml, size: 179,363 bytes, created on October 4, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Fibroblast activation protein (FAP) is a cell surface protein highly expressed on stromal cells in the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs). FAP can regulate the TME by remodeling the extracellular matrix (ECM), and overexpression of FAP in CAFs is associated with poor prognosis in various cancers. Summary of the Invention

[0004] The present disclosure is based, at least in part, on the discovery that genetically modified cells (e.g., iNKT cells) expressing chimeric antigen receptors that target fibroblast activation proteins (FAPs) can kill FAP-expressing cells (e.g., cancer-associated fibroblasts (CAFs)) in the tumor microenvironment (TME). In some embodiments, anti-FAP CAR iNKT cells are engineered to express arming molecules (e.g., soluble IL-15). In some embodiments, the anti-FAP CAR iNKT cells kill FAP-expressing cells (e.g., FAP-expressing tumor cells and / or FAP-expressing cancer-associated fibroblasts (CAFs)). In some embodiments, the anti-FAP CAR iNKT cells reduce immune suppression in the TME, thereby increasing the efficacy of other cancer therapies (e.g., CAR T cells that target tumor antigens). In some embodiments, the anti-FAP CAR iNKT cells described by the present disclosure exhibit improved properties compared to existing FAP-CAR cell therapies in the art, including, but not limited to, killing of FAP-expressing cells in vitro and in vivo, and improved persistence in subjects receiving the therapy.

[0005] In some aspects, the present disclosure provides invariant natural killer T (iNKT) cells comprising a chimeric antigen receptor (CAR) that specifically binds to fibroblast activation protein (FAP). In some embodiments, the chimeric antigen receptor of the iNKT cell comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having the amino acid sequence of SEQ ID NO: 2, a CDRH3 having the amino acid sequence of SEQ ID NO: 3, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 12, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the chimeric antigen receptor of iNKT cells comprises CDRH1 having the amino acid sequence of SEQ ID NO: 40, CDRH2 having the amino acid sequence of SEQ ID NO: 41, CDRH3 having the amino acid sequence of SEQ ID NO: 42, CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43. In some embodiments, the chimeric antigen receptor of iNKT cells comprises CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, CDRH3 having the amino acid sequence of SEQ ID NO: 42, CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43. In some embodiments, the chimeric antigen receptor of iNKT cells comprises CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, CDRH3 having the amino acid sequence of SEQ ID NO: 54, CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 55.

[0006] In some embodiments, the chimeric antigen receptor of iNKT cells comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the chimeric antigen receptor of iNKT cells comprises a VH comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the chimeric antigen receptor of iNKT cells comprises a VH comprising the amino acid sequence of SEQ ID NO: 44, and a VL comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the chimeric antigen receptor of iNKT cells comprises a VH comprising the amino acid sequence of SEQ ID NO: 47, and a VL comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the chimeric antigen receptor of iNKT cells comprises a VH comprising the amino acid sequence of SEQ ID NO: 56, and a VL comprising the amino acid sequence of SEQ ID NO: 57.

[0007] In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv comprising the amino acid sequence of SEQ ID NO: 49. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv comprising the amino acid sequence of SEQ ID NO: 58.

[0008] In some embodiments, the iNKT cells comprise a CAR that further comprises a hinge region. In some embodiments, the hinge region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 30. In some embodiments, the iNKT cells comprise a CAR that further comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 31. In some embodiments, the iNKT cells comprise a CAR that further comprises a hinge / transmembrane domain comprising the amino acid sequence of any one of SEQ ID NOs: 83, 85, or 87. In some embodiments, the iNKT cells comprise a CAR that further comprises one or more cytoplasmic domains. In some embodiments, the one or more cytoplasmic domains comprise the amino acid sequence of any one of SEQ ID NOs: 32, 33, 39, 90, 92, 94, 96, 98, 100, 102, or 105. In some embodiments, the cytoplasmic domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 32. In some embodiments, the iNKT cells comprise a CAR that further comprises an intracellular costimulatory domain. In some embodiments, the costimulatory domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 33. In some embodiments, the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 17, 18, 59, 60, 61, or 62.

[0009] In some embodiments, the anti-FAP CAR iNKT cells are engineered to express one or more immunomodulatory gene products (i.e., arming molecules). In some embodiments, the one or more immunomodulatory gene products include IL-15, IL-12, CD40L, or 4-1BB, IL-18, or IL-21. In some embodiments, the immunomodulatory gene product is soluble IL-15 (sIL-15).

[0010] In some embodiments, the CAR binds to the FAP with a KD ranging from 1E-10 M to 10E-7 M. In some embodiments, the CAR binds to the FAP with a KD ranging from 1E-8 M to 3E-8 M.

[0011] In some aspects, the present disclosure provides invariant natural killer T (iNKT) cells comprising a chimeric antigen receptor (CAR) that specifically binds to a fibroblast activation protein (FAP), wherein the iNKT cells express IL-15 (e.g., soluble IL-15). In some embodiments, the anti-FAP CAR iNKT cells comprise a CAR that comprises an antigen-binding portion that specifically binds to a FAP derived from the antigen-binding portion of any one of the anti-FAP antibodies listed in Tables 2 and 3.

[0012] In some aspects, the disclosure provides a composition comprising a FAP CAR iNKT cell described herein and a pharmaceutically acceptable carrier.

[0013] In some aspects, the present disclosure provides a method for killing a cell, the method comprising contacting the cell with an anti-FAP iNKT cell or composition thereof described herein. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is a lung cancer cell, a breast cancer cell, a colon cancer cell, a prostate cancer cell, a gastric cancer cell, a pancreatic cancer cell, a prostate cancer cell, a thyroid cancer cell, a cervical cancer cell, a urothelial cancer cell, or a sarcoma cell. In some embodiments, the lung cancer is a non-small cell lung cancer. In some embodiments, cells in the tumor microenvironment express a FAP.

[0014] In some aspects, the present disclosure provides a method for treating a tumor in a subject, the method comprising administering to a subject having or suspected of having cancer an anti-FAP iNKT or composition thereof described herein.

[0015] In some aspects, the present disclosure provides methods for reducing tumor growth, the methods comprising contacting a tumor in a subject with an anti-FAP iNKT or composition thereof described herein. In some embodiments, administration is by injection. In some embodiments, the injection is intraperitoneal, intravenous, or intratumoral. In some embodiments, the anti-FAP CAR iNKT cells are combined with a therapeutic agent. In some embodiments, the therapeutic agent is a CAR T cell specific for a tumor antigen. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor or agonist.

[0016] In some aspects, the present disclosure provides a method for treating a tumor in a subject, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells comprising a chimeric antigen receptor (CAR) having an anti-FAP binding moiety.

[0017] In some aspects, the present disclosure provides a method for killing tumor cells, the method comprising contacting the tumor cells with a composition comprising invariant natural killer T (iNKT) cells comprising a chimeric antigen receptor (CAR) having an anti-FAP binding moiety.

[0018] In some aspects, the present disclosure provides a method for reducing immunosuppression in a tumor microenvironment (TME) of a subject compared to immunosuppression in the subject prior to administration, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells comprising a chimeric antigen receptor (CAR) having an anti-FAP binding moiety.

[0019] In some embodiments, the anti-FAP CAR iNKT cells express an arming molecule. In some embodiments, the arming molecule is soluble IL-15 (sIL-15).

[0020] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is of epithelial origin. In some embodiments, the solid tumor is a lung cancer tumor, a breast cancer tumor, a colon cancer tumor, a prostate cancer tumor, a gastric cancer tumor, a pancreatic cancer tumor, a prostate cancer tumor, a thyroid cancer tumor, a cervical cancer tumor, a urothelial cancer tumor, or a sarcoma tumor.

[0021] In some embodiments, the anti-FAP CAR iNKT cells kill FAP-expressing cells in the TME. In some embodiments, the FAP-expressing cells in the TME comprise FAP-expressing tumor cells and / or cancer-associated fibroblasts. In some embodiments, the anti-FAP CAR iNKT cells directly kill the FAP-expressing cells. In some embodiments, the anti-FAP CAR iNKT cells indirectly kill the FAP-expressing cells. In some embodiments, killing the cancer-associated fibroblasts reduces immunosuppression in the TME compared to immunosuppression in the subject prior to administration. In some embodiments, killing the cancer-associated fibroblasts increases immune cell infiltration in the TME compared to immune cell infiltration in the subject prior to administration.

[0022] In some embodiments, the method further comprises administering to the subject CAR T cells that target a tumor antigen comprising NY-ESO-1, or BCMA.

[0023] In some embodiments, administration of the anti-FAP CAR iNKT cells reduces tumor burden compared to the tumor burden in the subject before administration.

[0024] In some embodiments, administration of anti-FAP CAR iNKT cells results in resistance to T cell exhaustion, enhanced tissue homing of anti-FAP iNKT cells, selective cytotoxicity against M2 macrophages, and / or stimulation of dendritic cell maturation. In some embodiments, the anti-FAP CAR iNKT cells retain their responsiveness to CD1d and / or NK receptor ligands.

[0025] In some embodiments, the anti-FAP CAR iNKT cells comprise a CAR comprising any one of the FAP-binding moieties listed in Tables 2 and 3.

[0026] In some embodiments, the anti-FAP CAR iNKT cells comprise a CAR comprising any one of the FAP antibodies listed in Table 2.

[0027] In some embodiments, the subject is a human. In some embodiments, the subject has been diagnosed with a solid tumor, including lung cancer, breast cancer, colon cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, urothelial cancer, or sarcoma.

[0028] In some aspects, the disclosure provides antibodies or antigen-binding fragments that specifically bind to an amino acid sequence having at least 85% identity to SEQ ID NOs: 35-38. In some embodiments, the antibody specifically binds to an amino acid sequence represented by SEQ ID NOs: 35-38.

[0029] In some aspects, the disclosure provides an antibody or antigen-binding fragment comprising a heavy chain variable region having the sequence set forth in SEQ ID NO:7 or 14.

[0030] In some embodiments, the antibody comprises a heavy chain variable region having the sequence set forth in SEQ ID NO:7 or 14 and a light chain variable region having the sequence set forth in SEQ ID NO:8 or 15.

[0031] In some embodiments, the antibody comprises (i) a heavy chain variable region having the sequence set forth in SEQ ID NO:7 and / or a light chain variable region having the sequence set forth in SEQ ID NO:8, or (ii) a heavy chain variable region having the sequence set forth in SEQ ID NO:14 and / or a light chain variable region having the sequence set forth in SEQ ID NO:15.

[0032] In some aspects, the disclosure provides an antibody or antigen-binding fragment comprising a variable heavy chain region comprising a complementarity determining region 3 (CDRH3) having the sequence set forth in SEQ ID NO: 3 or 12.

[0033] In some embodiments, the antibody or antigen-binding fragment further comprises a variable light chain region comprising complementarity determining region 3 (CDRL3) having the sequence set forth in SEQ ID NO: 6 or 13.

[0034] In some aspects, the disclosure provides an ab antibody or antigen-binding fragment comprising six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein (i) CDRH1 comprises the sequence set forth in SEQ ID NO: 1, CDRH2 comprises the sequence set forth in SEQ ID NO: 2, CDRH3 comprises the sequence set forth in SEQ ID NO: 3, CDRL1 comprises the sequence set forth in SEQ ID NO: 4, CDRL2 comprises the sequence set forth in SEQ ID NO: 5, and CDRL3 comprises the sequence set forth in SEQ ID NO: 6, or (ii) CDRH1 comprises the sequence set forth in SEQ ID NO: 10, CDRH2 comprises the sequence set forth in SEQ ID NO: 11, CDRH3 comprises the sequence set forth in SEQ ID NO: 12, CDRL1 comprises the sequence set forth in SEQ ID NO: 4, CDRL2 comprises the sequence set forth in SEQ ID NO: 5, and CDRL3 comprises the sequence set forth in SEQ ID NO: 13.

[0035] In some embodiments, the antibody or antigen-binding fragment is chimeric, hi some embodiments, the antibody or antigen-binding fragment is humanized.

[0036] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv). In some embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 9 or 16.

[0037] In some aspects, the present disclosure provides an isolated nucleic acid encoding an antibody or antigen-binding fragment described herein.

[0038] In some embodiments, the nucleic acid sequence encoding the anti-FAP scFv is represented by SEQ ID NO: 21 or 26. In some embodiments, the nucleic acid sequence encoding the anti-FAP CAR is represented by SEQ ID NO: 22 or 27.

[0039] In some aspects, the present disclosure provides a vector comprising the isolated nucleic acid described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector comprises a sequence represented by SEQ ID NO: 23 or 28.

[0040] In some aspects, the present disclosure provides a host cell comprising an antibody or antigen-binding fragment, isolated nucleic acid, or vector described herein. In some embodiments, the cell is a mammalian cell, a bacterial cell, a yeast cell, or an insect cell. In some embodiments, the cell is a hybridoma cell.

[0041] In some aspects, the present disclosure provides a chimeric antigen receptor (CAR) comprising the antigen-binding fragment described herein. In some embodiments, the CAR further comprises a hinge region. In some embodiments, the hinge region comprises an amino acid sequence at least 80% identical to SEQ ID NO: 30.

[0042] In some embodiments, the CAR further comprises a transmembrane domain, hi some embodiments, the transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:31.

[0043] In some embodiments, the CAR further comprises a cytoplasmic domain, hi some embodiments, the cytoplasmic domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:32.

[0044] In some embodiments, the CAR further comprises a costimulatory domain, hi some embodiments, the costimulatory domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:33.

[0045] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 17 or 18.

[0046] In some aspects, the present disclosure includes immune cells comprising a CAR described herein.

[0047] In some embodiments, the immune cells are natural killer (NK) cells or T cells. In some embodiments, the immune cells are invariant natural killer T (iNKT) cells.

[0048] In some embodiments, the immune cells are engineered to express one or more immunomodulatory gene products, hi some embodiments, the one or more immunomodulatory gene products include IL-15, IL-12, CD40L, or 4-1BB, IL-18, or IL-21.

[0049] In some aspects, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment or immune cell described herein and a pharmaceutically acceptable excipient.

[0050] In some aspects, the present disclosure provides methods for killing a cell, the method comprising contacting the cell with an antibody or antigen-binding fragment, immune cell, or pharmaceutical composition described herein.

[0051] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is lung cancer, breast cancer, colon cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, urothelial cancer, or sarcoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0052] In some aspects, the present disclosure provides methods for treating a tumor in a subject, the method comprising administering to a subject having or suspected of having cancer an immune cell, or pharmaceutical composition described herein.

[0053] In some aspects, the present disclosure provides a method for reducing tumor growth, the method comprising contacting a tumor in a subject with an antibody or antigen-binding fragment, immune cell, or pharmaceutical composition described herein.

[0054] In certain embodiments, the subject does not undergo lymphodepletion prior to or concurrently with treatment with the anti-FAP CAR iNKT cells or pharmaceutical compositions disclosed herein. Lymphodepletion is frequently performed prior to immunotherapy, such as CAR T therapy, for example, to eliminate tumors, alter tumor phenotype, modify the tumor microenvironment, remove cytokine sinks (e.g., make IL-2, IL-7, and IL-15 more available), and suppress the host immune system. However, lymphodepletion has several adverse effects, including neutropenia, anemia, thrombocytopenia, and immunosuppression, as well as the toxicities associated with lymphodepleting agents such as fludarabine and cyclophosphamide. In certain embodiments of the present invention, the subject does not undergo treatment with fludarabine or cyclophosphamide prior to or concurrently with administration of the anti-FAP CAR iNKT cells or pharmaceutical compositions disclosed herein.

[0055] In some embodiments, the subject is a human. In some embodiments, the tumor is a solid tumor. In some embodiments, the solid tumor is a lung cancer tumor, a breast cancer tumor, a colon cancer tumor, a prostate cancer tumor, a gastric cancer tumor, a pancreatic cancer tumor, a prostate cancer tumor, a thyroid cancer tumor, a cervical cancer tumor, a urothelial cancer tumor, or a sarcoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0056] In some embodiments, administration is by injection, hi some embodiments, the injection is intraperitoneal, intravenous, or intratumoral.

[0057] In some embodiments, the immune cells are combined with a therapeutic agent. In some embodiments, the therapeutic agent is a CAR T cell specific for a tumor antigen. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor or agonist.

[0058] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments and, together with the written specification, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein. [Brief explanation of the drawings]

[0059] [Figure 1] 1 shows an exemplary format of anti-FAP CAR iNKT cells.

[0060] [Figure 2] This figure shows a process for evaluating a therapeutic window targeting FAP-positive cells (e.g., cancer-associated fibroblasts (CAFs)) in the tumor microenvironment. FAP expression in different types of cancer-associated fibroblasts. Quantification of FAP molecule expression on the surface of different cell types was performed using the Quantibrite™ approach. CAFs derived from eight isolated frozen tumor samples (DTCs) from colorectal cancer (CRC) patients were evaluated. FAP expression in the CAF-S1 subpopulation was more than 10-fold higher than that in the CAF-S3 subpopulation. Similarly, FAP expression in CAF-S1 samples was 10-fold higher than that in thawed normal lung fibroblasts (NLFs). Passage of NLFs (P0, P3) increased FAP expression to levels similar to those of CAFs. Different FAP-expressing tumor cell lines obtained through multiple passages showed FAP expression ranging from that observed in primary healthy fibroblasts to CAF S3. In the case of U138 cells, there was variability based on passage. For discovery purposes, we transfected T2 cells with low or high amounts of FAP (T2low and T2high, respectively). Finally, for in vivo studies, we generated A375 cells expressing FAP at the same level as T2high.

[0061] [Figure 3-1] 1 is a graph showing the generation of anti-FAP CARs capable of binding to human and mouse FAP and killing FAP-expressing cells. 2 shows the process for identifying a panel of anti-FAP candidate scFvs. [Figure 3-2] (B) is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. (C) is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. (D) is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. (E) is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. (F) is a graph showing the expression levels of FAP in target T2 cells electroporated with either 0.3 μg or 10 μg of human or mouse FAP. [Figure 3-3] (D) is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. The graph shows binding of an exemplary pool of anti-FAP scFvs to human or mouse FAP. (E) is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. The graph shows target cell killing of an exemplary pool of anti-FAP CAR iNKT cells. [Figure 3-4] (F) is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. The binding of a pool of different candidate anti-FAP CAR candidates expressed in two different donors is shown. iNKT cells derived from donor 1 are shown binding to human FAP. (G) is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. The binding of a pool of different candidate anti-FAP CAR candidates expressed in two different donors is shown. A different pool of candidate anti-FAP CAR iNKT cells derived from donor 2 is shown in binding human FAP. [Figure 3-5]H is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. Target cell killing at an E:T ratio of 1:1 is shown. All anti-FAP candidate CAR iNKT candidates were able to kill more U87 and HS683 cells compared to the mock control. I is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. Target cell killing at an E:T ratio of 5:1 is shown. All anti-FAP candidate CAR iNKT candidates were able to kill more U87 and HS683 cells compared to the mock control. [Figure 3-6] J is a graph showing the generation of anti-FAP CARs capable of binding to human and mouse FAP and killing FAP-expressing cells. Target cell-induced iNKT cell activation is shown by measuring cytokine CD25 and CD69 markers at an E:T ratio of 1:1. K is a graph showing the generation of anti-FAP CARs capable of binding to human and mouse FAP and killing FAP-expressing cells. Target cell-induced iNKT cell activation is shown by measuring cytokine CD25 and CD69 markers at an E:T ratio of 5:1. [Figure 3-7] L is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. Target cell-induced iNKT cell activation is shown by measuring the 4-1bb marker at an E:T ratio of 1:1. M is a graph showing the generation of anti-FAP CARs that can bind to human and mouse FAP and kill FAP-expressing cells. Target cell-induced iNKT cell activation is shown by measuring the 4-1bb marker at an E:T ratio of 5:1. [Figure 3-8]N is a graph showing the generation of anti-FAP CARs capable of binding to human and mouse FAP and killing FAP-expressing cells. The candidate selection process identified two distinct groups of anti-FAP CARs. Left graph: Cytotoxicity of T2 cells expressing high levels of mouse FAP vs. cytotoxicity of T2 cells expressing high levels of human FAP. Top candidates selected by CARDIS were transiently transfected into iNKT cells. In parallel, T2 cells were transfected with high human or mouse FAP. The next day, CAR-iNKT cells were cocultured with mouse or human T2high at an E:T ratio of 5:1, and target cell killing was assessed 24 hours later. All candidates potently kill cells expressing high levels of mouse or human FAP. Right graph: Cytotoxicity of T2 cells expressing low levels of mouse FAP vs. cytotoxicity of T2 cells expressing low levels of human FAP. Transiently transfected CAR-iNKT cells were cocultured with mouse or human T2low cells. Assessment of target cell killing after 24 hours revealed two distinct cell populations: one retained the ability to potently kill T2low cells, while the second spared cells bearing FAP at levels similar to CAF-S3 or normal fibroblasts. Importantly, both populations recognized mouse and human FAP at comparable levels.

[0062] [Figure 4A] 1 shows an in vitro assay evaluating candidate anti-FAP CAR iNKT cells. FAP-CAR iNKT candidate responds to FAP+ tumors in vitro (donor 1). [Figure 4B] 1 shows an in vitro assay evaluating candidate anti-FAP CAR iNKT cells. FAP-CAR iNKT candidate responds to FAP+ tumors in vitro (donor 2). [Figure 4C] 1 shows an in vitro assay evaluating candidate anti-FAP CAR iNKT cells. Anti-FAP CAR iNKT cells were polyfunctional in response to A-375-FAP tumor cells in vitro (donor 1). [Figure 4D]1 shows an in vitro assay evaluating candidate anti-FAP CAR iNKT cells. Anti-FAP CAR iNKT cells were polyfunctional in response to A-375-FAP tumor cells in vitro (donor 2).

[0063] [Figure 5A] 1 shows in vivo tumor control by FAP-CAR iNKT against FAP-expressing tumors in a melanoma A-375-FAP+ cancer model. A schematic diagram of in vivo A-375-FAP tumor challenge with FAP-CAR iNKT cells is shown. [Figure 5B] 1 shows in vivo tumor control by FAP-CAR iNKT against FAP-expressing tumors in a melanoma A-375-FAP+ cancer model. A curve graph showing tumor growth is shown. [Figure 5C] 1 shows in vivo tumor control by FAP-CAR iNKT against FAP-expressing tumors in a melanoma A-375-FAP+ cancer model. Curve graphs representing tumor growth for individual mice in each group are shown. [Figure 5D] Figure 1 shows in vivo tumor control by FAP-CAR iNKT against FAP-expressing tumors in a melanoma A-375-FAP+ cancer model. Survival analysis of A-375-FAP tumor-bearing mice untreated, treated with MiNK FAP-CAR iNKT cells, treated with sibrotuzumab-CAR NKT cells, or treated with control BCMA-CAR iNKT cells is shown. [Figure 5E]

[0023] Figure 1 shows in vivo tumor control by FAP-CAR iNKT against FAP-expressing tumors in a melanoma A-375-FAP+ cancer model. Tumor growth weights are shown for each treatment group.

[0064] [Figure 6]Figures A-B show that FAP-CAR-IL-15 iNKT cells have the ability to enhance the cytotoxicity of T cells stimulated with repeated antigen. Figure A shows the cytotoxicity of NYESO-1-specific T cells exposed to antigen multiple times (up to 8 times [R#]) against tumor cells expressing HLA-A*02:01-SLLMWITQC. Figure B shows the cytotoxicity of NYESO-1-specific T cells exposed to antigen two (AE2), three (AE3), or six (AE6) times and co-cultured with medium alone or the supernatant of activated FAP-CAR-IL-15 iNKT cells.

[0065] [Figure 7] Figures A-B show that E07 FAP-CAR-IL-15 iNKT cells have the ability to target murine FAP. Figure A shows the cytotoxicity of FAP-CAR-IL-15 iNKT cells against MC38 tumor cells engineered to express murine FAP at different effector-to-target (E:T) ratios. Figure B shows the cytotoxicity of FAP-CAR-IL-15 iNKT cells against 4T1 tumor cells engineered to express murine FAP at different effector-to-target (E:T) ratios.

[0066] [Figure 8A] Figure 1 shows a schematic diagram of an in vivo NSCLC xenograft model developed to explain the mechanism of action of FAP-CAR-iNKT drugs, which target cancer-associated fibroblasts (CAFs) that express FAP, while tumor cells (A549) do not express FAP. Figure 2 shows the experimental design of an NSCLC xenograft tumor model treated with FAP-CAR iNKT cells. [Figure 8B] Figure 1 shows a schematic diagram of an in vivo NSCLC xenograft model developed to illustrate the mechanism of action of FAP-CAR-iNKT drugs, which target cancer-associated fibroblasts (CAFs) that express FAP, while tumor cells (A549) do not express FAP. Figure 2 shows a schematic diagram illustrating the drug mechanism of action of FAP-CAR-IL-15 iNKT cells. [Figure 8C]This figure shows a schematic diagram of an in vivo NSCLC xenograft model developed to explain the mechanism of action of FAP-CAR-iNKT drugs, which target cancer-associated fibroblasts (CAFs) that express FAP, while tumor cells (A549) do not express FAP. Bioluminescence imaging of NOG mice intravenously injected with 2 million A-549 tumor cells expressing HLA-A*02:01-SLLMWITQC and nanoluciferase demonstrates tumor formation within the lungs of the mice. The image shown is from an untreated animal and demonstrates robust tumor uptake within the lungs of the mice 7 days after tumor inoculation. [Figure 8D] Figure 6E shows a schematic of an in vivo NSCLC xenograft model developed to explain the mechanism of action of FAP-CAR-iNKT drugs, which target cancer-associated fibroblasts (CAFs) that express FAP, while tumor cells (A549) do not express FAP. H&E staining of the lungs of mice administered A-549 tumor cells 14 days after tumor implantation is shown. The left panel of Figure 6E shows the dynamics of tumor burden by bioluminescence imaging in tumor-bearing animals over a 21-day period. The right panel shows histological tumor area quantification in the lungs of tumor-bearing mice over the same 21-day period. Both quantification of tumor burden by bioluminescence imaging and histological characterization demonstrate progressive tumor growth in the untreated state. [Figure 8E] Figure 6E shows a schematic of an in vivo NSCLC xenograft model developed to explain the mechanism of action of FAP-CAR-iNKT drugs, which target cancer-associated fibroblasts (CAFs) that express FAP, while tumor cells (A549) do not express FAP. H&E staining of the lungs of mice administered A-549 tumor cells 14 days after tumor implantation is shown. The left panel of Figure 6E shows the dynamics of tumor burden by bioluminescence imaging in tumor-bearing animals over a 21-day period. The right panel shows histological tumor area quantification in the lungs of tumor-bearing mice over the same 21-day period. Both quantification of tumor burden by bioluminescence imaging and histological characterization demonstrate progressive tumor growth in the untreated state. [Figure 8F]A schematic diagram of an in vivo NSCLC xenograft model developed to demonstrate the mechanism of action of FAP-CAR-iNKT drugs, which target cancer-associated fibroblasts (CAFs) that express FAP, while tumor cells (A549) do not express FAP. FAP expression assessed by immunohistochemistry is shown. Three representative mice are shown for FAP expression 1-3 weeks after tumor formation, in whole lungs (left panel) and representative sections (right panel). Normal lungs and controls (no primary and isotype) are shown in the last three right panels. Graphical quantification of FAP expression is shown below. [Figure 8G] A schematic diagram of an in vivo NSCLC xenograft model developed to explain the mechanism of action of FAP-CAR-iNKT drugs targeting cancer-associated fibroblasts (CAFs), which express FAP, while tumor cells (A549) do not express FAP. Alpha-smooth muscle actin (α-SMA) expression is shown by immunohistochemistry at 1 and 3 weeks after tumor formation. Graphical quantification of α-SMA expression is shown on the right, demonstrating a slight increase in α-SMA expression within the tumor from 1 to 3 weeks after tumor formation. [Figure 8H] Figure 1 shows a schematic diagram of an in vivo NSCLC xenograft model developed to explain the mechanism of action of FAP-CAR-iNKT drugs, which target cancer-associated fibroblasts (CAFs) that express FAP, while tumor cells (A549) do not express FAP. Mouse lungs analyzed by qPCR for the expression of fap, col11A1, Il6, and Tgfb.

[0067] [Figure 9]Figures A-B show that FAP-CAR-IL-15 iNKT promotes NSCLC tumor control. Figure A shows individual tumor burdens from NOG mice challenged with A-549 tumor cells expressing HLA-A*02:01-SLLMWITQC. These cells were left untreated or treated with nonspecific CAR-IL-15 iNKT cells alone, FAP-CAR-IL-15 iNKT cells alone, T cells expressing the NYESO-1 TCR, or T cells expressing the FAP-CAR-IL-15 iNKT and NYESO TCRs. Figure B shows survival analysis of mice treated under the conditions described in Figure A.

[0068] [Figure 10A] Figure 1 shows that mice treated with MiNK FAP-CAR-IL-15 showed reduced tumor burden in lung tissue after administration of T cells or FAP-CAR-IL-15 plus T cells. Bioluminescence imaging of mice treated with FAP-CAR-IL-15 iNKT T cells (left panel) and T cells alone (right panel) at days 18 and 25 is shown. [Figure 10B] Figure 1 shows that mice treated with MiNK FAP-CAR-IL-15 had reduced tumor burden in lung tissue after administration of T cells or FAP-CAR-IL-15 plus T cells. Figure 2 shows the percent change in tumor burden from day 3 to day 24 after tumor injection in mice treated with either T cells alone or FAP-CAR-IL-15 iNKT cells plus T cells. [Figure 10C] Mice treated with MiNK FAP-CAR-IL-15 showed reduced tumor burden in lung tissue after administration of T cells or FAP-CAR-IL-15 plus T cells. H&E staining of lungs from mice treated with FAP-CAR-IL-15 iNKT T cells or T cells harvested on day 25 is shown (left panel). The right panel shows quantification of tumor surface area in lungs from mice treated with FAP-CAR-IL-15 iNKT T cells or T cells alone. [Figure 10D]Figure 1 shows that mice treated with MiNK FAP-CAR-IL-15 showed reduced tumor burden in lung tissue after administration of T cells or FAP-CAR-IL-15 plus T cells. Immunohistochemical staining of FAP in lungs of mice treated with T cells or FAP-CAR-IL-15 + T cells harvested on day 25 is shown (left panel). The right panel shows quantification of FAP expression in lungs of mice treated with FAP-CAR-IL-15 iNKT + T cells or T cells alone. [Figure 10E] Mice treated with MiNK FAP-CAR-IL-15 showed reduced tumor burden in lung tissue after administration of T cells or FAP-CAR-IL-15 plus T cells. Immunohistochemical staining of α-SMA in lungs of mice treated with T cells or FAP-CAR-IL-15 + T cells harvested on day 25 (left panel). The right panel shows quantification of α-SMA expression in lungs of mice treated with FAP-CAR-IL-15 iNKT + T cells or T cells alone.

[0069] [Figure 11A] 1 shows that animals treated with FAP-CAR-IL-15 iNKT exhibited higher levels of immune cell activation and tumor tissue infiltration. 1 shows measurements of IFN-γ in the serum of mice treated with FAP-CAR-IL-15 iNKT+T cells or T cells alone. [Figure 11B] Figure 1 shows that animals treated with FAP-CAR-IL-15 iNKT exhibited higher levels of immune cell activation and tumor tissue infiltration. Quantification of the number of iNKT cells detected by flow cytometry in lung tissue from the FAP-CAR-IL-15 iNKT+T cell and T cell only groups is shown. [Figure 11C] Figure 1 shows that animals treated with FAP-CAR-IL-15 iNKT exhibited higher levels of immune cell activation and tumor tissue infiltration. Quantification of the number of T cells detected by flow cytometry in lung tissue from the FAP-CAR-IL-15 iNKT+T cell and T cell only groups is shown. [Figure 11D]1 shows that animals treated with FAP-CAR-IL-15 iNKT exhibited higher levels of immune cell activation and tumor tissue infiltration. Measured proliferation of iNKT cells in blood, lung, spleen, and bone marrow tissues of animals treated with FAP-CAR-IL-15 iNKT T cells is shown. [Figure 11E] 1 shows that animals treated with FAP-CAR-IL-15 iNKT exhibited higher levels of immune cell activation and tumor tissue infiltration.Measurements of T cell activation and proliferation in blood, lung, spleen, and bone marrow tissue from animals treated with FAP-CAR-IL-15 iNKT+T cells compared to T cells alone are shown.

[0070] [Figure 12A] The figure shows the construction of second-generation CARs and the cell-killing assay used to test them. A modular FAP-CAR iNKT library was generated, consisting of four distinct modules based on the second-generation CAR structure (scFv, TM / hinge region, first ICD region, and second ICD region). Each region had a potential domain count of 3 scFv, 3 TM / hinge region, 24 first ICD region, and 24 second ICD region, resulting in a library with a total of 5,184 diverse combinations. This library was packaged as a lentivirus, transduced into iNKT cells, and sorted 17 days after transduction to enrich for FAP-CAR+iNKT. [Figure 12B] The construction of second-generation CARs and the cell killing assay for testing them are shown. At day 21 post-transduction, iNKT cells were co-cultured with FAP-expressing antigen-presenting cells to specifically activate FAP-CAR iNKTs and enrich for well-activated / expanded ICDs. At day 35 post-transduction, an additional round of co-culture with FAP-expressing antigen-presenting cells was performed for an additional 14 days, resulting in an enrichment of over 15,000 after 4 weeks of co-culture. [Figure 12C]Figure 1 shows the construction of a second-generation CAR and the cell killing assay in which it was tested. Figure 2 shows the results of nanopore sequencing of DNA recovered from the initial plasmid library and DNA extracted from aliquots taken 21 days after iNKT transduction and 14 days after the first and second enrichment rounds. [Figure 12D] Figure 1 shows the construction of second-generation CARs and the cell-killing assay in which they were tested. Results show cell killing by iNKT cells with different constructions. iNKT cells were electroporated with mRNA encoding the FAP-CAR construct (or mock water), with the different domains of the CAR tested listed on the x-axis. Target cells (T2) were electroporated with high (2µg mRNA) or low (100µg mRNA) levels of mRNA encoding the FAP, or mock (water). Twenty-four hours after electroporation, target cells were labeled with CFSE and then mixed with iNKT cells at an effector:target ratio of 5:1 and cocultured for 24 hours. Killing was then assessed by flow cytometry by analyzing the percentage of CFSE+ cells that stained positive with a live / dead stain. [Figure 12E] Figure 1 shows the construction of second-generation CARs and the cell-killing assay in which they were tested. Results show cell killing by iNKT cells with different constructions. iNKT cells were electroporated with mRNA encoding the FAP-CAR construct (or mock water), with the different domains of the CAR tested listed on the x-axis. Target cells (T2) were electroporated with high (2µg mRNA) or low (100µg mRNA) levels of mRNA encoding the FAP, or mock (water). Twenty-four hours after electroporation, target cells were labeled with CFSE and then mixed with iNKT cells at an effector:target ratio of 5:1 and cocultured for 24 hours. Killing was then assessed by flow cytometry by analyzing the percentage of CFSE+ cells that stained positive with a live / dead stain. [Figure 12F]Figure 1 shows the construction of second-generation CARs and the cell-killing assay in which they were tested. Results show cell killing by iNKT cells with different constructions. iNKT cells were electroporated with mRNA encoding the FAP-CAR construct (or mock water), with the different domains of the CAR tested listed on the x-axis. Target cells (T2) were electroporated with high (2µg mRNA) or low (100µg mRNA) levels of mRNA encoding the FAP, or mock (water). Twenty-four hours after electroporation, target cells were labeled with CFSE and then mixed with iNKT cells at an effector:target ratio of 5:1 and cocultured for 24 hours. Killing was then assessed by flow cytometry by analyzing the percentage of CFSE+ cells that stained positive with a live / dead stain.

[0071] [Figure 13A] Figure 1 shows Biacore binding kinetics and epitope mapping of anti-FAP antibodies. Figure 2 shows SPR binding of E07, D01, B08, A07, and D05 to human and mouse FAP. [Figure 13B] Biacore binding kinetics and epitope mapping of anti-FAP antibodies. Results of epitope binning using the "Tandem using dual" method are shown. White - no competition. Yellow - competition. Experiments were performed on a Biacore 8K. [Figure 13C] Figure 1 shows Biacore binding kinetics and epitope mapping of anti-FAP antibodies. Figure 2 shows the crystal structure of FAP and its related homolog DPP4. [Figure 13D] Figure 1 shows Biacore binding kinetics and epitope mapping of anti-FAP antibodies. Human FAP (hFAP) and DPP4 (hDPP4) chimeric constructs used for epitope mapping are shown. [Figure 13E] Figure 1 shows Biacore binding kinetics and epitope mapping of anti-FAP antibodies, demonstrating that D01, E07, and B08 do not bind to human DPP4 but bind to the human FAP domain of amino acids 141-290. [Figure 13F]Figure 1 shows Biacore binding kinetics and epitope mapping of anti-FAP antibodies, demonstrating that D01, E07, and B08 do not bind to human DPP4 but bind to the human FAP domain of amino acids 141-290. [Figure 13G] Figure 1 shows Biacore binding kinetics and epitope mapping of anti-FAP antibodies, demonstrating that D01, E07, and B08 do not bind to human DPP4 but bind to the human FAP domain of amino acids 141-290.

[0072] [Figure 14] Shows the proliferation of FAP-CAR-IL15-iNKT cells.

[0073] [Figure 15] Killing of FAP-expressing cancer cells by iNKT cells expressing CARs containing D01 or B08 is shown. iNKT cells were transiently transfected with either D01 or B08CAR and cocultured with T2 cells transfected to express low or high levels of FAP at a 5:1 effector:target ratio. After 24 hours, target cell killing was assessed by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0074] The present disclosure is based, at least in part, on the discovery that genetically modified immune cells (e.g., iNKT cells) expressing chimeric antigen receptors that target fibroblast activation proteins (FAPs) can kill FAP-expressing cells (e.g., cancer-associated fibroblasts (CAFs)) in the tumor microenvironment (TME). In some embodiments, anti-FAP CAR iNKT cells are engineered to express arming molecules (e.g., soluble IL-15). In some embodiments, anti-FAP CAR iNKT cells reduce immune suppression in the TME, thereby increasing the effectiveness of other cancer therapies (e.g., T cells that target tumor antigens). In some embodiments, the anti-FAP CAR iNKT cells described by the present disclosure exhibit improved properties compared to existing FAP-CAR cell therapies in the art, including killing of FAP-expressing cancer cells in vitro and in vivo, and improved persistence in subjects receiving the therapy.

[0075] The foregoing and other aspects, aspects, acts, functions, features, and embodiments of the present teachings can be more fully understood from the following description taken in conjunction with the accompanying drawings.

[0076] I. Definition Additional terms of this disclosure are defined throughout the specification.

[0077] Administer: As used herein, the terms "administer" or "administration" mean providing a conjugate to a subject in a physiologically and / or pharmacologically useful manner (e.g., to treat a condition in the subject).

[0078] Affinity matured antibody: As used herein, the term "affinity matured antibody" refers to an antibody that has undergone one or more modifications to one or more CDRs, resulting in an improved affinity (i.e., KD, kd, or ka) of the antibody for a target antigen compared to a parent antibody lacking the modification(s). In some embodiments, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Various procedures for generating affinity matured antibodies are known in the art, including screening combinatorial antibody libraries prepared using biodisplay. For example, Marks et al., BioTechnology, 10: 779-783 (1992) describe affinity maturation by shuffling VH and VL domains. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91: 3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol., 154(7): 3310-3319 (1995); and Hawkins et al., J. Mol. Biol., 226: 889-896 (1992). Selective mutagenesis positions and selective mutations at contact positions with activity-enhancing amino acid residues or hypermutation positions are described in U.S. Patent No. 6,914,128 B1.

[0079] Antibody: As used herein, the term "antibody" or "antibodies" refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope, that specifically binds to an antigen. Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody comprises a framework having a germline sequence from the species (e.g., a human germline sequence). In some embodiments, the antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the VH comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the heavy chain variable domains provided herein. In some embodiments, the VL comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the light chain variable domains provided herein.In some embodiments, an antibody comprises a constant domain, e.g., an Fc region. An immunoglobulin constant domain refers to the constant domain of a heavy or light chain. The amino acid sequences of the heavy and light chain constant domains of human IgG and their functional variations are known. In another embodiment, an antibody comprises a heavy chain constant domain selected from the group consisting of the constant domains of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In certain embodiments, the antibody described herein comprises the CH1, CH2, and / or CH3 domains of human gamma 1. In some embodiments, V. HThe amino acid sequence of the domain includes the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences are described in the art, see, e.g., U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) (supra). In some embodiments, the antibody is modified, for example, via glycosylation, phosphorylation, sumoylation, and / or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolation (GPI anchor attachment), and / or phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, or phospholipid units.

[0080] Approximately: As used herein, as applied to one or more subject values, the term "approximately" or "about" refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is included within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise apparent from the context (except where such number may exceed 100% of the possible values).

[0081] Binding affinity: As used herein, the term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an epitope). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium binding constant (K A It can be measured and / or expressed in several ways known in the art, including but not limited to, K D is k off / k on is calculated from the quotient of A is k on / k off It is calculated from the quotient of k on refers to the association rate constant of an antibody to an epitope, and k off For example, refers to the dissociation rate constant of an antibody to an epitope.

[0082] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance (SPR), fluorescence-activated cell sorting (FACS), or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for assessing binding affinity are HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20) and PBS buffer (10 mM PO4-3, 137 mM NaCl, and 2.7 mM KCl). Using these techniques, the concentration of bound protein can be measured as a function of target protein concentration. The concentration of bound protein ([bound]) is generally related to the concentration of free target protein ([free]) by the following formula: [Bound]=[Free] / (Kd+[Free])

[0083] However, it is not necessarily K AIt is not necessary to precisely determine K because it may be sufficient to have a quantitative measure of affinity determined using methods such as ELISA or FACS analysis, which is K A and can therefore be used in comparisons such as determining whether a higher affinity is, for example, 2-fold higher, to obtain a qualitative measure of affinity, or to obtain an inference of affinity by, for example, activity in a functional assay, e.g., an in vitro or in vivo assay.

[0084] Binding moiety: As used herein, the term "binding moiety" refers to a molecule or portion of a molecule that specifically binds to or interacts with a target molecule through covalent and / or non-covalent interactions. Binding moieties of the present disclosure include, but are not limited to, an antibody or antigen-binding fragment thereof (e.g., antibody, scFv, Fab, Fab', single-chain binding fragment), binding peptide, ligand, receptor, oligonucleotide, small molecule, or aptamer.

[0085] CDR: As used herein, the term "CDR" refers to a complementarity-determining region within an antibody variable sequence. A typical antibody molecule generally comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are responsible for antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as "complementarity-determining regions" ("CDRs"), and more conserved regions known as "framework regions" ("FRs"). Each VH and VL typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely defined using methodologies known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art.Furthermore, Kabat, EA, et al. ( 1991 ) Sequences of Proteins of Immunological Interest , Fifth Edition , US Department of Health and Human Services , NIH Publication No . 91-3242;IMGT(Manufacturing), the International ImMunoGeneTics Information System(Manufacturing) imgt.org, Lefranc, M.-P. http: / / dx.doi.org / 10.1037 / 0021-843X.112.2.213 Ruiz, M. et al., Nucleic Acids Res., 27:209-212 (1999); Nucleic Acids Res., 31:307–310 (2003);Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], 5:45-60 (2005);Lefranc, M.-P. et al., Nucleic Acids Res., 33:D593-597 (2005);Lefranc, M.-P. et al., Nucleic Acids Res., 37:D1006-1012 (2009);Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015);Chothia et al., (1989) Nature 342:877;Chothia, C. et al. (1987) J. Mol. Biol. 196:901–917, Al-Lazikani et al (1997) J. Molec.Biol. 273:927-948;Almagro, J. Mol. Recognize. 17:132-143 (2004) available at bioinf.org.uk / abs.As used herein, CDR can refer to CDRs defined by any method known in the art. Two antibodies having the same CDR means that the two antibodies have the same amino acid sequence of the CDR as determined by the same method, for example, the Kabat definition.

[0086] Generally, there are three CDRs in each heavy and light chain variable region, which are referred to as CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system that can be applied to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Subportions of the CDRs may be designated as L1, L2, and L3, or H1, H2, and H3, with "L" and "H" representing the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, and have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with the Kabat CDRs are described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly adhere to any of the above systems and may nevertheless overlap with the Kabat CDRs, but may be shortened or extended to account for predictions or experimental findings that certain residues or groups of residues, or entire CDRs, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems.

[0087] The CDRs of an antibody may have different amino acid sequences when different definition systems (e.g., the IMGT definition, the Kabat definition, or the Chothia definition) are used. The definition systems annotate each amino acid in a given antibody sequence (e.g., a VH or VL sequence listed in Table 2) with a number, and the numbers corresponding to the heavy and light chain CDRs are shown in Table 1. The anti-FAP antibodies provided herein (e.g., the CDRs listed in Table 2) are defined according to the Kabat definition. One of skill in the art can derive CDR sequences using the different numbering systems of the anti-FAP antibodies presented in Table 2. [Table 1]

[0088] Chimeric Antigen Receptor: As used herein, "chimeric antigen receptor" or "CAR" refers to an engineered receptor that confers specificity for an antigen (e.g., FAP) or other ligand or molecule to immune effector cells (e.g., T cells, NK cells, NKT cells, iNKT cells). Chimeric antigen receptors typically contain at least one extracellular ligand-binding domain or portion capable of specifically binding to an antigen, and an intracellular domain containing one or more signaling and / or costimulatory domains.

[0089] In some embodiments, the extracellular ligand-binding domain of the CAR is in the form of a binding protein, a small molecule, a peptide, a targeting agent, an agonist, or an antagonist. In some embodiments, the binding protein is an antibody, an antigen-binding fragment of an antibody (e.g., scFv), a ligand, a cytokine, or a receptor. In some embodiments, the antigen-binding fragment is an scFv (e.g., an scFv that targets a FAP).

[0090] In certain embodiments, the extracellular ligand-binding domain is in the form of a single-chain variable fragment (scFv) derived from an antibody (e.g., a monoclonal antibody), which provides specificity for a particular epitope or antigen (e.g., an epitope or antigen that is preferentially present on the surface of cells, such as cancer cells or other disease-causing cells or particles).

[0091] In some embodiments, the CAR comprises an intracellular signaling domain. The intracellular signaling domain is a cytoplasmic domain that transmits an activation signal to the cell after binding of the extracellular domain. The intracellular signaling domain can be any intracellular signaling domain of interest known in the art. Such cytoplasmic signaling domains can include, but are not limited to, CD3 zeta. In some embodiments, the intracellular domain also includes one or more intracellular costimulatory domains, such as those described herein, that transmit costimulatory signals that promote cell proliferation, cell survival, and / or cytokine secretion after binding of the extracellular domain. Such intracellular costimulatory domains may include, but are not limited to, any costimulatory domain disclosed herein or known in the art, such as CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, CD127, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, N1, N6, or any combination thereof. Additional suitable costimulatory domains are described in PCT International Application No. PCT / US2017 / 055133, which is incorporated herein by reference in its entirety. In some embodiments, the costimulatory domain is 4-1BB (CD137). In some embodiments, the chimeric antigen receptor further comprises an additional structural element, such as a transmembrane domain attached to the extracellular ligand-binding domain via a hinge or junction sequence. The transmembrane domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a CD protein such as a subunit of the T cell receptor (i.e., the α, β, γ, or ζ polypeptides that make up the CD3 complex), the IL2 receptor p55 (a chain), p75 (β chain) or γ chain, a subunit chain of the Fc receptor (e.g., Fcy receptor III), or the CD8 alpha chain. Alternatively, the transmembrane domain can be synthetic and comprise primarily hydrophobic residues such as leucine and valine. In some embodiments, the CAR comprises a CD8 transmembrane domain.Hinge region refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, the hinge region can contain up to 300 amino acids, 10-100 amino acids, and 25-50 amino acids. The hinge region can be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or a completely synthetic hinge sequence. In some embodiments, the hinge domain can comprise a portion of the human CD8 alpha chain, FcyR11a receptor, or IgG1. In some embodiments, the CAR comprises a CD8 hinge region.

[0092] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from one species, but in which the sequences of one or more of the CDR regions of the VH and / or VL have been replaced with CDR sequences from another species; for example, in an antibody having murine heavy and light chain variable regions, one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0093] Chimeric antibody: The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody in which murine heavy and light chain variable regions are linked to human constant regions.

[0094] Complementary: As used herein, the term "complementary" refers to the ability of precise pairing between two nucleotides or a set of two nucleotides. In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in binding between two nucleotides or a set of two nucleotides. For example, if a base at one position of an oligonucleotide can hydrogen bond with a base at the corresponding position of a target nucleic acid (e.g., mRNA), the bases are considered to be complementary to each other at that position. Base pairing can include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), and cytosine-type bases (C) are complementary to guanosine-type bases (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U, or T.

[0095] Conservative amino acid substitution: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, methods described in references that summarize such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0096] Costimulatory domain: As used herein, "costimulatory domain" refers to a polypeptide domain that transmits an intracellular growth signal and / or cell survival signal upon activation. Activation of a costimulatory domain can occur following homodimerization of two costimulatory domain polypeptides. Activation can also occur, for example, following activation of a construct (e.g., a chimeric antigen receptor or an inducible regulatory construct) that includes a costimulatory domain. Generally, a costimulatory domain can be derived from a transmembrane costimulatory receptor, particularly the intracellular portion of a costimulatory receptor. Non-limiting examples of costimulatory polypeptides include, but are not limited to, 4-1BB, CD28, ICOS, OX-40, and CD27, as well as any other costimulatory domain further described herein. In some embodiments, the CAR described herein comprises a 4-1BB costimulatory domain.

[0097] Costimulatory signal: As used herein, a "costimulatory signal" refers to an intracellular signal induced by a costimulatory domain that promotes cell proliferation, expansion of a cell population, promotes cell survival, regulates cytokine secretion (e.g., upregulation or downregulation), and / or regulates the production and / or secretion of other immunomodulatory molecules in vitro and / or in vivo. In some embodiments, a costimulatory signal is induced following homodimerization of two costimulatory domain polypeptides. In some embodiments, a costimulatory signal is induced following activation of a construct (e.g., a chimeric antigen receptor or an inducible regulatory construct) comprising a costimulatory domain.

[0098] Cross-reactivity: As used herein, and in the context of targeting agents (e.g., antibodies), the term "cross-reactivity" refers to the property of an agent that can specifically bind to multiple antigens of a similar type or class (e.g., multiple homologous, paralogous, or orthologous antigens) with similar affinity or avidity. For example, in some embodiments, an antibody that is cross-reactive to a similar type or class of human antigen and non-human primate antigen (e.g., a human FAP and a non-human primate FAP) can bind to the human antigen and the non-human primate antigen with similar affinity or avidity. In some embodiments, the antibody is cross-reactive to a similar type or class of human antigen and rodent antigen. In some embodiments, the antibody is cross-reactive to a similar type or class of rodent antigen and non-human primate antigen. In some embodiments, the antibody is cross-reactive to a similar type or class of human antigen, non-human primate antigen, and rodent antigen.

[0099] Effective amount: As used herein, "effective amount" refers to the amount of each active agent (e.g., iNKT cells having a FAP CAR-binding moiety) required, alone or in combination with one or more other active agents, to provide a therapeutic effect to a subject. In some embodiments, the therapeutic effect includes, but is not limited to, a reduction in tumor size, eradication of the tumor, or alleviation of symptoms associated with the tumor.

[0100] Epitope: As used herein, "epitope" is a term used in the art and refers to a localized region of an antigen (e.g., a peptide or peptide-MHC complex) to which an antibody or chimeric antigen receptor can bind. In certain embodiments, the epitope to which an antibody or chimeric antigen receptor binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), flow cytometry analysis, mutagenesis mapping (e.g., site-directed mutagenesis mapping), and / or structural modeling. For X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303 (each of which is incorporated herein by reference in its entirety)). Antibody:antigen crystals can be examined using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.).(See, for example, Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al.; US 20040014194) and computer software such as BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed. Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), all of which are incorporated herein by reference in their entireties. Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning, see, e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085, both of which are incorporated herein by reference in their entireties. In some embodiments, the epitope of an antigen is determined using alanine scanning mutagenesis. In some embodiments, the epitope of an antigen is determined using hydrogen / deuterium exchange in combination with mass spectrometry.

[0101] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of a variable region excluding the CDRs. The precise definition of a CDR sequence can be determined by various systems, and the meaning of a framework sequence is subject to different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 in the light chain and CDR-H1, CDR-H2, and CDR-H3 in the heavy chain) divide the framework regions of the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, with CDR1 located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying a particular subregion as FR1, FR2, FR3, or FR4, references to framework regions by others refer to the combined FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that make up a framework region. Acceptor sequences for human heavy and light chains are known in the art. In one embodiment, acceptor sequences known in the art can be used in the antibodies disclosed herein.

[0102] Fibroblast Activation Protein (FAP): As used herein, the term "fibroblast activation protein" or "FAP" refers to a 97 kDa type II transmembrane serine protease. FAP is a member of the prolyl peptidase family, which also includes dipeptidyl peptidase IV (DPPIV, CD26), DPP7 (DPPII, resting cell proline dipeptidase), DPP8, DPP9, and prolyl carboxypeptidase (PCP, angiotensinase C). FAP contains dipeptidyl peptidase enzyme activity and endopeptidase activity. An exemplary human FAP amino acid sequence is set forth in Accession No. AAB49652.1 (SEQ ID NO: 35). Accession numbers AAB44837.1, AAE30605.1, AAX04090.1, ADL88098.1, AQN54508.1, ATK13500.1, ATK18094.1, AWT87270.1, AYI13559.1, QBE27403.1, QFN60450.1, QNB70086.1, and QYQ07220.1 also describe the same FAP amino acid sequence of SEQ ID NO: 35. Exemplary mouse FAP amino acid sequences are described in accession numbers CAA71116.1 (SEQ ID NO: 36), AND76664.1 (SEQ ID NO: 37), and AAH19190.1 (SEQ ID NO: 38).

[0103] Fibroblast activation protein-α (FAP) is a cell surface antigen expressed on various cells (e.g., reactive stromal fibroblasts in the tumor microenvironment, soft tissue sarcomas, granulation tissue in wound healing, and certain fetal mesenchymal fibroblasts). The expression level of FAP on certain cells (e.g., fibroblasts in the tumor microenvironment) is higher than that on normal tissues. FAP can also be shed from the cell membrane to form soluble FAP (Lee et al., Antiplasmin-cleaving enzyme is a soluble form of fibroblast activation protein. Blood (2006) 107:1397-404. 10.118). Therefore, it is not limited to the cell surface.

[0104] Human antibody: As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may contain amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0105] Humanized antibody: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but H and / or V L "Humanized" refers to an antibody in which at least a portion of the sequence has been modified to be more "human-like," i.e., to resemble human germline variable sequences more closely. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-FAP antibodies and antigen-binding fragments thereof are provided. Such antibodies can be produced by obtaining a murine anti-FAP monoclonal antibody using conventional hybridoma technology, followed by humanization using in vitro genetic engineering, such as that disclosed in PCT Publication No. WO 2005 / 123126 A2 to Ksaian et al. In some embodiments, humanized antibodies comprise a human immunoglobulin (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are present neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically a human immunoglobulin constant region or domain (Fc). The antibody may have a modified Fc region as described in WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (1, 2, 3, 4, 5, 6) that are altered relative to the original antibody, also referred to as one or more CDRs derived from one or more CDRs of the original antibody. Humanized antibodies may also be affinity matured.

[0106] Invariant Natural Killer T Cells (iNKT): As used herein, the terms "invariant natural killer T cells," or "invariant NKT cells," "iNKT cells," or "type I NKT cells" refer to a population of T lymphocytes expressing a conserved, semi-invariant TCR specific for lipid antigens (Ag) restricted to the monomorphic MHC class I-associated molecule CD1d. Natural killer T cells (NKT cells) were originally characterized in mice as T cells expressing both a TCR and the C-type lectin NK receptor, NK1.1 (NKR-P1a-c or CD161). Invariant NKT (iNKT) cells express a semi-invariant αβ TCR (e.g., formed by the invariant TRAV11-TRAJ18 (4) rearrangement in mice and by the homologous invariant TRAV10-TRAJ18 chains in humans) paired with a limited set of diverse Vβ chains (primarily TRBV1, TRBV29, or TRBV13 (6) in mice and TRBV25 in humans) (see, e.g., Dellabona et al., An invariant V alpha 24-J alpha Q / V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med. (1994) 180:1171-1084). Semi-invariant TCRs recognize exogenous and endogenous lipid Ags presented by the monomorphic MHC class I-associated molecule CD1d (see, e.g., Brennan et al., Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat Rev Immunol. (2013) 13:101-17. 10.1038).Exogenous lipid Ags include the typical α-galactosylceramide (α-GalCer) (Kawano et al., CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science. (1997) 278:1626-9. 10.1126), and many bacterial-derived antigens can activate iNKT cells.

[0107] iNKT cells undergo a distinct developmental pathway compared to T cells, acquiring innate effector functions already present in the thymus. Thymic iNKT cells express markers typically upregulated by peripheral effector / memory T cells, such as CD44 and CD69. They also express distinctive NK differentiation markers, such as NK1.1 (in some mouse genetic backgrounds and CD161 in humans), CD122 (IL-2R / IL-15R β chain), CD94 / NKG2, and Ly49 (AJ), as well as a broad range of TH1 / 2 / 17 effector cytokines. Upon migration to the periphery, iNKT cells form tissue-resident populations, monitor cellular integrity, rapidly respond to local injury and inflammation, and initiate innate and adaptive immune responses.

[0108] iNKT cells, capable of rapidly producing IFNγ, IL-4, or both, have been found to play a role in various diseases by establishing context-dependent Th1- or Th2-based immune responses. In bacterial and viral infections, iNKT cells typically contribute to early pathogen control by establishing productive Th1 responses. Both mouse and human studies have demonstrated the role of iNKT cells in diseases associated with excessive Th1 responses, such as type 1 diabetes and chronic obstructive pulmonary disease. iNKT cells have also been shown to suppress Th1 responses and promote tolerogenic responses to grafts. For example, after hematopoietic stem cell transplantation, the presence of iNKT cells predicts survival by reducing graft-versus-host disease (GvHD) in patients and preclinical models.

[0109] Isolated antibody: As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a FAP is substantially free of antibodies that specifically bind to antigens other than the FAP). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0110] Percent identity: The determination of "percent identity" or "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, as modified by Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with NBLAST nucleotide program parameters set, for example, to score=100 and word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with XBLAST program parameters set, for example, to score=50 and word length=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules. Ibid. When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used.

[0111] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. Typically, only exact matches are counted when calculating percent identity.

[0112] Recombinant antibody: As used herein, the term "recombinant human antibody" refers to any human antibody that is prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (as described in more detail in this disclosure), antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), or antibodies isolated from an animal (e.g., a mouse) into which human immunoglobulin genes have been introduced (e.g., Taylor, LD, et al. (1992) Nucl. The term "recombinant human antibodies" is intended to include antibodies prepared, expressed, created, or isolated by human immunoglobulin gene sequences (see, for example, "Human Immunology Res. 20:6287-6295; Kellermann SA, and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or by other methods involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thus modifying the V and constant regions of the recombinant antibody. H Area and V L The amino acid sequence of the region is similar to that of human germline V H Sequence and V LThese sequences are derived from and related to sequences that may not naturally exist within the human antibody germline repertoire in vivo. One embodiment of the present disclosure provides fully human antibodies capable of binding to human FAPs. The fully human antibodies can be generated using techniques well known in the art, such as using human Ig phage libraries, such as those disclosed in PCT Publication No. WO 2005 / 007699 A2 to Jermuth et al.

[0113] Single-chain variable fragment (scFv): As used herein, the term "single-chain variable fragment (scFv)" refers to a fusion protein of the variable regions of an immunoglobulin heavy chain (VH) and light chain (VL) connected by a short linker peptide. The linker refers to a peptide or short oligopeptide sequence used to connect the two subunits into a single polypeptide. The linker may have a sequence found in a naturally occurring protein, or it may be an artificial sequence not found in any naturally occurring protein. The linker may be flexible and lacking secondary structure, or it may have a tendency to form specific three-dimensional structures under physiological conditions. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker is serine- or threonine-rich. In some embodiments, an scFv is a fusion protein in which the N-terminus of the VH is linked to the C-terminus of the VL. In some embodiments, an scFv is a fusion protein in which the N-terminus of the VL is linked to the C-terminus of the VH. An scFv retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker. In some embodiments, scFvs can be generated to facilitate phage display. In some embodiments, scFvs can be generated directly from subcloned heavy and light chains derived from hybridomas. ScFvs have many uses, including in flow cytometry, immunohistochemistry, and as antigen-binding domains in chimeric antigen receptors.

[0114] Specific Binding: As used herein, the term "specifically binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that can be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context. With respect to an antibody, the term "specifically binding" refers to the ability of the antibody to bind to a particular antigen with a degree of affinity or avidity relative to an appropriate reference antigen(s), such that the antibody can be used to distinguish the particular antigen from other antigens, for example, to preferentially target particular cells, such as muscle cells, via binding to the antigen, as described herein. In some embodiments, the K D But at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 The antibody specifically binds to the target if M or less. In some embodiments, the antibody specifically binds to a FAP.

[0115] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, e.g., a human patient, having or suspected of having a disease. In some embodiments, the subject is a human patient suffering from or suspected of having cancer (e.g., a FAP-expressing cancer) or other disease associated with FAP.

[0116] Treatment: As used herein, the term "treat" or "treatment" refers to the application or administration of a composition comprising one or more active agents (e.g., anti-FAP antibodies) to a subject with a disease or disorder of interest (e.g., cancer), a symptom of the disease / disorder (e.g., tumor growth, tumor metastasis, fatigue, weight change including unintentional weight loss or gain, pain, fever, sores that do not heal, persistent cough or hoarseness, abnormal bleeding, or anemia), or a predisposition to the disease / disorder (e.g., cancer), with the intent to cure, ameliorate, alleviate, palliate, alter, treat, ameliorate, or affect the disorder (e.g., cancer), a symptom of the disease (e.g., tumor growth, tumor metastasis, fatigue, weight change including unintentional weight loss or gain, pain, fever, sores that do not heal, persistent cough or hoarseness, abnormal bleeding, or anemia), or a predisposition to the disease or disorder. Alleviating the disease / disorder of interest includes delaying or preventing the onset or progression of the disease or reducing the severity of the disease.

[0117] Tumor microenvironment (TME): As used herein, the term "tumor microenvironment" or "TME" refers to the fluids, molecules, cells, and / or tissues surrounding and / or at the tumor site. The TME includes normal cells, tumor cells, tumor stromal cells (e.g., stromal fibroblasts), blood vessels, blood, immune cells, and noncellular components (e.g., extracellular matrix components such as collagen, fibronectin, hyaluronic acid, laminin, and secreted molecules such as cytokines).

[0118] II. Methods for Treating Cancer Using Anti-FAP CAR iNKT Cells Aspects of the present disclosure provide a plurality of genetically modified immune cells (e.g., iNKT cells) expressing anti-fibroblast-activating protein (FAP) chimeric antigen receptors for the treatment of cancer. In some embodiments, iNKT cells can be engineered to express a chimeric antigen receptor comprising any of the FAP-binding moieties described herein (e.g., the anti-FAP antibodies or antigen-binding fragments described in Table 2 and existing FAP-binding moieties provided in Table 3) as an extracellular ligand-binding domain. In some embodiments, genetically modified iNKT cells expressing an anti-FAP CAR of the present disclosure can be engineered to further express soluble IL-15. In some embodiments, the present disclosure relates to methods of treating diseases (e.g., cancer) using genetically modified immune cells (e.g., anti-FAP iNKT cells) described herein. As used herein, "engineered to express" means that the cell expresses or is capable of expressing the molecule (e.g., an immunomodulatory gene product or CAR) that it has been engineered to express.

[0119] In some embodiments, FAPs are associated with cancer. In some embodiments, FAPs have been previously described to promote tumor growth through multiple mechanisms, including, but not limited to, proliferation, invasion, angiogenesis, epithelial-to-mesenchymal transition, stem cell promotion, immunosuppression, and / or drug resistance (e.g., Fitzgerald et al., The role of fibroblast activation protein in health and malignancy, Cancer Metastasis Rev. 2020 Sep;39(3):783-803). In some embodiments, FAPs are highly expressed in stromal cells (e.g., cancer-associated fibroblasts (CAFs)) in the tumor microenvironment (TME). In some embodiments, FAPs play an immunosuppressive role in the TME. FAP-expressing cancers include, but are not limited to, breast cancer, colon cancer, pancreatic cancer, gastric cancer, brain cancer, ovarian cancer, myeloma, melanoma, or sarcoma. In some embodiments, FAP is associated with a non-cancer disease, such as multiple human conditions including fibrosis, arthritis, atherosclerosis, autoimmune diseases, metabolic diseases, and cancer. In some embodiments, FAP is associated with increased disease progression and severity (e.g., increased cancer and / or non-cancer disease progression and severity).

[0120] Therapeutic agents targeting FAP have been previously described (e.g., Schuberth et al., Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific redirected T cells. J Transl Med (2013) 11:1-11), including FAP inhibitors (e.g., talabostat), anti-FAP antibodies (e.g., sibrotuzumab), FAP vaccines, and FAP CAR T cells (e.g., Bughda et al., Fibroblast Activation Protein (FAP)-Targeted CAR-T Cells: Launching an Attack on Tumor Stroma, ImmunoTargets and Therapy 2021:10 313-323; Wang et al., Targeting Fibroblast Activation Protein in Tumor Stroma with Chimeric Antigen Receptor T Cells Can Inhibit Tumor Growth and Augment Host Immunity without Severe Toxicity, Cancer Immunol Res;2(2) February 2014). However, clinical use of FAP CAR T cells has not been successful. In some cases, FAP CAR T cells failed to control tumor growth and induced fatal bone toxicity and cachexia (e.g., via lysis of multipotent bone marrow stromal cells) (Tran et al., Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia. J Exp Med (2013) 210:1125-35. 10.1084).

[0121] In some embodiments, the genetically modified immune cells (e.g., iNKT cells) described herein express a CAR that combines an antigen recognition domain (e.g., an anti-FAP antibody or antigen-binding fragment described herein), a transmembrane domain (e.g., a CD8 transmembrane domain), a cytoplasmic domain (e.g., the intracellular domain of CD3 zeta, CD28, OX40, 4-1BB, or any combination thereof), and a costimulatory domain (e.g., a 4-1BB costimulatory domain). Thus, in some embodiments, the transduced immune cells (e.g., iNKT cells) can elicit a direct immune response (e.g., a CAR-mediated immune response) and / or an indirect immune response (e.g., an NK cell response) in a subject. In some embodiments, the present disclosure provides for the use of a CAR to redirect the specificity of primary iNKT cells to a tumor antigen (e.g., a FAP). Thus, in some embodiments, the present disclosure also provides a method of stimulating an iNKT cell-mediated immune response against a target cell population or tissue in a subject (e.g., a mammal), the method comprising administering to the subject a plurality of iNKT cells expressing a CAR (e.g., an anti-FAP CAR), wherein the CAR comprises a binding moiety that specifically interacts with a predetermined target (e.g., a FAP), a transmembrane domain (e.g., a CD8 transmembrane domain), a zeta chain portion comprising the intracellular domain of, for example, human CD3 zeta, and a costimulatory signaling region. In some embodiments, the present disclosure includes a type of cell therapy in which iNKT cells are genetically modified (i.e., engineered) to express a CAR (e.g., an anti-FAP CAR) and IL-15 (e.g., soluble IL-15 (sIL-15)). "Engineered to express" means that the cell expresses or is capable of expressing the molecule (e.g., an immunomodulatory gene product or CAR) that it has been engineered to express. In some embodiments, the present disclosure includes a type of cell therapy in which anti-FAP CAR iNKT cells of the present disclosure are administered (e.g., injected, infused, etc.) to a subject in need thereof. The infused cells can kill tumor cells in the recipient. Unlike antibody therapies, CAR iNKT cells can replicate in vivo, resulting in long-term persistence that can result in sustained tumor control.In some embodiments, the secretion of sIL-15 by iNKT cells expressing anti-FAP CARs enhanced the persistence of iNKT cells in a subject. Furthermore, previous studies have shown that the number of iNKT cells is reduced in cancer tissues (e.g., solid tumors such as lung cancer, head and neck cancer, colorectal cancer, and renal cancer) compared to normal tissues. In some embodiments, the function of iNKT cells is impaired in certain cancers. Thus, administration of the anti-FAP iNKT cells described herein may restore and enhance iNKT function in a subject, thereby improving prognosis.

[0122] In some embodiments, the antigen-binding domain in a CAR of the present disclosure targets a tumor antigen (e.g., a FAP) for the purpose of treating cancer. In some embodiments, the antigen-binding portion of a CAR of the present disclosure is designed to treat a specific cancer (e.g., a cancer that expresses a FAP).

[0123] In some embodiments, the anti-FAP CAR iNKT cells of the present disclosure kill FAP-expressing stromal cells in the TME (e.g., CAFs). In some embodiments, the anti-FAP CAR iNKT cells of the present disclosure do not kill normal cells that express lower levels of FAP compared to stromal cells (e.g., CAFs) in cancer. In some embodiments, FAP expression in cancer cells, including but not limited to breast cancer, lung cancer, colon cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, urothelial cancer, or sarcoma, is high compared to FAP expression in normal tissues. FAP expression levels can be measured by any suitable method known in the art, such as flow cytometry, immunoblotting, RT-PCR, etc.

[0124] FAP expression is a key characteristic of CAFs (Garin-Chesa et al., Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proc Natl Acad Sci USA. 1990;87(18):7235-7239). FAP activity influences the secreted CAF proteome, reducing levels of antiangiogenic factors (e.g., PEDF, angiopoietin-1, VEGFC) and modulating matrix-processing enzymes (e.g., Koczorowska et al., Fibroblast activation protein-alpha, a stromal cell surface protease, shapes key features of cancer-associated fibroblasts through proteome and degradome alterations. Mol Oncol. 2016;10(1):40-58). Furthermore, FAPs remodel the ECM by cleaving collagen through their endopeptidase activity and modifying bioactive signaling peptides in cancer (Kelly et al., Fibroblast activation protein-alpha and dipeptidyl peptidase IV (CD26): cell-surface proteases that activate cell signaling and are potential targets for cancer therapy. Drug Resist Update. 2005;8(1-2):51-58). FAP expression in cancer is thought to be associated with poor prognosis.In some embodiments, high-level expression of FAP in the TME plays an immunosuppressive role (Kraman et al., Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-alpha. Science. 2010;330(6005):827-830). Without wishing to be bound by any particular theory, FAP induces CAFs to mediate tumor immunosuppression by recruiting myeloid-derived suppressor cells (MDSCs) in the TME (e.g., Yang et al., FAP promotes immunosuppression by cancer-associated fibroblasts in the tumor microenvironment via STAT3-CCL2 signaling. Cancer Res. 2016;76(14):4124-4135).

[0125] FAP-targeted CAR-T cells have been engineered to target CAFs in various solid tumors, such as mesothelioma, lung cancer, and pancreatic cancer (e.g., Bughda et al., Fibroblast Activation Protein (FAP)-Targeted CAR-T Cells: Launching an Attack on Tumor Stroma, ImmunoTargets and Therapy 2021:10 313-323; Wang et al., Targeting Fibroblast Activation Protein in Tumor Stroma with Chimeric Antigen Receptor T Cells Can Inhibit Tumor Growth and Augment Host Immunity without Severe Toxicity, Cancer Immunol Res;2(2) February 2014; Rodriguez-Garcia A, Palazon A, Noguera-Ortega E, Powell DJ, Guedan S. CAR-T cells hit the tumor microenvironment: strategies to overcome tumor escape. Frontier Immunol. 2020;11). However, because FAP is highly expressed in these cancers but at low levels in normal cells (e.g., skeletal muscle, adipose tissue, and pancreas), on-target off-tumor toxicity was observed in these studies.

[0126] In some embodiments, the present disclosure provides anti-FAP CAR iNKT cells engineered to kill high FAP-expressing stromal cells (e.g., CAFs in the TME). In some embodiments, the anti-FAP CAR iNKT cells described herein exhibit reduced or absent on-target extratumoral toxicity. In some embodiments, the anti-FAP CAR iNKT cells kill FAP-expressing tumor cells and / or FAP-expressing CAFs. In some embodiments, depletion of FAP-expressing stromal cells (e.g., CAFs in the TME) by the anti-FAP CAR iNKT cells described herein reduces immune suppression in the TME. In some embodiments, depletion of FAP-expressing stromal cells (e.g., CAFs in the TME) by the anti-FAP CAR iNKT cells described herein increases immune cell infiltration into tumors (e.g., solid tumors). In some embodiments, the anti-FAP CAR iNKT cells described herein are armed to prolong the persistence of the CAR iNKT cells in the TME (e.g., secrete IL-15). In some embodiments, the anti-FAP CAR iNKT cells described herein are armed (e.g., secrete IL-15) to enhance a particular property of the anti-FAP CAR iNKT cells (e.g., improved killing efficiency, extended persistence in the subject, and / or enhanced tumor killing with additional cancer therapy (e.g., CAR T cells targeting a tumor antigen)) compared to iNKT cells lacking the arming molecule.

[0127] The CAR-modified iNKT cells of the present disclosure can also function as a type of vaccine for ex vivo immunization and / or in vivo therapy in a mammal. Preferably, the mammal is a human.

[0128] In some embodiments, the iNKT cells described herein can be used to treat and prevent diseases (e.g., cancer) that occur in immunocompromised individuals, such as individuals suffering from cancer. In particular, iNKT cells expressing an anti-FAP CAR of the present disclosure are used to treat cancer, such as FAP-positive cancer (e.g., breast cancer, lung cancer, head and neck cancer, colon cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, kidney cancer, urothelial cancer, or sarcoma). In certain embodiments, iNKT cells expressing an anti-FAP CAR of the present disclosure are used to treat patients at risk of developing cancer, such as FAP-positive cancer (e.g., breast cancer, lung cancer, head and neck cancer, colon cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, kidney cancer, urothelial cancer, or sarcoma). In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer.

[0129] In certain embodiments, the subject does not undergo lymphodepletion prior to or concurrently with treatment with the anti-FAP CAR iNKT cells or pharmaceutical compositions disclosed herein. Lymphodepletion is frequently performed prior to immunotherapy, such as adoptive cell therapy, such as CAR T therapy. In some cases, subjects undergoing adoptive cell therapy undergo chemotherapy to deplete T cells from the subject for purposes such as tumor debulking, modifying the tumor phenotype, modifying the tumor microenvironment, removing cytokine sinks (e.g., increasing the availability of IL-2, IL-7, and IL-15), and suppressing the host immune system. In some cases, lymphodepletion can effectively extend the duration of infused cells and enhance the efficacy of treatment. However, lymphodepletion has several adverse effects, such as neutropenia, anemia, thrombocytopenia, and immunosuppression, as well as the toxicities associated with lymphodepleting agents such as fludarabine and cyclophosphamide. In some embodiments, a subject receiving iNKT cell therapy (e.g., anti-FAP CAR iNKT cell therapy) does not require lymphodepletion therapy. In certain embodiments, the subject does not receive treatment with fludarabine or cyclophosphamide prior to or concomitantly with administration of the anti-FAP CAR iNKT cells or pharmaceutical compositions disclosed herein. In some embodiments, iNKT cell therapy without lymphodepletion (e.g., anti-FAP CAR iNKT cell therapy or composition thereof) is effective in reducing tumor burden and / or improving survival. In some embodiments, iNKT cell therapy without lymphodepletion (e.g., anti-FAP CAR iNKT cell therapy or composition thereof) exhibits long-term efficacy in killing cancer cells. In some embodiments, iNKT cell therapy without lymphodepletion (e.g., anti-FAP CAR iNKT cell therapy or composition thereof) does not induce graft-versus-host disease (GVHD).

[0130] In some embodiments, iNKT cells expressing an anti-FAP CAR of the present disclosure, or compositions comprising such cells, can be used to provide anti-tumor immunity, treat or prevent cancer, reduce immunosuppression in the TME, and / or enhance the therapeutic effect of other therapeutic agents (e.g., CAR-T cells or immune checkpoint inhibitors), or can be administered to a subject in need thereof. In some embodiments, the cancer is a FAP-expressing cancer. In some embodiments, the cancer is breast cancer, lung cancer, head and neck cancer, colon cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, kidney cancer, urothelial cancer, or sarcoma.

[0131] In some embodiments, iNKT cells expressing an anti-FAP CAR of the present disclosure may be administered alone or as a composition (e.g., a pharmaceutical composition) in combination with other components, such as a diluent and / or IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present disclosure may include a target cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer, such as neutral buffered saline, phosphate buffered saline, a carbohydrate, such as glucose, mannose, sucrose, or dextran, mannitol, a protein, a polypeptide, or an amino acid, such as glycine, an antioxidant, a chelating agent, such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide), and a preservative.

[0132] The pharmaceutical compositions of the present disclosure can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration are determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage may be determined by clinical trials. In some embodiments, the compositions of the present disclosure are formulated for intravenous administration.

[0133] When an "immunologically effective amount," "antitumor effective amount," "tumor suppression effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present disclosure to be administered can be determined by a physician, taking into account individual differences such as age, weight, tumor size, extent of infection or metastasis, and the condition of the patient (subject). Pharmaceutical compositions comprising CAR-modified immune cells (e.g., iNKT cells expressing anti-FAP CARs) described herein can be administered in doses up to 10 days. 4 ~10 9 cells / kg body weight, 10 5 ~10 6 It can generally be said that iNKT cell compositions can be administered at a dose of 1000 cells / kg body weight (including all integer values ​​within these ranges). iNKT cell compositions can also be administered multiple times at these doses. Cells can be administered using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

[0134] Administration of the compositions of the present invention can be carried out by any convenient method, including infusion, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (i.v.), or intraperitoneally. In some embodiments, the immune cell (e.g., iNKT cell) compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the immune cell (e.g., iNKT cell) compositions of the present disclosure are preferably administered by i.v. injection. The immune cell (e.g., iNKT cell) compositions may also be injected directly into a tumor, lymph node, or disease site.

[0135] In some embodiments, iNKT cells (e.g., anti-FAP CAR iNKT cells) activated and expanded using the methods described herein or other methods known in the art are expanded to therapeutic levels and administered to a patient in combination with (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients, efalizumab treatment for psoriasis patients, or other treatments for PML patients. In some embodiments, the iNKT cells of the present disclosure can be used in combination with chemotherapy, radiation therapy, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation therapy. In some embodiments, the cell compositions of the present disclosure are administered to patients in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablative therapy using chemotherapy agents such as fludarabine, external radiotherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In some embodiments, the cell compositions of the present disclosure are administered after B cell ablative therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in some embodiments, the subject may receive standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, following transplantation, the subject receives an infusion of expanded immune cells (e.g., iNKT cells) of the present disclosure. In additional embodiments, the iNKT cells are administered before or after surgery.

[0136] In some embodiments, the present disclosure also contemplates treating cancer using anti-FAP CAR iNKT cells in combination with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is a T cell (e.g., a T cell or a CAR T cell) that targets a tumor antigen, including ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), vascular endothelial growth factor receptor 2 (VEGFR2), IL13R, GD3, C-type lectin-like molecule 1 (CLL1), cholecytokinin B receptor (CCKBR), gonadotropin-releasing hormone receptor (GnRHR), somatostatin receptor 2 (SSRT2), gastrin-releasing peptide receptor (GRP), or other T cells. receptor (GRPR), neurokinin 1 receptor (NK1R), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, folate receptor, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, B-human chorionic gonadotropin, alpha-fetoprotein (AFP), neurotensin receptor 1 (NTSR1), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal Ductal carboxylesterase, muthsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-1, IGF-II, IGF1 receptor, mesothelin, tumor-specific peptide epitopes, melanocortin 1 receptor (MCR) 1R), 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, major histocompatibility complex (MHC) molecules presenting the extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the Al domain of tenascin-C (TnCAl), lineage-specific or tissue-specific antigens such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin,These include, but are not limited to, virus-specific surface antigens such as major histocompatibility complex (MHC) molecules, FAPs (CD269, TNFRSF17), CS1, or HIV-specific antigens (such as HIV gp120); EBV-specific antigens, CMV-specific antigens, HPV-specific antigens such as E6 or E7 oncoproteins, Russ virus-specific antigens, influenza virus-specific antigens, and derivatives or variants of these surface markers. In some embodiments, treatment with the anti-FAP CAR iNKT cells described herein reduces immunosuppression in the TME and increases CAR T cell infiltration into tumors, thereby increasing CAR T cell efficacy. In some embodiments, the anti-FAP CAR iNKT cells secrete IL-15, which enhances the cytotoxic activity of CAR T cells.

[0137] In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor, such as a PD1 / PD-L1 inhibitor or a CTLA-4 inhibitor. Any suitable known immune checkpoint inhibitor can be combined with the anti-FAP CAR iNKT cells described herein.

[0138] The dosage for humans can be adjusted according to art-recognized practices. For example, the dosage of CAMPATH for adult patients typically ranges from 1 to about 100 mg, and is typically administered daily for 1 to 30 days. A preferred daily dose is 1 to 10 mg / day, although larger doses up to 40 mg / day may be used in some cases (as described in U.S. Patent No. 6,120,766). Strategies for dosing and scheduling of CAR T cells have been discussed (Ertl et al., 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal of Translational Medicine, 8:55).

[0139] III. FAP-binding moieties as chimeric antigen receptors The present disclosure provides FAP-binding moieties (e.g., antibodies or antigen-binding fragments specific for fibroblast activation protein (FAP), FAP-binding peptides) that can be engineered to become extracellular ligand-binding domains of chimeric antigen receptors (CARs) expressed by genetically modified cells (e.g., iNKT cells).

[0140] In some embodiments, the present disclosure provides an anti-FAP antibody or antigen-binding fragment thereof as the FAP-binding moiety. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof provided herein is an antibody that binds to a FAP with high specificity and affinity. In some embodiments, the antigen-binding fragment of the anti-FAP antibody described herein specifically binds to an extracellular epitope of the FAP or an epitope exposed by the antibody. In some embodiments, the antigen-binding fragment of the anti-FAP antibody provided herein specifically binds to a FAP from a human, non-human primate, mouse, rat, etc. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof provided herein specifically binds to a human FAP and a mouse FAP. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof described herein specifically binds to an epitope on a human FAP and / or a mouse FAP. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof described herein can bind to a fragment of a human FAP and / or a mouse FAP. In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof described herein can bind to FAP fragments of about 5 to about 200 amino acids in length, about 10 to about 200 amino acids in length, about 20 to about 200 amino acids in length, about 30 to about 150 amino acids in length, about 30 to about 120 amino acids in length, about 30 to about 100 amino acids in length, about 30 to about 90 amino acids in length, about 30 to about 80 amino acids in length, about 30 to about 60 amino acids in length, about 30 to about 50 amino acids in length, about 40 to about 80 amino acids in length, or about 40 to about 60 amino acids in length (e.g., human FAP and / or mouse FAP, e.g., SEQ ID NOS: 35 to 38). In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof can bind to FAP fragments comprising a consecutive number of amino acids from human FAP protein and / or mouse FAP protein (e.g., human FAP and / or mouse FAP, e.g., SEQ ID NOS: 35 to 38).In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof described herein may bind to a fragment comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, or at least 180 consecutive amino acids of a human FAP protein and / or a mouse FAP protein. In some embodiments, the anti-FAP antibodies described herein bind to one or more consecutive amino acids in the amino acid fragment 141-290 of a human FAP (e.g., the human FAP represented by SEQ ID NO: 35). In some embodiments, the anti-FAP antibodies described herein bind to one or more consecutive amino acids in the amino acid fragment 141-290 of a human FAP (e.g., the human FAP represented by SEQ ID NO: 35). In some embodiments, the anti-FAP antibodies described herein bind to one or more non-contiguous amino acids in the amino acid fragment 141-290 of human FAP (e.g., the human FAP set forth in SEQ ID NO: 35). In some embodiments, the anti-FAP antibodies described herein bind to one or more contiguous and non-contiguous amino acids in the amino acid fragment 141-290 of human FAP (e.g., the human FAP set forth in SEQ ID NO: 35). In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof described herein may bind to fragments having an amino acid sequence that differs from a fragment of a human FAP protein and / or a mouse FAP protein by up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid. An exemplary human FAP amino acid sequence is set forth in Accession No. AAB49652.1 (SEQ ID NO: 35). Accession numbers AAB44837.1, AAE30605.1, AAX04090.1, ADL88098.1, AQN54508.1, ATK13500.1, ATK18094.1, AWT87270.1, AYI13559.1, QBE27403.1, QFN60450.1, QNB70086.1, and QYQ07220.1 also describe the same FAP amino acid sequence of SEQ ID NO:35.

[0141] An exemplary human FAP amino acid sequence is set forth in SEQ ID NO: 35 (amino acid fragment 141-290 in bold and underlined): [ka]

[0142] Exemplary mouse FAP amino acid sequences are set forth in Accession Nos. CAA71116.1 (SEQ ID NO: 36), AND76664.1 (SEQ ID NO: 37), and AAH19190.1 (SEQ ID NO: 38).

[0143] An exemplary mouse FAP amino acid sequence is set forth in SEQ ID NO: 36: MKTWLKTVFGVTTLALALVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGREN RIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYI KDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIA LVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD

[0144] An exemplary mouse FAP amino acid sequence is set forth in SEQ ID NO:37: LRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATK YALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSG KWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGT AFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD

[0145] An exemplary mouse FAP amino acid sequence is set forth in SEQ ID NO:38: MKTWLKTVFGVTTLALALVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGREN RIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYI KDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIA LVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGILSGRSQNHLYTHMTHFLKQCFSLSD

[0146] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof has a cytotoxicity of at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10-13 M or lower binding affinity (e.g., K D For example, the anti-FAP antibody or antigen-binding fragment thereof of the present disclosure specifically binds to a FAP protein (e.g., a human and / or mouse FAP set forth in any one of SEQ ID NOs: 35 to 38) at 5 pM to 500 nM, 10 pM to 450 nM, 20 pM to 400 nM, 30 pM to 350 nM, 40 pM to 300 nM, 50 pM to 250 nM, 60 pM to 200 nM, 70 pM to 150 nM, 80 pM to 100 nM, 80 pM to 90 nM, , 90pM to 80nM, 100pM to 70nM, 200pM to 60nM, 300pM to 50nM, 400pM to 40nM, 500pM to 30nM, 600pM to 20nM, 700pM to 10nM, 800pM to 5nM, or 900pM to 2nM, 30nM to 700nM, 50nM to 500nM, 100nM to 500nM, 100nM to 200nM, or 150nM to 200nM.

[0147] In some embodiments, the present disclosure provides a method for the treatment of rhodium in the range of 0.1 nM to 100 nM (e.g., 0.1 nM to 80 nM, 0.1 nM to 50 nM, 0.1 nM to 25 nM, 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 1 nM, 0.1 nM to 0.5 nM, 0.2 nM to 0.4 nM, 0.3 nM to 0.4 nM, 10 nM to 80 nM, 10 nM to 70 nM, 10 nM to 60 nM, 10 nM to 50 nM, 10 nM to 40 nM, 10 nM to 30 nM, 10 nM to 20 nM, 20 nM to 80 nM, 20 nM to 70 nM, 20 nM to 60 nM, 20 nM to 60 nM, 20 nM to 80 nM, 20 nM to 70 nM, 20 nM to 60 nM, 20 nM to 8 ...60 nM, 20 nM to 80 nM, 20 nM to 60 nM, 20 nM to 60 nM, 20 nM to 80 nM, 20 nM FAPs, including anti-FAP antibodies having a binding affinity for a FAP (e.g., a human FAP or a mouse FAP) of 100 nM to 50 nM, 20 nM to 40 nM, 20 nM to 30 nM, 10 nM to 15 nM, 30 nM to 80 nM, 30 nM to 70 nM, 30 nM to 60 nM, 30 nM to 50 nM, 30 nM to 40 nM, 40 nM to 80 nM, 40 nM to 70 nM, 40 nM to 60 nM, 40 nM to 50 nM, 50 nM to 80 nM, 50 nM to 70 nM, 50 nM to 60 nM, 60 nM to 80 nM, 60 nM to 70 nM, or 70 nM to 80 nM. They discovered that iNKT cells expressing a CAR are better at killing tumor cells expressing a FAP (e.g., a human or mouse FAP) compared to iNKT cells expressing a FAP CAR that contains an anti-FAP antibody with higher binding affinity.

[0148] The present disclosure also includes antibodies that compete with any of the antibodies described herein for binding to a FAP protein (e.g., a human and / or mouse FAP protein set forth in any one of SEQ ID NOS: 35-38) and have an affinity of 100 nM or less (e.g., 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of an anti-FAP antibody or antigen-binding fragment thereof can be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE). In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof has a K in the subnanomolar range. D It binds to FAP.

[0149] Non-limiting examples of anti-FAP antibodies are shown in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0150] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof of the present disclosure comprises one or more of the heavy chain CDR (e.g., CDRH1, CDRH2, or CDRH3) amino acid sequences from any one of the anti-FAP antibodies selected from Table 2. In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof of the present disclosure comprises CDRH1, CDRH2, and CDRH3 provided for any one of the antibodies selected from Table 2. In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof of the present disclosure comprises one or more of the light chain CDR (e.g., CDRL1, CDRL2, or CDRL3) amino acid sequences from any one of the anti-FAP antibodies selected from Table 2. In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof of the present disclosure comprises CDRL1, CDRL2, and CDRL3 provided for any one of the anti-FAP antibodies selected from Table 2.

[0151] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 as provided for any one of the anti-FAP antibodies selected from Table 2. In some embodiments, the CDR3 domains of the heavy and light chains of an antibody may play a particularly important role in the binding specificity / affinity of the antibody to an antigen. Thus, the anti-FAP antibody or antigen-binding fragment thereof may comprise at least the heavy and / or light chain CDR3 of any one of the anti-FAP antibodies selected from Table 2.

[0152] Functional variants of any of the exemplary anti-FAP antibodies or antigen-binding fragments thereof disclosed herein are also within the scope of the present disclosure. Functional variants have a V H and / or V L The reference antibody may contain mutations of one or more amino acid residues in one or more heavy chain CDRs and / or one or more light chain CDRs, but retain substantially similar binding activity and biological activity (e.g., substantially similar binding affinity, binding specificity, inhibitory activity, anti-inflammatory activity, or a combination thereof) as the reference antibody.

[0153] In some embodiments, any of the anti-FAP antibodies or antigen-binding fragments thereof of the present disclosure have one or more CDR (e.g., heavy chain CDR or light chain CDR) sequences substantially similar to any of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 sequences from one of the anti-FAP antibodies selected from Table 2. In some embodiments, the position of one or more CDRs along the VH (e.g., CDRH1, CDRH2, or CDRH3) and / or VL (e.g., CDRL1, CDRL2, or CDRL3) regions of an antibody described herein may be altered by 1, 2, 3, 4, 5, or 6 amino acid positions, as long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived is substantially maintained). For example, in some embodiments, the positions defining the CDRs of any antibody described herein can be altered by shifting the N-terminal and / or C-terminal boundaries of the CDRs by 1, 2, 3, 4, 5, or 6 amino acids relative to the CDR positions of any antibody described herein, so long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived is substantially maintained). In another embodiment, the length of one or more CDRs along the VH (e.g., CDRH1, CDRH2, or CDRH3) and / or VL (e.g., CDRL1, CDRL2, or CDRL3) regions of an antibody described herein can be altered (e.g., shortened or lengthened) by one, two, three, four, five, or more amino acids, as long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., substantially at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived is maintained).

[0154] Thus, in some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or antigen-binding fragment thereof may be 1, 2, 3, 4, 5, or more amino acids shorter than one or more of the CDRs described herein (e.g., a CDR from any anti-FAP antibody selected from Table 2), so long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained compared to the binding of the original antibody from which it is derived). In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or antigen-binding fragment thereof may be 1, 2, 3, 4, 5, or more amino acids longer than one or more of the CDRs described herein (e.g., a CDR from any anti-FAP antibody selected from Table 2), so long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained compared to the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or antigen-binding fragment thereof may be extended by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any anti-FAP antibody selected from Table 2), so long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained compared to the binding of the original antibody from which it was derived).In some embodiments, the carboxy portion of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or antigen-binding fragment thereof may be extended by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any anti-FAP antibody selected from Table 2), so long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained compared to the binding of the original antibody from which it was derived). In some embodiments, the amino portion of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or antigen-binding fragment thereof may be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any anti-FAP antibody selected from Table 2), as long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained compared to the binding of the original antibody from which it is derived). In some embodiments, the carboxy portion of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or antigen-binding fragment thereof may be shortened by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any anti-FAP antibody selected from Table 2), so long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained compared to the binding of the original antibody from which it was derived). Any method can be used to determine whether immunospecific binding to a FAP (e.g., a human FAP), including, for example, using binding assays and conditions described in the art.

[0155] In some examples, an anti-FAP antibody or antigen-binding fragment thereof comprises one or more CDR (e.g., heavy chain CDR or light chain CDR) sequences substantially similar to any of the anti-FAP antibodies selected from Table 2. For example, the antibody may comprise one or more CDR sequence(s) from any of the anti-FAP antibodies selected from Table 2, comprising up to 5, 4, 3, 2, or 1 amino acid residue mutations compared to the corresponding CDR region of any of the CDRs provided herein (e.g., a CDR from any of the anti-FAP antibodies selected from Table 2), so long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., substantially maintained, i.e., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained compared to the binding of the antibody from which it was derived). In some embodiments, any of the amino acid mutations in any of the CDRs provided herein may be conservative mutations. Conservative mutations can be introduced into CDRs at positions where the residues are unlikely to be involved in interactions with the FAP protein (e.g., human FAP), as determined, for example, based on a crystal structure.

[0156] Some aspects of the present disclosure provide anti-FAP antibodies comprising one or more of the heavy chain variable (VH) and / or light chain variable (VL) domains provided herein. In some embodiments, any of the VH domains provided herein comprise one or more of the heavy chain CDR sequences (e.g., CDRH1, CDRH2, and CDRH3) provided herein, e.g., any of the heavy chain CDR sequences provided in any one of the anti-FAP antibodies selected from Table 2. In some embodiments, any of the VL domains provided herein comprise one or more of the CDR-L sequences (e.g., CDRL1, CDRL2, and CDRL3) provided herein, e.g., any of the light chain CDR sequences provided in any one of the anti-FAP antibodies selected from Table 2.

[0157] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof includes any antibody comprising the heavy chain variable domain and / or the light chain variable domain of any one of the anti-FAP antibodies selected from Table 2, and variants thereof. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof includes any antibody comprising a pair of heavy chain variable region and light chain variable region of any anti-FAP antibody selected from Table 2.

[0158] Aspects of the present disclosure provide anti-FAP antibodies or antigen-binding fragments thereof comprising a heavy chain variable (VH) domain amino acid sequence and / or a light chain variable (VL) domain amino acid sequence that is homologous to any of the amino acid sequences described herein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH or VL that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH and / or any VL of any one of the anti-FAP antibodies selected from Table 2. In some embodiments, the homologous VH and / or VL amino acid sequences do not vary within any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) can occur within the VH and / or VL sequences excluding any of the CDR sequences provided herein. In some embodiments, any of the anti-FAP antibodies or antigen-binding fragments thereof provided herein comprise VH and VL sequences that comprise framework sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequences of any anti-FAP antibody selected from Table 2. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH of an anti-FAP antibody listed in Table 2. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL of any of the anti-FAP antibodies listed in Table 2.

[0159] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof is a humanized antibody (e.g., a humanized variant comprising one or more CDRs in Table 2). In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 that are the same as those shown in Table 2, and comprises a humanized VH and / or a humanized VL. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof is a humanized variant comprising one or more amino acid substitutions (e.g., in the VH framework regions) compared to any of the VHs listed in Table 2, and / or a humanized variant comprising one or more amino acid substitutions (e.g., in the VL framework regions) compared to any of the VLs listed in Table 2.

[0160] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 8.

[0161] In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 3. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having the amino acid sequence of SEQ ID NO: 2, and a CDRH3 having the amino acid sequence of SEQ ID NO: 3. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having the amino acid sequence of SEQ ID NO: 2, a CDRH3 having the amino acid sequence of SEQ ID NO: 3, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 6.

[0162] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which comprise a total of five or fewer amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 1, CDRH2 having the amino acid sequence of SEQ ID NO: 2, and CDRH3 having the amino acid sequence of SEQ ID NO: 3. As used anywhere in this disclosure, "collectively" means that the total number of amino acid mutations in all three heavy chain CDRs is within a defined range. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which comprise a total of five or fewer amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 6.

[0163] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1, a CDRH2, and a CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a CDRH1 having the amino acid sequence of SEQ ID NO:1, a CDRH2 having the amino acid sequence of SEQ ID NO:2, and a CDRH3 having the amino acid sequence of SEQ ID NO:3. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO:4, CDRL2 having the amino acid sequence of SEQ ID NO:5, and CDRL3 having the amino acid sequence of SEQ ID NO:6.

[0164] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 2, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 3. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 6.

[0165] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8.

[0166] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO:8.

[0167] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO:8.

[0168] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO:8. In some embodiments, a number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) may occur within the VH of SEQ ID NO: 7 and / or the VL of SEQ ID NO: 8, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO:7, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO:8.

[0169] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO:8. In some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 7 and / or the VL of SEQ ID NO: 8, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO:7, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO:8.

[0170] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 15.

[0171] In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 12. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and a CDRH3 having the amino acid sequence of SEQ ID NO: 12. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 12, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 13.

[0172] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 12. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 13.

[0173] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 12. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 13.

[0174] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 12. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 13.

[0175] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 14 and a VL comprising the amino acid sequence of SEQ ID NO: 15.

[0176] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 15.

[0177] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 15.

[0178] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 15. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 14 and / or the VL of SEQ ID NO: 15, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO: 14, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO: 15.

[0179] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 15. In some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 14 and / or the VL of SEQ ID NO: 15, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO: 14, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO: 15.

[0180] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 44. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 45.

[0181] In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 40, a CDRH2 having the amino acid sequence of SEQ ID NO: 41, and a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 40, a CDRH2 having the amino acid sequence of SEQ ID NO: 41, a CDRH3 having the amino acid sequence of SEQ ID NO: 42, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0182] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 40, CDRH2 having the amino acid sequence of SEQ ID NO: 41, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0183] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO: 40, CDRH2 having the amino acid sequence of SEQ ID NO: 41, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0184] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 40, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 41, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0185] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 44. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 45.

[0186] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 45.

[0187] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 45.

[0188] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 45. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 44 and / or the VL of SEQ ID NO: 45, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO: 44, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO: 45.

[0189] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 45. In some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 44 and / or the VL of SEQ ID NO: 45, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO: 44, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO: 45.

[0190] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 48.

[0191] In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 42, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0192] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0193] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0194] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0195] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 47 and a VL comprising the amino acid sequence of SEQ ID NO: 48.

[0196] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 48.

[0197] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 48.

[0198] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 48. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 47 and / or the VL of SEQ ID NO: 48, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO:47, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO:48.

[0199] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 48. In some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 47 and / or the VL of SEQ ID NO: 48, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO:47, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO:48.

[0200] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 52.

[0201] In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 50, and a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 50, a CDRH3 having the amino acid sequence of SEQ ID NO: 42, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0202] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0203] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 50, and a CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0204] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 50, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0205] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 51 and a VL comprising the amino acid sequence of SEQ ID NO: 52.

[0206] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 52.

[0207] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 52.

[0208] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 52. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 51 and / or the VL of SEQ ID NO: 52, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO: 51, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO: 52.

[0209] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 52. In some embodiments, the degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 51 and / or the VL of SEQ ID NO: 52, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO: 51, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO: 52.

[0210] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 56. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 57.

[0211] In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 54. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and a CDRH3 having the amino acid sequence of SEQ ID NO: 54. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 54, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 55.

[0212] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 54. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 55.

[0213] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 54. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 55.

[0214] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 54. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 55.

[0215] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 51 and a VL comprising the amino acid sequence of SEQ ID NO: 52.

[0216] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 52.

[0217] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 52.

[0218] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 52. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 51 and / or the VL of SEQ ID NO: 52, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO: 51, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO: 52.

[0219] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 52. In some embodiments, the degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO:51 and / or within the VL of SEQ ID NO:51, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO: 51, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO: 52.

[0220] The anti-FAP antibodies or antigen-binding fragments thereof described herein can be in any antibody format, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (Fab, F(ab'), F(ab')2, Fv, etc.), single-chain antibodies (e.g., scFv), bispecific antibodies, or nanobodies. In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof described herein are single-chain variable fragments (scFv). In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof described herein are scFv-Fabs (e.g., scFvs fused to a portion of the constant region).

[0221] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises the VL domain and / or VH domain of any one of the anti-FAP antibodies selected from Table 2, and comprises a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or of any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules. Non-limiting examples of human constant regions are described in the art; see, e.g., Kabat EA et al., (1991) (supra). Other antibody heavy and light chain constant regions are known in the art, such as those provided in the IMGT database (imgt.org) or vbased2.org / vbstat.phph, both of which are incorporated herein by reference.

[0222] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). In some embodiments, the anti-FAP scFv comprises the VH and VL of any one of the anti-FAP antibodies selected from Table 2. In some embodiments, the VH and VL of the anti-FAP scFv are joined together by a linker. In some embodiments, the linker can have a length of about 2-10 amino acids, 5-20 amino acids, 10-30 amino acids, 20-50 amino acids, 40-60 amino acids, 60-80 amino acids, or more than 80 amino acids. In some embodiments, the linker can comprise a sequence substantially comprising glycine and serine. An exemplary linker sequence is GGGGSGGGGSGGGAS (SEQ ID NO: 29). In some embodiments, the linker can include, but is not limited to, any of those encompassed by U.S. Patent Nos. 8,445,251 and 9,434,931. In some embodiments, the anti-FAP comprises a linker between the VH and VL, wherein the linker comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:29.

[0223] In some embodiments, the anti-FAP scFv comprises a VH and a VL, wherein the C-terminus of the VH is joined to the N-terminus of the VL via a linker (e.g., the linker set forth in SEQ ID NO: 29). In some embodiments, the anti-FAP scFv comprises a VH and a VL, wherein the C-terminus of the VL is joined to the N-terminus of the VH via a linker (e.g., the linker set forth in SEQ ID NO: 29).

[0224] In some embodiments, the anti-FAP scFv comprises a VH that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH of any anti-FAP antibody listed in Table 2. Alternatively or additionally, the anti-FAP scFv comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH of any anti-FAP antibody listed in Table 2. In some embodiments, an anti-FAP scFv comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of any anti-FAP antibody listed in Table 2. Alternatively or additionally, an anti-FAP scFv comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of any anti-FAP antibody listed in Table 2.

[0225] In some embodiments, the anti-FAP scFv comprises an amino acid sequence that includes 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to any of the anti-FAP scFvs listed in Table 2. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any of the anti-FAP scFvs listed in Table 2.

[0226] In some embodiments, the anti-FAP scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-FAP scFv comprises a VH that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 7, and / or a VL that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 8. In some embodiments, the anti-FAP scFv comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO:7, and / or a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO:8.

[0227] In some embodiments, the anti-FAP scFv comprises an amino acid sequence that contains 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO:9.

[0228] In some embodiments, the anti-FAP scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 14 and / or a VL comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-FAP scFv comprises a VH that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 14, and / or a VL that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 15. In some embodiments, the anti-FAP scFv comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 14, and / or a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 15.

[0229] In some embodiments, the anti-FAP scFv comprises an amino acid sequence that contains 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO: 16.

[0230] In some embodiments, the anti-FAP scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 44 and / or a VL comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-FAP scFv comprises a VH that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 44, and / or a VL that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 45. In some embodiments, the anti-FAP scFv comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO:44, and / or a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO:55.

[0231] In some embodiments, the anti-FAP scFv comprises an amino acid sequence that contains 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO:46.

[0232] In some embodiments, the anti-FAP scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 47 and / or a VL comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the anti-FAP scFv comprises a VH that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 47, and / or a VL that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 48. In some embodiments, the anti-FAP scFv comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO:47, and / or a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO:48.

[0233] In some embodiments, the anti-FAP scFv comprises an amino acid sequence that contains 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO: 49.

[0234] In some embodiments, the anti-FAP scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 51 and / or a VL comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-FAP scFv comprises a VH that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 51, and / or a VL that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 52. In some embodiments, the anti-FAP scFv comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO:51, and / or a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO:52.

[0235] In some embodiments, the anti-FAP scFv comprises an amino acid sequence that contains 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO: 53.

[0236] In some embodiments, the anti-FAP scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 56 and / or a VL comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the anti-FAP scFv comprises a VH that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 56, and / or a VL that comprises no more than 20 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 57. In some embodiments, the anti-FAP scFv comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO:56, and / or a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO:57.

[0237] In some embodiments, the anti-FAP scFv comprises an amino acid sequence that contains 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO:58.

[0238] In some embodiments, any of the anti-FAP antibodies or antigen-binding fragments described herein are modified, for example, via glycosylation, phosphorylation, sumoylation, and / or methylation. In some embodiments, the anti-FAP antibody or antigen-binding fragment is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolation (GPI anchor attachment), and / or phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules comprise a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules are present. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or intracellularly, optionally deficient in an enzyme in the N- or O-glycosylation pathway, e.g., a glycosyltransferase. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof," published May 1, 2014.

[0239] In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where the residue is unlikely to be involved in interactions with the target antigen (e.g., a FAP), as determined, for example, based on a crystal structure.

[0240] In some embodiments, any one of the anti-FAP antibodies or antigen-binding fragments described herein can include a signal peptide (e.g., an N-terminal signal peptide) in the heavy and / or light chain sequence. In some embodiments, the anti-FAP antibodies or antigen-binding fragments described herein include any one of the VH and VL sequences, any one of the IgG heavy and light chain sequences, or any one of the scFv sequences described herein, and further include a signal peptide (e.g., an N-terminal signal peptide).

[0241] In some embodiments, the present disclosure also contemplates engineering any of the FAP-binding moieties known in the art to be the extracellular ligand-binding domain of a CAR expressed by a genetically modified immune cell (e.g., an iNKT cell) described herein. Non-limiting examples of FAP-binding moieties are shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3]

[0242] Aspects of the present disclosure also provide chimeric antigen receptors (CARs) comprising an extracellular ligand-binding domain. In some embodiments, the selection of the ligand-binding domain depends on the type and number of ligands defining the surface of the target cell. For example, the ligand-binding domain can be selected to recognize one or more ligands that act as cell surface markers on target cells associated with a particular pathology. Thus, examples of cell surface markers that can act as ligands for the ligand-binding domain in a CAR of the present disclosure can include markers associated with viral, bacterial, or parasitic infections, autoimmune diseases, and, more preferably, cancer cells. In some embodiments, a CAR of the present disclosure is engineered to target one or more tumor antigens of interest by engineering a desired ligand-binding moiety that specifically binds to one or more antigens on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to or characteristic of a particular hyperproliferative disease, such as cancer. Generally, a CAR of the present disclosure (e.g., an anti-FAP CAR) comprises at least an extracellular domain and an intracellular domain. In some embodiments, the extracellular domain comprises a target-specific binding element (e.g., an scFv that specifically binds to a FAP (e.g., a FAP protein represented by SEQ ID NOs: 35-38)), otherwise referred to herein as a ligand-binding domain (also referred to herein as an antigen-binding domain). In some embodiments, the extracellular domain is an antigen-binding domain or a portion thereof. In some embodiments, the extracellular ligand-binding domain is a Fab. In some embodiments, the extracellular ligand-binding domain is an scFv. In some embodiments, the extracellular ligand-binding domain of a CAR described herein comprises an antigen-binding fragment that specifically binds to a FAP (e.g., a human FAP). In some embodiments, the extracellular ligand-binding domain of a CAR described herein comprises any one of a FAP antibody or antigen-binding fragment thereof (e.g., an anti-FAP scFv).

[0243] In some embodiments, the anti-FAP CAR of the present disclosure has a cytotoxicity of at least about 10 -4 M, 10 -5 M, 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or lower binding affinity (e.g., K D For example, the anti-FAP CAR of the present disclosure may bind to a FAP protein (e.g., a human FAP protein and / or a mouse FAP protein set forth in SEQ ID NOs: 35-38) at 5 pM to 500 nM, 10 pM to 450 nM, 20 pM to 400 nM, 30 pM to 350 nM, 40 pM to 300 nM, 50 pM to 250 nM, 60 pM to 200 nM, 70 pM to 150 nM, or , 80pM to 100nM, 80pM to 90nM, 90pM to 80nM, 100pM to 70nM, 200pM to 60nM, 300pM to 50nM, 400pM to 40nM, 500pM to 30nM, 600pM to 20nM, 700pM to 10nM, 800pM to 5nM, or 900pM to 2nM.

[0244] The present disclosure also includes CARs that compete with any of the CARs described herein for binding to a FAP protein (e.g., a human FAP protein and / or a mouse FAP protein set forth in SEQ ID NOs: 35-38) and have an affinity of 100 nM or less (e.g., 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of anti-FAP CARs can be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE). In some embodiments, the anti-FAP CARs described herein have a K in the subnanomolar range. D It binds to FAP.

[0245] In some embodiments, an anti-FAP CAR of the disclosure comprises one or more of the heavy chain CDR (e.g., CDRH1, CDRH2, or CDRH3) amino acid sequences from any one of the anti-FAP antibodies selected from Table 2. In some embodiments, an anti-FAP CAR of the disclosure comprises a CDRH1, CDRH2, and CDRH3 provided for any one of the antibodies selected from Table 2. In some embodiments, an anti-FAP CAR of the disclosure comprises one or more of the light chain CDR (e.g., CDRL1, CDRL2, or CDRL3) amino acid sequences from any one of the anti-FAP antibodies selected from Table 2. In some embodiments, an anti-FAP CAR of the disclosure comprises a CDRL1, CDRL2, and CDRL3 provided for any one of the anti-FAP antibodies selected from Table 2.

[0246] In some embodiments, an anti-FAP CAR comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 provided for any one of the anti-FAP antibodies selected from Table 2. In some embodiments, the CDR3 domains of the heavy and light chains of an antibody may play a particularly important role in the binding specificity / affinity of the antibody to an antigen. Thus, an anti-FAP CAR may comprise at least the heavy and / or light chain CDR3 of any one of the anti-FAP antibodies selected from Table 2.

[0247] In some embodiments, any of the anti-FAP CARs of the present disclosure have one or more CDR (e.g., heavy chain CDR or light chain CDR) sequences substantially similar to any of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 sequences from one of the anti-FAP antibodies selected from Table 2. In some embodiments, the position of one or more CDRs along the VH (e.g., CDRH1, CDRH2, or CDRH3) and / or VL (e.g., CDRL1, CDRL2, or CDRL3) regions of a chimeric antigen receptor described herein may be varied by 1, 2, 3, 4, 5, or 6 amino acid positions, so long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived is substantially maintained).

[0248] In some examples, an anti-FAP CAR comprises one or more CDR (e.g., heavy chain CDR or light chain CDR) sequences substantially similar to any of the anti-FAP antibodies selected from Table 2. For example, an anti-FAP CAR may comprise one or more CDR sequence(s) from any of the anti-FAP antibodies selected from Table 2 that comprise up to 5, 4, 3, 2, or 1 amino acid residue mutations compared to the corresponding CDR region of any of the CDRs provided herein (e.g., a CDR from any of the anti-FAP antibodies selected from Table 2), so long as immunospecific binding to a FAP (e.g., a human FAP) is maintained (e.g., substantially maintained, i.e., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained compared to the binding of the antibody from which it was derived).

[0249] Some aspects of the present disclosure provide anti-FAP CARs comprising one or more of the heavy chain variable (VH) and / or light chain variable (VL) domains provided herein. In some embodiments, any of the VH domains provided herein comprise one or more of the heavy chain CDR sequences (e.g., CDRH1, CDRH2, and CDRH3) provided herein, e.g., any of the heavy chain CDR sequences provided in any one of the anti-FAP antibodies selected from Table 2. In some embodiments, any of the VL domains provided herein comprise one or more of the CDR-L sequences (e.g., CDRL1, CDRL2, and CDRL3) provided herein, e.g., any of the light chain CDR sequences provided in any one of the anti-FAP antibodies selected from Table 2.

[0250] In some embodiments, an anti-FAP CAR comprises the heavy chain variable domain and / or the light chain variable domain of any one of the anti-FAP antibodies selected from Table 2, and variants thereof. In some embodiments, an anti-FAP CAR comprises a pair of heavy and light chain variable regions of any anti-FAP antibody selected from Table 2.

[0251] Aspects of the present disclosure provide anti-FAP CARs comprising a heavy chain variable (VH) domain amino acid sequence and / or a light chain variable (VL) domain amino acid sequence that is homologous to any of the amino acid sequences described herein. In some embodiments, the anti-FAP CAR comprises a VH or VL that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH and / or any VL of any one of the anti-FAP antibodies selected from Table 2. In some embodiments, the homologous VH and / or VL amino acid sequence of the anti-FAP CAR is unchanged within any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) can occur within the VH and / or VL sequence of the anti-FAP CAR, excluding any of the CDR sequences provided herein. In some embodiments, any of the anti-FAP CARs provided herein comprise a VH sequence and a VL sequence comprising framework sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequences of any anti-FAP antibody selected from Table 2. In some embodiments, the anti-FAP CAR comprises a VH that comprises 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH of an anti-FAP antibody listed in Table 2. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL of any of the anti-FAP antibodies listed in Table 2.

[0252] In some embodiments, an anti-FAP CAR comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 that are the same as those shown in Table 2, and comprises a humanized VH and / or a humanized VL. In some embodiments, an anti-FAP CAR is a humanized variant that comprises one or more amino acid substitutions (e.g., in the VH framework regions) compared to any of the VHs listed in Table 2, and / or a humanized variant that comprises one or more amino acid substitutions (e.g., in the VL framework regions) compared to any of the VLs listed in Table 2.

[0253] In some embodiments, the anti-FAP CAR comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 7. Alternatively or additionally, the anti-FAP CAR comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 8.

[0254] In some embodiments, an anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 3. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having the amino acid sequence of SEQ ID NO: 2, and a CDRH3 having the amino acid sequence of SEQ ID NO: 3. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having the amino acid sequence of SEQ ID NO: 2, a CDRH3 having the amino acid sequence of SEQ ID NO: 3, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 6.

[0255] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 1, CDRH2 having the amino acid sequence of SEQ ID NO: 2, and CDRH3 having the amino acid sequence of SEQ ID NO: 3. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 6.

[0256] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO:1, CDRH2 having the amino acid sequence of SEQ ID NO:2, and CDRH3 having the amino acid sequence of SEQ ID NO:3. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO:4, CDRL2 having the amino acid sequence of SEQ ID NO:5, and CDRL3 having the amino acid sequence of SEQ ID NO:6.

[0257] In some embodiments, an anti-FAP CAR comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 2, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 3. Alternatively or additionally, an anti-FAP CAR comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 6.

[0258] In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 7. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8.

[0259] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 7. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 8.

[0260] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 7. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 8.

[0261] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 7. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 8. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 7 and / or the VL of SEQ ID NO: 8, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO:7, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO:8.

[0262] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 7. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 8. In some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 7 and / or the VL of SEQ ID NO: 8, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO:7, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO:8.

[0263] In some embodiments, the anti-FAP CAR comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 14. Alternatively or additionally, the anti-FAP CAR comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 15.

[0264] In some embodiments, an anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 12. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and a CDRH3 having the amino acid sequence of SEQ ID NO: 12. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 12, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 13.

[0265] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 12. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 13.

[0266] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 12. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 13.

[0267] In some embodiments, an anti-FAP CAR comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 12. Alternatively or additionally, an anti-FAP CAR comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 13.

[0268] In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 14. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising the amino acid sequence of SEQ ID NO: 15.

[0269] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 14. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 15.

[0270] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 14. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 15.

[0271] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 14. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 15. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 14 and / or the VL of SEQ ID NO: 15, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO: 14, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO: 15.

[0272] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 14. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 15. In some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 14 and / or the VL of SEQ ID NO: 15, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO: 14, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO: 15.

[0273] In some embodiments, the anti-FAP CAR comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 44. Alternatively or additionally, the anti-FAP CAR comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 45.

[0274] In some embodiments, an anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 40, a CDRH2 having the amino acid sequence of SEQ ID NO: 41, and a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 40, a CDRH2 having the amino acid sequence of SEQ ID NO: 41, a CDRH3 having the amino acid sequence of SEQ ID NO: 42, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0275] In some embodiments, an anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which comprise a total of five or fewer amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 40, CDRH2 having the amino acid sequence of SEQ ID NO: 41, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, an anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3, which comprise a total of five or fewer amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0276] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO: 40, CDRH2 having the amino acid sequence of SEQ ID NO: 41, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0277] In some embodiments, an anti-FAP CAR comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 40, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 41, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, an anti-FAP CAR comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0278] In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 44. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 44, and a VL comprising the amino acid sequence of SEQ ID NO: 45.

[0279] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 45.

[0280] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 5.

[0281] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 45. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 44 and / or the VL of SEQ ID NO: 45, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO: 44, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO: 45.

[0282] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 45. In some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 44 and / or the VL of SEQ ID NO: 45, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO: 44, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO: 45.

[0283] In some embodiments, the anti-FAP CAR comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 48.

[0284] In some embodiments, an anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 42, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0285] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0286] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0287] In some embodiments, an anti-FAP CAR comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, an anti-FAP CAR comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0288] In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 47, and a VL comprising the amino acid sequence of SEQ ID NO: 48.

[0289] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 48.

[0290] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 48.

[0291] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 48. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 47 and / or the VL of SEQ ID NO: 48, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO: 47, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO: 48.

[0292] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 48. In some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 47 and / or the VL of SEQ ID NO: 48, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO:47, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO:48.

[0293] In some embodiments, the anti-FAP CAR comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 51. Alternatively or additionally, the anti-FAP CAR comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 52.

[0294] In some embodiments, an anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 50, and a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 50, a CDRH3 having the amino acid sequence of SEQ ID NO: 42, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0295] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 50, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3, which comprise a total of no more than five amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0296] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 50, and CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0297] In some embodiments, an anti-FAP CAR comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 50, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 42. Alternatively or additionally, an anti-FAP CAR comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 43.

[0298] In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 51. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 51, and a VL comprising the amino acid sequence of SEQ ID NO: 52.

[0299] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 52.

[0300] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 52.

[0301] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 52. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 51 and / or the VL of SEQ ID NO: 52, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO: 51, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO: 52.

[0302] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 51. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 52. In some embodiments, the degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 51 and / or the VL of SEQ ID NO: 52, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO: 51, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO: 52.

[0303] In some embodiments, the anti-FAP CAR comprises a heavy chain variable domain (VH) CDRH1, CDRH2, and CDRH3 having the amino acid sequence of SEQ ID NO: 56. Alternatively or additionally, the anti-FAP CAR comprises a light chain variable domain (VL) CDRL1, CDRL2, and CDRL3 having the amino acid sequence of SEQ ID NO: 57.

[0304] In some embodiments, an anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 54. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and a CDRH3 having the amino acid sequence of SEQ ID NO: 54. In some embodiments, an anti-FAP CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 54, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 55.

[0305] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which comprise a total of five or fewer amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 54. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3, which comprise a total of five or fewer amino acid mutations (e.g., no more than 5, 4, 3, 2, or 1 amino acid mutations) compared to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 55.

[0306] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRH1 having the amino acid sequence of SEQ ID NO: 10, CDRH2 having the amino acid sequence of SEQ ID NO: 11, and CDRH3 having the amino acid sequence of SEQ ID NO: 54. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 that are collectively at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 55.

[0307] In some embodiments, an anti-FAP CAR comprises a CDRH1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH2 having the amino acid sequence of SEQ ID NO: 11, and / or a CDRH3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRH3 having the amino acid sequence of SEQ ID NO: 54. Alternatively or additionally, an anti-FAP CAR comprises a CDRL1 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and / or a CDRL3 having three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to a CDRL3 having the amino acid sequence of SEQ ID NO: 55.

[0308] In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 56. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the anti-FAP CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 56, and a VL comprising the amino acid sequence of SEQ ID NO: 57.

[0309] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 56. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 57.

[0310] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 56. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 57.

[0311] In some embodiments, the anti-FAP CAR comprises a VH that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 56. Alternatively or additionally, the anti-FAP CAR comprises a VL that includes 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 57. In some embodiments, the number of amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) may occur within the VH of SEQ ID NO: 56 and / or the VL of SEQ ID NO: 57, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VH framework sequence of SEQ ID NO: 56, and / or a light chain variable sequence comprising a framework sequence that comprises no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to the VL framework sequence of SEQ ID NO: 57.

[0312] In some embodiments, the anti-FAP CAR comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH set forth in SEQ ID NO: 56. Alternatively or additionally, the anti-FAP CAR comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL set forth in SEQ ID NO: 57. In some embodiments, a degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within the VH of SEQ ID NO: 56 and / or the VL of SEQ ID NO: 57, excluding any of the CDR sequences therein. In some embodiments, the anti-FAP CAR comprises a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VH of SEQ ID NO: 56, and / or a light chain variable sequence comprising a framework sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of the VL of SEQ ID NO: 57.

[0313] Some aspects of the present disclosure provide anti-FAP CARs that comprise a FAP-binding agent known in the art, e.g., any of the FAP-binding agents presented in Table 3. In some embodiments, an anti-FAP CAR of the present disclosure comprises a FAP-binding peptide known in the art, e.g., a FAP-binding peptide presented in Table 3. In some embodiments, an anti-FAP CAR of the present disclosure comprises one or more heavy chain variable (VH) domains and / or light chain variable (VL) domains of an anti-FAP antibody known in the art, e.g., an anti-FAP antibody presented in Table 3. In some embodiments, an anti-FAP CAR of the present disclosure comprises a VH domain that includes one or more heavy chain CDR sequences (e.g., CDRH1, CDRH2, and CDRH3) of an anti-FAP antibody known in the art, e.g., an anti-FAP antibody presented in Table 3. In some embodiments, an anti-FAP CAP of the present disclosure comprises a VL domain comprising one or more of the light chain CDR sequences (e.g., CDRL1, CDRL2, and CDRL3) of an anti-FAP antibody known in the art, such as an anti-FAP antibody presented in Table 3.

[0314] In some embodiments, an anti-FAP CAR of the present disclosure comprises the heavy chain variable domain and / or the light chain variable domain of any one of the anti-FAP antibodies known in the art, e.g., an anti-FAP antibody selected from Table 3, and variants thereof. In some embodiments, an anti-FAP CAR comprises a pair of heavy chain variable regions and light chain variable regions of any anti-FAP antibody known in the art, e.g., an anti-FAP antibody selected from Table 3.

[0315] Aspects of the present disclosure provide anti-FAP CARs that comprise a heavy chain variable (VH) domain amino acid sequence and / or a light chain variable (VL) domain amino acid sequence that is homologous to any of the anti-FAP antibodies known in the art, e.g., the anti-FAP antibodies presented in Table 3. In some embodiments, the anti-FAP CAR comprises a VH or VL that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH and / or any VL of any of the anti-FAP antibodies known in the art, e.g., the anti-FAP antibodies presented in Table 3. In some embodiments, the homologous VH and / or VL amino acid sequences of the anti-FAP CAR are unchanged within the CDR sequences of any of the anti-FAP antibodies known in the art, e.g., the anti-FAP antibodies presented in Table 3. For example, in some embodiments, a degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) can occur within the VH and / or VL sequences of an anti-FAP CAR, excluding any of the CDR sequences of any of the anti-FAP antibodies known in the art, e.g., any of the anti-FAP antibodies presented in Table 3. In some embodiments, any of the anti-FAP CARs provided herein comprise VH and VL sequences that comprise framework sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequences of any of the anti-FAP antibodies known in the art, e.g., any of the anti-FAP antibodies presented in Table 3. In some embodiments, an anti-FAP CAR comprises a VH that comprises 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH of any anti-FAP antibody known in the art, such as any of the anti-FAP antibodies presented in Table 3. Alternatively or additionally, an anti-FAP CAR comprises a VL that comprises 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL of any anti-FAP antibody known in the art, such as any of the anti-FAP antibodies presented in Table 3.

[0316] In some embodiments, an anti-FAP CAR comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 that are the same as the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of an anti-FAP antibody known in the art, e.g., an anti-FAP antibody presented in Table 3, and comprises a humanized VH and / or a humanized VL. In some embodiments, an anti-FAP CAR is a humanized variant that comprises one or more amino acid substitutions (e.g., in the VH framework region) compared to an anti-FAP antibody known in the art, e.g., an anti-FAP antibody presented in Table 3, and / or a humanized variant that comprises one or more amino acid substitutions (e.g., in the VL framework region) compared to an anti-FAP antibody known in the art, e.g., an anti-FAP antibody presented in Table 3.

[0317] The anti-FAP antibodies or antigen-binding fragments thereof described herein can be grafted into chimeric antigen receptors, including, but not limited to, antigen-binding fragments thereof (Fab, F(ab'), F(ab')2, Fv, etc.), single-chain antibodies (e.g., scFv), bispecific antibodies, or nanobodies. In some embodiments, the anti-FAP CARs described herein comprise a single-chain variable fragment (scFv) as the extracellular ligand-binding domain.

[0318] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising a single-chain variable fragment (scFv). In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising the VH and VL of any one of the anti-FAP scFvs selected from Table 2. In some embodiments, the anti-FAP CAR comprises the VH and VL of any anti-FAP antibody known in the art, such as an anti-FAP antibody listed in Table 3. In some embodiments, the VH and VL of the anti-FAP CAR are joined to each other by a linker. In some embodiments, the linker can have a length of about 2-10 amino acids, 5-20 amino acids, 10-30 amino acids, 20-50 amino acids, 40-60 amino acids, 60-80 amino acids, or more than 80 amino acids. In some embodiments, the linker can comprise a sequence substantially comprising glycine and serine. An exemplary linker sequence is GGGGSGGGGSGGGAS (SEQ ID NO: 29). In some embodiments, the linker can include, but is not limited to, any of those encompassed by U.S. Patent Nos. 8,445,251 and 9,434,931. In some embodiments, the anti-FAP CAR comprises a linker between the VH and VL, wherein the linker comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:29.

[0319] In some embodiments, an anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising a VH and a VL, wherein the C-terminus of the VH is joined to the N-terminus of the VL via a linker (e.g., a linker set forth in SEQ ID NO: 29). In some embodiments, an anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising a VH and a VL, wherein the C-terminus of the VL is joined to the N-terminus of the VH via a linker (e.g., a linker set forth in SEQ ID NO: 29).

[0320] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising a VH that comprises 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH of an anti-FAP antibody listed in Table 2. Alternatively or additionally, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv that comprises a VL that comprises 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH of an anti-FAP antibody listed in Table 2. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising a VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH of any anti-FAP antibody listed in Table 2. Alternatively or additionally, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising a VL comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH of any anti-FAP antibody listed in Table 2.

[0321] In some embodiments, an anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that includes 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to any of the anti-FAP scFvs listed in Table 2. In some embodiments, an anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any of the anti-FAP scFvs listed in Table 2.

[0322] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that includes 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 9.

[0323] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that includes 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 16.

[0324] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 46.

[0325] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that includes 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 49.

[0326] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence containing 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 53.

[0327] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that includes 20 or fewer amino acid mutations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the scFv amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the scFv amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 58.

[0328] In some embodiments, an anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising a VH that comprises 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH of any anti-FAP antibody known in the art, such as an anti-FAP antibody presented in Table 3. Alternatively or additionally, an anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv that comprises a VL that comprises 20 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH of any anti-FAP antibody known in the art, such as an anti-FAP antibody presented in Table 3. In some embodiments, an anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising a VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH of any anti-FAP antibody known in the art, such as the anti-FAP antibodies presented in Table 3. Alternatively or additionally, an anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv comprising a VL comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH of any anti-FAP antibody known in the art, such as the anti-FAP antibodies presented in Table 3.

[0329] In some embodiments, the anti-FAP CAR of the present disclosure further comprises a hinge region. In some embodiments, the hinge region is a CD8 hinge region. An exemplary amino acid sequence of a CD8 hinge region is set forth in SEQ ID NO: 30: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0330] In some embodiments, an anti-FAP CAR of the present disclosure comprises a CD8 hinge region having the amino acid sequence set forth in SEQ ID NO: 30, or a variant thereof. In some embodiments, an anti-FAP CAR comprises a hinge region comprising an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the hinge region can be any suitable hinge region known in the art, such as a hinge region described in Guedan et al., Engineering and Design of Chimeric Antigen Receptors, Mol Ther Methods Clin Dev. 2019 Mar 15;12: 145-156, e.g., a hinge region derived from IgG1, IgG2, IgG4, CD28, CD8, or a hybrid thereof.

[0331] In some embodiments, the anti-FAP CAR of the present disclosure further comprises a transmembrane domain linking the extracellular ligand-binding domain with the intracellular signaling and costimulatory domains. Regarding the transmembrane domain, the CAR can be designed to include a transmembrane domain fused to the extracellular domain (e.g., the antigen-binding domain) of the CAR. Any transmembrane domain is contemplated for use herein, so long...

Claims

1. An invariant natural killer T (iNKT) cell comprising a chimeric antigen receptor (CAR) that specifically binds to fibroblast activation protein (FAP), wherein the chimeric antigen receptor is (a) a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having the amino acid sequence of SEQ ID NO: 2, a CDRH3 having the amino acid sequence of SEQ ID NO: 3, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 6; (b) a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 12, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 13; (c) a CDRH1 having the amino acid sequence of SEQ ID NO: 40, a CDRH2 having the amino acid sequence of SEQ ID NO: 41, a CDRH3 having the amino acid sequence of SEQ ID NO: 42, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 43; (d) a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 42, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 43; or (e) a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 54, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO: 55; The iNKT cell comprising:

2. The iNKT cell of claim 1, wherein the CAR comprises a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 11, a CDRH3 having the amino acid sequence of SEQ ID NO: 42, a CDRL1 having the amino acid sequence of SEQ ID NO: 4, a CDRL2 having the amino acid sequence of SEQ ID NO: 5, and a CDRL3 having the amino acid sequence of SEQ ID NO:

43.

3. The CAR is (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8; (b) a VH comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising the amino acid sequence of SEQ ID NO: 15; (c) a VH comprising the amino acid sequence of SEQ ID NO: 44, and a VL comprising the amino acid sequence of SEQ ID NO: 45; (d) a VH comprising the amino acid sequence of SEQ ID NO: 47 and a VL comprising the amino acid sequence of SEQ ID NO: 48; or (e) a VH comprising the amino acid sequence of SEQ ID NO: 56, and a VL comprising the amino acid sequence of SEQ ID NO: 57; The iNKT cell of claim 1 or 2, comprising:

4. The iNK cell according to any one of claims 1 to 3, wherein the CAR comprises a VH comprising the amino acid sequence of SEQ ID NO: 47 and a VL comprising the amino acid sequence of SEQ ID NO:

48.

5. The CAR is (a) an scFv comprising the amino acid sequence of SEQ ID NO: 9; (b) an scFv comprising the amino acid sequence of SEQ ID NO: 16; (c) an scFv comprising the amino acid sequence of SEQ ID NO: 46; (d) an scFv comprising the amino acid sequence of SEQ ID NO: 49; or (e) an scFv comprising the amino acid sequence of SEQ ID NO: 58; The iNKT cell according to any one of claims 1 to 4, comprising:

6. The iNKT cell according to any one of claims 1 to 6, wherein the CAR comprises an scFv comprising the amino acid sequence of SEQ ID NO:

49.

7. The iNKT cell of any one of claims 1 to 6, wherein the CAR further comprises a hinge region.

8. The iNKT cell of claim 7, wherein the hinge region comprises an amino acid sequence at least 80% identical to SEQ ID NO:

30.

9. The iNKT cell of any one of claims 1 to 8, wherein the CAR further comprises a transmembrane domain.

10. The iNKT cell of claim 9, wherein the transmembrane domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

31.

11. The iNKT cell of any one of claims 1 to 7, wherein the CAR comprises a hinge / transmembrane domain comprising the amino acid sequence of any one of SEQ ID NOs: 83, 85, or 87.

12. The iNKT cell of any one of claims 1 to 11, wherein the CAR further comprises one or more cytoplasmic domains.

13. The iNKT cell of claim 12, wherein the one or more cytoplasmic domains comprise the amino acid sequence of any one of SEQ ID NOs: 32, 33, 39, 90, 92, 94, 96, 98, 100, 102, or 105.

14. The iNKT cell of claim 12 or 13, wherein the cytoplasmic domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

32.

15. The iNKT cell of any one of claims 1 to 14, wherein the CAR further comprises an intracellular costimulatory domain.

16. The iNKT cell of claim 15, wherein the costimulatory domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

33.

17. The iNKT cell of any one of claims 1 to 16, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 17, 18, 59, 60, or 62.

18. The iNKT cell of any one of claims 1 to 17, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

60.

19. The iNKT cell of any one of claims 1 to 18, wherein the iNKT cell is engineered to express one or more immunomodulatory gene products.

20. 20. The iNKT cell of claim 19, wherein the one or more immunomodulatory gene products comprise IL-15, IL-12, CD40L, or 4-1BB, IL-18, or IL-21.

21. The iNKT cell of claim 19 or 20, wherein the immunomodulatory gene product is soluble IL-15 (sIL-15).

22. The iNKT cell of any one of claims 1 to 21, wherein the CAR binds to FAP with a KD in the range of 1E-10M to 10E-7M.

23. The iNKT cell of any one of claims 1 to 22, wherein the CAR binds to FAP with a KD ranging from 1E-8M to 3E-8M, or from 3E-8M to 4E-7M.

24. An invariant natural killer T (iNKT) cell comprising a chimeric antigen receptor (CAR) that specifically binds to fibroblast activation protein (FAP), wherein the iNKT cell is engineered to express IL-15.

25. A composition comprising the iNKT cell of any one of claims 1 to 24.

26. 27. The composition of claim 26, further comprising a pharmaceutically acceptable carrier.

27. 27. A method for killing a cell, comprising contacting the cell with an iNKT cell according to any one of claims 1 to 24, or a composition according to claim 25 or 26.

28. 27. The method of claim 26, wherein the cell is a cancer cell.

29. 29. The method of claim 28, wherein the cancer cells are lung cancer cells, breast cancer cells, colon cancer cells, prostate cancer cells, gastric cancer cells, pancreatic cancer cells, prostate cancer cells, thyroid cancer cells, cervical cancer cells, urothelial cancer cells, or sarcoma cells.

30. The method according to any one of claims 26 to 29, wherein the lung cancer is non-small cell lung cancer.

31. The method of any one of claims 26 to 30, wherein the cells of the tumor microenvironment express FAP.

32. A method for treating a tumor in a subject, the method comprising administering to a subject having or suspected of having cancer an iNKT described in any one of claims 1 to 24, or a composition described in claim 25 or 26.

33. 27. A method for reducing tumor growth, comprising contacting the tumor in a subject with an iNKT according to any one of claims 1 to 24, or a composition according to claim 25 or 26.

34. 34. The method of claim 32 or 33, wherein the subject is a human.

35. The method of any one of claims 32 to 34, wherein the tumor is a solid tumor.

36. 36. The method of claim 35, wherein the solid tumor is a lung cancer tumor, a breast cancer tumor, a colon cancer tumor, a prostate cancer tumor, a gastric cancer tumor, a pancreatic cancer tumor, a prostate cancer tumor, a thyroid cancer tumor, a cervical cancer tumor, a urothelial cancer tumor, or a sarcoma.

37. 37. The method of claim 36, wherein the lung cancer is non-small cell lung cancer.

38. The method of any one of claims 32 to 37, wherein the administration is by injection.

39. 39. The method of claim 38, wherein the injection is an intraperitoneal injection, an intravenous injection, or an intratumoral injection.

40. The method of any one of claims 32 to 39, wherein the iNKT cells are used in combination with a therapeutic agent.

41. 41. The method of claim 40, wherein the therapeutic agent is a CAR T cell specific for a tumor antigen.

42. 42. The method of claim 41, wherein the therapeutic agent is an immune checkpoint inhibitor or agonist.

43. 1. A method for treating a tumor in a subject, comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells comprising a chimeric antigen receptor (CAR) having an anti-FAP binding moiety.

44. 1. A method for killing tumor cells, comprising contacting the tumor cells with a composition comprising invariant natural killer T (iNKT) cells comprising a chimeric antigen receptor (CAR) having an anti-FAP binding moiety.

45. 1. A method for reducing immunosuppression in a tumor microenvironment (TME) of a subject relative to immunosuppression in the subject prior to administration, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells comprising a chimeric antigen receptor (CAR) having an anti-FAP binding moiety.

46. The method of any one of claims 43 to 45, wherein the anti-FAP CAR iNKT cells are engineered to express an arming molecule.

47. The method of claim 46, wherein the arming molecule is soluble IL-15 (sIL-15).

48. The method of any one of claims 43 to 47, wherein the tumor is a solid tumor.

49. The method of any one of claims 43 to 48, wherein the tumor is of epithelial cell origin.

50. 50. The method of claim 49, wherein the solid tumor is a lung cancer tumor, a breast cancer tumor, a colon cancer tumor, a prostate cancer tumor, a gastric cancer tumor, a pancreatic cancer tumor, a prostate cancer tumor, a thyroid cancer tumor, a cervical cancer tumor, a urothelial cancer tumor, or a sarcoma tumor.

51. The method of any one of claims 43 to 50, wherein the anti-FAP CAR iNKT cells kill FAP-expressing cells in the TME.

52. 52. The method of claim 51, wherein the FAP-expressing cells of the TME comprise FAP-expressing tumor cells and / or cancer-associated fibroblasts.

53. 53. The method of claim 51 or 52, wherein the anti-FAP CAR iNKT cells directly kill FAP-expressing cells.

54. 53. The method of claim 51 or 52, wherein the anti-FAP CAR iNKT cells indirectly kill FAP-expressing cells.

55. 55. The method of any one of claims 51-54, wherein killing of the cancer-associated fibroblasts reduces immunosuppression in the TME compared to immunosuppression in the subject prior to the administration.

56. 56. The method of any one of claims 52-55, wherein killing of the cancer-associated fibroblasts increases immune cell infiltration in the TME compared to immune cell infiltration in the subject prior to said administering.

57. 47. The method of any one of claims 43-46, further comprising administering to the subject CAR T cells that target a tumor antigen comprising NY-ESO-1, or BCMA.

58. 58. The method of any one of claims 43-57, wherein said administering said anti-FAP CAR iNKT cells reduces tumor burden compared to tumor burden in said subject before said administration.

59. 59. The method of any one of claims 43-58, wherein said administration of said anti-FAP CAR iNKT cells results in resistance to T cell exhaustion, enhanced tissue homing of anti-FAP iNKT cells, selective cytotoxicity against M2 macrophages, and / or stimulation of dendritic cell maturation.

60. The method of any one of claims 43 to 59, wherein the anti-FAP CAR iNKT cells retain their response to CD1d and / or NK receptor ligands.

61. 61. The method of any one of claims 43 to 60, wherein the anti-FAP CAR iNKT cells comprise a CAR comprising any one of the FAP-binding moieties derived from the anti-FAP antibodies listed in Table 3.

62. The method of any one of claims 43 to 61, wherein the subject is a human.

63. 64. The method of any one of claims 43-63, wherein the subject has been diagnosed with a solid tumor, including lung cancer, breast cancer, colon cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, urothelial cancer, or sarcoma.

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