A monoclonal antibody against canine fibroblast activation protein (FAP) cross-reacts with mouse and human fibroblast activation protein

Abstract

The present invention relates to antibodies, binding polypeptides, and scFvs specific for fibroblast activation protein (FAP) that are capable of cross-reacting with canine, murine, and human FAP.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is entitled to priority under 35 USC § 119(e) to U.S. Provisional Application No. 62 / 904,272, filed September 23, 2019, the entirety of which is incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. CA172921 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Tumors are composed of a heterogeneous population of cells, including cancer cells and a large number of non-cancerous cells. The occurrence of different cell types varies between tumors and at different stages of tumor progression, but they include infiltrating inflammatory and immune cells, endothelial cells, smooth muscle cells of mesenchymal origin, pericytes, and tumor-associated fibroblasts (TAFs), which are collectively referred to herein as stromal cells. TAFs are a heterogeneous population that are phenotypically distinct from normal fibroblasts. Fibroblast activation protein (FAP) has emerged as a marker of reactive fibroblasts in tumors and granulation tissues, as well as in fibrotic lesions.

[0004] FAP is a type II transmembrane cell surface protein belonging to the post-proline dipeptidyl aminopeptidase family, with the highest similarity to dipeptidyl peptidase IV (DPPIV / CD26). FAP is selectively expressed by TAFs and pericytes in more than 90% of human epithelial cancers examined. It is also expressed during embryonic development, in tissues of healing wounds, and in chronic inflammatory and fibrotic conditions, such as liver cirrhosis and idiopathic pulmonary fibrosis, as well as in bone and soft tissue sarcomas and some melanomas. However, FAP expression is not detected in benign lesions or normal adult tissues, whereas DPPIV is more widely expressed in various cell types. In vitro studies have shown that FAP has both dipeptidyl peptidase and endopeptidase activities, including collagenolytic activity capable of degrading gelatin and type I collagen, but its in vivo substrates have yet to be identified.

[0005] There is a need in the art for the development of antibodies capable of cross-reacting with canine, murine, and human fibroblast activation proteins. The present invention addresses this need. Summary of the Invention

[0006] As described herein, the present invention relates to antibodies, binding polypeptides, and scFvs specific for fibroblast activation protein (FAP) that are capable of cross-reacting with canine, murine, and human FAP.

[0007] In one aspect, the invention provides isolated binding polypeptides that include an antigen-binding domain that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP).

[0008] In certain embodiments, the antigen binding domain is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence a heavy chain variable region comprising the amino acid sequence TIFF0007603670000001.tif4128, and wherein HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0009] In certain embodiments, the binding polypeptide: (a) binds to fibroblast activation protein (FAP); and / or (b) comprises an antibody or antigen-binding fragment thereof; and / or (c) comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7; and / or (d) comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:7; and / or (e) comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7; and / or (f) comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9; and / or (g) comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9; and / or (h) comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9. It consists of a light chain variable region comprising the amino acid sequence set forth in ID NO:9.

[0010] In certain embodiments, (a) the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody; and / or (b) the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody, and the antibody is a full-length antibody; and / or (c) the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody, and the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0011] In another aspect, the present invention provides an isolated binding polypeptide comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9.

[0012] In another aspect, the present invention provides single chain variable fragments (scFv) that comprise an antigen-binding domain that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP).

[0013] In certain embodiments, the antigen binding domain is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence a heavy chain variable region comprising the amino acid sequence TIFF0007603670000002.tif4128, and wherein HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6), wherein the heavy chain variable region and the light chain variable region are separated by a linker.

[0014] In another aspect, the present invention provides a single chain variable fragment (scFv) comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, wherein the heavy chain variable region and the light chain variable region are separated by a linker, and optionally the linker comprises the amino acid sequence set forth in SEQ ID NO:15.

[0015] In another aspect, the present invention provides a single chain variable fragment (scFv) comprising the amino acid sequence set forth in SEQ ID NO:11 or 13; or consisting of the amino acid sequence set forth in SEQ ID NO:11 or 13.

[0016] In another aspect, the invention provides an isolated nucleic acid encoding any of the binding polypeptides or any of the scFvs contemplated herein.

[0017] In another aspect, the invention provides an isolated nucleic acid encoding a binding polypeptide comprising an antigen-binding domain that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP).

[0018] In certain embodiments, the antigen binding domain is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000003.tif4128, and wherein HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0019] In certain embodiments, (a) the binding polypeptide comprises an antibody or antigen-binding fragment thereof, optionally wherein the antibody is a full-length antibody; and / or (b) the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single-domain antibody; and / or (c) the antibody or antigen-binding fragment is a humanized antibody or fragment thereof.

[0020] In certain embodiments, (a) the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO:8; and / or (b) the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:8; and / or (c) the heavy chain variable region is encoded by a nucleic acid consisting of a polynucleotide sequence set forth in SEQ ID NO:8; and / or (d) the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:10; and / or (e) the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:11; and / or (f) the light chain variable region is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO:10; and / or (f) the light chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:10.

[0021] In another aspect, the invention provides an isolated nucleic acid encoding a binding polypeptide comprising: a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:8; and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:10.

[0022] In another aspect, the invention provides a heavy chain variable region comprising (a) three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6); or (b) a single chain variable fragment (scFv) comprising a heavy chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:8; and a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:10, wherein the heavy chain variable region and the light chain variable region are separated by a linker, and optionally the linker is and / or (c) encoding a single chain variable fragment (scFv) comprising the amino acid sequence set forth in SEQ ID NO:12 or 14; and / or (d) encoding a single chain variable fragment (scFv) consisting of the polynucleotide sequence set forth in SEQ ID NO:12 or 14.

[0023] In another aspect, the invention provides a vector comprising any of the isolated nucleic acids contemplated herein.

[0024] In certain embodiments, the vector is an expression vector; and / or the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector.

[0025] In another aspect, the present invention provides a host cell that (a) comprises any of the vectors contemplated herein; and / or (b) is of eukaryotic or prokaryotic origin; and / or (c) is of mammalian origin; and / or (d) is of bacterial origin.

[0026] In another aspect, the invention provides a method of producing a binding polypeptide or scFv that binds to a FAP, the method comprising culturing any of the host cells contemplated herein.

[0027] In another aspect, the invention provides a pharmaceutical composition comprising any of the binding polypeptides or any of the scFvs contemplated herein.

[0028] In another aspect, the present invention provides a method for identifying a subject suitable for adoptive cell therapy against a fibroblast activation protein (FAP), the method comprising the steps of (a) isolating diseased tissue from a subject, (b) contacting the isolated tissue with a binding polypeptide that specifically binds to the FAP, and (c) detecting FAP-expressing cells in the isolated tissue, thereby identifying a subject suitable for adoptive cell therapy.

[0029] In certain embodiments, the binding polypeptide is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000005.tif4128, and wherein HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0030] In certain embodiments, (a) the binding polypeptide comprises an antibody or antigen-binding fragment thereof; and / or (b) the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody, optionally wherein the antibody is a full-length antibody; and / or (c) the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof; and / or (d) the binding polypeptide is conjugated to a therapeutic or diagnostic molecule; and / or (e) the binding polypeptide is conjugated to a diagnostic molecule, wherein the diagnostic molecule comprises a detectable label; and / or (f) the binding polypeptide is conjugated to a diagnostic molecule, wherein the diagnostic molecule comprises a detectable label, and further wherein the detectable label is a radiolabel, a fluorophore, an enzyme, a hapten, biotin, or a chromophore.

[0031] In certain aspects, after a subject has been identified as a suitable subject, the subject is administered adoptive cellular therapy.

[0032] In certain embodiments, (a) the adoptive cell therapy comprises modified immune cells comprising a chimeric antigen receptor (CAR); and / or (b) the adoptive cell therapy comprises modified immune cells comprising a chimeric antigen receptor (CAR), where the immune cells are T lymphocytes; and / or (c) the adoptive cell therapy comprises modified immune cells comprising a chimeric antigen receptor (CAR), where the immune cells are NK cells; and / or (d) the adoptive cell therapy comprises modified immune cells comprising a chimeric antigen receptor (CAR), where the CAR specifically binds to a FAP.

[0033] In certain embodiments, the binding polypeptide: (a) comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7; and / or (b) comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:7; and / or (c) consists of a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7; and / or (d) comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9; and / or (e) comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:9; and / or (f) consists of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9.

[0034] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an isolated binding polypeptide comprising: a heavy chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7; and a light chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9.

[0035] In certain embodiments, (a) the cancer is associated with a fibroblast activation protein (FAP)-expressing cell; and / or (b) the FAP-expressing cell is a cancer-associated cell; and / or (c) the FAP-expressing cell is a cancer-associated cell, and the cancer-associated cell is a cancer-associated fibroblast (CAF); and / or (d) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a FAP-expressing adipocyte; and / or (e) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a tumor-associated macrophage (TAM); and / or (f) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a tumor-associated neutrophil (TAN); and / or (g) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a myeloid-derived suppressor cell (MDSC); and / or (h) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a cancer-initiating cell.

[0036] In certain embodiments, (a) the binding polypeptide specifically binds to a fibroblast activation protein (FAP); and / or (b) the binding polypeptide comprises an antibody or antigen-binding fragment thereof; and / or (c) the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody, optionally wherein the antibody is a full-length antibody; and / or (d) the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0037] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising the steps of: (a) identifying the subject as a suitable subject comprising: (i) isolating diseased tissue from the subject; (ii) contacting the isolated tissue with a binding polypeptide that specifically binds to a FAP; and (iii) detecting FAP-expressing cells in the isolated tissue; and (b) administering to the suitable subject an adoptive cell therapy comprising modified T cells comprising a chimeric antigen receptor (CAR) that specifically binds to a fibroblast activation protein (FAP). [The present invention 1001] An isolated binding polypeptide comprising an antigen-binding domain that specifically binds to an epitope of human and canine, and / or mouse fibroblast activation protein (FAP). [The present invention 1002] The antigen-binding domain comprises: (a) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000006.tif5128 and a heavy chain variable region comprising: (b) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). 1001. A binding polypeptide of the present invention comprising: [The present invention 1003] (a) binds to fibroblast activation protein (FAP); and / or (b) comprises an antibody or an antigen-binding fragment thereof; and / or (c) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7; and / or (d) comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and / or (e) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7; and / or (f) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9; and / or (g) comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9; and / or (h) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9; A binding polypeptide of the present invention 1001 or 1002. [The present invention 1004] (a) the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody; and / or (b) the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single domain antibody, and the antibody is a full-length antibody; and / or (c) the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single domain antibody, and the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof; A binding polypeptide according to any one of claims 1001 to 1003 of the present invention. [The present invention 1005] (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9. 2. An isolated binding polypeptide comprising: [The present invention 1006] A single-chain variable fragment (scFv) that contains an antigen-binding domain that specifically binds to an epitope of human, canine, and / or mouse fibroblast activation protein (FAP). [The present invention 1007] The antigen-binding domain comprises: (a) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000007.tif4128 and a heavy chain variable region comprising: (b) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). Including, the heavy chain variable region and the light chain variable region are separated by a linker; The scFv of the present invention 1006. [The present invention 1008] (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9. A single chain variable fragment (scFv) comprising: A single chain variable fragment (scFv), wherein the heavy chain variable region and the light chain variable region are separated by a linker, optionally, the linker comprising the amino acid sequence set forth in SEQ ID NO:15. [The present invention 1009] (a) comprises the amino acid sequence set forth in SEQ ID NO:11 or 13; or (b) consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 13; Single chain variable fragment (scFv). [The present invention 1010] An isolated nucleic acid encoding a binding polypeptide or scFv of any of claims 1001 to 1009. [The present invention 1011] An isolated nucleic acid encoding a binding polypeptide comprising an antigen-binding domain that specifically binds to an epitope of human and canine, and / or mouse fibroblast activation protein (FAP). [The present invention 1012] The antigen-binding domain comprises: (a) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000008.tif4128 and a heavy chain variable region comprising: (b) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). The nucleic acid of the present invention comprising: [The present invention 1013] (a) the binding polypeptide comprises an antibody or an antigen-binding fragment thereof, optionally wherein the antibody is a full-length antibody; and / or (b) the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single domain antibody; and / or (c) the antibody or antigen-binding fragment is a humanized antibody or fragment thereof; The nucleic acid of the present invention 1011 or 1012. [The present invention 1014] (a) the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO:8; and / or (b) the heavy chain variable region is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO:8; and / or (c) the heavy chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:8; and / or (d) the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:10; and / or (e) the light chain variable region is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO:10; and / or (f) the light chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:10; Any of 1011 to 1013 nucleic acids. [The present invention 1015] (a) a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:8; and (b) a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:10. An isolated nucleic acid encoding a binding polypeptide comprising: [The present invention 1016] (a)(i) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000009.tif4128 and a heavy chain variable region comprising: (ii) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). or (b)(i) a heavy chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:8, and (ii) a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:10. and encoding a single chain variable fragment (scFv) comprising: the heavy chain variable region and the light chain variable region are separated by a linker, optionally comprising the amino acid sequence set forth in SEQ ID NO. 15; and / or (c) encoding a single chain variable fragment (scFv) comprising a polynucleotide sequence set forth in SEQ ID NO: 12 or 14; and / or (d) encoding a single chain variable fragment (scFv) consisting of the polynucleotide sequence set forth in SEQ ID NO: 12 or 14; Isolated nucleic acid. [The present invention 1017] A vector comprising any one of the isolated nucleic acids of the present inventions 1010 to 1016. [The present invention 1018] (a) is an expression vector; and / or (b) selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector; The vector of the present invention 1017. [The present invention 1019] (a) comprises a vector of the invention 1017 or 1018; and / or (b) is of eukaryotic or prokaryotic origin; and / or (c) is of mammalian origin; and / or (d) is of bacterial origin; host cell. [The present invention 1020] A method for producing a binding polypeptide or scFv that binds to a FAP, comprising the step of culturing a host cell of the invention. [The present invention 1021] A pharmaceutical composition comprising a binding polypeptide or scFv of any of claims 1001 to 1009. [The present invention 1022] 1. A method for identifying a subject suitable for adoptive cellular therapy against fibroblast activation protein (FAP), comprising: (a) isolating diseased tissue from said subject; (b) contacting the isolated tissue with a binding polypeptide that specifically binds to a FAP; and (c) detecting FAP-expressing cells in the isolated tissue. Including, Thereby identifying suitable subjects for said adoptive cell therapy. [The present invention 1023] the binding polypeptide being (a) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000010.tif4128 and a heavy chain variable region comprising: (b) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). The method of the present invention 1022, comprising: [The present invention 1024] (a) the binding polypeptide comprises an antibody or an antigen-binding fragment thereof; and / or (b) the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single domain antibody, and optionally the antibody is a full-length antibody; and / or (c) the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof; and / or (d) the binding polypeptide is conjugated to a therapeutic or diagnostic molecule; and / or (e) the binding polypeptide is conjugated to a diagnostic molecule, the diagnostic molecule comprising a detectable label; and / or (f) the binding polypeptide is conjugated to a diagnostic molecule, the diagnostic molecule comprising a detectable label, and further, the detectable label is a radiolabel, a fluorophore, an enzyme, a hapten, biotin, or a chromophore. The method of the present invention 1022 or 1023. [The present invention 1025] The method of any one of claims 1022 to 1024, wherein after the subject has been identified as a suitable subject, the adoptive cell therapy is administered to the subject. [The present invention 1026] (a) the adoptive cell therapy comprises engineered immune cells comprising a chimeric antigen receptor (CAR); and / or (b) the adoptive cell therapy comprises an engineered immune cell comprising a chimeric antigen receptor (CAR), the immune cell being a T lymphocyte; and / or (c) the adoptive cell therapy comprises an engineered immune cell comprising a chimeric antigen receptor (CAR), the immune cell being a NK cell; and / or (d) the adoptive cell therapy comprises an engineered immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR specifically binds to a FAP. The method of the present invention 1025. [The present invention 1027] the binding polypeptide being (a) comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7; and / or (b) comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and / or (c) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7; and / or (d) a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9; and / or (e) comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9; and / or (f) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9; Any of the methods according to claims 1022 to 1026 of the present invention. [The present invention 1028] 1. A method for treating cancer in a subject in need thereof, comprising: (a) a heavy chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7; and (b) a light chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9. administering to said subject an isolated binding polypeptide comprising: [The present invention 1029] (a) the cancer is associated with fibroblast activation protein (FAP)-expressing cells; and / or (b) the FAP-expressing cell is a cancer-associated cell; and / or (c) the FAP-expressing cell is a cancer-associated cell, the cancer-associated cell being a cancer-associated fibroblast (CAF); and / or (d) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a FAP-expressing adipocyte; and / or (e) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a tumor-associated macrophage (TAM); and / or (f) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a tumor-associated neutrophil (TAN); and / or (g) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a myeloid-derived suppressor cell (MDSC); and / or (h) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a cancer-initiating cell; The method of the present invention 1028. [The present invention 1030] (a) the binding polypeptide specifically binds to fibroblast activation protein (FAP); and / or (b) the binding polypeptide comprises an antibody or an antigen-binding fragment thereof; and / or (c) the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single domain antibody, and optionally the antibody is a full-length antibody; and / or (d) the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof; The method of any one of claims 1028 to 1029. [The present invention 1031] 1. A method for treating cancer in a subject in need thereof, comprising: (a)(i) isolating diseased tissue from said subject; (ii) contacting the isolated tissue with a binding polypeptide that specifically binds to a FAP; and (iii) detecting FAP-expressing cells in the isolated tissue. identifying the subject as a suitable subject, comprising: (b) administering to the suitable subject an adoptive cell therapy comprising modified T cells comprising a chimeric antigen receptor (CAR) that specifically binds to a fibroblast activation protein (FAP). A method comprising: [Brief description of the drawings]

[0038] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0039] [Figure 1] Figure 1 is a sequence alignment between the canine FAP gene sequence listed in the NCBI database (XM_005640252.2) and the product of PCR amplification of the canine FAP gene using two primers generated using the NCBI sequence. At the nucleotide level, the product had two conserved base pair substitutions at T127 and A603 (boxed residues, left; insert, right). [Figure 2A]Figures 2A-2C are vector maps and graphs illustrating the generation of recombinant canine FAP-expressing cells. The PCR product of canine FAP was cloned into a eukaryotic expression plasmid (pcDNA3.1) (Figure 2A) and then subcloned into a lentiviral plasmid (pLenti6 / v5-D-TOPO) (Figure 2B). The lentiviral plasmid was transfected into HEK293 cells along with a packaging plasmid, which was used to generate virus-containing supernatants. The resulting virus particle-containing supernatants were used to transduce BALB / c 3T3 cells. Expression of recombinant canine FAP was confirmed by flow cytometry using sheep anti-human FAP antibody (Figure 2C). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Diagram 3] FIG. 3 is a flow cytometry plot demonstrating the generation of anti-FAP antibody-producing hybridoma cells. Canine FAP-transduced BALB / c 3T3 cells were used to immunize 14-week-old BALB / c mice. Spleen cells were fused with sp2 / 0 cells, and the resulting hybridomas were screened against PKH-labeled MC KOSA parental (FAP null) cells and MC KOSA.K9FAP cells expressing the canine FAP transgene. Primary staining was obtained with hybridoma-produced antibodies. Secondary staining was performed using a goat anti-mouse IgG secondary antibody, followed by reading by flow cytometry. [Figure 4] Figures 4A-4C illustrate the isotype characterization of the newly generated 4G5 anti-canine FAP antibody. An ELISA-based commercially available isotyping kit was used in these studies. Figure 4A shows the colorimetric data from the ELISA. The columns represent replicate wells in each row. Positive reactions are present in rows A and G. Figure 4B is an image of the ELISA plate showing significant positive signals in rows A and G. Figure 4C is a plate map of the study in which the 4G5 antibody tested positive for IgG1 and kappa isotype immunoglobulins. [Diagram 5]FIG. 5 depicts a protein gel demonstrating that the 4G5 antibody and a mouse IgG1 isotype control antibody produce similar banding patterns. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any method and material similar or equivalent to those described herein can be used in the practice of the present invention for testing, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms are used.

[0041] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0042] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0043] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for carrying out the disclosed methods.

[0044] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin from a natural source or from a recombinant source, or an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. Tetramers may be naturally occurring or reconstituted from single chain antibodies or antibody fragments. Antibodies also include dimers, which may be naturally occurring or constructed from single chain antibodies or antibody fragments. Antibodies of the present invention include, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab') 2 Antibodies can exist in a variety of forms, including fragments, fragments, and single chain antibodies (scFv), humanized antibodies, and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0045] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments, linear antibodies, scFv antibodies, single domain antibodies, such as camelid antibodies consisting of either the VL or VH domain that exhibit sufficient affinity for the target (Riechmann, 1999, Journal of Immunological Methods 231:25-38), as well as multispecific antibodies formed from antibody fragments. Antibody fragments also include human or humanized antibodies or portions of human or humanized antibodies.

[0046] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0047] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0048] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody that has been produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence that specifies the antibody, and which DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0049] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA that contains a nucleotide sequence or a partial nucleotide sequence that encodes a protein that elicits an immune response, as the term is used herein, therefore encodes an "antigen." Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0050] As used herein, the term "anti-tumor effect" refers to the biological effect manifested by the reduction of tumor volume, the reduction of tumor cell number, the reduction of metastasis number, the increase of life span, or the improvement of various physiological symptoms associated with cancerous conditions. "Anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention in preventing the development of tumors in the first place.

[0051] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is subsequently reintroduced into the individual.

[0052] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0053] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0054] As used herein, the term "cancer" is defined as a disease characterized by rapid and uncontrolled proliferation of abnormal cells.Cancer cells can spread locally or through bloodstream and lymphatic system to other parts of the body.Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain tumor, lymphoma, leukemia, lung cancer, etc.

[0055] As used herein, the term "conserved sequence modification" is intended to refer to an amino acid modification that does not significantly affect or change the binding properties of the antibody that contains the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for their ability to bind to a FAP using the functional assays described herein.

[0056] "Costimulatory ligand," as that term is used herein, includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to a cognate costimulatory molecule on a T cell, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by engagement of the TCR / CD3 complex with, for example, a peptide-loaded MHC molecule. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind B7-H3. Costimulatory ligands also include, in particular, costimulatory molecules presented on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, antibodies that specifically bind B7-H3, and ligands that specifically bind CD83.

[0057] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0058] The term "dysregulated," when used in the context of the level of expression or activity of a FAP, refers to a level of expression or activity that is different from the level of expression or activity of the FAP in an otherwise identical healthy animal, organism, tissue, cell, or component thereof. The term "dysregulated" also refers to an altered regulation of the levels of expression and activity of a FAP compared to the regulation in an otherwise identical healthy animal, organism, tissue, cell, or component thereof.

[0059] "Encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of the gene or cDNA.

[0060] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs can contain introns.

[0061] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result, including, but not limited to, inhibition of viral infection as determined by any means suitable in the art.

[0062] As used herein, "endogenous" refers to any material from or produced within an organism, cell, tissue, or system.

[0063] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0064] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0065] "Expression vector" refers to a vector that contains a recombinant polynucleotide that contains an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses that incorporate recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0066] As used herein, "homologous" refers to the identity of subunit sequences between two polymer molecules, for example, between two nucleic acid molecules, for example, two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer that is 10 subunits long) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matching or homologous, the two sequences are 90% homologous.

[0067] "Humanized" and "chimeric" forms of non-human (e.g., murine) antibodies are immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')) that contain minimal sequence derived from non-human immunoglobulin. 2, or other antigen-binding subsequences of antibodies). For the most part, humanized and chimeric antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some cases, residues from the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized and chimeric antibodies can include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to refine and optimize antibody performance. In general, humanized and chimeric antibodies will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. Humanized and chimeric antibodies will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The World Health Organization (WHO) International Nonproprietary Nomenclature (INN) Expert Group has defined the requirements for a non-human derived antibody to be considered "humanized". According to the guidelines, a comparison of the candidate antibody to human sequences must be performed by the International Immunogenetics Information System® (IMGT®) DomainGapAlign tool (www.imgt.org). This tool queries the IMGT® database of antibody germline variable region genes, where an alignment score is generated only for germline sequence variable region exons, thus omitting CDR3 and part of the J region from the analysis. For an antibody to be "humanized", in addition to being "closer to human than other species", the top "hit" must be human and the identity to the human sequence must be at least 85%; otherwise, the antibody will be designated as "chimeric".For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0068] "Fully human" refers to an immunoglobulin, eg, an antibody, whose entire molecule is of human origin or consists of an amino acid sequence identical to a human form of an antibody.

[0069] As used herein, "instruction material" includes publications, recordings, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The instruction material of the kits of the invention can be, for example, affixed to a container containing the nucleic acids, peptides, and / or compositions of the invention or shipped with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instruction material can be shipped separately from the container with the intention that the instruction material and the compound will be used jointly by the recipient.

[0070] As used herein, "identity" refers to the identity of subunit sequences between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that match or are identical; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer that is 10 amino acids long) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are identical or matched, the two amino acid sequences are 90% identical.

[0071] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0072] In the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0073] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence encoding a protein can include introns in some versions.

[0074] As used herein, "lentivirus" refers to a genus of the Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; it can deliver a significant amount of genetic information to the DNA of host cells, making it one of the most efficient methods of gene delivery vectors.HIV, SIV, and FIV are all examples of lentiviruses.Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0075] The term "operably linked" refers to the functional link between a control sequence and a heterologous nucleic acid sequence, which allows the latter to be expressed. For example, a first nucleic acid sequence is functionally linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Generally, the DNA sequences that are functionally linked are adjacent and, where necessary to join two protein coding regions, are in the same reading frame.

[0076] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0077] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, those that are synthesized by recombinant means, i.e., conventional cloning techniques and PCR. (商標) These include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including cloning of nucleic acid sequences from recombinant libraries or cell genomes, using nucleic acid sequences such as those described above, as well as by synthetic means.

[0078] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, of which there are many types, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0079] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate the specific transcription of a polynucleotide sequence.

[0080] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence that is required for the expression of a gene product that is operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements that are required for the expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0081] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a cell to produce the gene product under most or all physiological conditions of the cell.

[0082] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be substantially produced in a cell only if an inducer corresponding to the promoter is present in the cell.

[0083] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes a gene product to be substantially produced in a cell only if the cell is a cell of the tissue type corresponding to the promoter.

[0084] A "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the plasma membrane of a cell.

[0085] "Single chain antibody" refers to an antibody formed by recombinant DNA technology in which the heavy and light fragments of immunoglobulin chains are linked together using an engineered amino acid span to reproduce the Fv region of the antibody as a single polypeptide.Various methods for producing single chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0086] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. As used herein, a "subject" or "patient" may be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0087] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that is separated from other cell types that it normally associates with in its naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous population of cells. In other cases, the term simply refers to a cell that is separated from the cells that it naturally associates with in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0088] As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0089] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0090] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct position and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0091] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0092] As used herein, the term "specifically binds" refers to an antibody or ligand that recognizes and binds to its cognate binding partner (e.g., a stimulatory and / or costimulatory molecule present on a T cell) protein present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0093] The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., the TCR / CD3 complex) with its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex. Stimulation can mediate changes in expression of certain molecules, such as, for example, downregulation of TGF-β, and / or remodeling of cytoskeletal structure.

[0094] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell and / or a tumor cell.

[0095] As used herein, "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) or tumor cell, can specifically bind to a cognate binding partner (herein referred to as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, among others, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0096] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose not only the individual numerical values ​​within the range, but also all possible subranges. For example, a description of a range such as 1-6 should be considered to specifically disclose not only the individual numbers within the range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, but also subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. This applies regardless of the breadth of the range.

[0097] B. Binding Polypeptides, Antibodies, and scFvs The binding polypeptides and antibodies of the present invention are characterized by certain functional features or properties of the antibody. In such an embodiment, the antigen-binding domain can be referred to as cross-species reactive. For example, the binding polypeptides and antibodies specifically bind to canine fibroblast activation protein (FAP) and also cross-react with mouse and human FAP. Preferably, the binding polypeptides and antibodies of the present invention bind to canine, mouse, and human FAP with high affinity. Preferably, the binding polypeptides and antibodies of the present invention specifically recognize the canine FAP protein naturally expressed on a cell and do not cross-react with other surface molecules on the cell.

[0098] In certain aspects, the invention provides isolated binding polypeptides comprising an antigen-binding domain that specifically binds to an epitope of human and canine and / or murine fibroblast activation protein (FAP). In certain embodiments, the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs).

[0099] In certain embodiments, the present invention provides an isolated binding polypeptide comprising a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs). In certain embodiments, HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and / or HCDR2 comprises the amino acid sequence and / or HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3). In certain embodiments, the antigen binding domain comprises a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0100] In certain aspects, the present invention provides an isolated binding polypeptide comprising an HCDR1 comprising the amino acid sequence YTITSYSLH (SEQ ID NO:1). Also provided is an isolated binding polypeptide comprising an HCDR1 comprising the amino acid sequence GYTITSYSLH (SEQ ID NO:17). Also provided is an isolated binding polypeptide comprising an HCDR2 comprising the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3). Also provided is an isolated binding polypeptide comprising an HCDR3 comprising TIFF0007603670000013.tif4128. Also provided is an isolated binding polypeptide comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence TASSSVSYMY (SEQ ID NO:4). Also provided is an isolated binding polypeptide comprising an LCDR2 comprising the amino acid sequence LTSNLA (SEQ ID NO:5). Also provided is an isolated binding polypeptide comprising an LCDR2 comprising the amino acid sequence LTSNLAS (SEQ ID NO:20). Also provided is an isolated binding polypeptide comprising an LCDR3 comprising the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0101] In certain embodiments, the present invention provides a method for the preparation of a polypeptide comprising a polypeptide comprising: TIFF0007603670000014.tif4128 and / or HCDR3 contains the amino acid sequence The present invention provides an isolated binding polypeptide comprising a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) comprising TIFF0007603670000015.tif4128. In certain embodiments, the isolated binding polypeptide comprises a light chain variable region comprising three light chain complementarity determining regions (LCDRs), in which LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0102] In one particular aspect, the present invention provides a method for the preparation of a polypeptide comprising the amino acid sequence GYTITSYSLH (SEQ ID NO:17), HCDR1 comprising the amino acid sequence GYTITSYSLH (SEQ ID NO:17), An isolated binding polypeptide is provided, comprising an HCDR2 comprising TIFF0007603670000016.tif4128, an HCDR3 comprising the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3), an LCDR1 comprising the amino acid sequence TASSSVSYMY (SEQ ID NO:4), an LCDR2 comprising the amino acid sequence LTSNLAS (SEQ ID NO:20), and an LCDR3 comprising the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0103] In certain embodiments, the present invention provides isolated binding polypeptides comprising a heavy chain variable region comprising any of the three heavy chain complementarity determining regions described herein, i.e., HCDR1, HCDR2, and HCDR3. In certain embodiments, the isolated binding polypeptide comprises a light chain variable region comprising any of the three light chain complementarity determining regions described herein, i.e., LCDR1, LCDR2, and LCDR3. In certain embodiments, the isolated binding polypeptide comprises any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described herein. Those skilled in the art will be able to easily determine the appropriate complementarity determining region based on amino acid numbering in view of the heavy and light chain variable region sequences provided herein.

[0104] Permissible variations in complementarity determining region (CDR) sequences will be known to those of skill in the art. For example, in some embodiments, a polypeptide comprises a complementarity determining region (HCDR or LCDR) comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:1, 2, 3, 4, 5, 6, 17, 18, 19, or 20.

[0105] In some embodiments, the binding polypeptide binds to fibroblast activation protein (FAP). In some embodiments, the binding polypeptide comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody. In further embodiments, the antibody is a full-length antibody. In yet other embodiments, the antibody or antigen-binding fragment is a murine antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0106] In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7. In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, a binding polypeptide consists of a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7.

[0107] In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, a binding polypeptide consists of a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:9.

[0108] Also provided is an isolated binding polypeptide comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9.

[0109] In certain embodiments, the present invention includes antibodies that bind to the same epitope on human, mouse, or dog FAP as the antibodies of the present invention (i.e., antibodies that have the ability to cross-compete with any of the antibodies of the present invention for binding to dog FAP). In a preferred embodiment, the reference antibody for cross-competition studies can be one of the antibodies described herein (e.g., 4G5). For example, Biacore analysis, ELISA assay, or flow cytometry can be used to demonstrate cross-competition with the antibodies of the present invention. The ability of the test antibody to inhibit the binding of, for example, 4G5 to dog FAP demonstrates that the test antibody can compete with 4G5 for binding to dog, mouse, and human FAP, and thus is believed to bind to the same epitope of FAP as 4G5.

[0110] The antibodies of the present invention can be prepared using an antibody having one or more of the VH and / or VL sequences disclosed herein as starting material for engineering an engineered antibody, which may have altered properties compared to the starting antibody. The antibody can be engineered by modifying one or more amino acids in one or both variable regions (i.e., VH and / or VL), for example, in one or more CDR regions and / or in one or more framework regions. Additionally or alternatively, the antibody can be engineered by modifying residues in the constant region, for example, to change the effector function of the antibody.

[0111] Also provided are single chain variable fragments (scFv) that comprise an antigen binding domain that specifically binds to an epitope of human and / or canine and / or murine fibroblast activation protein (FAP).

[0112] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a peptide-encoding linker that connects the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., FAP-binding domain) comprises an scFv with the following configuration from N-terminus to C-terminus: VH-linker-VL. In some embodiments, the antigen-binding domain comprises an scFv with the following configuration from N-terminus to C-terminus: VL-linker-VH. Those skilled in the art will be able to select the appropriate configuration for use in the present invention.

[0113] The linker is usually rich in glycine for flexibility and serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in the art, including, but not limited to, glycine serine (GS) linkers, e.g., (GS) n , (GSGGS) n (SEQ ID NO:21), (GGGS) n (SEQ ID NO:22), and (GGGGS) n(SEQ ID NO:23), where n represents an integer of at least 1. Exemplary linker sequences can include amino acid sequences, including, but not limited to, GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO:27), GGGSG (SEQ ID NO:28), GSSSG (SEQ ID NO:29), GGGGS (SEQ ID NO:30), GGGSGGGGSGGGGS (SEQ ID NO:15), and the like. One of skill in the art will be able to select an appropriate linker sequence for use in the present invention. In one aspect, the scFv of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), where the VH and VL are represented by the nucleic acid sequence The amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO:15) can be encoded by TIFF0007603670000017.tif12128.

[0114] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing sequences encoding VH and VL, as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 136(8):2252-61). 2(10:31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0115] In certain aspects, the present invention provides single-chain variable fragments (scFv) comprising an antigen-binding domain that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP), the antigen-binding domain comprising a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs).

[0116] In certain embodiments of the scFv, HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and / or HCDR2 comprises the amino acid sequence and / or HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3), and / or LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). The heavy chain variable region and the light chain variable region are separated by a linker.

[0117] In certain embodiments of the scFv, HCDR1 comprises the amino acid sequence GYTITSYSLH (SEQ ID NO:17) and / or HCDR2 comprises the amino acid sequence and / or HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3), and / or LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLAS (SEQ ID NO:20), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). The heavy chain variable region and the light chain variable region are separated by a linker.

[0118] Also provided is a single chain variable fragment (scFv) comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 and / or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9. The heavy and light chain variable regions are separated by a linker.

[0119] In another aspect, there is provided a single chain variable fragment (scFv) comprising the amino acid sequence set forth in SEQ ID NO:11 or 13. In another aspect, there is provided a single chain variable fragment (scFv) consisting of the amino acid sequence set forth in SEQ ID NO:11 or 13.

[0120] Acceptable variations in scFv sequences will be known to those of skill in the art. For example, in some embodiments, the scFv comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 9, 11, 13, 15, 17, 18, 19, or 20.

[0121] Table 1. Amino acid and nucleotide sequences TIFF0007603670000020.tif24168TIFF0007603670000021.tif226168TIFF0007603670000022.tif229168TIFF0007603670000023.tif127168

[0122] C. Nucleic Acids and Expression Vectors The present disclosure provides isolated nucleic acids encoding binding polypeptides (e.g., antibodies or fragments thereof, such as scFvs) that comprise an antigen-binding domain that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP). The nucleic acids of the present disclosure can include a polynucleotide sequence that encodes any one of the binding polypeptides, scFvs, or antibodies disclosed herein.

[0123] In certain embodiments, the binding polypeptide has HCDR1 comprising the amino acid sequence YTITSYSLH (SEQ ID NO:1) and / or HCDR2 comprising the amino acid sequence TIFF0007603670000024.tif4128, and / or a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) where HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3). In certain embodiments, the antigen binding domain also comprises a light chain variable region comprising three light chain complementarity determining regions (LCDRs) where LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0124] In certain embodiments, the binding polypeptide has HCDR1 comprising the amino acid sequence GYTITSYSLH (SEQ ID NO:17) and / or HCDR2 comprising the amino acid sequence and / or a heavy chain variable region wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLAS (SEQ ID NO:20), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0125] In certain embodiments, the binding polypeptide has HCDR1 comprising the amino acid sequence YTITSYSLH (SEQ ID NO:1) and / or HCDR2 comprising the amino acid sequence TIFF0007603670000026.tif4128 and / or HCDR3 contains the amino acid sequence a heavy chain variable region comprising the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or a light chain variable region comprising the amino acid sequence LTSNLA (SEQ ID NO:5), and / or an antigen binding domain comprising the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0126] In certain embodiments, the nucleic acid encoding the binding polypeptide comprises an antigen-binding domain comprising a heavy chain variable region comprising any of the three heavy chain complementarity determining regions described herein, i.e., HCDR1, HCDR2, and HCDR3. In certain embodiments, the antigen-binding domain comprises a light chain variable region comprising any of the three light chain complementarity determining regions described herein, i.e., LCDR1, LCDR2, and LCDR3. In certain embodiments, the antigen-binding domain comprises any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, as described herein. Those skilled in the art will be able to easily determine the appropriate complementarity determining region based on the amino acid numbering in view of the heavy and light chain variable region sequences provided herein.

[0127] In certain embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a humanized antibody or a fragment thereof.

[0128] Also provided are nucleic acids encoding binding polypeptides comprising an antigen binding domain that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP), wherein the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO:8. In certain embodiments, the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:8. In certain embodiments, the heavy chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:8.

[0129] In certain embodiments, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 10. In certain embodiments, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO: 10. In certain embodiments, the light chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO: 10.

[0130] Also provided is an isolated nucleic acid encoding a binding polypeptide comprising a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:8 and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:10.

[0131] Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs). In certain embodiments, HCDR1 comprises the amino acid sequence GYTITSYSLH (SEQ ID NO:17) and / or HCDR2 comprises the amino acid sequence TIFF0007603670000028.tif4128, and / or HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3), and / or LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLAS (SEQ ID NO:20), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0132] In certain embodiments, the nucleic acid has HCDR1 comprising the amino acid sequence YTITSYSLH (SEQ ID NO:1) and / or HCDR2 comprising the amino acid sequence TIFF0007603670000029.tif4128, and / or the light chain variable region comprises three light chain complementarity determining regions (LCDRs) in which LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0133] In certain embodiments, the nucleic acid has HCDR1 comprising the amino acid sequence GYTITSYSLH (SEQ ID NO:17) and / or HCDR2 comprising the amino acid sequence TIFF0007603670000030.tif4128, and / or HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3). In certain embodiments, the antigen binding domain comprises a light chain variable region comprising three light chain complementarity determining regions (LCDRs) where LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLAS (SEQ ID NO:20), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0134] In certain embodiments, the nucleic acid has HCDR1 comprising the amino acid sequence YTITSYSLH (SEQ ID NO:1) and / or HCDR2 comprising the amino acid sequence TIFF0007603670000031.tif4128 and / or HCDR3 contains the amino acid sequence TIFF0007603670000032.tif4128. In certain embodiments, the single chain variable fragment also comprises a light chain variable region comprising three light chain complementarity determining regions (LCDRs), in which LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0135] In certain embodiments, the nucleic acid comprising the single chain variable fragment comprises a heavy chain variable region comprising any of the three heavy chain complementarity determining regions described herein, i.e., HCDR1, HCDR2, and HCDR3. In certain embodiments, the single chain variable fragment comprises a light chain variable region comprising any of the three light chain complementarity determining regions described herein, i.e., LCDR1, LCDR2, and LCDR3. In certain embodiments, the single chain variable fragment comprises any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, as described herein. Those skilled in the art will be able to easily determine the appropriate complementarity determining region based on the amino acid numbering in view of the heavy and light chain variable region sequences provided herein.

[0136] Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising a heavy chain variable region encoded by the polynucleotide sequence set forth in SEQ ID NO:8; and / or a light chain variable region encoded by the polynucleotide sequence set forth in SEQ ID NO:10. The heavy chain variable region and the light chain variable region are separated by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO.15.

[0137] Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv), the nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO:12 or 14. Also provided is an isolated nucleic acid encoding a single chain variable fragment (scFv), the nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:12 or 14.

[0138] Acceptable variations in nucleic acid sequences will be known to those of skill in the art. For example, in some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NOs:8, 10, 12, or 14.

[0139] In certain embodiments, the nucleic acid of the present disclosure comprises a first polynucleotide sequence and a second polynucleotide sequence.In certain embodiments, the first polynucleotide sequence comprises a polynucleotide sequence encoding the FAP targeting binding polypeptide of the present disclosure.In some embodiments, the FAP targeting binding polypeptide of the present disclosure can be used in combination with other therapeutic agents, such as, but not limited to, immunotherapy, such as cancer immunotherapy and checkpoint blockade, or CAR-T cell therapy.Thus, in such embodiments, the second polynucleotide sequence can comprise a polynucleotide sequence encoding an anti-cancer antibody, a checkpoint blockade molecule, or a CAR.

[0140] The first and second polynucleotide sequences can be separated by a linker. For example, in certain embodiments, the heavy chain variable region and the light chain variable region of the scFv are separated by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO.15. A linker for use in the present disclosure allows multiple proteins to be encoded in the same nucleic acid sequence (e.g., a multicistronic or bicistronic sequence) that is translated as a polyprotein that is dissociated into separate protein components. In certain embodiments, the nucleic acid comprises, from 5' to 3', a first polynucleotide sequence, a linker, and a second polynucleotide sequence. In certain embodiments, the nucleic acid comprises, from 5' to 3', a second polynucleotide sequence, a linker, and a first polynucleotide sequence.

[0141] In some embodiments, the linker comprises a nucleic acid sequence that codes for an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into the initiation codon, such as ATG, of a protein coding region, thereby resulting in cap-independent translation of the gene. A variety of internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRES available from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES available from, for example, cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select the appropriate IRES for use in the present invention.

[0142] In some embodiments, the linker comprises a nucleic acid sequence that encodes a self-cleaving peptide. As used herein, "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows multiple proteins to be encoded as a polyprotein that dissociates into component proteins upon translation. The use of the term "self-cleaving" is not intended to imply a proteolytic cleavage reaction. A variety of self-cleaving or 2A peptides are known to those of skill in the art, including, but not limited to, those found in members of the Picornaviridae virus family, such as foot and mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-1); and carioviruses, such as theirovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. One of skill in the art would be able to select an appropriate self-cleaving peptide for use in the present invention.

[0143] In some embodiments, the linker further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-Golgi and processes protein precursors prior to their secretion. Furin cleaves at the COOH-terminus of its consensus recognition sequence. A variety of furin consensus recognition sequences (or "furin cleavage sites") are known to those of skill in the art, including, but not limited to, Arg-X1-Lys-Arg (SEQ ID NO:32) or Arg-X1-Arg-Arg (SEQ ID NO:33), X2-Arg-X1-X3-Arg (SEQ ID NO:34), and Arg-X1-X1-Arg (SEQ ID NO:35), e.g., Arg-Gln-Lys-Arg (SEQ ID NO:36), where X1 is any naturally occurring amino acid, X2 is Lys or Arg, and X3 is Lys or Arg. Those of skill in the art will be able to select a suitable furin cleavage site for use in the present invention.

[0144] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, a linker comprising a nucleic acid sequence encoding a furin cleavage site and F2A, a linker comprising a nucleic acid sequence encoding a furin cleavage site and E2A, a linker comprising a nucleic acid sequence encoding a furin cleavage site and P2A, and a linker comprising a nucleic acid sequence encoding a furin cleavage site and T2A. Those skilled in the art will be able to select the appropriate combination for use in the present invention. In such embodiments, the linker can further comprise a spacer sequence between the furin cleavage site and the 2A peptide. In some embodiments, the linker comprises a furin cleavage site 5' to the 2A peptide. In some embodiments, the linker comprises a 2A peptide 5' to the furin cleavage site. A variety of spacer sequences are known in the art, including, but not limited to, glycine serine (GS) spacers, such as (GS)n, (GSGGS)n (SEQ ID NO:21), and (GGGS)n (SEQ ID NO:22), where n represents an integer of at least 1. Exemplary spacer sequences can include amino acid sequences, including, but not limited to, GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO:27), GGGSG (SEQ ID NO:28), GSSSG (SEQ ID NO:29), and the like. One of skill in the art would be able to select an appropriate spacer sequence for use in the present invention.

[0145] Another aspect of the present invention provides a vector comprising any one of the isolated nucleic acids disclosed herein.In certain embodiments, the vector is selected from the group consisting of DNA vector, RNA vector, plasmid, lentivirus vector, adenovirus vector, adeno-associated virus vector and retrovirus vector.In certain embodiments, the vector is an expression vector.

[0146] Also provided is a host cell comprising any of the vectors or nucleic acids disclosed herein.Host cell can be of eukaryotic, prokaryotic, mammalian or bacterial origin.Also provided herein is a method for producing binding polypeptide or scFv that binds to FAP, the method comprising culturing host cell.

[0147] In some aspects, the nucleic acids of the disclosure may be operably linked to transcriptional control elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those of skill in the art.

[0148] In certain embodiments, the nucleic acid is operably linked to a promoter. In certain embodiments, the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

[0149] For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoter; promoters present in retroviral long terminal repeats; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, for example, eukaryotes and prokaryotes. For example, modification of reversible promoters derived from a first organism for use in a second organism, such as a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, is well known in the art.Such reversible promoters and systems based on such reversible promoters, but also including additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (A1cR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-associated regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0150] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK cell-specific promoter.For example, the CD4 gene promoter can be used; see, for example, Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416.As another example, the CD8 gene promoter can be used.NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al. Blood (2011) 117:1565.

[0151] For expression in yeast cells, suitable promoters are constitutive promoters such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, etc.; or regulatable promoters such as the GAL1 promoter, the GAL10 promoter, the ADH2 promoter, the PHOS promoter, the CUP1 promoter, the GALT promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 (e.g., for use in Pichia). The selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Suitable promoters for use in prokaryotic host cells include, but are not limited to, the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, and the like; the araBAD promoter; in vivo regulated promoters, such as the ssaG promoter or related promoters (see, e.g., U.S. Patent Application Publication No. 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813) and the like (e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol.(1992) 10:888-892); sigma 70 promoters, e.g., consensus sigma 70 promoters (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); stationary phase promoters, e.g., dps promoters, spv promoters, and the like; promoters from pathogenicity island SPI-2 (see, e.g., WO96 / 17951); actA promoters (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoters (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); tet promoters (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res.(1984) 12:7035). Suitable strong promoters for use in prokaryotes, such as Escherichia coli, include, but are not limited to, Trc, Tac, T5, T7, and Plambda. Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25).

[0152] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably linked to it. Other constitutive promoter sequences can also be used, including but not limited to the Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, EF-1 alpha promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0153] In some embodiments, the locus or construct or transgene containing a suitable promoter is irreversibly switched through the induction of an inducible system.Suitable systems for the induction of irreversible switching are well known in the art, for example, the induction of irreversible switching can use Cre-lox mediated recombination (see, for example, Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference).Any suitable combination of recombinase, endonuclease, ligase, recombination site, etc., known in the art, can be used in the generation of irreversibly switchable promoter. The methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein are used in the generation of irreversibly switched promoters and are well known in the art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0154] The nucleic acids of the present disclosure may be present in an expression vector and / or a cloning vector. Expression vectors may include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. Numerous suitable vectors and promoters are known to those of skill in the art, and many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example and should in no way be construed as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0155] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker that is functional in the expression host may also be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984, and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci.USA (1993) 90: 10613-10617); SV40; herpes simplex virus; viral vectors based on human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0156] Further suitable expression vectors for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0157] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0158] In some embodiments, an expression vector (e.g., lentiviral vector) can be used to introduce a nucleic acid into a host cell. Thus, an expression vector (e.g., lentiviral vector) of the present invention can include a nucleic acid encoding a polypeptide. In some embodiments, an expression vector (e.g., lentiviral vector) includes additional elements that aid in the functional expression of a polypeptide encoded therein. In some embodiments, an expression vector including a nucleic acid encoding a polypeptide further includes a mammalian promoter. In one embodiment, the vector further includes an elongation factor 1 alpha promoter (EF-1α promoter). Use of the EF-1α promoter can increase the efficiency of expression of downstream transgenes. Physiological promoters (e.g., EF-1α promoter) may be less likely to induce integration-mediated genotoxicity and can abrogate the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those of skill in the art and can be incorporated into the vectors of the present invention. In some embodiments, a vector (e.g., lentiviral vector) further includes non-essential cis-acting sequences that can improve titer and gene expression. One non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear import. Other non-essential cis-acting sequences are known to those skilled in the art and can be incorporated into the vectors of the present invention (e.g., lentiviral vectors). In some embodiments, the vector further comprises a post-transcriptional regulatory element. The post-transcriptional regulatory element can improve the translation of RNA, improve the expression of the transgene, and stabilize the RNA transcript. One example of a post-transcriptional regulatory element is the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vector for the present invention further comprises a WPRE sequence. A variety of post-transcriptional regulatory elements are known to those skilled in the art and can be incorporated into the vectors of the present invention (e.g., lentiviral vectors).The vector of the present invention can further comprise additional elements, such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTR). The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of retroviral DNA, including the U3, R, and U5 regions. LTRs generally provide functions required for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and for viral replication. In one embodiment, the vector of the present invention (e.g., lentiviral vector) comprises a 3'U3 deleted LTR. Thus, the vector of the present invention (e.g., lentiviral vector) can comprise any combination of elements described herein to enhance the efficiency of functional expression of the transgene. For example, the vector of the present invention (e.g., lentiviral vector) can comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3'U3 deleted LTR' in addition to the nucleic acid encoding the CAR.

[0159] The vector of the present invention may be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3'LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). The self-inactivating vector can prevent viral transcription beyond the first round of viral replication. Therefore, the self-inactivating vector may infect and then be integrated into the host genome (e.g., mammalian genome) only once, and cannot be passed on further. Therefore, the self-inactivating vector can greatly reduce the risk of producing a replication-competent virus.

[0160] In some embodiments, the nucleic acid of the present invention may be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art; any known method can be used to synthesize RNA comprising a sequence encoding the polypeptide of the present disclosure. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15: 9053. The introduction of RNA comprising a nucleotide sequence encoding the polypeptide of the present disclosure into host cells can be performed in vitro, ex vivo, or in vivo. For example, RNA comprising a nucleotide sequence encoding the polypeptide of the present disclosure can be used to electroporate host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) in vitro or ex vivo.

[0161] To evaluate the expression of a polypeptide or a part thereof, the expression vector introduced into the cell can also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that are to be transfected or infected by the viral vector. In some embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be flanked by the appropriate regulatory sequences to allow expression in the host cell. Useful selection markers include, but are not limited to, antibiotic resistance genes.

[0162] Reporter genes are used to identify cells that may be transfected and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by recipient organisms and tissues, and code for polypeptides whose expression is manifested by some easily detectable characteristic, such as enzyme activity. The expression of reporter genes is evaluated at a suitable time after DNA is introduced into recipient cells. Suitable reporter genes can include, but are not limited to, genes that code for luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0163] In some aspects, the nucleic acids of the disclosure provide for the production of a polypeptide described herein, e.g., in a host cell. In some aspects, the nucleic acids of the disclosure provide for the amplification of nucleic acids encoding the polypeptides.

[0164] D.How to use The anti-FAP antibodies, binding polypeptides, and scFvs disclosed herein can also be used for diagnostic and imaging applications.

[0165] For example, the anti-FAP antibodies described herein can be used to assay FAP protein levels in biological samples using classical immunohistological methods known to those skilled in the art, including immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Western blotting, or immunohistochemistry. Suitable antibody assay labels are known in the art and include, but are not limited to, enzyme labels, e.g., glucose oxidase, alkaline phosphatase, and horseradish peroxidase; radioisotopes, e.g., iodine ( 125 I, 121 I), Carbon (14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99m Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Such labels can be used to label the binding polypeptides, antibodies or antigen-binding fragments thereof (e.g., scFv) described herein.

[0166] Alternatively, a secondary antibody that recognizes the anti-FAP antibody or antigen-binding fragment thereof described herein can be labeled and used in combination with the anti-FAP antibody or antigen-binding fragment thereof to detect FAP protein levels. In one aspect, the invention relates to the use of the anti-FAP antibody of the invention to assay and / or detect FAP protein levels in a biological sample in vitro or in a subject in vivo.

[0167] Assaying for expression level of FAP protein is intended to include qualitatively or quantitatively measuring or estimating the level of FAP protein in a first biological sample, either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing with the level of disease-related protein in a second biological sample). The expression level of FAP polypeptide in a first biological sample can be measured or estimated and compared with a standard FAP protein level. The standard can be taken from a second biological sample obtained from an individual without the disorder, or can be determined by averaging the levels from a population of individuals without the disorder. As recognized in the art, once a "standard" FAP polypeptide level is known, it can be used repeatedly as a standard for comparison.

[0168] The anti-FAP antibodies or antigen-binding fragments thereof described herein can be used for prognostic, diagnostic, monitoring, or screening applications, including in vitro and in vivo applications based herein, that are well known and standard to those of skill in the art.

[0169] Prognostic, diagnostic, monitoring and screening assays and kits for in vitro evaluation and assessment of immune system status and / or immune response can be used to predict, diagnose and monitor or assess patient samples, including those known to have or suspected to have immune system dysfunction or disease or condition, or for predicted or desired immune system, antigen or vaccine responses associated with disease treatment of the condition. Evaluation and assessment of immune system status and / or immune response is also useful to determine a patient's suitability for drug clinical trials or for administration of a particular chemotherapeutic agent or antibody or antigen-binding fragment thereof, including combinations of the following, versus different agents or antibodies or antigen-binding fragments thereof. This type of prognostic and diagnostic monitoring and assessment has already been demonstrated in breast cancer using antibodies against the HER2 protein (HercepTest (商標) (Dako), the assay has also been used to evaluate patients for antibody therapy using Herceptin®. In vivo applications include directed cell therapy and modulation of the immune system and radioimaging of immune responses.

[0170] In one aspect, the invention relates to an anti-FAP antibody and / or pharmaceutical composition of the invention for use as a diagnostic agent.

[0171] In one aspect, the invention relates to an anti-FAP antibody and / or pharmaceutical composition of the invention for use in a method for the prediction, diagnosis, and / or monitoring of immune system dysfunction and / or cancer.

[0172] In one aspect, the invention relates to the use of an anti-FAP antibody of the invention to predict, diagnose, and / or monitor immune system dysfunction and / or cancer in a subject by assaying and / or detecting FAP protein levels in a subject's biological sample in vitro.

[0173] In one embodiment, anti-FAP antibody or its antigen-binding fragment can be used in immunohistochemistry of biopsy samples.In another embodiment, anti-FAP antibody or its antigen-binding fragment can be used to detect the level of FAP or the level of cells that contain FAP on their membrane surface, and then correlate the level with a certain disease condition.Anti-FAP antibody or its antigen-binding fragment described herein can carry a detectable or functional label.When fluorescent label is used, it can utilize currently available microscopy and fluorescence-activated cell sorter analysis (FACS) or a combination of both method procedures known in the art to identify and quantify specific binding members.

[0174] The anti-FAP antibody or antigen-binding fragment thereof described herein can carry a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes, such as aminocoumarin, fluorescein, and Texas red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. The anti-FAP antibody or antigen-binding fragment thereof can carry a radioactive label, such as an isotope. 3 H, 14 C. 32 P, 35 S, 36 C1, 51 Cr, 52 Co, 57 Co, 59 Fe, 67 Cu, 90 Y, 99M Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au,211 At, 213 Bi, 225 Ac, and 186 Re can be carried. When a radioactive label is used, currently available counting procedures known in the art can be utilized to identify and quantify the specific binding of the anti-FAP antibody or antigen-binding fragment thereof to the FAP. When the label is an enzyme, detection can be accomplished by any of the currently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques known in the art. This can be accomplished by contacting the sample or control sample with the anti-FAP antibody or antigen-binding fragment thereof under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and the FAP. Any complexes formed between the antibody or antigen-binding fragment thereof and the FAP in the sample and control are detected and compared. In light of the specific binding of the antibodies described herein to the FAP, the antibodies or antigen-binding fragment thereof can be used to specifically detect FAP expression on the surface of cells. The antibodies or antigen-binding fragments thereof described herein can also be used to purify the FAP by immunoaffinity purification. Also included herein is an assay system that can be prepared in the form of a test kit for quantitative analysis of the extent of the presence of, for example, the FAP or the CTLA-4 / FAP ligand complex. The system or test kit can include a labeled component, such as a labeled antibody, and one or more additional immunochemical reagents.

[0175] In one aspect, the invention relates to an in vitro method for assaying and / or detecting FAP protein levels in a biological sample, comprising the steps of: (1) contacting the sample and, optionally, a control sample, with an anti-FAP antibody, or antigen-binding fragment thereof, of the invention under conditions that allow for the formation of a complex between the antibody, or antigen-binding fragment thereof, and the FAP; and (2) detecting and comparing the complex formed in the sample and, optionally, the control.

[0176] In certain embodiments, the level and / or distribution of FAP is determined in vivo (e.g., non-invasively) by detecting the detectably labeled antibody disclosed herein using a suitable imaging technique, such as positron emission tomography (PET) scan. For example, targeted antibody PET or immuno-PET (e.g., anti-FAP PET) can be used to detect the level and / or distribution (e.g., tumor localization) of target FAP-expressing cells in vivo. Techniques for antibody imaging (e.g., antibody PET imaging) are known in the art, as described, for example, by Lamberts, LE et al. (2015) J. Clin. Oncol. 33 (DOI: 10.1200 / JCO.2014.57.8278); Tavare, R. et al. (2014) PNAS 111(3):1108-1113; Pampaloni et al., J Clin Oncol 32:5s, 2014 (suppl; abstr 3084); and Boerman and Oyen (2011) The Journal of Nuclear Medicine 52 (8):1171-72; U.S. Patent No. 5,192,525, U.S. Patent No. 5,219,548, U.S. Patent No. 5,399,338, which are incorporated herein by reference.

[0177] In one embodiment, the level and / or distribution of FAPs can be determined by, for example, measuring the level and / or distribution of FAPs, e.g., as described in Tavare, R. et al. (2014) PNAS 111(3):1108-1113, e.g., 64The FAP level and / or distribution is determined in vivo by detecting an anti-FAP antibody detectably labeled with a PET reagent conjugated to 5-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for Cu radiolabeling. In another embodiment, the level and / or distribution of the FAP is determined in vivo, for example, by detecting the fluorine-18 label of an anti-FAP antibody detectably labeled with a PET reagent, for example, an antibody, an antibody fragment, or a FAP targeting polypeptide. In yet another embodiment, the FAP level and / or distribution is determined in vivo by detecting an anti-FAP antibody detectably labeled with a PET reagent, for example, as described in U.S. Pat. No. 9,988,452. In yet another embodiment, the FAP targeting polypeptide is covalently linked to desferoxamine for chelation to a suitable radioisotope.

[0178] In other embodiments, the level of FAP is determined (eg, using immunohistochemical techniques) in a sample obtained from the subject (eg, a tumor biopsy).

[0179] Detection reagents are also within the scope of the present invention. For example, immunoPET reagents are provided that include the anti-FAP antibody molecules described herein. Exemplary labeling reagents include, but are not limited to, astatine-211 (211At), bromine-76 ( 76 Br), Calcium-47( 47 Ca), Carbon-11( 11 C), carbon-14( 14 C), Chromium-51( 51 Cr), Cobalt-57( 57 Co), Cobalt-58( 58 Co), Copper-64( 64 Cu), Erbium-169( 169 Er), Fluorine-18( 18 F), fluorodeoxyglucose ( 18 F-FDG), Gallium-67( 67 Ga), Gallium-68( 68 Ga), hydrogen-3( 3H), Indium-111( 111 In), iodine-123( 123 I), iodine-124( 124 I), iodine-125( 125 I), iodine-131( 131 I), iron-59( 59 Fe), krypton-81m ( 81m Kr), Lutetium-177( 177 Lu), Nitrogen-13( 13 N), oxygen-15( 15 O), phosphorus-32( 32 P), Samarium-153( 153 Sm), Selenium-75( 75 Se), Strontium-89( 89 Sr), Thallium-201( 201 Tl), sodium-22( 22 Na), sodium-24( 24 Na), Technetium 99m ( 99m Tc), xenon-133( 133 Xe), Yttrium-86( 86 Y), Yttrium-88( 88 Y), Yttrium-90 ( 90 Y), and zirconium-89 ( 89 Zr). Further exemplary labeling reagents and their applications in immunoPET are described, for example, in Lamberts, LE et al. (2015) J. Clin. Oncol. 33 (DOI: 10.1200 / JCO.2014.57.8278), and Boerman and Oyen (2011) The Journal of Nuclear Medicine 52 (8):1171-72.

[0180] In one aspect, the invention provides a method for identifying a subject suitable for adoptive cell therapy against a fibroblast activation protein (FAP), the method comprising the steps of (a) isolating diseased tissue from a subject, (b) contacting the isolated tissue with a binding polypeptide that specifically binds to the FAP, and (c) detecting FAP-expressing cells in the isolated tissue, thereby identifying a subject suitable for adoptive cell therapy.

[0181] In certain embodiments, the binding polypeptide is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYTITSYSLH (SEQ ID NO:17) and HCDR2 comprises the amino acid sequence TIFF0007603670000033.tif4128, and wherein HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLAS (SEQ ID NO:20), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0182] In certain embodiments, the isolated binding polypeptide comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1), and / or HCDR2 comprises the amino acid sequence and / or a heavy chain variable region comprising the amino acid sequence TIFF0007603670000034.tif4128, and / or HCDR3 comprising the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0183] In certain embodiments, the isolated binding polypeptide comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence GYTITSYSLH (SEQ ID NO:17) and / or HCDR2 comprises the amino acid sequence and / or a heavy chain variable region comprising the amino acid sequence TIFF0007603670000035.tif5128, and / or HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence LTSNLAS (SEQ ID NO:20), and / or LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0184] In certain embodiments, the isolated binding polypeptide comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1), and / or HCDR2 comprises the amino acid sequence TIFF0007603670000036.tif5128 and / or HCDR3 contains the amino acid sequence a heavy chain variable region comprising the amino acid sequence TASSSVSYMY (SEQ ID NO:4), and / or an LCDR2 comprising the amino acid sequence LTSNLA (SEQ ID NO:5), and / or an LCDR3 comprising the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0185] In certain embodiments, the isolated binding polypeptide comprises a heavy chain variable region that comprises any of the three heavy chain complementarity determining regions described herein, i.e., HCDR1, HCDR2, and HCDR3. In certain embodiments, the antigen binding domain comprises a light chain variable region that comprises any of the three light chain complementarity determining regions described herein, i.e., LCDR1, LCDR2, and LCDR3. In certain embodiments, the antigen binding domain comprises any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, as described herein. Those skilled in the art will be able to easily determine the appropriate complementarity determining region based on amino acid numbering in view of the heavy and light chain variable region sequences provided herein.

[0186] In certain embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof. In certain embodiments, the binding polypeptide is conjugated to a therapeutic or diagnostic molecule. In certain embodiments, the diagnostic molecule comprises a detectable label. In certain embodiments, the detectable label is a radiolabel, a fluorophore, an enzyme, a hapten, biotin, or a chromophore.

[0187] In certain embodiments, after the subject is identified as a suitable subject, the subject is administered adoptive cell therapy.In certain embodiments, the adoptive cell therapy comprises modified immune cells that comprise chimeric antigen receptors (CARs).In certain embodiments, the immune cells are T lymphocytes.In certain embodiments, the immune cells are NK cells.In certain embodiments, the CAR specifically binds to FAP.

[0188] In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7. In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:9.

[0189] E. Treatment Method The antibody, binding polypeptide and scFv described herein can be included in a composition for treating disease or condition in a subject in need thereof.The composition can include a pharmaceutical composition and can further include a pharma- ceutically acceptable carrier.A therapeutically effective amount of the pharmaceutical composition can be administered to the subject.

[0190] In one aspect, the present invention provides a method for treating cancer in a subject in need thereof. The method comprises administering to the subject an isolated binding polypeptide comprising an antigen-binding domain that specifically binds to an epitope of human and canine, and / or mouse fibroblast activation protein (FAP). In certain embodiments, the binding polypeptide comprises any of the HCDRs or LCDRs contemplated herein. In certain embodiments, the binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7 and / or a light chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9.

[0191] In certain embodiments, the cancer is associated with fibroblast activation protein (FAP)-expressing cells. In certain embodiments, the FAP-expressing cells are cancer-associated cells. In certain embodiments, the cancer-associated cells are cancer-associated fibroblasts (CAFs). In certain embodiments, the FAP-expressing cancer-associated cells are FAP-expressing adipocytes. In certain embodiments, the FAP-expressing cancer-associated cells are tumor-associated macrophages (TAMs). In certain embodiments, the FAP-expressing cancer-associated cells are tumor-associated neutrophils (TANs). In certain embodiments, the FAP-expressing cancer-associated cells are myeloid-derived suppressor cells (MDSCs). In certain embodiments, the FAP-expressing cancer-associated cells are cancer-initiating cells.

[0192] In certain embodiments, the binding polypeptide specifically binds to fibroblast activation protein (FAP). In certain embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof.

[0193] In some embodiments, the FAP targeting binding polypeptides of the present disclosure can be used in combination with other therapeutic agents, such as, but not limited to, immunotherapy, such as cancer immunology antibody therapy and checkpoint blockade, or CAR-T therapy. Thus, in such embodiments, the second polynucleotide sequence can include a polynucleotide sequence encoding an anti-cancer antibody, a checkpoint blockade molecule, or a CAR.

[0194] Another aspect of the invention includes a method for treating cancer in a subject in need thereof, comprising the steps of: (a) identifying the subject as a suitable subject comprising: (i) isolating diseased tissue from the subject; (ii) contacting the isolated tissue with a binding polypeptide that specifically binds to a FAP; and (iii) detecting FAP-expressing cells in the isolated tissue; and (b) administering to the suitable subject an adoptive cell therapy comprising modified T cells comprising a chimeric antigen receptor (CAR) that specifically binds to a fibroblast activation protein (FAP).

[0195] In another aspect of the present invention, a method for treating fibrosis is provided herein.Fibrosis is the formation of excess fibrous connective tissue in organs or tissues in repair or reaction process.This deposition of connective tissue by stimulated fibroblasts can hinder or completely inhibit the normal construction and function of underlying organs or tissues.In certain embodiments, the fibrosis that can be treated includes, but is not limited to, pulmonary fibrosis, cardiac fibrosis, liver fibrosis, skin fibrosis (including keloid and scleroderma), intestinal fibrosis, and renal fibrosis.In certain embodiments, the fibrosis is cardiac fibrosis.

[0196] The composition of the present invention can be administered at a dosage and route and time determined by appropriate preclinical and clinical experiments and tests.The composition can be administered multiple times at dosages within these ranges.The administration of the composition can be combined with other methods useful for treating the desired disease or condition, as determined by those skilled in the art.

[0197] F. Pharmaceutical Compositions and Formulations Also provided is a pharmaceutical composition comprising any one of the binding polypeptides, scFvs, antibodies, or antigen-binding fragments disclosed herein.Among the compositions are pharmaceutical compositions and formulations for administration, such as for treating disease or disorder.Also provided is a therapeutic method for administering the pharmaceutical composition to a subject, such as a patient.

[0198] Pharmaceutical compositions and formulations generally include one or more optional pharma- ceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

[0199] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation will be administered. "Pharmaceutically acceptable carrier" refers to ingredients other than the active ingredient in a pharmaceutical formulation that are not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some aspects, the choice of carrier is determined in part by the particular composition and / or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition can include a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001 to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (approximately polypeptides (fewer than 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).

[0200] In some aspects, the composition includes a buffering agent. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001 to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0201] The formulation may comprise an aqueous solution. The formulation or composition may comprise more than one active ingredient useful for a particular indication, disease, or condition treated with the composition, preferably one that has complementary activities in the composition, where the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma-ceutical active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. In some embodiments, the pharmaceutical composition comprises the composition in an amount effective to treat or prevent a disease or condition, such as a therapeutically or prophylactically effective amount. In some embodiments, therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the subject undergoing treatment. The desired dosage can be delivered by administration of a single bolus of the composition, by administration of multiple boluses of the composition, or by administration of a continuous infusion of the composition.

[0202] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the composition is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the composition is administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. In some embodiments, the composition is provided as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0203] Sterile injectable solutions can be prepared, for example, by incorporating the composition into a vehicle by mixing with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard texts can be consulted to prepare appropriate preparations.

[0204] Various additives can be added to enhance the stability and sterility of the composition, including antibacterial preservatives, antioxidants, chelating agents, and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of injectable pharmaceutical dosage forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0205] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0206] The contents of the articles, patents, and patent applications, and all other literature and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0207] G. Aspects of the Disclosure In one aspect, the invention provides isolated binding polypeptides that include an antigen-binding domain that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP).

[0208] In certain embodiments, the antigen binding domain is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000038.tif5128, and wherein HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0209] In certain embodiments, the binding polypeptide binds to fibroblast activation protein (FAP). In certain embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof.

[0210] In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7. In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, a binding polypeptide consists of a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7.

[0211] In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, a binding polypeptide consists of a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:9.

[0212] In another aspect, the present invention provides an isolated binding polypeptide comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9.

[0213] In another aspect, the invention provides single chain variable fragments (scFv) that comprise an antigen-binding domain that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP).

[0214] In certain embodiments of the scFv, the antigen binding domain is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence a heavy chain variable region comprising the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3), and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6), wherein the heavy chain variable region and the light chain variable region are separated by a linker.

[0215] In another aspect, the present invention provides a single chain variable fragment (scFv) comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, wherein the heavy chain variable region and the light chain variable region are separated by a linker.

[0216] In certain embodiments of the scFv, the linker comprises the amino acid sequence set forth in SEQ ID NO:15.

[0217] In another aspect, the present invention provides a single chain variable fragment (scFv) comprising the amino acid sequence set forth in SEQ ID NO:11 or 13. In another aspect, the present invention provides a single chain variable fragment (scFv) consisting of the amino acid sequence set forth in SEQ ID NO:11 or 13.

[0218] In another aspect, the invention provides an isolated nucleic acid encoding any of the binding polypeptides or scFvs contemplated herein.

[0219] In another aspect, the invention provides an isolated nucleic acid encoding a binding polypeptide comprising an antigen-binding domain that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP).

[0220] In certain embodiments of the nucleic acid, the antigen binding domain is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000040.tif5128, and wherein HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0221] In certain embodiments of the nucleic acid, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In certain embodiments of the nucleic acid, the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody. In certain embodiments of the nucleic acid, the antibody is a full-length antibody. In certain embodiments of the nucleic acid, the antibody or antigen-binding fragment is a humanized antibody or a fragment thereof.

[0222] In certain embodiments of the nucleic acid, the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO:8. In certain embodiments of the nucleic acid, the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO:8. In certain embodiments of the nucleic acid, the heavy chain variable region is encoded by a nucleic acid consisting of a polynucleotide sequence set forth in SEQ ID NO:8.

[0223] In certain embodiments of the nucleic acid, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 10. In certain embodiments of the nucleic acid, the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NO: 10. In certain embodiments of the nucleic acid, the light chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO: 10.

[0224] In another aspect, the invention provides an isolated nucleic acid encoding a binding polypeptide comprising: a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:8; and a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:10.

[0225] In another aspect, the invention provides a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0226] In another aspect, the present invention provides an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising a heavy chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:8; and a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:10, wherein the heavy chain variable region and the light chain variable region are separated by a linker. In one particular embodiment, the linker comprises the amino acid sequence set forth in SEQ ID NO.15.

[0227] In another aspect, the present invention provides an isolated nucleic acid encoding a single chain variable fragment (scFv) comprising the polynucleotide sequence set forth in SEQ ID NO: 12 or 14. In another aspect, the present invention provides an isolated nucleic acid encoding a single chain variable fragment (scFv) consisting of the polynucleotide sequence set forth in SEQ ID NO: 12 or 14.

[0228] In another aspect, the present invention provides a vector comprising any of the isolated nucleic acids contemplated herein.In certain embodiments, the vector is an expression vector.In certain embodiments, the vector is selected from the group consisting of DNA vector, RNA vector, plasmid, lentivirus vector, adenovirus vector, adeno-associated virus vector and retrovirus vector.

[0229] In another aspect, the present invention provides a host cell comprising any of the vectors contemplated herein. In certain embodiments, the host cell is of eukaryotic or prokaryotic origin. In certain embodiments, the host cell is of mammalian origin. In certain embodiments, the host cell is of bacterial origin.

[0230] In another aspect, the invention provides a method of producing a binding polypeptide or scFv that binds to a FAP, the method comprising culturing any of the host cells contemplated herein.

[0231] In another aspect, the invention provides a pharmaceutical composition comprising any of the binding polypeptides or any of the scFvs contemplated herein.

[0232] In another aspect, the present invention provides a pharmaceutical composition comprising any of the antibodies or antigen-binding fragments contemplated herein.

[0233] In another aspect, the invention provides a method for identifying a subject suitable for adoptive cell therapy against a fibroblast activation protein (FAP), comprising the steps of: (a) isolating diseased tissue from a subject; (b) contacting the isolated tissue with a binding polypeptide that specifically binds to the FAP; and (c) detecting FAP-expressing cells in the isolated tissue, thereby identifying a subject suitable for adoptive cell therapy.

[0234] In certain aspects of the method, the binding polypeptide is a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF0007603670000042.tif4128, and wherein HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

[0235] In certain embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof.

[0236] In certain embodiments, the binding polypeptide is conjugated to a therapeutic or diagnostic molecule. In certain embodiments, the diagnostic molecule comprises a detectable label. In certain embodiments, the detectable label is a radiolabel, a fluorophore, an enzyme, a hapten, biotin, or a chromophore.

[0237] In certain embodiments, after a subject is identified as a suitable subject, the subject is administered adoptive cell therapy, hi certain embodiments, the adoptive cell therapy comprises engineered immune cells comprising a chimeric antigen receptor (CAR).

[0238] In certain embodiments, the immune cell is a T lymphocyte. In certain embodiments, the immune cell is a NK cell.

[0239] In certain embodiments, the CAR specifically binds to a FAP.

[0240] In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7. In certain embodiments, a binding polypeptide comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, a binding polypeptide consists of a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:7.

[0241] In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, a binding polypeptide comprises a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, a binding polypeptide consists of a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:9.

[0242] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an isolated binding polypeptide comprising a heavy chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7; and a light chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9.

[0243] In certain embodiments, the cancer is associated with fibroblast activation protein (FAP)-expressing cells. In certain embodiments, the FAP-expressing cells are cancer-associated cells. In certain embodiments, the cancer-associated cells are cancer-associated fibroblasts (CAFs). In certain embodiments, the FAP-expressing cancer-associated cells are FAP-expressing adipocytes. In certain embodiments, the FAP-expressing cancer-associated cells are tumor-associated macrophages (TAMs). In certain embodiments, the FAP-expressing cancer-associated cells are tumor-associated neutrophils (TANs). In certain embodiments, the FAP-expressing cancer-associated cells are myeloid-derived suppressor cells (MDSCs). In certain embodiments, the FAP-expressing cancer-associated cells are cancer-initiating cells.

[0244] In certain embodiments, the binding polypeptide specifically binds to fibroblast activation protein (FAP). In certain embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single chain variable fragment (scFv), or a single domain antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof.

[0245] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising the steps of: (a) identifying the subject as a suitable subject comprising: (i) isolating diseased tissue from the subject; (ii) contacting the isolated tissue with a binding polypeptide that specifically binds to a FAP; and (iii) detecting FAP-expressing cells in the isolated tissue; and (b) administering to the suitable subject an adoptive cell therapy comprising modified T cells comprising a chimeric antigen receptor (CAR) that specifically binds to a fibroblast activation protein (FAP).

[0246] Although the present invention has been described in relation to its specific embodiments, those skilled in the art will understand that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step, or step to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Although certain embodiments have been described in detail herein, the same will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0247] Experimental Example The present invention will be further described in detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be interpreted as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0248] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. Accordingly, the following working examples specifically demonstrate preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0249] The materials and methods used in carrying out the experiments disclosed herein are described below.

[0250] Bioinformatic prediction of the canine FAP gene sequence: The NCBI predicted sequence for canine FAP (XM_005640252.2) was used to design PCR primers. The PCR products obtained were 100% identical to the predicted sequence at the protein level. At the nucleotide level, there were two conserved base pair substitutions at T127 and A603.

[0251] PCR, subcloning, and expression of canine FAP cDNA: Confluent 10 cm dishes of canine SK osteosarcoma cells expressing endogenous FAP were treated with 1 ml TRIzol (Life Technologies, #15596-026) and total RNA was extracted according to the manufacturer's protocol. cDNA was reverse transcribed from 5 μg of total RNA using the SuperScript First-Strand Synthesis Kit (Life Technologies, #11904-018). The following primers were used: forward: TIFF0007603670000043.tif4128;Reverse This cDNA was used as a template for touchdown PCR using TIFF0007603670000044.tif4128. An amplification product of 2291 bp was detected on a 1% agarose gel.

[0252] The resulting PCR products were purified, cloned into pGEM-T Easy (Promega) and sequenced. This shuttle vector is linearized and contains a single "T" overhang. This allows for simple non-directional cloning of the PCR products by using the 3' "A" overhang added to the PCR product by Taq polymerase. From here, the cDNA was non-directionally cloned into the plasmid pcDNA3.1 using the EcoRI cloning site.

[0253] The canine FAP cDNA was subcloned from pcDNA3.1 into a lentiviral plasmid (pLenti6 / v5-D-TOPO) using SpeI to excise the cDNA and XbaI to cleave pLenti6 / v5-D-TOPO. These restriction sites have compatible ends. This resulted in canine FAP.pLenti6 / v5-D-TOPO.

[0254] Canine FAP.pLenti / v5-D-TOPO was co-transfected with packaging plasmids (pMD2.G, pCMVΔR8.2) into HEK 293 cells. Virus-containing supernatants were collected 48 h later. Virus titers were determined by p24 ELISA and transduced into Balb / C 3T3 fibroblasts at different MOIs ranging from 0.5:1 to 10:1.

[0255] Flow cytometry was used to confirm the expression of the transgene in Balb / C 3T3. canine FAP cells. The primary antibody used was biotinylated sheep anti-huFAP polyclonal antibody (5 μg / ml) from R&D Systems. The secondary antibody was APC-streptavidin (1 μg / ml) from Biolegend.

[0256] Immunization and hybridoma generation: Balb / C 3T3 cells expressing full-length canine FAP were used to immunize 14-week-old Balb / C.FAP -\- Mice were immunized. All injections were given intraperitoneally with 1 × 10 7The primary immunization consisted of MC KOSA parental (FAP null) versus MC KOSA.K9FAP expressing the canine FAP transgene using hybridoma supernatants as the primary antibody and AF488 goat anti-mouse IgG as the secondary antibody. To identify clone 4G5 as reactive to transduced cells but not parental cells, and to confirm reactivity with FAP expressing cells but not FAP negative cells, the cells were screened against the following additional cells: human primary fibroblasts, BALB / c 3T3 expressing mouse or human FAP transgene, canine primary fibroblasts, SK KOSA (FAP expressing), and BALB / c 3T3 (FAP negative) cells. 4G5 was then determined to be an IgG1k isotype antibody using (Thermo Fisher Rapid ELISA Mouse mAb Isotyping Kit #37503).

[0257] Example 1: Bioinformatic prediction of the canine FAP gene sequence used to design PCR primers As a first step, the sequence of the canine FAP gene was amplified by PCR. Using the NCBI predicted sequence for canine FAP (XM_005640252.2), PCR primers specific for canine FAP were designed. The resulting PCR product was 100% identical to the sequence of canine FAP at the protein level (Figure 1). Subsequent sequencing revealed that canine FAP has two conserved base pair substitutions at T127 and A603 at the nucleotide level.

[0258] Example 2: PCR, subcloning, sequencing, and expression of canine FAP cDNA As a first step towards the generation of anti-canine FAP antibodies, a construct capable of producing recombinant canine FAP protein was created. To prepare canine FAP cDNA, canine SK osteosarcoma cells expressing endogenous FAP were subjected to TRIzol-based RNA extraction, followed by reverse transcription of the isolated RNA into cDNA. This cDNA was used as a template for touchdown PCR, which yielded an amplification product of 2291 bp. The amplified PCR product was purified and cloned into a shuttle vector that allows simple non-directional cloning of PCR products. From here, the cDNA was cloned into a eukaryotic expression plasmid (Figure 2A). The canine FAP cDNA was then subcloned into a lentiviral plasmid to allow transduction into mammalian cell lines (Figure 2B). The resulting canine FAP-lentiviral construct was then co-transfected with a packaging plasmid into HEK 293 cells. Virus-containing supernatants were then collected after 48 hours. Virus titers were determined by p24 ELISA and transduced into BALB / c-derived 3T3 fibroblasts at various MOIs ranging from 0.5:1 to 10:1. Flow cytometry was used to confirm transgene expression in 3T3-canine FAP cells (Figure 2C).

[0259] Example 3. Immunization, fusion, and screening BALB / c 3T3 fibroblasts transduced to express full-length canine FAP were then used to generate 14-week-old BALB / c FAP -\- Mice were immunized. All injections were given intraperitoneally with 1 × 10 7The immunization consisted of 100% IgG4-heptaerythrocytes. The initial immunization was followed by six boosts at regular intervals over the next 8 months. At the end of the study, spleens were harvested and the resulting single cell suspension was used to generate hybridomas via fusion with sp2 / 0 cells. The resulting hybridomas were then screened for those producing anti-canine FAP antibodies. As an initial screen, cells producing immunoglobulins capable of staining MC KOSA cells expressing the canine FAP transgene but not the FAP-null MC KOSA parental cells were identified by flow cytometry (Figure 3). As a result, the 4G5 clone was identified as a potential candidate. Follow-up studies further revealed that the immunoglobulins produced by this clone were capable of staining FAP-expressing human primary fibroblasts, BALB / c 3T3 cells expressing mouse or human FAP transgenes, canine primary fibroblasts, and FAP-expressing SK KOSA cells. Similarly, 4G5 immunoglobulins were unable to stain the FAP-negative parental BALB / c 3T3 cells, further demonstrating its specificity. Finally, a commercially available ELISA-based antibody isotyping kit was used to further characterize the immunoglobulins produced by 4G5. These results demonstrated that 4G5 is a mouse-IgG1-kappa isotype antibody (Figures 4A-4C). A follow-up protein gel comparing 4G5 to a mouse IgG1 isotype control showed similar patterns of heavy and light chain bands, further demonstrating the isotype identity of 4G5 (Figure 5).

[0260] Other Aspects The recitation of a list of elements in any definition of a variable herein includes definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0261] The disclosures of any and all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed in connection with certain embodiments, it is clear that other embodiments and modifications of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. An isolated binding polypeptide comprising an antigen-binding domain that specifically binds to an epitope of human and canine and / or murine fibroblast activation protein (FAP), The antigen-binding domain comprises: (a) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence EINPANGDHNFSEKFEIK (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and (b) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). A binding polypeptide comprising:

2. (a) the binding polypeptide binds to human, canine, and murine fibroblast activation protein (FAP); (b) the binding polypeptide comprises an antibody or an antigen-binding fragment thereof; (c) the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:7; (d) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7; (e) the heavy chain variable region consists of the amino acid sequence set forth in SEQ ID NO:7; (f) the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9; (g) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9; or (h) the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO:9; 2. The binding polypeptide of claim 1.

3. the binding polypeptide comprises an antibody or an antigen-binding fragment thereof; and (a) the antigen-binding fragment is a Fab or a single chain variable fragment (scFv); (b) the antibody comprises a full-length antibody; or (c) the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment.

3. A binding polypeptide according to claim 1 or 2.

4. (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

9.

2. An isolated binding polypeptide that specifically binds to an epitope of human and canine, and / or mouse fibroblast activation protein (FAP), comprising:

5. A single-chain variable fragment (scFv) that contains an antigen-binding domain that specifically binds to an epitope of human, canine, and / or mouse fibroblast activation protein (FAP).

6. The antigen-binding domain comprises: (a) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence EINPANGDHNFSEKFEIK (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and (b) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). Including, the heavy chain variable region and the light chain variable region are separated by a linker; The scFv of claim 5.

7. (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

9. A single chain variable fragment (scFv) that specifically binds to an epitope of human and canine, and / or murine fibroblast activation protein (FAP), comprising: A single chain variable fragment (scFv), wherein the heavy chain variable region and the light chain variable region are separated by a linker, optionally, the linker comprising the amino acid sequence set forth in SEQ ID NO:

15.

8. (a) comprises the amino acid sequence set forth in SEQ ID NO:11 or 13; or (b) consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 13; A single-chain variable fragment (scFv) that specifically binds to an epitope of human, canine, and / or mouse fibroblast activation protein (FAP).

9. 9. An isolated nucleic acid encoding a binding polypeptide or scFv according to any one of claims 1 to 8.

10. An isolated nucleic acid encoding a binding polypeptide comprising an antigen-binding domain that specifically binds to an epitope of human and canine and / or murine fibroblast activation protein (FAP), comprising: The antigen-binding domain comprises: (a) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence EINPANGDHNFSEKFEIK (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and (b) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). A nucleic acid comprising:

11. (a) the binding polypeptide comprises an antibody or an antigen-binding fragment thereof; (b) the binding polypeptide is an antibody or antigen-binding fragment thereof, and the antigen-binding fragment is a Fab or a single chain variable fragment (scFv); or (c) the binding polypeptide is an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment; The nucleic acid of claim 10.

12. (a) the heavy chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO:8; (b) the heavy chain variable region is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO:8; (c) the heavy chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:8; (d) the light chain variable region is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:10; (e) the light chain variable region is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO:10; or (f) the light chain variable region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:10; 12. The nucleic acid according to 10 or 11.

13. (a) a heavy chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:8; and (b) a light chain variable region encoded by a nucleic acid sequence comprising the polynucleotide sequence set forth in SEQ ID NO:

10.

2. An isolated nucleic acid encoding a binding polypeptide that specifically binds to an epitope of human and canine, and / or mouse fibroblast activation protein (FAP), comprising:

14. (a) (i) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence EINPANGDHNFSEKFEIK (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3), and (ii) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6). or encoding a single chain variable fragment (scFv) that specifically binds to an epitope of human, canine, and / or mouse fibroblast activation protein (FAP), comprising: (b) (i) a heavy chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:8, and (ii) a light chain variable region comprising the nucleotide sequence set forth in SEQ ID NO:

10. and encoding a single chain variable fragment (scFv) that specifically binds to an epitope of human, canine, and / or mouse fibroblast activation protein (FAP), comprising: the heavy chain variable region and the light chain variable region are separated by a linker; or (c) encoding a single chain variable fragment (scFv) that specifically binds to an epitope of human, canine, and / or mouse fibroblast activation protein (FAP), comprising a polynucleotide sequence set forth in SEQ ID NO:12 or 14; or (d) encoding a single chain variable fragment (scFv) that specifically binds to an epitope of human, canine, and / or mouse fibroblast activation protein (FAP), comprising the polynucleotide sequence set forth in SEQ ID NO:12 or 14; Isolated nucleic acid.

15. A vector comprising the isolated nucleic acid of any one of claims 9 to 14.

16. (a) is an expression vector; and / or (b) selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector; The vector of claim 15.

17. A host cell comprising the vector of claim 15 or 16, (a) is of eukaryotic or prokaryotic origin; (b) is of mammalian origin; or (c) is of bacterial origin; host cell.

18. 20. A method for producing a binding polypeptide or scFv that binds to a FAP, comprising culturing the host cell of claim 17.

19. A pharmaceutical composition comprising a binding polypeptide or scFv according to any one of claims 1 to 8.

20. 1. A medicament for treating cancer in a subject in need thereof, comprising: (a) a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence YTITSYSLH (SEQ ID NO:1), HCDR2 comprises the amino acid sequence EINPANGDHNFSEKFEIK (SEQ ID NO:2), and HCDR3 comprises the amino acid sequence LDDSRFHWYFDV (SEQ ID NO:3); and (b) a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises the amino acid sequence TASSSVSYMY (SEQ ID NO:4), LCDR2 comprises the amino acid sequence LTSNLA (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQWSGYPPIT (SEQ ID NO:6).

23. An agent comprising an isolated binding polypeptide that specifically binds to an epitope of human and canine, and / or mouse fibroblast activation protein (FAP), comprising:

21. 1. A medicament for treating cancer in a subject in need thereof, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:9 23. An agent comprising an isolated binding polypeptide that specifically binds to an epitope of human and canine, and / or mouse fibroblast activation protein (FAP), comprising:

22. the cancer is associated with fibroblast activation protein (FAP)-expressing cells; and (a) the FAP-expressing cell is a cancer-associated cell, and the cancer-associated cell is a cancer-associated fibroblast (CAF); (b) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a FAP-expressing adipocyte; (c) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a tumor-associated macrophage (TAM); (d) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a tumor-associated neutrophil (TAN); (e) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a myeloid-derived suppressor cell (MDSC); or (f) the FAP-expressing cell is a cancer-associated cell, and the FAP-expressing cancer-associated cell is a cancer-initiating cell; The drug of claim 21.

23. (a) the binding polypeptide specifically binds to human, canine, and mouse fibroblast activation protein (FAP); (b) the binding polypeptide is an antibody or an antigen-binding fragment thereof; (c) the binding polypeptide comprises an antibody or an antigen-binding fragment thereof, and the antigen-binding fragment is a Fab or a single chain variable fragment (scFv); or (d) the binding polypeptide comprises an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment; A drug according to any one of claims 20 to 22.

Images