Fibroblast activating protein (FAP) CAR-constant natural killer t cells and uses thereof

Abstract

The present disclosure is based, at least in part, on the discovery of novel anti-fibroblast activating protein (FAP) antibodies or antigen-binding fragments thereof, and genetically modified cells (e.g., iNKT cells) expressing chimeric antigen receptors comprising anti-FAP antibodies or antigen-binding fragments thereof exhibit improved properties, the methods include increased binding to FAP, killing of cancer cells expressing FAP in vitro and in vivo, and enhanced persistence in a subject receiving treatment.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 413,236, filed Oct. 4, 2022, under 35 U.S.C. 119(e), the entire content of which is incorporated herein by reference.

[0003] Reference to Electronic Sequence Listing

[0004] The content of the electronic sequence listing (A132770004WO00-SEQ-LGE.xml; size: 179,363 bytes; and created on Oct. 4, 2023) is incorporated herein by reference in its entirety. Background Art

[0005] Fibroblast activation protein (FAP) is a cell surface protein that is highly expressed on stromal cells such as cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME). FAP can regulate the TME by remodeling the extracellular matrix (ECM), and its overexpression on CAFs is associated with poor prognosis in various cancers. Summary of the Invention

[0006] This disclosure is at least partially based on the discovery that genetically modified cells (e.g., iNKT cells) expressing a chimeric antigen receptor targeting fibroblast activation protein (FAP) are capable of killing 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 an armored molecule (e.g., soluble IL-15). In some embodiments, 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, anti-FAP CAR iNKT cells reduce immunosuppression in the TME, thereby increasing the efficacy of other cancer therapies (e.g., CAR T cells targeting tumor antigens). In some embodiments, the anti-FAP CAR iNKT cells described in this disclosure exhibit improved properties compared to existing FAP-CAR cell therapies in the art, including but not limited to killing FAP-expressing cells in vitro and in vivo; and enhanced persistence in treated subjects.

[0007] In some aspects, the present disclosure provides an invariant natural killer T (iNKT) cell comprising a chimeric antigen receptor (CAR) that specifically binds fibroblast activation protein (FAP). In some embodiments, the chimeric antigen receptor of the iNKT cell comprises CDRH1 having the amino acid sequence of SEQ ID NO:1, CDRH2 having the amino acid sequence of SEQ ID NO:2, CDRH3 having the amino acid sequence of SEQ ID NO:3, 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. In some embodiments, the chimeric antigen receptor of the iNKT cell 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:12, 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. In some embodiments, the chimeric antigen receptor of the iNKT cell 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 the iNKT cell 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 the iNKT cell 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.

[0008] In some embodiments, the chimeric antigen receptor of the iNKT cell comprises a variable heavy chain (VH) having the amino acid sequence of SEQ ID NO:7 and a variable light chain (VL) having the amino acid sequence of SEQ ID NO:8. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises a VH having the amino acid sequence of SEQ ID NO:14 and a VL having the amino acid sequence of SEQ ID NO:15. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises a VH having the amino acid sequence of SEQ ID NO:44 and a VL having the amino acid sequence of SEQ ID NO:45. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises a VH having the amino acid sequence of SEQ ID NO:47 and a VL having the amino acid sequence of SEQ ID NO:48. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises a VH having the amino acid sequence of SEQ ID NO:56 and a VL having the amino acid sequence of SEQ ID NO:57.

[0009] In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv having the amino acid sequence of SEQ ID NO:9. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv having the amino acid sequence of SEQ ID NO:16. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv having the amino acid sequence of SEQ ID NO:46. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv having the amino acid sequence of SEQ ID NO:49. In some embodiments, the chimeric antigen receptor of the iNKT cell comprises an scFv having the amino acid sequence of SEQ ID NO:58.

[0010] In some embodiments, the CAR comprised by the iNKT cells further comprises a hinge region. In some embodiments, the hinge region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:30. In some embodiments, the CAR comprised by the iNKT cells further comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:31. In some embodiments, the CAR comprised by the iNKT cells further comprises a hinge / transmembrane domain, which comprises the amino acid sequence of any one of SEQ ID NO:83, 85 or 87. In some embodiments, the CAR comprised by the iNKT cells 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 NO:32, 33, 39, 90, 92, 94, 96, 98, 100, 102 or 105. In some embodiments, the cytoplasmic domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:32. In some embodiments, the CAR comprised by the iNKT cells further comprises an intracellular co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises an amino acid sequence that is 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 NO:17, 18, 59, 60, 61 or 62.

[0011] In some embodiments, the anti-FAP CAR iNKT cells are engineered to express one or more immunomodulatory gene products (i.e., armoring molecules). In some embodiments, the one or more immunomodulatory gene products comprise 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).

[0012] In some embodiments, the CAR binds to FAP with a KD in the range of 1E-10M and 10E-7M. In some embodiments, the CAR binds to FAP with a KD in the range of 1E-8M and 3E-8M.

[0013] In some aspects, the present disclosure provides an invariant natural killer T (iNKT) cell that comprises a chimeric antigen receptor (CAR) that specifically binds fibroblast activation protein (FAP), wherein the iNKT cell expresses IL-15 (e.g., soluble IL-15). In some embodiments, the anti-FAP CAR iNKT cell comprises a CAR that comprises an antigen-binding portion that specifically binds to FAP, and the antigen-binding portion is derived from the antigen-binding portion of any one of the anti-FAP antibodies set forth in Tables 2 and 3.

[0014] In some aspects, the present disclosure provides a composition that comprises the FAP CAR iNKT cells described herein and a pharmaceutically acceptable carrier.

[0015] In some aspects, the present disclosure provides a method of killing cells that comprises contacting the cells with the anti-FAP iNKT cells or a composition thereof described herein. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are lung cancer cells, breast cancer cells, colorectal 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. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the cells in the tumor microenvironment express FAP.

[0016] In some aspects, the present disclosure provides a method for treating a tumor in a subject that comprises administering to a subject having or suspected of having cancer the anti-FAP iNKT or a composition thereof described herein.

[0017] In some aspects, the present disclosure provides a method for reducing tumor growth that comprises contacting a tumor of a subject with the anti-FAP iNKT or a composition thereof described herein. In some embodiments, the administration is by injection. In some embodiments, the injection is intraperitoneal injection, intravenous injection, or intratumoral injection. In some embodiments, the anti-FAP CAR iNKT cells are administered in combination 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.

[0018] In some aspects, the present disclosure provides a method for treating a tumor in a subject that comprises administering to the subject a composition comprising invariant natural killer T (iNKT) cells that comprise a chimeric antigen receptor (CAR) having an anti-FAP binding portion.

[0019] 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, the composition comprising a chimeric antigen receptor (CAR) having an anti-FAP binding moiety.

[0020] In some aspects, the present disclosure provides a method for reducing immunosuppression in the tumor microenvironment (TME) of a subject relative to the immunosuppression of the subject prior to administration, the method comprising administering to the subject a composition comprising invariant natural killer T (iNKT) cells, the composition comprising a chimeric antigen receptor (CAR) having an anti-FAP binding moiety.

[0021] In some embodiments, the anti-FAP CAR iNKT cells express an armoring molecule. In some embodiments, the armoring molecule is soluble IL-15 (sIL-15).

[0022] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is derived from epithelial cells. In some embodiments, the solid tumor is a lung cancer tumor, a breast cancer tumor, a colorectal 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.

[0023] In some embodiments, the anti-FAP CAR iNKT cells kill cells expressing FAP in the TME. In some embodiments, the cells expressing FAP in the TME comprise tumor cells expressing FAP and / or cancer-associated fibroblasts. In some embodiments, the anti-FAP CAR iNKT cells directly kill cells expressing FAP. In some embodiments, the anti-FAP CAR iNKT cells indirectly kill cells expressing FAP. In some embodiments, killing cancer-associated fibroblasts reduces immunosuppression in the TME relative to the immunosuppression in the subject prior to administration. In some embodiments, killing cancer-associated fibroblasts increases immune cell infiltration in the TME relative to the immune cell infiltration in the subject prior to administration.

[0024] In some embodiments, the method further comprises administering to the subject CAR T cells targeting a tumor antigen, the tumor antigen comprising NY-ESO-1 or BCMA.

[0025] In some embodiments, administering the anti-FAP CAR iNKT cells reduces the tumor burden in the subject relative to the tumor burden prior to administration.

[0026] In some embodiments, administration of anti-FAP CAR iNKT cells elicits 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, anti-FAP CAR iNKT cells retain responsiveness to CD1d and / or NK receptor ligands.

[0027] In some embodiments, the anti-FAP CAR iNKT cells comprise a CAR that comprises any one of the FAP-binding moieties listed in Tables 2 and 3.

[0028] In some embodiments, the anti-FAP CAR iNKT cells comprise a CAR that comprises any one of the FAP antibodies listed in Table 2.

[0029] In some embodiments, the subject is a human. In some embodiments, the subject is diagnosed with a solid tumor, including lung cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, urothelial cancer, or sarcoma.

[0030] In some aspects, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to an amino acid sequence having at least 85% identity to SEQ ID NOs: 35-38. In some embodiments, the antibody specifically binds to the amino acid sequence shown below: SEQ ID NOs: 35-38.

[0031] In some aspects, the present disclosure provides an antibody or antigen-binding fragment that comprises a heavy chain variable region having the sequence shown in SEQ ID NO: 7 or 14.

[0032] In some embodiments, the antibody comprises: a heavy chain variable region having the sequence shown in SEQ ID NO: 7 or 14 and a light chain variable region having the sequence shown in SEQ ID NO: 8 or 15.

[0033] In some embodiments, wherein the antibody comprises: (i) a heavy chain variable region having the sequence shown in SEQ ID NO: 7 and / or a light chain variable region having the sequence shown in SEQ ID NO: 8; or (ii) a heavy chain variable region having the sequence shown in SEQ ID NO: 14 and / or a light chain variable region having the sequence shown in SEQ ID NO: 15.

[0034] In some aspects, the present disclosure provides an antibody or antigen-binding fragment that comprises a variable heavy chain region that comprises a complementarity-determining region 3 (CDRH3) having the sequence shown in SEQ ID NO: 3 or 12.

[0035] In some embodiments, the antibody or antigen-binding fragment further comprises a variable light chain region that comprises complementarity determining region 3 (CDRL3) having the sequence shown in SEQ ID NO: 6 or 13.

[0036] In some aspects, the present disclosure provides an antibody or antigen-binding fragment that comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein (i) CDRH1 comprises the sequence shown in SEQ ID NO: 1, CDRH2 comprises the sequence shown in SEQ ID NO: 2, CDRH3 comprises the sequence shown in SEQ ID NO: 3, CDRL1 comprises the sequence shown in SEQ ID NO: 4, CDRL2 comprises the sequence shown in SEQ ID NO: 5, and CDRL3 comprises the sequence shown in SEQ ID NO: 6; or (ii) CDRH1 comprises the sequence shown in SEQ ID NO: 10, CDRH2 comprises the sequence shown in SEQ ID NO: 11, CDRH3 comprises the sequence shown in SEQ ID NO: 12, CDRL1 comprises the sequence shown in SEQ ID NO: 4, CDRL2 comprises the sequence shown in SEQ ID NO: 5, and CDRL3 comprises the sequence shown in SEQ ID NO: 13.

[0037] In some embodiments, the antibody or antigen-binding fragment is chimeric. In some embodiments, the antibody or antigen-binding fragment is humanized.

[0038] 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 shown in SEQ ID NO: 9 or 16.

[0039] In some aspects, the present disclosure provides an isolated nucleic acid that encodes an antibody or antigen-binding fragment described herein.

[0040] In some embodiments, the nucleic acid sequence encoding the anti-FAP scFv is as shown in SEQ ID NO: 21 or 26. In some embodiments, the nucleic acid sequence encoding the anti-FAP CAR is as shown in SEQ ID NO: 22 or 27.

[0041] In one aspect, 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 viral vector is a lentiviral vector. In some embodiments, the lentiviral vector comprises the sequence shown in SEQ ID NO: 23 or 28.

[0042] In some aspects, the present disclosure provides a host cell comprising the antibody or antigen-binding fragment, isolated nucleic acid, or vector described herein. In some embodiments, the cell is a mammalian cell, bacterial cell, yeast cell, or insect cell. In some embodiments, the cell is a hybridoma cell.

[0043] 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 that is at least 80% identical to SEQ ID NO: 30.

[0044] In some embodiments, the CAR further comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 31.

[0045] In some embodiments, the CAR further comprises a cytoplasmic domain. In some embodiments, the cytoplasmic domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 32.

[0046] In some embodiments, the CAR further comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 33.

[0047] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 17 or 18.

[0048] In some aspects, the present disclosure encompasses an immune cell comprising the CAR described herein.

[0049] In some embodiments, the immune cell is a natural killer (NK) cell or a T cell. In some embodiments, the immune cell is an invariant natural killer T (iNKT) cell.

[0050] In some embodiments, the immune cell is engineered to express one or more immune regulatory gene products. In some embodiments, the one or more immune regulatory gene products comprise IL-15, IL-12, CD40L or 4-1BB, IL-18, or IL-21.

[0051] 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.

[0052] In some aspects, the present disclosure provides a method for killing cells, the method comprising contacting the cells with an antibody or antigen-binding fragment, immune cell or pharmaceutical composition described herein.

[0053] In certain embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are cancer cells. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is lung cancer, breast cancer, colorectal 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.

[0054] 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 immune cell or pharmaceutical composition described herein.

[0055] In some aspects, the present disclosure provides a method for reducing tumor growth, the method comprising contacting the cells with an antibody or antigen-binding fragment, immune cell or pharmaceutical composition described herein.

[0056] In certain embodiments, the subject does not undergo lymphodepletion before or concurrently with treatment with the anti-FAP CAR iNKT cells or pharmaceutical composition disclosed herein. Lymphodepletion is often performed prior to immunotherapy (e.g., CAR T therapy) to, for example, reduce tumor volume, alter tumor phenotype, alter the tumor microenvironment, deplete cytokine reservoirs (e.g., make IL-2, IL-7 and IL-15 more available) and suppress the host immune system. However, lymphodepletion has a variety of negative effects, including neutropenia, anemia, thrombocytopenia and immunosuppression, as well as toxicities associated with lymphodepleting agents such as fludarabine and cyclophosphamide. In certain embodiments of the present invention, the subject does not receive fludarabine or cyclophosphamide treatment before or concurrently with administration of the anti-FAP CAR iNKT cells or pharmaceutical composition disclosed herein.

[0057] 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, breast cancer tumor, colorectal cancer tumor, prostate cancer tumor, gastric cancer tumor, pancreatic cancer tumor, prostate cancer tumor, thyroid cancer tumor, cervical cancer tumor, urothelial cancer tumor, or sarcoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0058] In some embodiments, the administration is by injection. In some embodiments, the injection is intraperitoneal, intravenous, intratumoral injection.

[0059] In some embodiments, the immune cells are administered in combination 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The drawings incorporated herein and constituting a part of this specification illustrate certain embodiments and, together with the written description, are used to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

[0061] Figure 1 Exemplary forms of anti-FAP CAR iNKT cells are shown.

[0062] Figure 2 Methods for evaluating the therapeutic window for targeting FAP-positive cells (e.g., cancer-associated fibroblasts (CAFs)) in the tumor microenvironment are shown. FAP expression in different types of cancer-associated fibroblasts. Quantification of the expression of FAP molecules on the surface of different cell types using the Quantibrite TM method. CAFs in 8 dissociated tumor samples (DTCs) from colorectal cancer (CRC) patients were evaluated. The expression of FAP in the CAF-S1 subset was more than 10-fold higher than the expression of FAP in the CAF-S3 subset. Similarly, after thawing, the expression of FAP in the CAF-S1 samples was more than 10-fold higher than the expression of FAP in normal lung fibroblasts (NLFs). Passage of NLF (P0, P3) increased FAP expression to a level similar to that of CAFs. The FAP expression levels exhibited by different tumor cell lines expressing FAP obtained from multiple passages were between the FAP expression levels observed for primary healthy fibroblasts to CAF S3. For U138 cells, there were differences according to passage. For discovery purposes, we transfected T2 cells with a lower or higher amount of FAP (T2low and T2high, respectively). Finally, for in vivo studies, A375 cells with an FAP expression level identical to that in T2high were generated.

[0063] Figures 3A - 3N It is a figure showing the generation of anti-FAP CARs that can bind to human and murine FAP and kill FAP-expressing cells. Figure 3A It shows a method for identifying a group of anti-FAP candidate scFvs. Figure 3B It shows an exemplary workflow for a transient anti-FAP iNKT assay. Figure 3C It shows the expression levels of FAP in target T2 cells electroporated with 0.3 μg or 10 μg of human or murine FAP. Figure 3D It shows the binding of human or murine FAP to a group of exemplary anti-FAP scFvs. Figure 3E It shows the target cell killing by a group of exemplary anti-FAP CAR iNKT cells. Figure 3F And G respectively show the binding of a different group of candidate anti-FAP CAR candidates expressed in 2 different donors. Figure 3F It shows the binding of iNKT cells derived from donor 1 to human FAP. Figure 3G It shows the binding of a different group of candidate anti-FAP CAR iNKT cells derived from donor 2 to human FAP. Figures 3H - 3I It shows the killing of target cells at an E:T ratio of 1:1 ( Figure 3H ) or 5:1 ( Figure 3I ). Compared with the mock control, all anti-FAP candidate CAR iNKT candidates were able to kill more U87 and HS683 cells. Figures 3J - 3K It shows the activation of target cell-induced iNKT cells determined by measuring the cytokine CD25 and CD69 markers at an E:T ratio of 1:1 ( Figure 3J ) or 5:1 ( Figure 3K ). Figures 3L - 3M It shows the activation of target cell-induced iNKT cells determined by measuring the 4-1bb marker at an E:T ratio of 1:1 ( Figure 3K ) or 5:1 ( Figure 3M ). Figure 3NShown is that the candidate selection method identified two different anti-FAP CARs. Left panel. Cytotoxicity of T2 cells highly expressing murine FAP and highly expressing human FAP. The best candidates selected by CARDIS were transiently transfected into iNKT cells. Meanwhile, T2 cells were transfected with a higher amount of human or murine FAP. The next day, the CAR-iNKT cells were co-cultured with murine or human T2high at an E:T ratio of 5:1, and the killing of target cells was evaluated after 24 hours. All candidates were able to potently kill cells highly expressing murine or human FAP. Right panel. Cytotoxicity of T2 cells lowly expressing murine FAP and lowly expressing human FAP. The transiently transfected CAR-iNKT cells were co-cultured with murine or human T2low cells. The killing of target cells was evaluated after 24 hours. Two groups of cells were disclosed: the first group retained its ability to potently kill T2low cells, while the second group retained those cells carrying a level of FAP similar to that of CAF-S3 or normal fibroblasts. Importantly, both groups recognized murine and human FAP at the same level.

[0064] Figures 4A - 4D Shown is an in vitro assay evaluating candidate anti-FAP CAR iNKT cells. Figures 4A - 4B Shown is that FAP-CAR iNKT candidates respond to FAP+ tumors in vitro ( Figure 4A (Donor 1) and Figure 4B (Donor 2)). Figure 4C And 4D Shown is that anti-FAP CAR iNKT cells have multiple functions in response to A-375-FAP tumor cells in vitro ( Figure 4C (Donor 1) and Figure 4D (Donor 2)).

[0065] Figures 5A - 5E Shown is that FAP-CAR iNKT exhibits in vivo tumor control against FAP-expressing tumors in the melanoma A-375-FAP+ cancer model. Figure 5A Shown is a schematic diagram of an in vivo A-375-FAP tumor challenge using FAP-CAR iNKT cells. Figure 5B Shown is a graph depicting tumor growth. Figure 5C Shown is a graph representing the tumor growth of each individual mouse in each group. Figure 5D Shown is the survival analysis of mice bearing A-375-FAP tumors untreated, treated with MiNK FAP-CAR iNKT cells, treated with sibrotuzumab-CAR NKT cells, or treated with control BCMA-CAR iNKT cells. Figure 5E Shown is the weight of tumor growth in each treatment group.

[0066] Figures 6A - 6B It is shown that FAP-CAR-IL-15iNKT cells have the ability to enhance the cytotoxicity of T cells that have experienced repeated antigen stimulation. Figure 6A The cytotoxicity of NYESO-1-specific T cells that have undergone multiple rounds of antigen exposure (up to 8 rounds [R#]) against tumor cells expressing HLA-A*02:01-SLLMWITQC is shown. Figure 6B The cytotoxicity of NYESO-1-specific T cells against tumor cells expressing NYESO-1 that have been exposed to the antigen two times (AE2), three times (AE3), or six times (AE6) and co-cultured in either a separate medium or with the supernatant from activated FAP-CAR-IL-15iNKT cells is shown.

[0067] Figures 7A - 7B It is shown that E07 FAP-CAR-IL-15iNKT cells have the ability to target murine FAP. Figure 7A The cytotoxicity of FAP-CAR-IL-15iNKT cells in causing cytotoxicity against MC38 tumor cells engineered to express murine FAP at different effector-to-target (E:T) ratios is shown. Figure 7B The cytotoxicity of FAP-CAR-IL-15iNKT cells in causing cytotoxicity against 4T1 tumor cells engineered to express murine FAP at different effector-to-target (E:T) ratios is shown.

[0068] Figures 8A - 8H A schematic diagram of an in vivo NSCLC xenograft model is shown, which was developed to illustrate the mode of action of the FAP-CAR-iNKT drug that targets cancer-associated fibroblasts (CAFs) expressing FAP, while the tumor cells (A549) do not express FAP. Figure 8A The experimental design of an NSCLC xenograft tumor model treated with FAP-CAR iNKT cells is shown. Figure 8B A schematic diagram illustrating the mode of action of FAP-CAR-IL-15iNKT cell drug is shown. Figure 8C The establishment of tumors in the lungs of mice determined by bioluminescence imaging of NOG mice that were intravenously injected with 2 million A-549 tumor cells expressing HLA-A*02:01-SLLMWITQC and nano-luciferase is shown. The images shown are from untreated animals, indicating the presence of a robust tumor mass in the lungs of the mice seven days after tumor inoculation. Figure 8DH&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 tumor burden kinetics determined by bioluminescence imaging of tumor-bearing animals over 21 days. The right panel shows the quantification of tumor areas in the lungs of tumor-bearing mice over the same 21 days by histological analysis. Both the quantification of tumor burden by bioluminescence imaging and histological characterization indicate that tumors grow progressively under untreated conditions. Figure 8F FAP expression evaluated by immunohistochemistry is shown. The expression of FAP in the whole lungs (left panel) and representative sections (right panel) of three representative mice at weeks 1 - 3 after tumor establishment is shown. The last three panels on the right show normal lungs and controls (no primary tumor and isotype). The graphical quantification of FAP expression is shown below. Figure 8G α - smooth muscle actin (α - SMA) shown by immunohistochemistry at weeks 1 and 3 after tumor establishment is shown. The graphical quantification of α - SMA expression is shown on the right, where there is a moderate but small increase in the expression of α - SMA in tumors from weeks 1 - 3 after tumor establishment. Figure 8H Expression of fap, col11A1, Il6, and Tgfb in murine lungs analyzed by qPCR is shown.

[0069] Figures 9A - 9B FAP - CAR - IL - 15iNKT promotes NSCLC tumor control is shown. Figure 9A Tumor burdens of NOG mice attacked with A - 549 tumor cells expressing HLA - A*02:01 - SLLMWITQC are shown, which were untreated or treated with non - specific CAR - IL - 15iNKT cells alone, FAP - CAR - IL - 15iNKT cells alone, T cells expressing NYESO - 1 TCR, or FAP - CAR - IL - 15iNKT and T cells expressing NYESO TCR. Figure 9B Shown at Figure 9A Survival analysis of mice treated under the conditions described is shown.

[0070] Figures 10A - 10E Mice treated with MiNK FAP - CAR - IL - 15 are shown, demonstrating a reduction in tumor burden in lung tissue after receiving T cells or FAP - CAR - IL - 15 plus T cells. Figure 10A Bioluminescence imaging of mice treated with FAP - CAR - IL - 15iNKT + T cells (left panel) and T cells alone (right panel) at days 18 and 25 is shown. Figure 10B The percentage change in tumor burden from day 3 to day 24 after tumor injection in mice treated with T cells alone or FAP - CAR - IL - 15iNKT cells + T cells is shown.Figure 10C Shows H&E staining of the lungs of mice treated with FAP-CAR-IL-15iNKT+T cells or T cells harvested on day 25 (left panel). The right panel shows quantification of the lung tumor surface area in mice treated with FAP-CAR-IL-15iNKT cells+T cells or T cells only. Figure 10D Shows immunohistochemical staining of FAP in the lungs of mice treated with T cells or FAP-CAR-IL-15+T cells collected on day 25 (left panel). The right panel shows quantification of FAP expression in the lungs of mice treated with FAP-CAR-IL-15iNKT+T cells or T cells only. Figure 10E Shows immunohistochemical staining of α-SMA in the lungs of mice treated with T cells or FAP-CAR-IL-15iNKT+T cells collected on day 25 (left panel). The right panel shows quantification of the lung tumor surface area in mice treated with FAP-CAR-IL-15iNKT cells+T cells or T cells only.

[0071] Figures 11A - 11E Shows that animals treated with FAP-CAR-IL-15iNKT showed higher levels of immune cell activation and tumor tissue infiltration. Figure 11A Shows measurements of IFN-γ in the sera of mice treated with FAP-CAR-IL-15iNKT+T cells or T cells only. Figure 11B Shows quantification of the number of iNKT cells detected by flow cytometry in lung tissues from the FAP-CAR-IL-15iNKT+T cell group and the T cell only group. Figure 11C Shows quantification of the number of T cells detected by flow cytometry in lung tissues from the FAP-CAR-IL-15iNKT+T cell group and the T cell only group. Figure 11D Shows measurements of iNKT cell proliferation in the blood, lungs, spleen, and bone marrow tissues of animals treated with FAP-CAR-IL-15iNKT+T cells. Figure 11E Shows measurements of T cell activation and proliferation in the blood, lungs, spleen, and bone marrow tissues of animals treated with FAP-CAR-IL-15iNKT+T cells compared to animals treated with T cells only.

[0072] Figures 12A - 12F Shows the configuration of the second-generation CAR and the cell killing assay testing this CAR. Figure 12AShows a modular FAP-CAR iNKT library generated based on the structure of the second-generation CAR, containing four different modules (scFv, TM / hinge region, the first ICD region, and the second ICD region). Each region has the following number of possible domains: scFv (3), TM / hinge (3), the first ICD region (24), the second ICD region (24), resulting in a library with a total of 5,184 possible combinations. This library was packaged as lentivirus and transduced into iNKT cells, and FAP-CAR+iNKT was enriched by sorting on the 17th day after transduction. Figure 12B Shows that on the 21st day after transduction, iNKT cells were co-cultured with FAP-expressing antigen-presenting cells to specifically activate FAP-CAR iNKT and enrich ICDs with good activation / proliferation. On the 35th day after transduction, another round of co-culture with FAP-expressing antigen-presenting cells was carried out for 14 days, and more than 15,000 cells were enriched after 4 weeks of co-culture. Figure 12C Shows the nanopore sequencing results of the DNA recovered from the initial plasmid library and the DNA extracted from aliquots obtained on the 21st day after iNKT transduction and on the 14th day after the first and second rounds of enrichment. Figures 12D - 12F Shows the results of iNKT cells killing cells with different configurations. mRNA encoding the FAP-CAR construct (or water for mock) was introduced into iNKT cells by electroporation, and different domains of the tested CAR are listed on the x-axis. High-level (2 μg mRNA) or low-level (100 ng mRNA) FAP-encoding mRNA or blank (water) was introduced into target cells (T2) by electroporation. 24 hours after electroporation, the target cells were labeled with CFSE, then mixed with iNKT cells at an effector:target cell ratio of 5:1, and co-cultured for 24 hours. Then, the killing effect was evaluated by analyzing the percentage of CFSE+ cells stained positive with the live / dead dye by flow cytometry.

[0073] Figures 13A - 13G Shows the Biacore binding kinetics and epitope mapping of the anti-FAP antibody. Figure 13A Shows the SPR binding of E07, D01, B08, A07, and D05 to human and mouse FAP. Figure 13B Shows the results of epitope grouping using the 'Tandem using dual' method. White - no competition. Yellow - with competition. The experiment was operated on a Biacore 8K. Figure 13C Shows the crystal structures of FAP and its related homolog DPP4. Figure 13D Shows the human FAP (hFAP) and DPP4 (hDPP4) chimeric constructs for epitope mapping. Figures 13E - 13GIt is shown that D01, E07, and B08 do not bind to human DPP4, but bind to amino acids 141 to 290 in the human FAP domain.

[0074] Figure 14 Show the expansion of FAP-CAR-IL15-iNKT cells.

[0075] Figure 15 It is shown that iNKT cells expressing a CAR comprising D01 or B08 kill cancer cells expressing FAP. iNKT cells were transiently transfected with D01 CAR or B08 CAR and co-cultured with T2 cells transfected to express FAP at low or high levels at an effector:target ratio of 5:1. After 24 hours, the killing of target cells was assessed by flow cytometry. Detailed Description

[0076] The present disclosure is at least in part based on the discovery that genetically modified cells (e.g., iNKT cells) expressing a chimeric antigen receptor targeting fibroblast activation protein (FAP) are capable of killing cells expressing FAP in the tumor microenvironment (TME) (e.g., cancer-associated fibroblasts (CAFs)). In some embodiments, anti-FAP CAR iNKT cells are engineered to express an armored molecule (e.g., soluble IL-15). In some embodiments, anti-FAP CAR iNKT cells reduce immunosuppression in the TME, thereby increasing the efficacy of other cancer therapies (e.g., T cells targeting tumor antigens). In some embodiments, the anti-FAP CAR iNKT cells described in the present disclosure exhibit improved properties relative to existing FAP-CAR cell therapies in the art, including killing cancer cells expressing FAP in vitro and in vivo; and enhanced persistence in subjects receiving the therapy.

[0077] The foregoing and other aspects, embodiments, operations, functions, features, and implementations of the present teachings can be more fully understood by the following description in conjunction with the accompanying drawings.

[0078] I. Definitions

[0079] Additional terms of the present disclosure are defined throughout this specification.

[0080] Administering: As used herein, the terms "administering" or "administration" refer to providing a composition to a subject in a physiologically and / or pharmacologically useful manner (e.g., to treat a disorder of the subject).

[0081] Affinity matured antibody: “Affinity matured antibody” is used herein to refer to an antibody having one or more alterations in one or more CDRs, which alterations improve the affinity (i.e., KD, kd, or ka) of the antibody for a target antigen as compared to a parental antibody that does not have said one or more alterations. In some embodiments, the affinity matured antibody will have a nanomolar or even picomolar affinity for the target antigen. A variety of procedures are known in the art for generating affinity matured antibodies, including screening of combinatorial antibody libraries prepared by phage display. For example, Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation achieved by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in the following: 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). U.S. Patent No. 6,914,128 B1 describes selective mutagenesis of amino acid residues having enhanced activity at selected mutagenesis positions and at contact or hypermutable positions.

[0082] Antibody: As used herein, the term "antibody" or "antibodies" refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigen determinant (e.g., a complementarity-determining region that specifically binds 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 chains 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, intracellular antibodies, heteroconjugate antibodies, antibody - drug conjugates, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above antibodies. 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, an F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from a camel antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a bifunctional antibody. In some embodiments, the antibody comprises a framework having a germline sequence from a particular 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, VH comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any heavy chain variable domain provided herein. In some embodiments, VH comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any light chain variable domain provided herein. In some embodiments, the antibody comprises a constant domain, such as an Fc region. An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of human IgG heavy chain and light chain constant domains and their functional variants are known. In another embodiment, the antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. With respect to the heavy chain, in some embodiments, the heavy chain of the antibody described herein can be an α (alpha), δ (delta), ε (epsilon), γ (gamma), or μ (mu) heavy chain.In some embodiments, the heavy chain of the antibodies described herein can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In a particular embodiment, the antibodies described herein comprise human γ1 CH1, CH2, and / or CH3 domains. In some embodiments, the V. H The amino acid sequence of the domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any sequence known in the art. Non-limiting examples of human constant region sequences have been described in the art, see, for example, U.S. Patent 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, which is 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, glypiation (GPI anchor linkage), and / or phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or polysaccharides. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, or phospholipid units.

[0083] About: As used herein, the term "about" or "approximately" when used in reference to one or more values of interest refers to a value similar to the specified reference value. In certain embodiments, the term "about" or "approximately" refers to a range of values within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the reference value, unless otherwise stated or apparent from the context (unless the value exceeds 100% of the possible value).

[0084] 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 (such as an antibody) and its binding partner (such as an epitope). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair. The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (K D )). Affinity can be measured and / or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (KD ), and the equilibrium association constant (K A ). K D is calculated from the quotient of k off / k on , while K A is calculated from the quotient of k on / k off . k on refers to, for example, the association rate constant of an antibody with an epitope, while k off refers to, for example, the dissociation rate constant of an antibody with an epitope.

[0085] 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 fluorescence assays). Exemplary conditions for evaluating binding affinity are in 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). These techniques can be used to measure the change in the concentration of the binding protein as a function of the concentration of the target protein. The concentration of the binding protein ([Bound]) is generally related to the concentration of the free target protein ([Free]) and is represented by the following formula:

[0086] [Bound]=[Free] / (Kd + [Free])

[0087] However, it is not always necessary to precisely determine K A , because sometimes it is sufficient to obtain a quantitative measure of the affinity, for example, the affinity determined by methods such as ELISA or FACS analysis is proportional to K A and can therefore be used for comparison, e.g., to determine whether a higher affinity, such as being 2-fold higher, can obtain a qualitative measure of the affinity, or to obtain an inference of the affinity, such as through activity in a functional assay, e.g., in vitro or in vivo assays.

[0088] Binding moiety: As used herein, the term "binding moiety" refers to a molecule or a part of a molecule that specifically binds or interacts with a target molecule via covalent and / or non-covalent interactions. The binding moieties of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof (e.g., antibodies, scFv, Fab, Fab', single-chain binding fragments), binding peptides, ligands, receptors, oligonucleotides, small molecules, or aptamers.

[0089] CDR: As used herein, the term "CDR" refers to the complementarity determining regions within the variable sequences of an antibody. A typical antibody molecule comprises a variable heavy chain region (VH) and a variable light chain region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into several hypervariable regions, also known as "complementarity determining regions" ("CDR"), interspersed with more conserved regions called "framework regions" ("FR"). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The boundaries of the framework regions and CDRs can be precisely identified using methods known in the art, such as 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. See, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; International Immunogenetics Information System imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., Nucleic Acids Res., 29:207-209 (2001); Lefranc, M.-P., 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; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also bioinf.org.uk / abs. As used herein, a CDR can refer to a CDR defined by any method known in the art. Two antibodies having the same CDR means that, by the same method, e.g., Kabat definition determination, these two antibodies have the same amino acid sequence of the said CDR.

[0090] In general, each of the variable regions of the heavy and light chains has three CDRs, which for each variable region are designated CDR1, CDR2, and CDR3, respectively. As used herein, the term "CDR set" refers to a set of three CDRs that occur in a single variable region capable of binding an antigen. The exact boundaries of these CDRs are defined differently according to different systems. 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 a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Sub-portions of the CDRs may be designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" denote the light chain region and heavy chain region, respectively. These regions may be referred to as Chothia CDRs, the boundaries of which overlap with the Kabat CDRs. Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)) described other boundaries defining CDRs that overlap with the Kabat CDRs. Still other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with the Kabat CDRs, although they may be shortened or lengthened based on prediction or experimental findings, i.e., specific residues or groups of residues or even entire CDRs will not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems.

[0091] When using different definition systems (e.g., IMGT definition, Kabat definition, or Chothia definition), the CDRs of an antibody may have different amino acid sequences. The definition system numbers each amino acid in a given antibody sequence (e.g., the VH or VL sequences listed in Table 2), and the numbering corresponding to the heavy and light chain CDRs is provided in Table 1. The CDRs of the anti-FAP antibodies provided herein (e.g., the CDRs listed in Table 2) are defined according to the Kabat definition. Those skilled in the art can derive the CDR sequences using the different numbering systems for the anti-FAP antibodies provided in Table 2.

[0092] Table 1. CDR Definitions

[0093]

[0094]

[0095] 1 imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999)

[0096] 2 Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242

[0097] 3 Chothia et al., J. Mol. Biol. 196:901-917 (1987))

[0098] Chimeric antigen receptor: As used herein, "chimeric antigen receptor" or "CAR" refers to an engineered receptor that has binding specificity for an antigen (e.g., FAP) or other ligand or molecule on an immune effector cell (e.g., T cell, NK cell, NKT cell, iNKT cell). A chimeric antigen receptor generally comprises at least an extracellular ligand-binding domain or portion capable of specifically binding an antigen and an intracellular domain comprising one or more signaling domains and / or co-stimulatory domains.

[0099] In some embodiments, the extracellular ligand-binding domain of the CAR is in the form of a binding protein, small molecule, peptide, targeting agent, agonist or 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 targeting FAP).

[0100] In a particular embodiment, 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 preferentially present on the cell surface, such as a cancer cell or other pathogenic cell or particle).

[0101] 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 upon 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, CD3ζ. In some embodiments, the intracellular domain further comprises one or more intracellular co-stimulatory domains, such as those described herein, that transmit co-stimulatory signals that promote cell proliferation, cell survival, and / or cytokine secretion upon binding of the extracellular domain. Such intracellular co-stimulatory domains can include, but are not limited to, any co-stimulatory domain disclosed herein or domains 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. Other suitable co-stimulatory domains are described in PCT International Application No. PCT / US2017 / 055133, which is incorporated herein by reference in its entirety. In some embodiments, the co-stimulatory domain is 4-1BB (CD137). In some embodiments, the chimeric antigen receptor further comprises additional structural elements, including a transmembrane domain linked to the extracellular ligand-binding domain via a hinge or linker sequence. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a subunit of the T cell receptor (i.e., the α, β, γ, or ζ polypeptide that makes up the CD3 complex), the IL2 receptor p55 (α chain), p75 (β chain), or γ chain, an Fc receptor subunit chain (e.g., Fcγ receptor III), or a CD protein, such as the CD8α chain. Alternatively, the transmembrane domain can be synthetic and can primarily comprise hydrophobic residues, such as leucine and valine. In some embodiments, the CAR comprises a CD8 transmembrane domain. The hinge region refers to any oligopeptide or polypeptide that serves to link the transmembrane domain to the extracellular ligand-binding domain. For example, the hinge region can comprise up to 300 amino acids, 10 to 100 amino acids, and 25 to 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 can be a completely synthetic hinge sequence. In some embodiments, the hinge domain can comprise a portion of the human CD8α chain, the FcγRIIIa receptor, or IgG1. In some embodiments, the CAR further comprises a CD8 hinge region.

[0102] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species, but in which the sequence of one or more CDR regions of VH and / or VL has been replaced by CDR sequences from another species. For example, an antibody having murine heavy and light chain variable regions in which one or more murine CDRs (e.g., CDR3) have been replaced by human CDR sequences.

[0103] 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. For example, an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0104] Complementary: As used herein, the term "complementary" refers to the ability of two nucleotides or two sets of nucleotides to pair precisely. Specifically, complementary is a term that characterizes the degree of hydrogen bond pairing that enables binding between two nucleotides or two sets of nucleotides. For example, if the base at a position on an oligonucleotide can form a hydrogen bond with the base at the corresponding position on a target nucleic acid (e.g., mRNA), the two bases are considered complementary at that position. Base pairing can include 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, an adenine-type base (A) is complementary to a thymine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanine-type base (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U, or T and are considered complementary to any A, C, U, or T. Inosine (I) is also regarded as a universal base in the art and is considered complementary to any A, C, U, or T.

[0105] 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 altering polypeptide sequences known to those of ordinary skill in the art, such as those found in the following references that compile such methods: for example, Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012; or Current Protocols in Molecular Biology, edited by F. M. Ausubel et al., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids 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.

[0106] Co-stimulatory domain: As used herein, "co-stimulatory domain" refers to a polypeptide domain that transmits intracellular proliferation and / or cell survival signals upon activation. Activation of the co-stimulatory domain can occur after homodimerization of two co-stimulatory domain polypeptides. Activation can also occur, for example, after activation of a construct that contains a co-stimulatory domain (such as a chimeric antigen receptor or an inducible regulatory construct). Generally, the co-stimulatory domain can be derived from a transmembrane co-stimulatory receptor, particularly the intracellular portion of a co-stimulatory receptor. Non-limiting examples of co-stimulatory polypeptides include, but are not limited to, 4-1BB, CD28, ICOS, OX-40, and CD27, and any other co-stimulatory domain further described herein. In some embodiments, the CARs described herein contain a 4-1BB co-stimulatory domain.

[0107] Co-stimulatory signal: As used herein, "co-stimulatory signal" refers to an intracellular signal induced by a co-stimulatory domain that promotes cell proliferation, in vitro and / or in vivo expansion of a cell population, promotes cell survival, regulates (such as upregulates or downregulates) the secretion of cytokines, and / or regulates the production and / or secretion of other immunomodulatory molecules. In some embodiments, the co-stimulatory signal is induced after homodimerization of two co-stimulatory domain polypeptides. In some embodiments, the co-stimulatory signal is induced after activation of a construct that contains a co-stimulatory domain (such as a chimeric antigen receptor or an inducible regulatory construct).

[0108] Cross-reactivity: As used herein, and in the context of a targeting agent (e.g., an antibody), the term "cross-reactivity" refers to the ability of the agent to specifically bind more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity. For example, in some embodiments, an antibody that is cross-reactive with human and non-human primate antigens of a similar type or class (e.g., human FAP and non-human primate FAP) is capable of binding the human antigen and the non-human primate antigen with similar affinity or avidity. In some embodiments, the antibody is cross-reactive with human and rodent antigens of a similar type or class. In some embodiments, the antibody is cross-reactive with rodent and non-human primate antigens of a similar type or class. In some embodiments, the antibody is cross-reactive with human, non-human primate, and rodent antigens of a similar type or class.

[0109] Effective amount: As used herein, "effective amount" refers to the amount of each active agent (e.g., iNKT cells having an FAP CAR binding moiety) required, alone or in combination with one or more other active agents, to produce a therapeutic effect in a subject. In some embodiments, the therapeutic effect includes, but is not limited to, reducing tumor size, eradicating the tumor, or alleviating tumor-related symptoms.

[0110] Epitope: As used herein, "epitope" is a term in the art and refers to a local region of an antigen (e.g., a peptide or a peptide-MHC complex) to which an antibody or a chimeric antigen receptor can bind. In certain embodiments, the epitope of an antibody or a chimeric antigen receptor can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange combined 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., Giegé R et al. (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303, each of which is incorporated herein by reference in its entirety). Antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software, such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) Volumes 114 and 115, edited by Wyckoff HW et al.; U.S. 20040014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, edited by Carter CW; Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323), each of which is incorporated herein by reference in its entirety). Mutagenesis mapping studies can be done using any method known to those of skill in the art. For descriptions of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, e.g., Champe M et al. (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085, each of which is incorporated herein by reference in its entirety. In some embodiments, alanine scanning mutagenesis studies are used to determine the epitope of an antigen. In some embodiments, hydrogen / deuterium exchange combined with mass spectrometry is used to determine the epitope of an antigen.

[0111] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of the CDR sequences can be determined by different systems, the meaning of the framework sequences will correspondingly have different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain, and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. In the absence of designating a particular subregion as FR1, FR2, FR3, or FR4, the framework regions referred to by others represent the combined FR within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four subregions, while FRs represents two or more of the four subregions that make up the framework region. Human heavy and light chain receptor sequences are known in the art. In one embodiment, receptor sequences known in the art can be used in the antibodies disclosed herein.

[0112] 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 (DPP II, quiescent cell proline dipeptidase), DPP8, DPP9, and prolyl carboxypeptidase (PCP, angiotensinase C). FAP contains both dipeptidase and endopeptidase activities. An exemplary human FAP amino acid sequence is shown in accession number 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 shown in accession numbers CAA71116.1 (SEQ ID NO:36), AND76664.1 (SEQ ID NO:37), and AAH19190.1 (SEQ ID NO:38).

[0113] Fibroblast activation protein-α (FAP) is a cell surface antigen expressed on a variety of cells such as 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 such as fibroblasts in the tumor microenvironment is higher than the expression level of FAP on normal tissues. FAP can also be shed from the plasma 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). Thus, it is not restricted to the cell surface.

[0114] 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 include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation), such as amino acid residues in the CDRs and particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which the CDR sequences from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0115] Humanized antibody: The term "humanized antibody" refers to an antibody that contains the heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which the V H and / or V LAt least a portion of the sequence has been altered to be more "human-like", i.e., more similar to antibodies of the human germline variable sequences. 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 murine anti-FAP monoclonal antibodies using conventional hybridoma techniques and subsequently humanizing them using in vitro genetic engineering, such as those disclosed in PCT Publication No. WO 2005 / 123126A2 by Kasaian et al. In some embodiments, the humanized antibody comprises a human immunoglobulin (acceptor antibody) having a desired specificity, affinity, and capacity in which residues from the acceptor complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species, such as a mouse, rat, or rabbit (donor antibody). In some embodiments, residues of the Fv framework regions (FRs) of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, the humanized antibody may comprise residues not found in either the acceptor antibody or the imported CDR or framework sequences, but including such residues is for further improving and optimizing antibody performance. Generally, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of the FR regions are the FRs of the human immunoglobulin consensus sequence. The humanized antibody optionally will also comprise at least a portion of the immunoglobulin constant region or domain (Fc), usually at least a portion of the constant region or domain (Fc) of a human immunoglobulin. The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) altered relative to the original antibody, and these CDRs are also referred to as CDRs derived from one or more CDRs of the original antibody. Affinity maturation may also be involved in humanized antibodies.

[0116] Invariant natural killer T cells (iNKT): As used herein, the term "invariant natural killer T cells" or "invariant NKT cells", "iNKT cells" or "type I NKT cells" refers to a population of T lymphocytes that express a conserved semi-invariant TCR specific for lipid antigens (Ag), which is restricted to the monomorphic class I MHC-related molecule CD1d. Natural killer T cells (NKT cells) were initially characterized in mice as T cells that express TCR and NK1.1 (NKR-P1a-c or CD161, a C-type lectin NK receptor). Invariant NKT (iNKT) cells express a semi-invariant αβ TCR (formed, for example, by the invariant TRAV11-TRAJ18(4) rearrangement in mice or the homologous invariant TRAV10-TRAJ18 chain in humans), which pairs with a limited diversity of Vβ chains that are predominantly TRBV1, TRBV29 or TRBV13(6) in mice and mainly TRBV25 in humans (see, for example, 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) 180:1171–6. 10.1084). The semi-invariant TCR recognizes exogenous and endogenous lipid antigens presented by the monomorphic class I MHC-related molecule CD1d (see, for example, 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 Ag includes classical 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 bacterially derived Ag can activate iNKT cells.

[0117] Compared with T cells, iNKT cells undergo a unique developmental pathway to acquire innate effector functions that are already present in the thymus. Thymic iNKT cells do express markers that are typically upregulated on peripheral effector / memory T cells (such as CD44 and CD69) as well as unique NK differentiation markers, such as NK1.1 (in some mouse genetic backgrounds, and CD161 in humans), CD122 (IL-2R / IL-15Rβ chain), CD94 / NKG2, and Ly49(AJ), as well as broad-spectrum TH1 / 2 / 17 effector cytokines. After migrating to peripheral tissues, iNKT cells form a population of tissue-resident cells that are responsible for monitoring cellular integrity and rapidly responding to local injury and inflammation, thereby initiating responses in innate and adaptive immune response cells.

[0118] Since iNKT cells are capable of rapidly producing IFNγ, IL-4, or both, they have been found to play a role in various diseases by establishing environment-dependent Th1 or Th2 immune responses. During bacterial and viral infections, iNKT cells generally help to control pathogens early by establishing an effective Th1 response. In mouse and human studies, the role of iNKT cells has been described in diseases associated with excessive Th1 responses, such as type 1 diabetes and chronic obstructive pulmonary disease. The role of iNKT cells in helping to suppress Th1 responses and drive tolerance responses to grafts has also been described. For example, after hematopoietic stem cell transplantation, the presence of iNKT cells predicts improved survival and reduced graft-versus-host disease (GvHD) in patients and preclinical models.

[0119] Isolated antibody: As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds FAP is substantially free of antibodies that specifically bind antigens other than FAP). In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0120] Percent Identity: A “percent identity” or “percent homology” between two sequences (e.g., an amino acid sequence or a nucleic acid sequence) can be determined using a mathematical algorithm. A particular non-limiting example of a mathematical algorithm used for comparing two sequences is the algorithm of Karlin S and Altschul SF (1993) PNAS 90:5873-5877, modified from Karlin S and Altschul SF (1990) PNAS 87:2264-2268, 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 are incorporated herein by reference in their entirety. One can perform a BLAST nucleotide search using the NBLAST nucleotide program parameters settings, e.g., score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. One can perform a BLAST protein search using the XBLAST program parameters settings, e.g., score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul SF et al. (1997) Nuc Acids Res 25:3389-3402 can be utilized, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterative search to detect distant relationships between molecules. Id. When using the BLAST, gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another particular non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0121] Techniques similar to those described above can be used to determine the percent identity between two sequences, whether or not gaps are present. When calculating percent identity, only exact matches are typically counted.

[0122] Recombinant Antibodies: As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail in the present disclosure); antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H.R., (1997) TIBTech. 15:62-70; Azzazy H. and Highsmith W.E., (2002) Clin. Biochem. 35:425-445; Gavilondo J.V. and Larrick J.W. (2002) BioTechniques 29:128-145; Hoogenboom H. and Chames P. (2000) Immunology Today 21:371-378); antibodies isolated from transgenic animals (e.g., mice) that are engineered to express human immunoglobulin genes (see, e.g., Taylor, L.D. et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A. and Green L.L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al. (2000) Immunology Today 21:364-370); or antibodies prepared, expressed, produced, or isolated by any other means that involve splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions that are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when transgenic animals that express human Ig are used), and thus the amino acid sequences of the V H region and the V L region, although derived from and related to human germline V H and V L sequences, may not be present in the human antibody germline repertoire that exists in nature. One embodiment of the present disclosure provides fully human antibodies that are capable of binding to human FAP, which can be generated using techniques well known in the art, such as, but not limited to, using human Ig phage libraries, such as those disclosed by Jermutus et al. in PCT Publication No. WO 2005 / 007699 A2.

[0123] Single-chain variable fragment (scFv): As used herein, the term "single-chain variable fragment (scFv)" refers to a fusion protein of the variable region of an immunoglobulin heavy chain (VH) and the variable region of a light chain (VL) linked by a short linker peptide. A linker refers to a peptide or short oligopeptide sequence used to join two subunits into a single polypeptide. The linker can have a sequence found in natural proteins or can be an artificial sequence not found in any natural protein. The linker can be flexible and lack secondary structure or can have a tendency to form a specific three-dimensional structure under physiological conditions. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker is rich in serine or threonine. In some embodiments, the scFv is a fusion protein with the N-terminus of VH linked to the C-terminus of VL. In some embodiments, the scFv is a fusion protein with the N-terminus of VL linked to the C-terminus of VH. Despite the removal of the constant regions and the introduction of a linker, the scFv retains the specificity of the original immunoglobulin. In some embodiments, scFv can be produced to facilitate phage display. In some embodiments, scFv can be directly produced from the heavy and light chains of a clone derived from a hybridoma. ScFv has many uses, such as flow cytometry, immunohistochemistry, and as the antigen-binding domain of a chimeric antigen receptor.

[0124] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity such that the molecule can be used to distinguish the binding partner from a suitable control in a binding assay or other binding context. In the case of an antibody, the term "specific binding" means that the antibody binds to a particular antigen with a degree of affinity or avidity such that the antibody can be used to distinguish the particular antigen from other antigens, for example, to a degree that allows preferential targeting of certain cells, such as muscle cells, by binding to the antigen, as described herein. In some embodiments, if the K D of the antibody for the target is 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, then the antibody specifically binds to the target. In some embodiments, the antibody specifically binds to FAP.

[0125] Subjects: 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, such as a human patient suffering from or suspected of suffering from a disease. In some embodiments, the subject is a human patient suffering from or suspected of suffering from cancer (e.g., cancer expressing FAP) or other diseases associated with FAP.

[0126] Treatment: As used herein, the term "treating" or "treatment" refers to administering or applying a composition comprising one or more active agents (e.g., an anti-FAP antibody) to a subject suffering from a target disease or disorder (e.g., cancer), having symptoms of the disease / disorder (e.g., tumor growth, tumor metastasis, fatigue, weight changes including unexpected weight loss or gain, pain, fever, non-healing sores, persistent cough or hoarseness, abnormal bleeding or anemia) or having a predisposition to the disease / disorder (e.g., cancer), with the aim of curing, healing, alleviating, reducing, altering, remedying, improving, enhancing or affecting the disorder (e.g., cancer), the symptoms of the disease (e.g., tumor growth, tumor metastasis, fatigue, weight changes including unexpected weight loss or gain, pain, fever, non-healing sores, persistent cough or hoarseness, abnormal bleeding or anemia) or the predisposition to the disease / disorder (e.g., cancer). Alleviating the target disease / disorder includes delaying or preventing the development or progression of the disease, or reducing the severity of the disease.

[0127] Tumor microenvironment (TME): As used herein, the term "tumor microenvironment" or "TME" refers to the fluid, molecules, cells and / or tissues around and / or at the tumor site. The TME can include normal cells, tumor cells, tumor stromal cells (e.g., stromal fibroblasts), blood vessels, blood, immune cells and acellular components (e.g., extracellular matrix such as collagen, fibronectin, hyaluronic acid, laminin and secreted molecules such as cytokines, etc.).

[0128] II. Methods of treating cancer using anti-FAP CAR iNKT cells

[0129] Aspects of the present disclosure provide a variety of genetically modified immune cells (e.g., iNKT cells) expressing chimeric antigen receptors against fibroblast activation protein (FAP) for cancer treatment. In some embodiments, iNKT cells can be engineered to express a chimeric antigen receptor that comprises any of the FAP-binding moieties described herein (e.g., the anti-FAP antibodies or antigen-binding fragments described in Table 2, and the existing FAP-binding moieties provided in Table 3) as an extracellular ligand-binding domain. In some embodiments, the genetically modified iNKT cells expressing 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 a disease (e.g., cancer) using the 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 an armored molecule) that the cell has been engineered to express.

[0130] In some embodiments, FAP is associated with cancer. In some embodiments, FAP has previously been described as promoting tumor growth via multiple mechanisms, including but not limited to: promoting proliferation, invasion, angiogenesis, epithelial-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. September 2020; 39(3):783–803). In some embodiments, FAP is highly expressed in stromal cells (e.g., cancer-associated fibroblasts (CAFs)) in the tumor microenvironment (TME). In some embodiments, FAP plays an immunosuppressive role in the TME. Cancers expressing FAP include but are not limited to: breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, brain cancer, ovarian cancer, myeloma, melanoma, or sarcoma. In some embodiments, FAP is associated with non-cancer diseases, such as various human pathologies, 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 disease progression and severity of cancer and / or non-cancer diseases).

[0131] Therapeutic methods for FAP have been previously described (e.g., Schuberth et al., Treatment of malignant pleural mesothelioma by fibroblast activation protein - specific re - directed T cells. J Transl Med (2013) 11:1–11), such as FAP inhibitors (e.g., Talabostat), anti - FAP antibodies (e.g., sibrotuzumab), FAP vaccines, 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, the clinical application of FAP CAR T cells has not been successful. In some cases, FAP CAR T cells are unable to regulate tumor growth and induce lethal bone toxicity and cachexia (e.g., by lysing 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).

[0132] 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., the CD8 transmembrane domain), a cytoplasmic domain (e.g., the intracellular domain of CD3-ζ, CD28, OX40, 4-1BB, or any combination thereof), and a co-stimulatory domain (e.g., the 4-1BB co-stimulatory 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 redirecting the specificity of primary iNKT cells to a tumor antigen (e.g., FAP) using a CAR. 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 the step of administering to the subject a plurality of iNKT cells expressing a CAR (e.g., an anti-FAP CAR), wherein the CAR comprises a binding portion that specifically interacts with a predetermined target (e.g., FAP), a transmembrane domain (e.g., the CD8 transmembrane domain), a ζ-chain portion comprising an intracellular domain such as human CD3ζ, and a co-stimulatory 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 that the cell is engineered to express (e.g., an immunomodulatory gene product or an armored molecule). In some embodiments, the present disclosure includes a type of cell therapy in which the 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 are capable of killing tumor cells in the recipient's body. Different from antibody therapy, CAR iNKT cells can replicate in vivo, achieving long-term persistence, thereby causing continuous tumor control. In some embodiments, the iNKT cells expressing the anti-FAP CAR secrete sIL-15, which will enhance the persistence of iNKT cells in the subject. In addition, previous studies have shown that the number of iNKT cells in cancer tissues (e.g., solid tumors such as lung cancer, head and neck cancer, colorectal cancer, kidney cancer, etc.) is reduced compared to normal tissues. In some embodiments, in certain cancers, the function of iNKT cells is impaired. Thus, administering the anti-FAP iNKT cells described herein can restore and enhance the iNKT function of the subject, thereby leading to a better prognosis.

[0133] In some embodiments, the antigen-binding domain in the CARs of the present disclosure targets tumor antigens (such as FAP) for the purpose of treating cancer. In some embodiments, the antigen-binding portion in the CARs of the present disclosure is designed for treating a specific cancer (such as a cancer expressing FAP).

[0134] In some embodiments, the anti-FAP CAR iNKT cells of the present disclosure kill stromal cells expressing FAP (such as CAF) in the TME. In some embodiments, the anti-FAP CAR iNKT cells of the present disclosure do not kill normal cells with an FAP expression level lower than that of stromal cells (such as CAF) in cancer. In some embodiments, the expression level of FAP in cancer cells is higher than that in normal tissues, and the cancers include but are not limited to breast cancer, lung cancer, colorectal cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, urothelial cancer or sarcoma. The FAP expression level can be measured by any suitable method known in the art, such as by flow cytometry, immunoblotting, RT-PCR, etc.

[0135] The expression of FAP is an important feature 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 affects the secreted CAF proteome, resulting in reduced levels of anti-angiogenic factors (such as PEDF, angiopoietin-1, VEGFC), and regulates matrix-processing enzymes (such as 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). In addition, FAP remodels the ECM through its endopeptidase activity, i.e., by cleaving collagen and modifying bioactive signaling peptides in cancer (Kelly et al., Fibroblast activation protein-alpha and dipeptidylpeptidase IV (CD26): cell-surface proteases that activate cell signaling and are potential targets for cancer therapy. Drug Resist Updat. 2005;8(1–2):51–58). FAP expression in cancer is thought to be associated with poor prognosis. In some embodiments, high levels of FAP expressed in the TME play 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 drives CAF-mediated 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).

[0136] CAR-T cells targeting FAP have been engineered to target CAFs in various solid cancers 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. Front Immunol. 2020;11). However, these studies observed off-tumor toxicity because although FAP is highly expressed in these cancers, it was also found to have low-level expression in normal cells (e.g., skeletal muscle, adipose tissue, and pancreas, etc.).

[0137] In some embodiments, the present disclosure provides anti-FAP CAR iNKT cells that are engineered to kill stromal cells that highly express FAP (e.g., CAFs in the TME). In some embodiments, the anti-FAP CAR iNKT cells described herein exhibit reduced or absent off-tumor toxicity. In some embodiments, the anti-FAP CAR iNKT cells kill tumor cells that express FAP and / or CAFs that express FAP. In some embodiments, depletion of stromal cells that express FAP (e.g., CAFs in the TME) by the anti-FAP CAR iNKT cells described herein will reduce immunosuppression in the TME. In some embodiments, depletion of stromal cells that express FAP (e.g., CAFs in the TME) by the anti-FAP CAR iNKT cells described herein enables more immune cells to infiltrate into the tumor (e.g., solid tumor). In some embodiments, the anti-FAP CAR iNKT cells described herein are armored (e.g., secrete IL-15) to extend the persistence of the CAR iNKT cells in the TME. In some embodiments, the anti-FAP CAR iNKT cells described herein are armored (e.g., secrete IL-15) to enhance certain properties of the anti-FAP CAR iNKT cells (e.g., enhanced killing efficiency, extended persistence in a subject, and / or enhanced tumor killing of additional cancer therapies (e.g., CAR T cells targeting tumor antigens) relative to iNKT cells without the armored molecule).

[0138] The CAR-modified iNKT cells of the present disclosure can also be used as a vaccine for mammalian ex vivo immunization and / or in vivo treatment. Preferably, the mammal is a human.

[0139] In some embodiments, the iNKT cells described herein can be used to treat and prevent diseases (e.g., cancer) that occur in immunocompromised individuals (e.g., individuals with cancer). Specifically, iNKT cells expressing the anti-FAP CAR of the present disclosure are used to treat cancer, such as FAP-positive cancers (e.g., breast cancer, lung cancer, head and neck cancer, colorectal cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, renal cancer, urothelial cancer, or sarcoma). In certain embodiments, iNKT cells expressing the anti-FAP CAR of the present disclosure are used to treat patients at risk of developing cancer, which is, for example, an FAP-positive cancer (e.g., breast cancer, lung cancer, head and neck cancer, colorectal cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, renal cancer, urothelial cancer, or sarcoma). In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer.

[0140] In certain embodiments, the subject does not undergo lymphodepletion before or concurrently with treatment with the anti-FAP CAR iNKT cells or pharmaceutical composition disclosed herein. Lymphodepletion is typically performed prior to immunotherapy, such as adoptive cell therapy, like CAR T therapy. In some cases, subjects receiving adoptive cell therapy receive a course of chemotherapy to deplete the subject's T cells, e.g., thereby shrinking tumors, altering tumor phenotypes, altering the tumor microenvironment, removing cytokine reservoirs (e.g., making IL-2, IL-7, and IL-15 more available) and suppressing the host immune system. In some cases, lymphodepletion can effectively prolong the persistence of the infused cells and improve treatment utility. However, lymphodepletion has a variety of negative effects, including neutropenia, anemia, thrombocytopenia, and immunosuppression, as well as toxicities associated with lymphodepleting agents such as fludarabine and cyclophosphamide. In some embodiments, subjects receiving iNKT cell therapy (e.g., anti-FAP CAR iNKT cell therapy) do not require lymphodepletion therapy. In certain embodiments, the subject does not receive fludarabine or cyclophosphamide treatment before or concurrently with administration of the anti-FAP CAR iNKT cells or pharmaceutical composition disclosed herein. In some embodiments, iNKT cell therapy (e.g., anti-FAP CAR iNKT cell therapy or its composition) without lymphodepletion can effectively reduce tumor burden and / or increase survival rate. In some embodiments, iNKT cell therapy (e.g., anti-FAP CAR iNKT cell therapy or its composition) without lymphodepletion exhibits long-term efficacy in killing cancer cells. In some embodiments, iNKT cell therapy (e.g., anti-FAP CAR iNKT cell therapy or its composition) without lymphodepletion does not induce graft-versus-host disease (GVHD).

[0141] In some embodiments, iNKT cells expressing the anti-FAP CAR of the present disclosure or a composition comprising such cells can be used or administered to a subject in need thereof to provide anti-tumor immunity; treat or prevent cancer; reduce immunosuppression in the TME; and / or increase the therapeutic efficacy of other therapeutic agents (e.g., CAR-T cells or immune checkpoint inhibitors). In some embodiments, the cancer is a cancer expressing FAP. In some embodiments, the cancer is breast cancer, lung cancer, head and neck cancer, colorectal cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, urothelial cancer, or sarcoma.

[0142] In some embodiments, the iNKT cells expressing the anti-FAP CAR of the present disclosure can be administered alone, or in combination with a diluent and / or other components (such as IL-2 or other cytokines or cell populations) in the form of a composition (such as a pharmaceutical composition). Briefly, the pharmaceutical compositions of the present disclosure can comprise the target cell population described herein, as well as one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline or phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives.

[0143] The pharmaceutical compositions of the present disclosure can be administered in a manner suitable for the disease to be treated (or prevented). Although appropriate dosages can be determined by clinical trials, the amount and frequency of administration will be determined according to factors such as the condition of the patient and the type and severity of the patient's disease. In some embodiments, the compositions of the present disclosure are formulated for intravenous administration.

[0144] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount", the precise amount of the composition of the present disclosure administered can be determined by a physician based on the individual differences in the age, weight, tumor size, degree and condition of infection or metastasis of the patient (subject). Generally, a pharmaceutical composition comprising the CAR-modified immune cells described herein (such as iNKT cells expressing the anti-FAP CAR) can be administered at a dose of 10 4 to 10 9 cells / kg body weight, 10 5 to 10 6 cells / kg body weight, including all integer values within these ranges. The iNKT cell compositions can also be administered multiple times at these doses. The cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0145] The subject compositions can be administered by any convenient means, including infusion, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous (iv) injection, or intraperitoneal administration. In some embodiments, the compositions of immune cells (e.g., iNKT cells) of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the compositions of immune cells (e.g., iNKT cells) of the present disclosure are preferably administered by intravenous injection. The compositions of immune cells (e.g., iNKT cells) can also be directly injected into a tumor, lymph node, or site of disease.

[0146] 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 any number of associated therapeutic modalities (e.g., before, concurrently with, or after administration of the associated therapeutic modality), including but not limited to treatment with antiviral therapies, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or treatment with natalizumab for MS patients or efalizumab for psoriasis patients or other treatments for PML patients and the like. In some embodiments, the iNKT cells of the present disclosure can be used in combination with: chemotherapy; radiation; immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506; antibodies or other immunotoxic agents, such as CAMPATH, anti-CD3 antibodies, or other antibody therapies; cytotoxins; fludarabine; cyclosporin; FK506; rapamycin; mycophenolic acid; steroids; FR901228; cytokines; and irradiation. In some embodiments, the cell compositions of the present disclosure are administered to a patient in combination with (e.g., before, concurrently with, or after these therapies) bone marrow transplantation, T cell ablation therapy, which uses chemotherapeutic agents, such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies (e.g., OKT3 or CAMPATH). In some embodiments, the cell compositions of the present disclosure are administered to a patient after B cell ablation therapy (e.g., an agent reactive with CD20, such as Rituxan). For example, in some embodiments, a subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells (e.g., iNKT cells) of the present disclosure. In another embodiment, the iNKT cells are administered before or after surgery.

[0147] In some embodiments, the present disclosure also encompasses treating cancer using a combination of anti-FAP CAR iNKT cells and 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) targeting a tumor antigen, including but not limited to: ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGF RvIII), vascular endothelial growth factor receptor 2 (VEGFR2), IL13R, GD3, C-type lectin-like molecule 1 (CLL1), cholecystokinin B receptor (CCKBR), gonadotropin-releasing hormone receptor (GnRHR), somatostatin receptor 2 (SSRT2), gastrin-releasing peptide receptor (GRP R), neurokinin 1 receptor (NK1R), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, folate receptor, gp36, TAG-72, glycosphingolipid, glioma-associated antigen, B-human chorionic gonadotropin, alpha-fetoprotein (AFP), neurotensin receptor 1 (NTSR1), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostate-specific protein (prostein), PSMA, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF 2M, neutrophil elastase, ephrin B2, CD22, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, melanocortin 1 receptor (MC1R), 5T4, ROR 1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnC A1), lineage-specific or tissue-specific antigens, such as CD3, CD4, CD8, CD24, CD25, CD 33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, major histocompatibility complex (MHC) molecules, FAP (CD269, TN FRSF 17), CS1, or virus-specific surface antigens, such as HIV-specific antigens (e.g., HIV gpl20); EBV-specific antigens, CMV-specific antigens, HPV-specific antigens (e.g., E6 or E7 oncoproteins), Lassa Virus-specific antigens, influenza virus-specific antigens, and any 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, increases infiltration of CAR T cells into the tumor, thereby enhancing the efficacy of CAR T cells. In some embodiments, the anti-FAP CAR iNKT cells secrete IL-15, thereby enhancing the cytotoxic activity of CAR T cells.

[0148] 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.

[0149] Doses for human administration can be adjusted according to practices generally accepted in the art. For example, for adult patients, the dose of CAMPATH is generally in the range of 1 to about 100 mg, usually administered daily for a period of 1 to 30 days. The preferred daily dose is 1 to 10 mg per day, but in some cases larger doses of up to 40 mg per day can be used (as described in U.S. Patent No. 6,120,766). Strategies for CAR T cell dosing and scheduling have been discussed (Ertl et al., 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal of Translational Medicine, 8:55).

[0150] III. FAP-Binding Portion as a Chimeric Antigen Receptor

[0151] The present disclosure provides an FAP-binding portion (e.g., an antibody or antigen-binding fragment specific for fibroblast activation protein (FAP), an FAP-binding peptide), which can be engineered as the extracellular ligand-binding domain of a chimeric antigen receptor (CAR) expressed by a genetically modified cell (e.g., an iNKT cell).

[0152] In some embodiments, the present disclosure provides an anti-FAP antibody or an antigen-binding fragment thereof as an FAP-binding moiety. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof provided herein is an antibody that binds FAP with high specificity and affinity. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof described herein specifically binds to an extracellular epitope of FAP or an epitope that becomes exposed to the antibody. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof provided herein specifically binds FAP from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof provided herein specifically binds human FAP and mouse FAP. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof described herein specifically binds to an epitope on human FAP and / or mouse FAP. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof described herein can bind to a fragment of human FAP and / or mouse FAP. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof described herein can bind to a fragment of FAP (e.g., human FAP and / or mouse FAP, e.g., SEQ ID NOs: 35-38) that is between about 5 and about 200 amino acids in length, between about 10 and about 200 amino acids in length, between about 20 and about 200 amino acids in length, between about 30 and about 150 amino acids in length, between about 30 and about 120 amino acids in length, between about 30 and about 100 amino acids in length, between about 30 and about 90 amino acids in length, between about 30 and about 80 amino acids in length, between about 30 and about 60 amino acids in length, between about 30 and about 50 amino acids in length, between about 40 and about 80 amino acids in length, or between about 40 and about 60 amino acids in length. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof can bind to an FAP fragment (e.g., human FAP and / or mouse FAP, e.g., SEQ ID NOs: 35-38) that contains a consecutive number of amino acids from the human FAP protein and / or the mouse FAP protein. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof described herein can bind to a fragment that contains 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 the human FAP protein and / or the mouse FAP protein. In some embodiments, the anti-FAP antibody described herein binds to one or more amino acids in amino acid fragment 141-290 of human FAP (e.g., human FAP as shown in SEQ ID NO: 35).In some embodiments, the anti-FAP antibodies described herein bind to one or more contiguous amino acids in amino acid fragment 141-290 of human FAP (e.g., human FAP as shown in SEQ ID NO:35). In some embodiments, the anti-FAP antibodies described herein bind to one or more non-contiguous amino acids in amino acid fragment 141-290 of human FAP (e.g., human FAP as shown 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 amino acid fragment 141-290 of human FAP (e.g., human FAP as shown in SEQ ID NO:35). In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof described herein can bind to a fragment having an amino acid sequence that differs from a fragment of the human FAP protein and / or the mouse FAP protein by at most five amino acids, at most four amino acids, at most three amino acids, at most two amino acids, or at most one amino acid. Exemplary human FAP amino acid sequences are shown in accession number 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 as SEQ ID NO:35.

[0153] An exemplary human FAP amino acid sequence is shown in SEQ ID NO:35 (amino acid fragment 141-290 is bolded and underlined):

[0154] MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEF VRGNEL PRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLPTKYALWWSPNGKFLAY AEFNDKDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSRPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD

[0155] Exemplary mouse FAP amino acid sequences are shown in accession numbers CAA71116.1 (SEQ ID NO:36), AND76664.1 (SEQ ID NO:37), and AAH19190.1 (SEQ ID NO:38).

[0156] An exemplary mouse FAP amino acid sequence is shown in SEQ ID NO:36:

[0157] MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD

[0158] The exemplary mouse FAP amino acid sequence is shown in SEQ ID NO:37:

[0159] LRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGN SPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD

[0160] The exemplary mouse FAP amino acid sequence is shown in SEQ ID NO:38:

[0161] MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGILSGRSQNHLYTHMTHFLKQCFSLSD

[0162] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof specifically binds to FAP (e.g., any one of the human and / or murine FAPs shown in any of SEQ ID NOs: 35-38), and its binding affinity (e.g., expressed as K D is 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. For example, the anti-FAP antibody or antigen-binding fragment thereof of the present disclosure can bind to the FAP protein (e.g., human and / or mouse FAP as shown in any one of SEQ ID NOs: 35-38) with the following affinities: between 5 pM and 500 nM, between 10 pM and 450 nM, between 20 pM and 400 nM, between 30 pM and 350 nM, between 40 pM and 300 nM, between 50 pM and 250 nM, between 60 pM and 200 nM, between 70 pM and 150 nM, between 80 pM and 100 nM, between 80 pM and 90 nM, between 90 pM and 80 nM, between 100 pM and 70 nM, between 200 pM and 60 nM, between 300 pM and 50 nM, between 400 pM and 40 nM, between 500 pM and 30 nM, between 600 pM and 20 nM, between 700 pM and 10 nM, between 800 pM and 5 nM, or between 900 pM and 2 nM, between 30 nM and 700 nM, between 50 nM and 500 nM, between 100 nM and 500 nM, between 100 nM and 200 nM, or between 150 nM and 200 nM.

[0163] In some embodiments, the present disclosure has found that iNKT cells expressing FAP CAR contain anti-FAP antibodies (e.g., human FAP or murine FAP) with a binding affinity for FAP 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 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), and these iNKT cells perform better in killing tumor cells expressing FAP (e.g., human or murine FAP) than iNKT cells expressing FAP CAR containing anti-FAP antibodies with higher binding affinity.

[0164] The present disclosure also includes antibodies that compete with any of the antibodies described herein for binding to the FAP protein (e.g., the human and / or murine FAP proteins shown in any of SEQ ID NOs: 35 - 38) and have an affinity of 100 nM or lower (e.g., 80 nM or lower, 50 nM or lower, 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower). The affinity and binding kinetics of the anti-FAP antibodies or their antigen-binding fragments can be tested using any suitable method, including but not limited to biosensor technologies (e.g., OCTET or BIACORE). In some embodiments, the anti-FAP antibody or its antigen-binding fragment binds to FAP at a sub-nanomolar concentration range of K D Bind to FAP.

[0165] Table 2 provides non-limiting examples of anti-FAP antibodies.

[0166] Table 2. Exemplary Anti-FAP Antibodies (CDRs Defined According to Kabat)

[0167]

[0168]

[0169]

[0170]

[0171] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof of the present disclosure comprises one or more heavy chain CDR (e.g., CDRH1, CDRH2, or CDRH3) amino acid sequences selected from any of the anti-FAP antibodies in Table 2. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof of the present disclosure comprises CDRH1, CDRH2, and CDRH3 provided for any of the antibodies selected from Table 2. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof of the present disclosure comprises one or more light chain CDR (e.g., CDRL1, CDRL2, or CDRL3) amino acid sequences selected from any of the anti-FAP antibodies in Table 2. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof of the present disclosure comprises CDRL1, CDRL2, and CDRL3 provided for any of the anti-FAP antibodies selected from Table 2.

[0172] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 provided for any of the anti-FAP antibodies selected from Table 2. In some embodiments, the CDR3 domains of the antibody heavy and light chains can play a particularly important role in the binding specificity / affinity of the antibody for the antigen. Thus, the anti-FAP antibody or antigen-binding fragment thereof can comprise at least the heavy chain and / or light chain CDR3 of any of the anti-FAP antibodies selected from Table 2.

[0173] 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. A functional variant can contain one or more amino acid residue changes in the V H and / or V L region, or in one or more heavy chain CDRs and / or one or more light chain CDRs, while retaining binding and biological activity that is substantially similar to the reference antibody (e.g., substantially similar binding affinity, binding specificity, inhibitory activity, anti-inflammatory activity, or a combination thereof).

[0174] In some embodiments, any anti-FAP antibody or antigen-binding fragment thereof of the present disclosure has one or more CDR (e.g., heavy chain CDR or light chain CDR) sequences that are substantially similar to any one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 sequences of an anti-FAP antibody selected from Table 2. In some embodiments, the position of one or more CDRs along the VH region (e.g., CDRH1, CDRH2, or CDRH3) and / or VL region (e.g., CDRL1, CDRL2, or CDRL3) of the antibodies described herein may differ by one, two, three, four, five, or six amino acid positions, provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintaining the binding effect of the original antibody from which it is derived, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in some embodiments, the position of the CDRs defining any antibody described herein can be varied by moving the N-terminal and / or C-terminal boundaries of the CDR by one, two, three, four, five, or six amino acids relative to the CDR positions of any antibody described herein, provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintaining the binding effect of the original antibody from which it is derived, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the length of one or more CDRs along the VH region (e.g., CDRH1, CDRH2, or CDRH3) and / or VL region (e.g., CDRL1, CDRL2, or CDRL3) of the antibodies described herein can be varied (e.g., shortened or lengthened) by one, two, three, four, five, or more amino acids, provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintaining the binding effect of the original antibody from which it is derived, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0175] Thus, in some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or an antigen-binding fragment thereof can be one, two, three, four, five, or more amino acids shorter than one or more CDRs described herein (e.g., CDRs in any anti-FAP antibody selected from Table 2), provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., relative to the binding of the original antibody from which it is derived, substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding). In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or an antigen-binding fragment thereof can be one, two, three, four, five, or more amino acids longer than one or more CDRs described herein (e.g., CDRs in any anti-FAP antibody selected from Table 2), provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., relative to the binding of the original antibody from which it is derived, substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding). In some embodiments, the amino portion of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or an antigen-binding fragment thereof can be extended by one, two, three, four, five, or more amino acids compared to one or more CDRs described herein (e.g., CDRs in any anti-FAP antibody selected from Table 2), provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., relative to the binding of the original antibody from which it is derived, substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding). In some embodiments, the carboxyl portion of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or an antigen-binding fragment thereof can be extended by one, two, three, four, five, or more amino acids compared to one or more CDRs described herein (e.g., CDRs in any anti-FAP antibody selected from Table 2), provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., relative to the binding of the original antibody from which it is derived, substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding).In some embodiments, the amino portion of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or an antigen-binding fragment thereof can be shortened by one, two, three, four, five, or more amino acids compared to one or more CDRs described herein (e.g., the CDRs in any anti-FAP antibody selected from Table 2), provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., relative to the binding of the original antibody from which it is derived, substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding). In some embodiments, the carboxyl portion of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of an anti-FAP antibody or an antigen-binding fragment thereof can be shortened by one, two, three, four, five, or more amino acids compared to one or more CDRs described herein (e.g., the CDRs in any anti-FAP antibody selected from Table 2), provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., relative to the binding of the original antibody from which it is derived, substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding). Any method can be used to determine whether immunospecific binding to FAP (e.g., human FAP) is maintained, such as using the binding assays and conditions described in the art.

[0176] In some instances, an anti-FAP antibody or an antigen-binding fragment thereof comprises one or more CDR (e.g., heavy-chain CDR or light-chain CDR) sequences that are substantially similar to any of the anti-FAP antibodies selected from Table 2. For example, the antibody can include one or more CDR sequences from any of the anti-FAP antibodies selected from Table 2, wherein the CDR sequences contain up to 5, 4, 3, 2, or 1 amino acid residue changes compared to the corresponding CDR regions in any of the CDRs provided herein (e.g., the CDRs in any anti-FAP antibody selected from Table 2), provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., relative to the binding of the original antibody from which it is derived, substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding). In some embodiments, any amino acid changes in any of the CDRs provided herein can be conservative changes. Conservative changes can be introduced at certain positions in the CDRs where the residues are, for example, determined based on crystal structures to be less likely to be involved in the interaction with the FAP protein (e.g., human FAP).

[0177] Some aspects of the present disclosure provide anti-FAP antibodies comprising one or more heavy chain variable (VH) domains and / or light chain variable (VL) domains provided herein. In some embodiments, any VH domain provided herein comprises one or more of the heavy chain CDR sequences provided herein (e.g., CDRH1, CDRH2, and CDRH3), such as any heavy chain CDR sequence provided in any anti-FAP antibody selected from Table 2. In some embodiments, any VL domain provided herein comprises one or more of the CDR-L sequences provided herein (e.g., CDRL1, CDRL2, and CDRL3), such as any light chain CDR sequence provided in any anti-FAP antibody selected from Table 2.

[0178] In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises any antibody comprising a heavy chain variable domain and / or a light chain variable domain selected from any anti-FAP antibody of Table 2, and variants thereof. In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises any antibody comprising a heavy chain variable domain and a light chain variable domain pair selected from any anti-FAP antibody of Table 2.

[0179] Aspects of the present disclosure provide anti-FAP antibodies or antigen-binding fragments thereof that comprise heavy chain variable (VH) domain and / or light chain variable (VL) domain amino acid sequences that are homologous to any of the antibodies or antigen-binding fragments thereof 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 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, 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%) 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 a VH sequence and a VL sequence 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 selected from Table 2. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (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 change) compared to the VH of any of the anti-FAP antibodies listed in Table 2. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (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 change) compared to the VL of any of the anti-FAP antibodies listed in Table 2.

[0180] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof is a humanized antibody (e.g., a humanized variant containing one or more CDRs of Table 2). In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 that are identical to 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 that comprises one or more amino acid substitutions (e.g., in the VH framework region) as compared to any of the VHs listed in Table 2, and / or comprises one or more amino acid substitutions (e.g., in the VL framework region) as compared to any of the VLs listed in Table 2.

[0181] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:8.

[0182] In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises 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 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. In some embodiments, according to the Kabat definition system, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1 having the amino acid sequence of SEQ ID NO:1, CDRH2 having the amino acid sequence of SEQ ID NO:2, CDRH3 having the amino acid sequence of SEQ ID NO:3, 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.

[0183] In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which collectively contain no more than 5 amino acid changes (e.g., no more than 5, 4, 3, 2, or 1 amino acid changes) 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. "Collectively" as used anywhere in the present disclosure refers to the total number of amino acid changes in all three heavy-chain CDRs being within the defined range. Alternatively or additionally, the anti-FAP antibody or an antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which collectively contain no more than 5 amino acid changes (e.g., no more than 5, 4, 3, 2, or 1 amino acid changes) 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.

[0184] In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which 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 antibody or an antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which 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.

[0185] In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises: a CDRH1 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to the CDRH1 having the amino acid sequence of SEQ ID NO:1; a CDRH2 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to the CDRH2 having the amino acid sequence of SEQ ID NO:2; and / or a CDRH3 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to the CDRH3 having the amino acid sequence of SEQ ID NO:3. Alternatively or additionally, the anti-FAP antibody or an antigen-binding fragment thereof comprises: a CDRL1 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to the CDRL1 having the amino acid sequence of SEQ ID NO:4; a CDRL2 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to the CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or a CDRL3 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to the CDRL3 having the amino acid sequence of SEQ ID NO:6.

[0186] In some embodiments, the anti-FAP antibody or an 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 an 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 an 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.

[0187] In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises a VH having no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:7. Alternatively or additionally, the anti-FAP antibody or an antigen-binding fragment thereof comprises a VL having no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:8.

[0188] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:8.

[0189] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:8. In some embodiments, the number of amino acid changes (such as 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 change) can occur within the VH of SEQ ID NO:7 and / or the VL of SEQ ID NO:8, excluding any CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: a heavy chain variable sequence that comprises a framework sequence that contains 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 change relative to the framework sequence of the VH of SEQ ID NO:7; and / or a light chain variable sequence that comprises a framework sequence that contains 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 change relative to the framework sequence of the VL of SEQ ID NO:8.

[0190] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that 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 VH shown in SEQ ID NO:7. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that 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 VL shown in SEQ ID NO:8. 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:7 and / or within the VL of SEQ ID NO:8, excluding any 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 that comprises 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.

[0191] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:15.

[0192] 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.

[0193] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which in total contain no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change) compared to 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. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which in total contain no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change) compared to 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.

[0194] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which are in total 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, which are in total 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.

[0195] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:10; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:11; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to 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 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:13.

[0196] 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.

[0197] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:15.

[0198] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:15.

[0199] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:15. In some embodiments, the number of amino acid changes (such as 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 change) can occur within the VH of SEQ ID NO:14 and / or the VL of SEQ ID NO:15, excluding any CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: a heavy chain variable sequence that comprises a framework sequence having 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 change compared to the framework sequence of the VH of SEQ ID NO:14; and / or a light chain variable sequence that comprises a framework sequence having 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 change compared to the framework sequence of the VL of SEQ ID NO:15.

[0200] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that 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 VH shown in SEQ ID NO:14. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that 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 VL shown in SEQ ID NO:15. 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%) can occur within the VH of SEQ ID NO:14 and / or the VL of SEQ ID NO:15, excluding any 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 that comprises 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.

[0201] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:44. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:45.

[0202] In some embodiments, according to the Kabat definition system, the anti-FAP antibody or an 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 an 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 an 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.

[0203] In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises a CDRH1, a CDRH2, and a CDRH3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change). Alternatively or additionally, the anti-FAP antibody or an antigen-binding fragment thereof comprises a CDRL1, a CDRL2, and a CDRL3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change).

[0204] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 that are in total 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 in total 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.

[0205] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:40; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:41; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to 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 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:43.

[0206] In some embodiments, the anti-FAP antibody or an 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 an 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 an 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.

[0207] In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises a VH that comprises no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP antibody or an antigen-binding fragment thereof comprises a VL that comprises no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO: 45.

[0208] In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP antibody or an antigen-binding fragment thereof comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 45.

[0209] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO: 45. In some embodiments, the number of amino acid changes (such as 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 change) can occur within the VH of SEQ ID NO: 44 and / or the VL of SEQ ID NO: 45, excluding any CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: a heavy chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VH of SEQ ID NO: 44; and / or a light chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VL of SEQ ID NO: 45.

[0210] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that 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 VH shown in SEQ ID NO:44. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that 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 VL shown in SEQ ID NO:45. 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%) can occur within the VH of SEQ ID NO:44 and / or the VL of SEQ ID NO:45, excluding any 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 that comprises 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.

[0211] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:48.

[0212] 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.

[0213] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which in total contain no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change) compared to 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. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3, which in total contain no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change) compared to 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.

[0214] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which are in total 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, which are in total 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.

[0215] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:10; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:11; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to 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 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:43.

[0216] 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.

[0217] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO: 48.

[0218] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 48.

[0219] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO: 48. In some embodiments, the number of amino acid changes (such as 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 change) can occur within the VH of SEQ ID NO: 47 and / or the VL of SEQ ID NO: 48, excluding any CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: a heavy chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VH of SEQ ID NO: 47; and / or a light chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VL of SEQ ID NO: 48.

[0220] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:47. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:48. In some embodiments, the degree of sequence variation (such as 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 of SEQ ID NO:47 and / or the VL of SEQ ID NO:48, excluding any 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% (such as 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 that comprises a framework sequence that is at least 80% (such as 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.

[0221] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:52.

[0222] 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.

[0223] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which in total contain no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change) compared to a CDRH1 having the amino acid sequence of SEQ ID NO: 10, a CDRH2 having the amino acid sequence of SEQ ID NO: 5, 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, which in total contain no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change) compared to 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.

[0224] 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:50, 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.

[0225] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:10; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:50; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to 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 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:43.

[0226] 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.

[0227] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:52.

[0228] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:52.

[0229] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (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 change) compared to the VH shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (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 change) compared to the VL shown in SEQ ID NO:52. In some embodiments, the number of amino acid changes (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 change) can occur within the VH of SEQ ID NO:51 and / or the VL of SEQ ID NO:52, excluding any CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: a heavy chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VH of SEQ ID NO:51; and / or a light chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VL of SEQ ID NO:52.

[0230] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that 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 VH shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that 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 VL shown 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%) can occur within the VH of SEQ ID NO:51 and / or the VL of SEQ ID NO:52, excluding any 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 that comprises 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.

[0231] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:56. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:57.

[0232] In some embodiments, according to the Kabat definition system, the anti-FAP antibody or an 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 an 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 an 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.

[0233] In some embodiments, the anti-FAP antibody or an antigen-binding fragment thereof comprises a CDRH1, a CDRH2, and a CDRH3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change). Alternatively or additionally, the anti-FAP antibody or an antigen-binding fragment thereof comprises a CDRL1, a CDRL2, and a CDRL3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change).

[0234] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, and CDRH3, which are in total 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, which are in total 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.

[0235] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:10; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:11; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to 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 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:55.

[0236] 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.

[0237] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:52.

[0238] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:52.

[0239] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:52. In some embodiments, the number of amino acid changes (such as 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 change) may occur within the VH of SEQ ID NO:51 and / or the VL of SEQ ID NO:52, excluding any CDR sequences therein. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises: a heavy chain variable sequence that comprises a framework sequence having 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 change compared to the framework sequence of the VH of SEQ ID NO:51; and / or a light chain variable sequence that comprises a framework sequence having 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 change compared to the framework sequence of the VL of SEQ ID NO:52.

[0240] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH that 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 VH shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL that 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 VL shown 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%) can occur within the VH of SEQ ID NO:51 and / or the VL of SEQ ID NO:52, excluding any 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 that comprises 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.

[0241] The anti-FAP antibodies or antigen-binding fragments thereof described herein can be in any antibody form, including but not limited to intact (i.e., full-length) antibodies, antigen-binding fragments thereof (e.g., Fab, F(ab'), F(ab')2, Fv), 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-Fab (e.g., scFv fused to a portion of the constant region).

[0242] In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises the VL domain and / or VH domain of any of the anti-FAP antibodies selected from Table 2, and comprises a constant region that comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. 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 well known in the art, such as those provided in the IMGT database (imgt.org) or vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0243] 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 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 length of the linker can be about 2 to 10 amino acids, 5 to 20 amino acids, 10 to 30 amino acids, 20 to 50 amino acids, 40 to 60 amino acids, 60 to 80 amino acids or more than 80 amino acids. In some embodiments, the linker can comprise a sequence consisting essentially of 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 linker contained in U.S. Patent Nos. 8,445,251 and 9,434,931. In some embodiments, the anti-FAP comprises a linker between VH and VL, and 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.

[0244] In some embodiments, the anti-FAP scFv comprises VH and VL, and the C-terminus of VH is joined to the N-terminus of VL via a linker (such as the linker shown in SEQ ID NO:29). In some embodiments, the anti-FAP scFv comprises VH and VL, and the C-terminus of VL is joined to the N-terminus of VH via a linker (such as the linker shown in SEQ ID NO:29).

[0245] In some embodiments, the anti-FAP scFv comprises a VH that contains no more than 20 amino acid changes (such as 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 change) 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 no more than 20 amino acid changes (such as 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 change) compared to the VH of any anti-FAP antibody listed in Table 2. In some embodiments, the anti-FAP scFv comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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, the anti-FAP scFv comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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.

[0246] In some embodiments, the anti-FAP scFv comprises an amino acid sequence that contains no more than 20 amino acid changes (such as 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 change) compared to any anti-FAP scFv listed in Table 2. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (such as 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 anti-FAP scFv listed in Table 2.

[0247] 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 having no more than 20 amino acid changes (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 change) compared to the VH shown in SEQ ID NO:7, and / or a VL having no more than 20 amino acid changes (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 change) compared to the VL shown in SEQ ID NO:8. In some embodiments, the anti-FAP scFv comprises: a VH that 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 VH shown in SEQ ID NO:7; and / or a VL that 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 VL shown in SEQ ID NO:8.

[0248] In some embodiments, the anti-FAP scFv comprises an amino acid sequence having no more than 20 amino acid changes (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 change) compared to the scFv amino acid sequence shown 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 shown in SEQ ID NO:9. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO:9.

[0249] 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 having no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:14, and / or a VL having no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:15. In some embodiments, the anti-FAP scFv comprises: a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:14; and / or a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:15.

[0250] In some embodiments, the anti-FAP scFv comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO:16. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (such as 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 amino acid sequence of the scFv shown in SEQ ID NO:16. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO:16.

[0251] 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 having no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO: 44, and / or a VL having no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO: 45. In some embodiments, the anti-FAP scFv comprises: a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 44; and / or a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 55.

[0252] In some embodiments, the anti-FAP scFv comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO: 46. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 46. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO: 46.

[0253] 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 having no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO: 47, and / or a VL having no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO: 48. In some embodiments, the anti-FAP scFv comprises: a VH, wherein the VH comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 47; and / or a VL, wherein the VL comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 48.

[0254] In some embodiments, the anti-FAP scFv comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 49. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO: 49.

[0255] 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 having no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:51, and / or a VL having no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:52. In some embodiments, the anti-FAP scFv comprises: a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:51; and / or a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:52.

[0256] In some embodiments, the anti-FAP scFv comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO:53. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:53. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO:53.

[0257] 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 having no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:56, and / or a VL having no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:57. In some embodiments, the anti-FAP scFv comprises: a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:56; and / or a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:57.

[0258] In some embodiments, the anti-FAP scFv comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO:58. In some embodiments, the anti-FAP scFv comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:16. In some embodiments, the anti-FAP scFv comprises the amino acid sequence of SEQ ID NO:58.

[0259] In some embodiments, any anti-FAP antibody or antigen-binding fragment described herein is modified, e.g., 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, glycosylphosphatidylinositolization (GPI anchor linkage), and / or phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or polysaccharides. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or enzymatically. In some embodiments, the antibody is glycosylated in vitro or intracellularly, and the cells may optionally lack enzymes in the N-glycosylation or O-glycosylation pathway, such as glycosyltransferases. In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules as described in International Patent Application Publication WO2014065661, published May 1, 2014, titled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0260] In some embodiments, conservative mutations can be introduced at certain positions in the antibody sequence (e.g., CDR or framework sequences) where the residues are less likely to participate in the interaction with the target antigen (e.g., FAP), e.g., as determined based on the crystal structure.

[0261] In some embodiments, any anti-FAP antibody or antigen-binding fragment described herein can include a signal peptide (e.g., an N-terminal signal peptide) in the heavy chain and / or light chain sequence. In some embodiments, the anti-FAP antibody or antigen-binding fragment described herein includes any one of the VH and VL sequences described herein, any one of the IgG heavy and light chain sequences, or an scFv sequence, and further includes a signal peptide (e.g., an N-terminal signal peptide).

[0262] In some embodiments, the present disclosure also encompasses engineering any FAP-binding moiety known in the art to be the extracellular ligand-binding domain of a CAR expressed by a genetically modified immune cell (e.g., iNKT cell) described herein. Non-limiting examples of FAP-binding moieties are shown in Table 3.

[0263] Table 3. List of Known FAP-Binding Moieties

[0264]

[0265]

[0266]

[0267]

[0268] Aspects of the present disclosure also provide chimeric antigen receptors (CARs) comprising an extracellular ligand-binding domain. In some embodiments, the choice of ligand-binding domain depends on the type and number of ligands that define the target cell surface. For example, the ligand-binding domain selected can recognize one or more ligands that serve as cell surface markers on target cells associated with a particular disease state. Thus, examples of cell surface markers that can serve as ligands for the ligand-binding domain in the CARs of the present disclosure can include cell surface markers associated with viral, bacterial, and parasitic infections, autoimmune diseases, and more preferably cancer cells. In some embodiments, the CARs of the present disclosure are 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, a "tumor antigen" refers to an antigen that is common or specific to a particular hyperproliferative disorder such as cancer. Generally, the CARs of the present disclosure (e.g., anti-FAP CARs) will comprise 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 FAP (e.g., the FAP protein shown in SEQ ID NO: 35-38)), also 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 the CARs described herein comprises an antigen-binding fragment that specifically binds to FAP (e.g., human FAP). In some embodiments, the extracellular ligand-binding domain of the CARs described herein comprises any FAP antibody or an antigen-binding fragment thereof (e.g., an anti-FAP scFv).

[0269] In some embodiments, the anti-FAP CAR of the present disclosure comprises an extracellular ligand-binding domain that specifically binds to FAP with a binding affinity (e.g., expressed as K D ) 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. For example, the anti-FAP CAR of the present disclosure can bind to the FAP protein (e.g., the human FAP protein and / or the mouse FAP protein shown in SEQ ID NOs: 35-38) with the following affinities: between 5 pM and 500 nM, between 10 pM and 450 nM, between 20 pM and 400 nM, between 30 pM and 350 nM, between 40 pM and 300 nM, between 50 pM and 250 nM, between 60 pM and 200 nM, between 70 pM and 150 nM, between 80 pM and 100 nM, between 80 pM and 90 nM, between 90 pM and 80 nM, between 100 pM and 70 nM, between 200 pM and 60 nM, between 300 pM and 50 nM, between 400 pM and 40 nM, between 500 pM and 30 nM, between 600 pM and 20 nM, between 700 pM and 10 nM, between 800 pM and 5 nM, or between 900 pM and 2 nM.

[0270] The present disclosure also includes a CAR that competes with any of the CARs described herein for binding to the FAP protein (e.g., the human FAP protein and / or the mouse FAP protein shown in SEQ ID NOs: 35-38) and has an affinity of 100 nM or lower (e.g., 80 nM or lower, 50 nM or lower, 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower). The affinity and binding kinetics of the anti-FAP CAR can be tested using any suitable method, including but not limited to biosensor technologies (e.g., OCTET or BIACORE). In some embodiments, the anti-FAP CARs described herein bind to FAP with a K D in the sub-nanomolar concentration range.

[0271] In some embodiments, the anti-FAP CAR of the present disclosure comprises the amino acid sequence of one or more heavy chain CDRs (e.g., CDRH1, CDRH2, or CDRH3) selected from any of the anti-FAP antibodies in Table 2. In some embodiments, the anti-FAP CAR of the present disclosure comprises CDRH1, CDRH2, and CDRH3 provided by any antibody selected from Table 2. In some embodiments, the anti-FAP CAR of the present disclosure comprises the amino acid sequence of one or more light chain CDRs (e.g., CDRL1, CDRL2, or CDRL3) selected from any of the anti-FAP antibodies in Table 2. In some embodiments, the anti-FAP CAR of the present disclosure comprises CDRL1, CDRL2, and CDRL3 provided by any anti-FAP antibody selected from Table 2.

[0272] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 provided by any anti-FAP antibody selected from Table 2. In some embodiments, the antibody heavy and light chain CDR3 domains can play a particularly important role in the binding specificity / affinity of the antibody for the antigen. Thus, the anti-FAP CAR can comprise at least the heavy chain and / or light chain CDR3 selected from any of the anti-FAP antibodies in Table 2.

[0273] In some embodiments, any anti-FAP CAR of the present disclosure has one or more CDR (e.g., heavy chain CDR or light chain CDR) sequences that are substantially similar to any one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 sequences of any of the anti-FAP antibodies in Table 2. In some embodiments, the position of one or more CDRs along the VH region (e.g., CDRH1, CDRH2, or CDRH3) and / or VL region (e.g., CDRL1, CDRL2, or CDRL3) of the chimeric antigen receptor described herein can vary by one, two, three, four, five, or six amino acid positions, provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintaining the binding effect of the original antibody from which it is derived, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0274] In some instances, the anti-FAP CAR comprises one or more CDR (e.g., heavy chain CDR or light chain CDR) sequences that are substantially similar to any of the anti-FAP antibodies selected from Table 2. For example, the anti-FAP CAR can include one or more CDR sequences selected from any of the anti-FAP antibodies in Table 2, which contain up to 5, 4, 3, 2, or 1 amino acid residue changes compared to the corresponding CDR region in any of the CDRs provided herein (e.g., CDRs selected from any of the anti-FAP antibodies in Table 2), provided that immunospecific binding to FAP (e.g., human FAP) is maintained (e.g., relative to the binding of the original antibody from which it is derived, substantially maintaining at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding).

[0275] Some aspects of the disclosure provide anti-FAP CARs comprising one or more heavy chain variable (VH) domains and / or light chain variable (VL) domains provided herein. In some embodiments, any VH domain provided herein includes one or more of the heavy chain CDR sequences provided herein (e.g., CDRH1, CDRH2, and CDRH3), such as any heavy chain CDR sequence provided in any of the anti-FAP antibodies selected from Table 2. In some embodiments, any VL domain provided herein includes one or more of the CDR-L sequences provided herein (e.g., CDRL1, CDRL2, and CDRL3), such as any light chain CDR sequence provided in any of the anti-FAP antibodies selected from Table 2.

[0276] In some embodiments, the anti-FAP CAR comprises a heavy chain variable domain and / or a light chain variable domain of any of the anti-FAP antibodies selected from Table 2, and variants thereof. In some embodiments, the anti-FAP CAR comprises a pair of heavy chain variable region and light chain variable region of any of the anti-FAP antibodies selected from Table 2.

[0277] Aspects of the present disclosure provide anti-FAP CARs that include a heavy chain variable (VH) domain and / or a light chain variable (VL) domain amino acid sequence that is homologous to any of the anti-FAP CARs described herein. In some embodiments, the anti-FAP CAR includes 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 anti-FAP antibody selected from Table 2. In some embodiments, the homologous VH and / or VL amino acid sequences of the anti-FAP CAR do not vary within any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) can occur within the VH and / or VL sequences of the anti-FAP CAR, excluding any of the CDR sequences provided herein. In some embodiments, any anti-FAP CAR provided herein includes a VH sequence and a VL sequence that include 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 includes a VH that contains no more than 20 amino acid changes (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 change) compared to the VH of any anti-FAP antibody listed in Table 2. Alternatively or additionally, the anti-FAP CAR includes a VL that contains no more than 20 amino acid changes (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 change) compared to the VL of any anti-FAP antibody listed in Table 2.

[0278] In some embodiments, the anti-FAP CAR includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 that are identical to those shown in Table 2, and includes a humanized VH and / or a humanized VL. In some embodiments, the anti-FAP CAR is a humanized variant that includes one or more amino acid substitutions (e.g., in the VH framework region) compared to any VH listed in Table 2, and / or one or more amino acid substitutions (e.g., in the VL framework region) compared to any VL listed in Table 2.

[0279] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:7. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:8.

[0280] In some embodiments, the anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO:3. In some embodiments, the 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, the 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.

[0281] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which in total contain no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change) 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 in total contain no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change) 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.

[0282] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which together are 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, which together are 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.

[0283] In some embodiments, the anti-FAP CAR comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:1; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:2; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH3 having the amino acid sequence of SEQ ID NO:3. Alternatively or additionally, the anti-FAP CAR comprises: CDRL1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:6.

[0284] 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.

[0285] In some embodiments, the anti-FAP CAR comprises: a VH having no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:7, and / or a VL having no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:8.

[0286] In some embodiments, the anti-FAP CAR comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:7. Alternatively or additionally, the anti-FAP CAR comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:8.

[0287] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (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 change) compared to the VH shown in SEQ ID NO:7. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (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 change) compared to the VL shown in SEQ ID NO:8. In some embodiments, the number of amino acid changes (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 change) can occur within the VH of SEQ ID NO:7 and / or the VL of SEQ ID NO:8, excluding any CDR sequences therein. In some embodiments, the anti-FAP CAR comprises: a heavy chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VH of SEQ ID NO:7; and / or a light chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VL of SEQ ID NO:8.

[0288] In some embodiments, the anti-FAP CAR comprises a VH that 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 VH shown in SEQ ID NO:7. Alternatively or additionally, the anti-FAP CAR comprises a VL that 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 VL shown in SEQ ID NO:8. 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:7 and / or the VL of SEQ ID NO:8, excluding any 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 that comprises 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.

[0289] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:14. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:15.

[0290] In some embodiments, the anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO:12. In some embodiments, the 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, the 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.

[0291] In some embodiments, the anti-FAP CAR comprises a CDRH1, a CDRH2, and a CDRH3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change). Alternatively or additionally, the anti-FAP CAR comprises a CDRL1, a CDRL2, and a CDRL3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change).

[0292] 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.

[0293] In some embodiments, the anti-FAP CAR comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:10; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:11; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH3 having the amino acid sequence of SEQ ID NO:12. Alternatively or additionally, the anti-FAP CAR comprises: CDRL1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:13.

[0294] 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.

[0295] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:14. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:15.

[0296] In some embodiments, the anti-FAP CAR comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:14. Alternatively or additionally, the anti-FAP CAR comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:15.

[0297] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:14. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:15. In some embodiments, the number of amino acid changes (such as 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 change) may occur within the VH of SEQ ID NO:14 and / or the VL of SEQ ID NO:15, excluding any CDR sequences therein. In some embodiments, the anti-FAP CAR comprises: a heavy chain variable sequence that comprises a framework sequence having 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 change compared to the framework sequence of the VH of SEQ ID NO:14; and / or a light chain variable sequence that comprises a framework sequence having 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 change compared to the framework sequence of the VL of SEQ ID NO:15.

[0298] In some embodiments, the anti-FAP CAR comprises a VH that 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 VH shown in SEQ ID NO:14. Alternatively or additionally, the anti-FAP CAR comprises a VL that 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 VL shown in SEQ ID NO:15. 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%) can occur within the VH of SEQ ID NO:14 and / or the VL of SEQ ID NO:15, excluding any 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 that comprises 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.

[0299] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:44. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:45.

[0300] In some embodiments, the anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, the 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, the 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.

[0301] In some embodiments, the anti-FAP CAR comprises a CDRH1, a CDRH2, and a CDRH3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change). Alternatively or additionally, the anti-FAP CAR comprises a CDRL1, a CDRL2, and a CDRL3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change).

[0302] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which are in total at least 80% (such as 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, which are in total at least 80% (such as 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.

[0303] In some embodiments, the anti-FAP CAR comprises: CDRH1 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:40; CDRH2 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:41; and / or CDRH3 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to CDRH3 having the amino acid sequence of SEQ ID NO:42. Alternatively or additionally, the anti-FAP CAR comprises: CDRL1 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (such as no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:43.

[0304] 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.

[0305] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO: 45.

[0306] In some embodiments, the anti-FAP CAR comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP CAR comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 5.

[0307] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (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 change) compared to the VH shown in SEQ ID NO:44. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (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 change) compared to the VL shown in SEQ ID NO:45. In some embodiments, the number of amino acid changes (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 change) can occur within the VH of SEQ ID NO:44 and / or the VL of SEQ ID NO:45, excluding any CDR sequences therein. In some embodiments, the anti-FAP CAR comprises: a heavy chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VH of SEQ ID NO:44; and / or a light chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VL of SEQ ID NO:45.

[0308] In some embodiments, the anti-FAP CAR comprises a VH that 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 VH shown in SEQ ID NO: 44. Alternatively or additionally, the anti-FAP CAR comprises a VL that 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 VL shown in SEQ ID NO: 45. 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%) can occur within the VH of SEQ ID NO: 44 and / or the VL of SEQ ID NO: 45, excluding any 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 that comprises 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.

[0309] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 48.

[0310] In some embodiments, the anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO:42. In some embodiments, the 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, the 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.

[0311] In some embodiments, the anti-FAP CAR comprises a CDRH1, a CDRH2, and a CDRH3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change). Alternatively or additionally, the anti-FAP CAR comprises a CDRL1, a CDRL2, and a CDRL3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change).

[0312] 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.

[0313] In some embodiments, the anti-FAP CAR comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:10; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:11; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH3 having the amino acid sequence of SEQ ID NO:42. Alternatively or additionally, the anti-FAP CAR comprises: CDRL1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:43.

[0314] 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.

[0315] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO: 48.

[0316] In some embodiments, the anti-FAP CAR comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO: 48.

[0317] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:47. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:48. In some embodiments, the number of amino acid changes (such as 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 change) can occur within the VH of SEQ ID NO:47 and / or the VL of SEQ ID NO:48, excluding any CDR sequences therein. In some embodiments, the anti-FAP CAR comprises: a heavy chain variable sequence that comprises a framework sequence that contains 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 change relative to the framework sequence of the VH of SEQ ID NO:47; and / or a light chain variable sequence that comprises a framework sequence that contains 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 change relative to the framework sequence of the VL of SEQ ID NO:48.

[0318] In some embodiments, the anti-FAP CAR comprises a VH that 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 VH shown in SEQ ID NO: 47. Alternatively or additionally, the anti-FAP CAR comprises a VL that 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 VL shown in SEQ ID NO: 48. 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%) can occur within the VH of SEQ ID NO: 47 and / or the VL of SEQ ID NO: 48, excluding any 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 that comprises 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.

[0319] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 51. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 52.

[0320] In some embodiments, the anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, the 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, the 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.

[0321] In some embodiments, the anti-FAP CAR comprises a CDRH1, a CDRH2, and a CDRH3 that, compared 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change). Alternatively or additionally, the anti-FAP CAR comprises a CDRL1, a CDRL2, and a CDRL3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change).

[0322] 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.

[0323] In some embodiments, the anti-FAP CAR comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:10; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:50; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH3 having the amino acid sequence of SEQ ID NO:42. Alternatively or additionally, the anti-FAP CAR comprises: CDRL1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:43.

[0324] 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.

[0325] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:52.

[0326] In some embodiments, the anti-FAP CAR comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP CAR comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:52.

[0327] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:52. In some embodiments, the number of amino acid changes (such as 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 change) may occur within the VH of SEQ ID NO:51 and / or the VL of SEQ ID NO:52, excluding any CDR sequences therein. In some embodiments, the anti-FAP CAR comprises: a heavy chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VH of SEQ ID NO:51; and / or a light chain variable sequence that comprises a framework sequence that contains 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 change compared to the framework sequence of the VL of SEQ ID NO:52.

[0328] In some embodiments, the anti-FAP CAR comprises a VH that 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 VH shown in SEQ ID NO:51. Alternatively or additionally, the anti-FAP CAR comprises a VL that 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 VL shown 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%) can occur within the VH of SEQ ID NO:51 and / or the VL of SEQ ID NO:52, excluding any 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 that comprises 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.

[0329] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3 of a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO:56. Alternatively or additionally, the anti-FAP CAR comprises CDRL1, CDRL2, and CDRL3 of a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO:57.

[0330] In some embodiments, the anti-FAP CAR comprises a CDRH3 having the amino acid sequence of SEQ ID NO:54. In some embodiments, the 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, the 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.

[0331] In some embodiments, the anti-FAP CAR comprises a CDRH1, a CDRH2, and a CDRH3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change). Alternatively or additionally, the anti-FAP CAR comprises a CDRL1, a CDRL2, and a CDRL3 that, compared to 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, contain in total no more than 5 amino acid changes (such as no more than 5, 4, 3, 2, or 1 amino acid change).

[0332] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, and CDRH3, which 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, which 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.

[0333] In some embodiments, the anti-FAP CAR comprises: CDRH1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH1 having the amino acid sequence of SEQ ID NO:10; CDRH2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH2 having the amino acid sequence of SEQ ID NO:11; and / or CDRH3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRH3 having the amino acid sequence of SEQ ID NO:54. Alternatively or additionally, the anti-FAP CAR comprises: CDRL1 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL1 having the amino acid sequence of SEQ ID NO:4; CDRL2 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL2 having the amino acid sequence of SEQ ID NO:5; and / or CDRL3 having no more than 3 amino acid changes (e.g., no more than 3, 2, or 1 amino acid change) compared to CDRL3 having the amino acid sequence of SEQ ID NO:55.

[0334] 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.

[0335] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:56. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:57.

[0336] In some embodiments, the anti-FAP CAR comprises a VH that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:56. Alternatively or additionally, the anti-FAP CAR comprises a VL that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:57.

[0337] In some embodiments, the anti-FAP CAR comprises a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH shown in SEQ ID NO:56. Alternatively or additionally, the anti-FAP CAR comprises a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL shown in SEQ ID NO:57. In some embodiments, the number of amino acid changes (such as 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 change) may occur within the VH of SEQ ID NO:56 and / or the VL of SEQ ID NO:57, excluding any CDR sequences therein. In some embodiments, the anti-FAP CAR comprises: a heavy chain variable sequence that comprises a framework sequence having 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 change compared to the framework sequence of the VH of SEQ ID NO:56; and / or a light chain variable sequence that comprises a framework sequence having 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 change compared to the framework sequence of the VL of SEQ ID NO:57.

[0338] In some embodiments, the anti-FAP CAR comprises a VH that 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 VH shown in SEQ ID NO:56. Alternatively or additionally, the anti-FAP CAR comprises a VL that 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 VL shown in SEQ ID NO:57. 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%) can occur within the VH of SEQ ID NO:56 and / or the VL of SEQ ID NO:57, excluding any 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 that comprises 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.

[0339] Some aspects of the present disclosure provide anti-FAP CARs that include FAP binders known in the art, such as any of the FAP binders provided in Table 3. In some embodiments, the anti-FAP CARs of the present disclosure include FAP binding peptides known in the art, such as the FAP binding peptides provided in Table 3. In some embodiments, the anti-FAP CARs of the present disclosure include one or more of the heavy chain variable (VH) domains and / or light chain variable (VL) domains of anti-FAP antibodies known in the art (such as the anti-FAP antibodies provided in Table 3). In some embodiments, the anti-FAP CAPs of the present disclosure include a VH domain that includes one or more heavy chain CDR sequences (such as CDRH1, CDRH2, and CDRH3) of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3). In some embodiments, the anti-FAP CAPs of the present disclosure include a VL domain that includes one or more light chain CDR sequences (such as CDRL1, CDRL2, and CDRL3) of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3).

[0340] In some embodiments, the anti-FAP CARs of the present disclosure include the heavy chain variable domains and / or light chain variable domains of any anti-FAP antibody known in the art (such as the anti-FAP antibodies selected from Table 3) and variants thereof. In some embodiments, the anti-FAP CARs include pairs of heavy chain variable regions and light chain variable regions of any anti-FAP antibody known in the art (such as the anti-FAP antibodies selected from Table 3).

[0341] Aspects of the present disclosure provide anti-FAP CARs that include heavy chain variable (VH) domain and / or light chain variable (VL) domain amino acid sequences that are homologous to any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3). In some embodiments, the anti-FAP CAR includes a VH or VL that is at least 75% (such as 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH and / or any VL of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3). In some embodiments, the homologous VH and / or VL amino acid sequences of the anti-FAP CAR do not vary within any CDR sequence of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3). For example, in some embodiments, the degree of sequence variation (such as 75%, 80%, 85%, 90%, 95%, 98%, or 99%) can occur within the VH and / or VL sequences of the anti-FAP CAR, excluding any CDR sequences of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3). In some embodiments, any anti-FAP CAR provided herein includes a VH sequence and a VL sequence that include framework sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequences of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3). In some embodiments, the anti-FAP CAR includes a VH that contains no more than 20 amino acid changes (such as 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 change) compared to the VH of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3). Alternatively or additionally, the anti-FAP CAR includes a VL that contains no more than 20 amino acid changes (such as 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 change) compared to the VL of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3).

[0342] In some embodiments, the anti-FAP CAR comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 that are identical to those of any known anti-FAP antibody in the art (such as the anti-FAP antibodies provided in Table 3), and comprises a humanized VH and / or a humanized VL. In some embodiments, the anti-FAP CAR is a humanized variant that comprises one or more amino acid substitutions (such as in the VH framework region) compared to any known anti-FAP antibody in the art (such as the anti-FAP antibodies provided in Table 3), and / or one or more amino acid substitutions (such as in the VL framework region) compared to any known anti-FAP antibody in the art (such as the anti-FAP antibodies provided in Table 3).

[0343] The anti-FAP antibodies or antigen-binding fragments thereof described herein can be grafted into a chimeric antigen receptor, including but not limited to its antigen-binding fragments (such as Fab, F(ab'), F(ab')2, Fv), single-chain antibodies (such as scFv), bispecific antibodies, or nanobodies. In some embodiments, the anti-FAP CAR described herein comprises a single-chain variable fragment (scFv) as the extracellular ligand-binding domain.

[0344] 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 anti-FAP scFv 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 the anti-FAP antibodies provided in Table 3). In some embodiments, the VH and VL of the anti-FAP CAR are joined together by a linker. In some embodiments, the length of the linker can be about 2 to 10 amino acids, 5 to 20 amino acids, 10 to 30 amino acids, 20 to 50 amino acids, 40 to 60 amino acids, 60 to 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 linker contained in U.S. Patent Nos. 8,445,251 and 9,434,931. In some embodiments, the anti-FAP CAR comprises a linker between VH and VL, and 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.

[0345] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv containing VH and VL, and the C-terminus of VH is joined to the N-terminus of VL via a linker (such as the linker shown in SEQ ID NO:29). In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain comprising an scFv containing VH and VL, and the C-terminus of VL is joined to the N-terminus of VH via a linker (such as the linker shown in SEQ ID NO:29).

[0346] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv, and the scFv comprises a VH that has no more than 20 amino acid changes (such as 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 change) compared to the VH of any of the anti-FAP antibodies listed in Table 2. Alternatively or additionally, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv, and the scFv comprises a VL that has no more than 20 amino acid changes (such as 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 change) compared to the VH of any of the anti-FAP antibodies listed in Table 2. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv, and the scFv comprises a VH that has an amino acid sequence that is at least 80% (such as 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH of any of the anti-FAP antibodies listed in Table 2. Alternatively or additionally, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv, and the scFv comprises a VL that has an amino acid sequence that is at least 80% (such as 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH of any of the anti-FAP antibodies listed in Table 2.

[0347] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv, and the scFv comprises an amino acid sequence that has no more than 20 amino acid changes (such as 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 change) compared to any of the anti-FAP scFvs listed in Table 2. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv, and the scFv comprises an amino acid sequence that is at least 80% (such as 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.

[0348] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv that comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO:9. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:9. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv that comprises the amino acid sequence of SEQ ID NO:9.

[0349] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv that comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO:16. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:16. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises an scFv that comprises the amino acid sequence of SEQ ID NO:16.

[0350] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO:46. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:46. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises the amino acid sequence of SEQ ID NO:46.

[0351] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO:49. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:49. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises the amino acid sequence of SEQ ID NO:49.

[0352] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO:53. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:53. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises the amino acid sequence of SEQ ID NO:53.

[0353] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises an amino acid sequence having no more than 20 amino acid changes (such as 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 change) compared to the scFv amino acid sequence shown in SEQ ID NO:58. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises an amino acid sequence that is at least 80% (such as 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 shown in SEQ ID NO:58. In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv that comprises the amino acid sequence of SEQ ID NO:58.

[0354] In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv, wherein the scFv comprises a VH that has no more than 20 amino acid changes (such as 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 change) compared to the VH of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3). Alternatively or additionally, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv, wherein the scFv comprises a VL that has no more than 20 amino acid changes (such as 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 change) compared to the VH of any anti-FAP antibody known in the art (such as the anti-FAP antibodies provided in Table 3). In some embodiments, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv, wherein the scFv comprises a VH that has an amino acid sequence that is at least 80% (such as 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 provided in Table 3). Alternatively or additionally, the anti-FAP CAR comprises an extracellular ligand-binding domain that comprises a scFv, wherein the scFv comprises a VL that has an amino acid sequence that is at least 80% (such as 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 provided in Table 3).

[0355] 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 the CD8 hinge region is shown in SEQ ID NO:30:

[0356] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0357] In some embodiments, the anti-FAP CAR of the present disclosure comprises a CD8 hinge region having the amino acid sequence shown in SEQ ID NO: 30, or a variant thereof. In some embodiments, the anti-FAP CAR comprises a hinge region that comprises 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 98%, 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 the hinge regions described by Guedan et al. in Engineering and Design of Chimeric Antigen Receptors, Mol Ther Methods Clin Dev. Mar 15, 2019; 12:145–156, such as hinge regions derived from IgG1, IgG2, IgG4, CD28, CD8 or hybrids thereof.

[0358] In some embodiments, the anti-FAP CAR of the present disclosure further comprises a transmembrane domain that connects the extracellular ligand-binding domain to the intracellular signaling and co-stimulatory domains. For the transmembrane domain, the CAR can be designed to comprise a transmembrane domain fused to the extracellular domain of the CAR (such as the antigen-binding domain). Any transmembrane domain is expected to be useful herein, so long as the domain is capable of anchoring the CAR comprising the domain to the cell membrane. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid the binding of these domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. Those skilled in the art will understand that the complete transmembrane domain or portions thereof function together with the cytoplasmic domain or portions thereof. The transmembrane domain can be of natural origin or synthetic origin. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain can be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine is present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker (preferably between 2 and 10 amino acids in length) can form the connection between the transmembrane domain of the CAR and the cytoplasmic signaling domain. A glycine-serine doublet provides a particularly suitable linker.

[0359] In some embodiments, the transmembrane domain is the CD8 transmembrane domain. An exemplary amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO: 31:

[0360] IYIWAPLAGTCGVLLLSLVITLYC

[0361] In some embodiments, the anti-FAP CAR of the present disclosure comprises a CD8 hinge region having the amino acid sequence shown in SEQ ID NO:31, or a variant thereof. In some embodiments, the anti-FAP CAR comprises a transmembrane domain, the transmembrane domain 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 98%, at least 99% or 100% identical to SEQ ID NO:31. In some embodiments, the transmembrane domain can be any suitable transmembrane domain known in the art, such as a transmembrane domain derived from: TCRα, TCRβ, TCRζ, CD2, CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD32, CD33, CD34, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, SLAMF4 or inducible T cell co-stimulator (ICOS). However, any transmembrane domain is expected to be useful herein, provided that the domain is capable of anchoring the CAR comprising the domain to the cell membrane. The transmembrane domain can be identified using any method known in the art or described herein, such as by using the UniProt database. In some embodiments, the transmembrane domain is linked to the hinge region. Exemplary pairs of hinge region and transmembrane domain include CD8 hinge / TM, IgG1 hinge / CD2 TM or IgG1 hinge / SLAMF4 TM. Table 6 shows the amino acid sequences and nucleotide coding sequences of exemplary hinge / TM domains.

[0362] Table 6 Exemplary hinge / TM domains

[0363]

[0364]

[0365] In some embodiments, the anti-FAP CAR further comprises one or more intracellular (or cytoplasmic) domains (ICDs). In some embodiments, one or more intracellular domains of the CAR (such as ICD1 and ICD2) are responsible for activating at least one normal effector function of the immune cell on which the CAR is placed. The term "effector function" refers to the specialized function of a cell (such as an iNKT cell). The effector function of a cell (such as an iNKT cell) can be, for example, cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the part of a protein that transduces the signal of an effector function and directs the cell to perform a specialized function. Although the entire intracellular signaling domain of a molecule can generally be used, it is not necessary to use the entire domain in many cases. In the case of using a truncated portion of the intracellular signaling domain, such a truncated portion can be used in place of the full domain as long as it can transduce the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain that is sufficient to transduce the effector function signal. In some embodiments, intracellular domains derived from different molecules can be used to form the intracellular domain of the anti-FAP CAR described herein. In certain embodiments, the intracellular (or cytoplasmic) domain of the chimeric antigen receptor disclosed herein can include, but is not limited to, the 4-1BB intracellular domain, the OX40 intracellular domain, the CD30 intracellular domain, the CD40 intracellular domain, the ICOS intracellular domain, the LFA-1 intracellular domain, the CD2 intracellular domain, the CD3ζ intracellular domain, the CD3γ intracellular domain, the CD3δ intracellular domain, the CD3ε intracellular domain, and the CD7 intracellular domain, the CD27 intracellular domain, the CD28 / IL15RA intracellular domain, the DAP10 intracellular domain, the DAP12 intracellular domain, the DNAM1 intracellular domain, the SLAMF6 intracellular domain, and the CD22 intracellular domain. In some embodiments, the anti-FAP CAR comprises the CD3ζ intracellular domain. An exemplary amino acid sequence of the CD3ζ intracellular domain is shown in SEQ ID NO:32:

[0366] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0367] In some embodiments, the anti-FAP CAR of the present disclosure comprises a CD3ζ intracellular domain (e.g., ICD2) having the amino acid sequence shown in SEQ ID NO: 32, or a variant thereof. In some embodiments, the anti-FAP CAR comprises an intracellular domain 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 98%, at least 99% or 100% identical to SEQ ID NO: 32.

[0368] In some embodiments, the intracellular domain further comprises one or more intracellular co-stimulatory domains (i.e., ICD1), such as those described herein, which transmit co-stimulatory signals that promote cell proliferation, cell survival, and / or cytokine secretion upon binding of the extracellular domain. In some embodiments, such intracellular co-stimulatory domains include, but are not limited to, any co-stimulatory domain disclosed herein or those known in the art, including, but not limited to, CD28, ICOS, 4-1BB, OX40 or CD27. In some embodiments, the anti-FAP CAR comprises a 4-1BB co-stimulatory domain. An exemplary amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO: 33:

[0369] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0370] In some embodiments, the anti-FAP CAR of the present disclosure comprises a 4-1BB co-stimulatory domain having the amino acid sequence shown in SEQ ID NO: 33, or a variant thereof. In some embodiments, the anti-FAP CAR comprises a 4-1BB co-stimulatory domain 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 98%, at least 99% or 100% identical to SEQ ID NO: 33.

[0371] Table 6 lists exemplary amino acid sequences and coding sequences of intracellular domains (e.g., ICD2 and ICD2)

[0372] Table 7 Intracellular Domains of Anti-FAP CAR

[0373]

[0374]

[0375]

[0376]

[0377]

[0378] In some embodiments, the anti-FAP CAR of the present disclosure comprises an extracellular domain selected from any one of the anti-FAP scFvs in Table 2, a transmembrane domain selected from any one of the hinge / TM domains liste...

Claims

1. A constant natural killer T (iNKT) cell, wherein the iNKT cell comprises a chimeric antigen receptor (CAR) that specifically binds to fibroblast activation protein (FAP), and wherein the chimeric antigen receptor comprises: (a) CDRH1 having the amino acid sequence of SEQ ID NO:1, CDRH2 having the amino acid sequence of SEQ ID NO:2, CDRH3 having the amino acid sequence of SEQ ID NO:3, 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; (b) 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:12, 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; (c) 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; (d) 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; or (e) 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.

2. The iNKT cell according to claim 1, wherein the CAR 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.

3. The iNKT cell according to claim 1 or 2, wherein the CAR comprises: (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.

4. The iNK cell according to any one of claims 1-3, 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 iNKT cell according to any one of claims 1-4, wherein the CAR comprises: (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.

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

49.

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

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

30.

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

10. The iNKT cell according to 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 according to any one of claims 1-7, wherein the CAR comprises a hinge / transmembrane domain, and the hinge / transmembrane domain comprises the amino acid sequence of any one of SEQ ID NO: 83, 85 or 87.

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

13. The iNKT cell according to claim 12, wherein the one or more cytoplasmic domains comprise the amino acid sequence of any one of SEQ ID NO: 32, 33, 39, 90, 92, 94, 96, 98, 100, 102 or 105.

14. The iNKT cell according to 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 according to any one of claims 1-14, wherein the CAR further comprises an intracellular co-stimulatory domain.

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

33.

17. The iNKT cell according to any one of claims 1-16, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NO: 17, 18, 59, 60 or 62.

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

60.

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

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

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

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

24. An invariant natural killer T (iNKT) cell, the 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, the composition comprising the iNKT cell according to any one of claims 1-24.

26. The composition according to claim 26, the composition further comprising a pharmaceutically acceptable carrier.

27. A method for killing cells, the method comprising contacting the cells with the iNKT cells of any one of claims 1-24 or the composition of claim 25 or 26.

28. The method according to claim 26, wherein the cells are cancer cells.

29. The method according to claim 28, wherein the cancer cells are lung cancer cells, breast cancer cells, colorectal 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 26-29, wherein the lung cancer is non-small cell lung cancer.

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

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

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

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

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

36. The method according to claim 35, wherein the solid tumor is a lung cancer tumor, a breast cancer tumor, a colorectal 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. The method according to any one of claims 36, wherein the lung cancer is non-small cell lung cancer.

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

39. The method according to claim 38, wherein the injection is intraperitoneal injection, intravenous injection or intratumoral injection.

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

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

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

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

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

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

46. The method according to any one of claims 43-45, wherein the anti-FAP CAR iNKT cells are engineered to be armored molecules.

47. The method according to claim 46, wherein the armored molecule is soluble IL-15 (sIL-15).

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

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

50. The method according to claim 49, wherein the solid tumor is a lung cancer tumor, a breast cancer tumor, a colorectal 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 according to any one of claims 43-50, wherein the anti-FAP CAR iNKT cells kill cells expressing FAP in the TME.

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

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

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

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

56. The method according to any one of claims 52-55, wherein killing the cancer-associated fibroblasts increases immune cell infiltration in the TME relative to the immune cell infiltration in the subject before the administration.

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

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

59. The method according to any one of claims 43-58, wherein the administration of the anti-FAP CAR iNKT cells elicits 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 according to any one of claims 43-59, wherein the anti-FAP CAR iNKT cells retain responsiveness to CD1d and / or NK receptor ligands.

61. The method according to any one of claims 43-60, wherein the anti-FAP CAR iNKT cells comprise a CAR that comprises any one of the FAP-binding portions derived from the anti-FAP antibodies shown in Table 3.

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

63. The method according to any one of claims 43-63, wherein the subject is diagnosed with a solid tumor, the solid tumor comprising lung cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, pancreatic cancer, prostate cancer, thyroid cancer, cervical cancer, urothelial cancer, or sarcoma.

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