Novel antibody sequences for diagnostics and therapeutics

Abstract

Aspects of the present disclosure generally relate to novel antigen-binding sequences and their uses. More specifically, certain features of the present disclosure relate to antibodies, minibodies, and cys-diabodies for use in targeting protein FAPs. The antigen-binding sequences herein can be used as part of a treatment to provide health benefits to subjects in need thereof. The antigen-binding sequences can also be used for detection, targeting, and imaging.

Description

[Technical field]

[0001] Aspects of the present disclosure relate to novel antigen-binding sequences and uses of various antigen-binding sequences. Certain features of the present disclosure relate to antibodies, minibodies, and cys-diabodies for use in targeting protein FAPs.

[0002] Sequence Listing Reference This application has been submitted with a Sequence Listing in electronic format. The Sequence Listing is provided in a file named IGNAB055WOv3.xml, which was created on July 7, 2022 and is 285000 bytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Fibroblast activation protein (FAP) is a cell surface glycoprotein serine protease that has been shown to play a role in extracellular matrix degradation, cell invasion, cell adhesion, wound healing, tissue remodeling, fibrosis, inflammation, and possibly tumor growth. Both membrane-bound and soluble forms of FAP exhibit post-proline cleaving endopeptidase activity with a marked preference for the Ala / Ser-Gly-Pro-Ser / Asn / Ala consensus sequence. FAP also possesses dipeptidyl peptidase activity and exhibits the ability to hydrolyze prolyl bonds between proline and the second residue on the N-terminus, with a preference for Ala-Pro, Ile-Pro, Gly-Pro, Arg-Pro, and Pro-Pro. The enzymatic activity of human FAP is believed to be highest in monoreactive fibroblasts, glucagon-producing A cells of pancreatic islets, and endometrial cells of healthy tissues. In mice, the highest FAP enzyme activity was detected in the uterus, pancreas, submandibular gland, and skin, and the lowest levels in the brain, prostate, leukocytes, and testes. FAP enzyme activity is influenced by cell surface DPP4 (dipeptidyl peptidase IV) and possibly also by TERT, TWIST1, and TCF15. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Pat. No. 5,059,523 [Patent Document 2] U.S. Patent No. 6,455,677 [Patent Document 3] US Patent Publication No. 20120128591 [Patent Document 4] U.S. Pat. No. 4,946,778 [Patent Document 5] International Publication No. 2017027325 A1 Brochure [Patent Document 6] International Publication No. 2018147960 A1 Brochure [Patent Document 7] US Patent Publication No. 20170357015 [Patent Document 8] US Patent Publication No. 20170153337 [Patent Document 9] US Patent Publication No. 20150196266 [Patent Document 10] US Patent Publication No. 20150087974 [Patent Document 11] US Patent Publication No. 20120318988 [Patent Document 12] US Patent Publication No. 20090159804 [Non-patent literature]

[0005] [Non-Patent Document 1] Brocks et al. (2001) Phage Display Selection of FAP-Specific scFv. Molec. Med, 7(7), 461-469 [Non-Patent Document 2] Creighton, Proteins (1984) [Non-Patent Document 3] Agnew, Chem Intl. Ed. Engl. 33:183-186 (1994) [Non-Patent Document 4] Yang Liu, Yinping Dong, Li Kong, Fang Shi, Hui Zhu & Jinming Yu; “Abscopal effect of radiotherapy combined with immune checkpoint inhibitors”; Journal of Hematology & Oncology, Volume 11, Paper number: 104 (2018) [Non-Patent Document 5] Melek Tugce Yilmaz, Aysenur Elmali, and Gozde Yazici; "Abscopal Effect, From Myth to Reality: From Radiation Oncologists' Perspective"; Cureus, January 2019; Volume 11(Issue 1) [Non-Patent Document 6] Kerr and Chisolm (2019) The Journal of Immunology, 2019, 202:11-19 [Non-Patent Document 7] Galon and Bruni (2019) Nature Reviews Drug Discovery, Volume 18, Pages 197-218 [Non-Patent Document 8] Kuby, Immunology, 4th ed., Chapter 4, WH Freeman & Co., New York, 2000. [Non-Patent Document 9] Wu, TT, EA Kabat, 1970. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132:211-250

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[0006] Some aspects of the present disclosure relate to isolated antigen-binding constructs. In some embodiments, the isolated antigen-binding construct comprises an HCDR1 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:1 (EYTIH), an HCDR2 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:2 (GINPNNGIPNYNQKFKG), an HCDR3 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:3 (RRIAYGYDEGHAMDY), an LCDR1 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:4 (KSSQSLLYSRNQKNYLA), an LCDR2 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:5 (WASTRES), and an LCDR3 comprising amino acids having the amino acid sequence of SEQ ID NO:6 (QQYYSYPLT). In some embodiments, the isolated antigen-binding construct further comprises a heavy chain having at least 80% identity with the amino acid sequence of SEQ ID NO:7. In some embodiments, the isolated antigen-binding construct comprises a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO:7. In some embodiments, the isolated antigen-binding construct comprises a heavy chain having at least 95% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 95% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 95% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the isolated antigen-binding construct comprises at least one VH framework residue selected from the group consisting of an alanine at position 24 of the sequence of SEQ ID NO:7 or a glycine at position 26 of the sequence of SEQ ID NO:7.In some embodiments, the isolated antigen-binding construct comprises at least one VL framework residue selected from the group consisting of a serine at position 73 of sequence SEQ ID NO:8, an arginine at position 83 of sequence SEQ ID NO:8, a glutamic acid at position 85 of sequence SEQ ID NO:8, a proline at position 86 of sequence SEQ ID NO:8, a phenylalanine at position 89 of sequence SEQ ID NO:8, or a tyrosine at position 98 of sequence SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises an alanine at position 24 and a glycine at position 26 of the heavy chain sequence of SEQ ID NO:7. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 and a serine at position 73 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 and a tyrosine at position 98 of the sequence of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the sequence of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the sequence of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the sequence of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a variable heavy domain (VH) of SEQ ID NO: 7 and a variable light domain (VL) of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a variable heavy domain (VH) of SEQ ID NO: 7 and a variable light domain (VL) of SEQ ID NO: 9.

[0007] Some embodiments of the present disclosure relate to an isolated antigen-binding construct specific for FAP alpha, comprising a CDR3 comprising amino acids having at least 90% identity with the amino acid sequence of SEQ ID NO:6 (QQYYSYPLT). Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a light chain CDR3 comprising amino acids having at least 100% identity with the amino acid sequence of SEQ ID NO:6. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising an LCDR1 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:4 (KSSQSLLYSRNQKNYLA), an LCDR2 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:5 (WASTRES), and an LCDR3 comprising amino acids having the amino acid sequence of SEQ ID NO:6 (QQYYSYPLT). Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a heavy chain comprising at least 99% identity with the amino acid sequence of SEQ ID NO:7. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 7, comprising at least one VH framework residue selected from the group consisting of an alanine at position 24 of the sequence of SEQ ID NO: 7 and a glycine at position 26 of the sequence of SEQ ID NO: 7. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 8, comprising at least one VL framework residue selected from the group consisting of a serine at position 73 of the sequence of SEQ ID NO: 8, an arginine at position 83 of the sequence of SEQ ID NO: 8, a glutamic acid at position 85 of the sequence of SEQ ID NO: 8, a proline at position 86 of the sequence of SEQ ID NO: 8, a phenylalanine at position 89 of the sequence of SEQ ID NO: 8, and a tyrosine at position 98 of the sequence of SEQ ID NO: 8. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a light chain comprising an amino acid at least 95% identity to the amino acid sequence of SEQ ID NO: 9. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a light chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:8.Some aspects of the present disclosure relate to an isolated antigen-binding construct comprising a variable heavy domain (VH) comprising amino acids having at least 95% identity to the amino acid sequence of SEQ ID NO: 7, and a variable light domain (VL) comprising amino acids having at least 95% identity to the amino acid sequence of SEQ ID NO: 8. Some aspects of the present disclosure relate to an isolated humanized antigen-binding construct comprising a variable heavy domain (VH) comprising amino acids having at least 95% identity to the amino acid sequence of SEQ ID NO: 7, and a variable light domain (VL) comprising amino acids having at least 89% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the isolated antigen-binding construct comprises at least one VH framework residue selected from the group consisting of an alanine at position 24 of the sequence of SEQ ID NO: 7 and a glycine at position 26 of the sequence of SEQ ID NO: 7. In some embodiments, the isolated antigen-binding construct comprises at least one VL framework residue selected from the group consisting of a serine at position 73 of sequence SEQ ID NO:8, an arginine at position 83 of sequence SEQ ID NO:8, a glutamic acid at position 85 of sequence SEQ ID NO:8, a proline at position 86 of sequence SEQ ID NO:8, a phenylalanine at position 89 of sequence SEQ ID NO:8, and a tyrosine at position 98 of sequence SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 and a serine at position 73 of the light chain sequence of SEQ ID NO:4. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO: 8.In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO: 8.

[0008] Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising amino acids having at least 80% identity to an amino acid sequence selected from the group of SEQ ID NOs: 23 to 44. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising amino acids having at least 90% identity to an amino acid sequence selected from the group of SEQ ID NOs: 23 to 44. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising amino acids having at least 100% identity to an amino acid sequence selected from the group of SEQ ID NOs: 23 to 44, 12 to 43, 12 to 45, 87 to 96, 110 to 115, 122 to 125, or 140 to 143.

[0009] Some aspects of the present disclosure relate to isolated antigen-binding constructs. In some embodiments, the isolated antigen-binding constructs comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 81 (NYDIN) or a sequence having 3 or less point mutations thereto, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN) or a sequence having 3 or less point mutations thereto, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY) or a sequence having 3 or less point mutations thereto, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA) or a sequence having 3 or less point mutations thereto, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 85 (SASYRYT) or a sequence having 3 or less point mutations thereto, and / or an LCDR3 comprising the amino acid sequence of SEQ ID NO: 86 (QQHYSNPRT) or a sequence having 3 or less point mutations thereto.

[0010] Also disclosed herein is an isolated antigen-binding construct comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO:81 (NYDIN) or a sequence having three or less point mutations thereto, an HCDR2 comprising an amino acid sequence of SEQ ID NO:82 (LIWTGGGTN) or a sequence having three or less point mutations thereto, and / or an HCDR3 comprising an amino acid sequence of SEQ ID NO:83 (GGPLVWYALDY) or a sequence having three or less point mutations thereto. Also disclosed herein is an isolated antigen-binding construct comprising an LCDR1 comprising an amino acid sequence of SEQ ID NO:84 (KASQDVSTAVA) or a sequence having three or less point mutations thereto, an LCDR2 comprising an amino acid sequence of SEQ ID NO:85 (SASYRYT) or a sequence having three or less point mutations thereto, and / or an LCDR3 comprising an amino acid sequence of SEQ ID NO:86 (QQHYSNPRT) or a sequence having three or less point mutations thereto. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 81 (NYDIN) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR1 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR2 comprising an amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR2 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR3 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR1 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for FAP, comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 85 (SASYRYT), or a sequence having three or fewer point mutations thereto.In some embodiments, the CDR2 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising the amino acid sequence of SEQ ID NO:86 (QQHYSNPRT) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR3 is part of a light chain. In some embodiments, there are no more than two point mutations. In some embodiments, there are no more than one point mutation. In some embodiments, there are no point mutations. In some embodiments, the isolated antigen-binding construct further comprises a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO:87. In some embodiments, the isolated antigen-binding construct further comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:88. In some embodiments, the antigen-binding construct further comprises a variable heavy chain domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO:87, and / or a variable light chain domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO:88. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 89, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 91, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 93, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 95, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 96.In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 42, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 43.

[0011] Also disclosed herein is an isolated antigen-binding construct thereof, comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 87, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 88. Also disclosed herein is an isolated antigen-binding construct thereof, comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 89, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 90. Also disclosed herein is an isolated antigen-binding construct thereof, comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 91, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 92. Also disclosed herein is an isolated antigen-binding construct thereof, comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 93, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 94. Also disclosed herein is an isolated antigen-binding construct thereof, comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 95, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 96. Also disclosed herein is an isolated antigen-binding construct thereof, comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 42, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 43.

[0012] Also disclosed herein is an isolated antigen-binding construct thereof comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 104 (SYVMH) or a sequence having three or less point mutations thereto, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 105 (YINPHNDGTK) or a sequence having three or less point mutations thereto, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 106 (ARWGIYYGYGAWFAY) or a sequence having three or less point mutations thereto, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 107 (KASQNVGTYVA) or a sequence having three or less point mutations thereto, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 108 (YSASNRYS) or a sequence having three or less point mutations thereto, and / or an LCDR3 comprising the amino acid sequence of SEQ ID NO: 109 (QQYNTYPYT) or a sequence having three or less point mutations thereto. Also disclosed herein is an isolated antigen-binding construct comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 104 (SYVMH) or a sequence having three or less point mutations thereto, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 105 (YINPHNDGTK) or a sequence having three or less point mutations thereto, and / or an HCDR3 comprising an amino acid sequence of SEQ ID NO: 106 (ARWGIYYGYGAWFAY) or a sequence having three or less point mutations thereto. Also disclosed herein is an isolated antigen-binding construct comprising an LCDR1 comprising an amino acid sequence of SEQ ID NO: 107 (KASQNVGTYVA) or a sequence having three or less point mutations thereto, an LCDR2 comprising an amino acid sequence of SEQ ID NO: 108 (YSASNRYS) or a sequence having three or less point mutations thereto, and / or an LCDR3 comprising an amino acid sequence of SEQ ID NO: 109 (QQYNTYPYT) or a sequence having three or less point mutations thereto. Also disclosed herein is an isolated antigen-binding construct specific for FAP, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 104 (SYVMH), or a sequence having three or fewer point mutations thereto. In some embodiments, the CDR1 is part of a heavy chain.Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR2 comprising an amino acid sequence of SEQ ID NO: 105 (YINPHNDGTK) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR2 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 106 (ARWGIYYGYGAWFAY) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR3 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 107 (KASQNVGTYVA) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR1 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR2 comprising an amino acid sequence of SEQ ID NO: 108 (YSASNRYS) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR2 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 109 (QQYNTYPYT) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR3 is part of a light chain. In some embodiments, there are no more than two point mutations thereto in any of the CDR regions of the antigen-binding constructs disclosed herein. In some embodiments, there are no point mutations thereto in any of the CDR regions of the antigen-binding constructs disclosed herein. In some embodiments, the antigen-binding construct further comprises a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the antigen-binding construct further comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 111.In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 110, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 111. In some embodiments, the antigen-binding construct further comprises a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the antigen-binding construct further comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 112, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the antigen-binding construct further comprises a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the antigen-binding construct further comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO:91, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO:92. In some embodiments, the antigen-binding construct further comprises a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO:114. In some embodiments, the antigen-binding construct further comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:115. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO:114, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO:115. In some embodiments, the antigen-binding construct further comprises a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO:95. In some embodiments, the antigen-binding construct further comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:96.In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 95, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antigen-binding construct further comprises a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antigen-binding construct is not the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antigen-binding construct further comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 84, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 45.

[0013] Also disclosed herein is an isolated antigen-binding construct thereof, comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 110, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 111. Also disclosed herein is an isolated antigen-binding construct thereof, comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 112, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 113. Also disclosed herein is an isolated antigen-binding construct thereof, comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 114, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 115. Also disclosed herein is an isolated antigen-binding construct thereof comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 44 and / or a variable light domain (VL) having at least 80% identity to the amino acid sequence of SEQ ID NO: 45.

[0014] Also disclosed herein is an isolated antigen-binding construct thereof comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 134 (SYTMS) or a sequence having three or less point mutations thereto, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 135 (TISSGGSYTY) or a sequence having three or less point mutations thereto, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 136 (TRDQVGYAMDY) or a sequence having three or less point mutations thereto, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 137 (TRDQVGYAMDY) or a sequence having three or less point mutations thereto, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 138 (YWASTRHT) or a sequence having three or less point mutations thereto, and / or an LCDR3 comprising the amino acid sequence of SEQ ID NO: 139 (QQYSRYPYT) or a sequence having three or less point mutations thereto. Also disclosed herein is an isolated antigen-binding construct comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 134 (SYTMS) or a sequence having 3 or less point mutations thereto, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 135 (TISSGGSYTY) or a sequence having 3 or less point mutations thereto, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 136 (TRDQVGYAMDY) or a sequence having 3 or less point mutations thereto. Also disclosed herein is an isolated antigen-binding construct comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 137 (TRDQVGYAMDY) or a sequence having 3 or less point mutations thereto, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 138 (YWASTRHT) or a sequence having 3 or less point mutations thereto, and / or an LCDR3 comprising the amino acid sequence of SEQ ID NO: 139 (QQYSRYPYT) or a sequence having 3 or less point mutations thereto. Also disclosed herein is an isolated antigen-binding construct specific for FAP, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 134 (SYTMS), or a sequence having three or fewer point mutations thereto. In some embodiments, the CDR1 is part of a heavy chain.Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR2 comprising an amino acid sequence of SEQ ID NO: 135 (TISSGGSYTY) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR2 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 136 (TRDQVGYAMDY) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR3 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 137 (TRDQVGYAMDY) or a sequence having no more than three point mutations thereto. In some embodiments, the CDR1 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR2 comprising an amino acid sequence of SEQ ID NO: 138 (YWASTRHT) or a sequence having no more than three point mutations thereto. In some embodiments, CDR2 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 139 (QQYSRYPYT) or a sequence having three or less point mutations thereto. In some embodiments, CDR3 is part of a light chain. In some embodiments, there are two or less point mutations thereto. In some embodiments, there are one or less point mutations thereto. In some embodiments, there are no point mutations thereto.

[0015] In some embodiments, the isolated antigen-binding construct further comprises a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the isolated antigen-binding construct further comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 141. In some embodiments, the isolated antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 140, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 141. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 142, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antigen-binding construct further comprises a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 143, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 94.

[0016] Also disclosed herein is an isolated antigen-binding construct comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 140, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 141. Also disclosed herein is an isolated antigen-binding construct comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 142, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 90. Also disclosed herein is an isolated antigen-binding construct comprising a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 143, and / or a variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 94.

[0017] In some embodiments, the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, V L , V HIn some embodiments, the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, V H , V L It is.

[0018] Also disclosed herein is an isolated antigen-binding construct comprising an amino acid sequence having at least 98% identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 97-103 and 126.

[0019] Also disclosed herein is an isolated antigen-binding construct comprising an amino acid sequence having at least 98% identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 116-120 and 127-133.

[0020] Also disclosed herein is an isolated antigen-binding construct comprising an amino acid sequence having at least 98% identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 144-146.

[0021] In some embodiments, the antigen-binding construct is mammalian. In some embodiments, the antigen-binding construct is murine. In some embodiments, the antigen-binding construct is human. In some embodiments, the isolated antigen-binding construct of any one of the above embodiments is an antibody. In some embodiments, the isolated antigen-binding construct is an scFV. In some embodiments, the isolated antigen-binding construct is a Fab. In some embodiments, the isolated antigen-binding construct is a Fab2. In some embodiments, the isolated antigen-binding construct is a nanobody. In some embodiments, the isolated antigen-binding construct is a minibody. In some embodiments, the isolated antigen-binding construct is a cys-diabody. In some embodiments, the isolated antigen-binding construct is part of a composition comprising one or more of an antibody, scFV, Fab, Fab2, nanobody, minibody, cys-diabody, or any combination thereof.

[0022] Some embodiments of the present disclosure relate to minibody antigen-binding constructs that include amino acids having at least about 80%, at least about 90%, or at least about 99% identity to an amino acid sequence selected from the group of SEQ ID NOs: 11-22. Some embodiments of the present disclosure relate to cys-diabody antigen-binding constructs that include amino acids having at least about 80%, at least about 90%, or at least about 99% identity to an amino acid sequence selected from the group of SEQ ID NOs: 11-22.

[0023] Some aspects of the present disclosure relate to minibody antigen-binding constructs that include amino acids having at least about 80%, at least about 90%, or at least about 99% identity to at least one of the amino acid sequences selected from the group of SEQ ID NOs: 97-103, 116-120, 126-133, and 144-146. Also disclosed herein are cys-diabody antigen-binding constructs that include amino acids having at least about 80%, at least about 90%, or at least about 99% identity to at least one of the amino acid sequences selected from the group of SEQ ID NOs: 97-103, 116-120, 126-133, and 144-146.

[0024] In some embodiments, the isolated antigen-binding construct is specific for human FAP. In some embodiments, the isolated antigen-binding construct according to any one of the above embodiments is specific for FAP alpha. In some embodiments, the isolated antigen-binding construct does not bind to DPP4.

[0025] In some embodiments, the minibody constructed with sibrotuzumab CDR domains is IAB16M1-12 and / or IAB16M2-13. Such minibodies may be referred to herein as "sibrotuzumab minibodies." In some embodiments, the isolated antigen-binding construct exhibits at least 2-fold greater expression in mammalian cells than that of sibrotuzumab minibodies. In some embodiments, the isolated antigen-binding construct exhibits at least 6-fold greater expression in mammalian cells than that of sibrotuzumab minibodies (FIG. 1). In some embodiments, the sibrotuzumab minibody is IAB16M1-12 and / or IAB16M2-13. In some embodiments, the isolated antigen-binding construct has a KD of less than 2×10^-9M. In some embodiments, the isolated antigen-binding construct has a KD of less than 1×10^-9M. In some embodiments, the isolated antigen-binding construct has an on-rate (k) of greater than 8.0 (1 / Ms). on In some embodiments, the isolated antigen-binding construct has an on-rate (k) greater than 9.0 (1 / Ms). on In some embodiments, the isolated antigen-binding construct has an off rate (k) of less than 2.5×10^-3 (1 / s). off In some embodiments, the isolated antigen-binding construct has an off rate (k) of less than 1.5×10^-3 (1 / s). off ).

[0026] In some embodiments, the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, V L , V H In some embodiments, the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, V H , V L It is.

[0027] In some embodiments, the isolated antigen-binding construct further comprises a payload. In some embodiments, the payload is fluorescent. In some embodiments, the payload is luminescent. In some embodiments, the payload is chromogenic. In some embodiments, the payload is radioactive. In some embodiments, the payload is non-radioactive. In some embodiments, the payload is chemically reactive. In some embodiments, the payload is a detectable marker. In some embodiments, the combination of a minibody or cys-diabody and a payload is an antibody drug conjugate (ADC). In some embodiments, the isolated antigen-binding construct is humanized.

[0028] Some aspects of the present disclosure include the amino acid sequence of any one of embodiments 1 to 66, 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy,166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th, and 225 The present invention relates to a composition comprising a composition further comprising at least one payload selected from the group consisting of: A, B, C, D ...

[0029] In some embodiments, the amino acid sequence of the antigen-binding construct further comprises at least one metal binding site. In some embodiments, the amino acid sequence of the antigen-binding construct further comprises a histidine (His) tag sequence. In some embodiments, the amino acid sequence of the antigen-binding construct further comprises 99m It further comprises a Tc-carbonyl radiolabel.

[0030] Some aspects of the present disclosure relate to an expression vector capable of expressing any one of the sequences of the embodiments disclosed herein. Also disclosed herein is an expression vector configured to express any one of the sequences of the antigen-binding constructs described herein. In some embodiments, the vector is a viral vector selected from a lentiviral vector or an adenoviral vector. In some embodiments, the vector is a vector for transfection in mammalian cells. In some embodiments, the vector comprises a sequence encoding a cleavable signal peptide having at least 99% identity to the amino acid sequence of SEQ ID NOs: 46 and 121 (METDTLLLWVLLLWVPGSTG).

[0031] Some aspects of the present disclosure relate to a method for introducing the amino acid sequence or expression vector of any of the preceding embodiments into a host cell, comprising performing electroporation, viral infection, and / or at least one chemical method. Also disclosed herein is a host cell comprising the amino acid sequence or expression vector of any one of the antigen-binding constructs described herein.

[0032] Some aspects of the present disclosure relate to the method for using the amino acid sequence or expression vector of any one of the preceding claims as pretargeting modality.In some embodiments, the method comprises adding a non-radioactive sequence, vector or antigen-binding construct to the system, and adding a rapid clearance radiolabeled product that recognizes the protein product of the sequence or vector.In some embodiments, the rapid clearance radiolabeled product comprises a small molecule and / or a peptide.Some aspects of the present disclosure relate to a host cell that comprises the amino acid sequence or expression vector of any one of the above embodiments disclosed herein.

[0033] Some aspects of the present disclosure relate to compositions comprising any one of the amino acid sequences of the above embodiments disclosed herein and at least one chelating agent. In some embodiments, the at least one chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NOGADA, NETA, deferoxamine (DFO), porphyrin, polyamine, crown ether, bis-thiosemicarbazone, polyoxime, dimercaprol, penicillamine, trientine, zinc, deferasirox, deferiprone, deferoxamine, succimer, pyrophosphate, tripolyphosphate, citric acid, tartaric acid, glycine, DMPS, DMSA, NTA, calcium, sodium, desferioxamine, dicobalt EDTA, dimercarpol, BAL, and demercaptosuccinic acid, or any combination thereof. In some embodiments, the composition further comprises a dual or triple chelator. In some embodiments, the dual or triple chelator is64 Cu / 67 Cu, or 89 Zr / 177 Lu, or 89 Zr / 227 In some embodiments, the dual chelator is a chelator for both metals: 64 Cu and 67 In some embodiments, the dual chelator is capable of capturing both metals: 89 Zr and 177 In some embodiments, the composition further comprises at least one optical probe. In some embodiments, the composition further comprises at least one payload. In some embodiments, the at least one payload is 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy,166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th, and 225 Ac, or any combination thereof.

[0034] Some aspects of the present disclosure relate to the use of the compositions disclosed herein as a medicament. Some aspects of the present disclosure relate to the use of the compositions disclosed herein in the manufacture of a medicament for administration to a subject. Some aspects of the present disclosure relate to the use of the compositions disclosed herein for imaging a cell, tissue, organ, and / or subject. Some aspects of the present disclosure relate to the use of the compositions disclosed herein for identifying a disease in a subject. In some embodiments, the disease is a cancer or tumor. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is a fibrosis. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is a cardiovascular disease. In some embodiments, the disease is not a hematological cancer or tumor. In some embodiments, the disease relates to the presence, abundance, and / or variation of a FAP.

[0035] Some aspects of the present disclosure relate to a method for identifying a disease in a subject, the method comprising administering to the subject at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies of any one of the embodiments disclosed herein; screening for binding of the at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to a FAP; and determining whether the subject has the disease based on the presence or absence of binding to the FAP. In some embodiments, the disease is a cancer or tumor. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is fibrosis. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is cardiovascular. In some embodiments, the subject is a mammal or human. In some embodiments, at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies binds to tumor stroma. In some embodiments, at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies binds to fibroblasts. In some embodiments, at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies binds to cancer-associated fibroblasts. In some embodiments, the disease is epithelial. In some embodiments, at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies further comprises a payload. In some embodiments, the payload comprises 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc,99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th, and 225In some embodiments, the payload is selected from the group consisting of: A, B, C, D, E, F, F, FAP ... In some embodiments, the method further comprises identifying the subject as having the disease if there is significant binding of the at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP.

[0036] Some aspects of the present disclosure relate to a pharmaceutical composition comprising an amount of at least one antibody, antigen-binding construct thereof, minibody, and / or cys-diabody of any one of the embodiments disclosed herein effective for treating a subject having cancer and / or a tumor; and a pharma- ceutically acceptable carrier. In some embodiments, the amount of at least one of the antibody, antigen-binding construct, minibody, and / or cys-diabody is about 0.01 mg / kg to about 25 mg / kg. In some embodiments, the amount of at least one of the antibody, antigen-binding construct, minibody, and / or cys-diabody is about 1 mg / kg to about 20 mg / kg. In some embodiments, the pharmaceutical composition further comprises at least one known small molecule, therapeutic agent, or antigen-binding construct effective for treating a disease. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective for treating a disease is an antibody. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective for treating a disease is used as part of a chemotherapy. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating the disease is an immuno-oncology drug. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating the disease is a DNA repair inhibitor. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating cancer and / or tumors is selected from the group consisting of alkylating agents, metabolic inhibitors, radiosensitizers, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, nitrosoureas, corticosteroids, antiangiogenic agents, apoptosis inducers, antimicrotubule agents, vinca alkaloids, taxanes, anthracyclines, antiandrogens, VEGF pathway inhibitors, VEGF pathway inhibitors, MAPK / Ras / Raf pathway inhibitors, and EGFR pathway inhibitors, KRAS pathway inhibitors. In some embodiments, the disease is fibrosis. In some embodiments, the disease is cancer or tumor. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is cardiovascular.

[0037] Some aspects of the present disclosure relate to a method for treating, inhibiting, or ameliorating a disease in a subject, comprising administering to a subject in need thereof a pharmaceutical composition of any one of the embodiments disclosed herein. In some embodiments, the disease is fibrosis. In some embodiments, the disease is cancer or a tumor. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is cardiovascular. In some embodiments, the method further comprises imaging the disease using a composition of any one of the embodiments disclosed herein. In some embodiments, the method further comprises imaging the disease using an antigen-binding construct of any one of the embodiments disclosed herein.

[0038] Some aspects of the present disclosure relate to a method for targeting FAP protein on fibroblasts of a subject, comprising administering to the subject any one of the pharmaceutical compositions of the embodiments disclosed herein. In some embodiments, the fibroblasts are cancer-associated fibroblasts. In some embodiments, following targeting of the fibroblasts, cancer cells and / or tumor-associated macrophages are damaged or killed.

[0039] Some aspects of the present disclosure relate to a method for inhibiting, ameliorating, impairing, or inducing apoptosis in cancer or tumor associated macrophages in a subject, comprising administering to a subject in need thereof a pharmaceutical composition of any one of the embodiments disclosed herein. In some embodiments, the subject is a mammal or human. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is epithelial. In some embodiments, the cancer or tumor is selected from the group consisting of bone cancer, osteosarcoma, breast cancer, carcinoid, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, epithelial ovarian cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, glioma, head and neck cancer, hepatocellular carcinoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, medullary thyroid carcinoma, melanoma, non-small cell lung cancer, osteosarcoma, oral squamous cell carcinoma, oral cancer, ovarian cancer, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, gastric cancer, testicular cancer, thyroid cancer, and urothelial cancer.

[0040] In some embodiments, the antigen-binding constructs or compositions disclosed herein are humanized.

[0041] Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein in the manufacture of a medicament for administration to a subject. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein for photodynamic therapy. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein for theranostics. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein for diagnosis. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein for molecular imaging. In some embodiments, the imaging is one or more of the group consisting of photoacoustic, MR imaging, magnetic nanoparticles, spectroscopy, optical probes, and / or any other standard imaging method. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein for therapy. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein for diagnosing cancer or tumor in a subject. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein to determine stratification of a subject for a therapy or treatment. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein to monitor a subject's response to a therapy or treatment. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein to provide information regarding altered therapy decisions in a subject in need thereof. Some aspects of the present disclosure relate to the use of any of the compositions or antigen-binding constructs disclosed herein to assist in surgery or a surgical procedure. [Brief description of the drawings]

[0042] [Figure 1]FIG. 1 shows the expression of different antigen-binding constructs in Expi293™ cells in mg / L. [Diagram 2] FIG. 1 shows the crystal structure of FAP alpha from the Protein Data Bank database 1Z68. [Diagram 3] FIG. 2 shows embodiments of the heavy chain (H) and light chain (L) CDR sequences of SEQ ID NOs: 1 to 6. [Figure 4] FIG. 2 shows embodiments of the heavy chain (VH) and light chain (VL) sequences of SEQ ID NOs: 7 to 11. [Diagram 5] FIG. 1 shows the cleavable signal peptide sequences of SEQ ID NOs: 46 and 121. [Figure 6] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-8 amino acid sequence of SEQ ID NO:12. [Figure 7] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-9 amino acid sequence of SEQ ID NO:13. [Figure 8] FIG. 1 shows an embodiment of the antigen-binding construct IAB16C2-18 amino acid sequence of SEQ ID NO: 14. [Figure 9] FIG. 1 shows an embodiment of the antigen-binding construct IAB16C4-19 amino acid sequence of SEQ ID NO: 15. [Figure 10] FIG. 1 shows an embodiment of the antigen-binding construct IAB16C1-22 amino acid sequence of SEQ ID NO: 16. [Figure 11] FIG. 1 shows an embodiment of the antigen-binding construct IAB16C3-23 amino acid sequence of SEQ ID NO: 17. [Figure 12] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-10 amino acid sequence of SEQ ID NO: 18. [Figure 13] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-11 amino acid sequence of SEQ ID NO: 19. [Figure 14] FIG. 2 shows an embodiment of the antigen-binding construct IAB16C1-24 amino acid sequence of SEQ ID NO:20. [Figure 15] FIG. 2 shows an embodiment of the antigen-binding construct IAB16C2-25 amino acid sequence of SEQ ID NO:21. [Figure 16] FIG. 2 shows an embodiment of the antigen-binding construct IAB16C3-26 amino acid sequence of SEQ ID NO:22. [Figure 17] FIG. 2 shows an embodiment of the antigen-binding construct IAB16C4-27 amino acid sequence of SEQ ID NO:23. [Figure 18] FIG. 2 shows an embodiment of the antigen-binding construct IAB16M1-12 amino acid sequence of SEQ ID NO:24. [Figure 19] FIG. 2 shows an embodiment of the antigen-binding construct IAB16M2-13 amino acid sequence of SEQ ID NO:25. [Figure 20] FIG. 2 shows an embodiment of the antigen-binding construct IAB16C2-16 amino acid sequence of SEQ ID NO:26. [Figure 21] FIG. 2 shows an embodiment of the antigen-binding construct IAB16C4-17 amino acid sequence of SEQ ID NO:27. [Figure 22] FIG. 2 shows an embodiment of the antigen-binding construct IAB16C1-20 amino acid sequence of SEQ ID NO:28. [Diagram 23] FIG. 2 shows an embodiment of the antigen-binding construct IAB16C3-21 amino acid sequence of SEQ ID NO:29. [Figure 24] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-28 amino acid sequence of SEQ ID NO:30. [Diagram 25] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-29 amino acid sequence of SEQ ID NO:31. [Figure 26] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-30 amino acid sequence of SEQ ID NO:32. [Figure 27] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-31 amino acid sequence of SEQ ID NO:33. [Figure 28] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-32 amino acid sequence of SEQ ID NO:34. [Figure 29] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-33 amino acid sequence of SEQ ID NO:35. [Diagram 30]FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-34 amino acid sequence of SEQ ID NO:36. [Diagram 31] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-35 amino acid sequence of SEQ ID NO:37. [Diagram 32] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-36 amino acid sequence of SEQ ID NO:38. [Diagram 33] FIG. 2 shows an embodiment of the antigen-binding construct IAB16M2-37 amino acid sequence of SEQ ID NO:39. [Diagram 34] FIG. 1 shows an embodiment of the antigen-binding construct IAB16C1-38 amino acid sequence of SEQ ID NO:40. [Diagram 35] FIG. 1 shows an embodiment of the antigen-binding construct IAB16C2-39 amino acid sequence of SEQ ID NO:41. [Diagram 36] FIG. 1 shows an embodiment of the antigen-binding construct IAB16C3-40 amino acid sequence of SEQ ID NO:42. [Figure 37] FIG. 1 shows an embodiment of the antigen-binding construct IAB16C4-41 amino acid sequence of SEQ ID NO:43. [Figure 38] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-14 amino acid sequence of SEQ ID NO:44. [Figure 39] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-15 amino acid sequence of SEQ ID NO:45. [Diagram 40] FIG. 1 shows an embodiment of a portion of a nucleic acid sequence encoding the antigen-binding constructs of SEQ ID NOs: 47-80. [Diagram 41] FIG. 1 shows some embodiments of novel antigen-binding constructs, parent constructs, structural classes, and corresponding amino acid and nucleotide sequences. [Figure 42A] 1 is a graph showing the binding kinetics of the antigen-binding construct IAB16M1-12 to FAP. [Figure 42B] FIG. 1 is a graph showing the binding kinetics of the deimmunized antigen-binding construct IAB16B2-37 to FAP. [Diagram 43]FIG. 1 shows a sequence alignment of the heavy and light chains of sibrotuzumab (SEQ ID NOs: 7 and 187) with six exemplary antigen-binding construct sequences (SEQ ID NOs: 24, 30, 32, 34, 36, and 38). [Diagram 44] FIG. 1 shows a sequence alignment of the heavy and light chains of sibrotuzumab (SEQ ID NOs: 7 and 187) with six exemplary antigen-binding construct sequences (SEQ ID NOs: 25, 31, 33, 35, 37, and 39). [Diagram 45] FIG. 1 shows a sequence alignment of the heavy and light chains of sibrotuzumab (SEQ ID NOs: 7 and 187) with four exemplary antigen-binding construct sequences (SEQ ID NOs: 28, 40, 29, and 42). [Figure 46] FIG. 1 shows a sequence alignment of the heavy and light chains of sibrotuzumab (SEQ ID NOs: 7 and 187) with four exemplary antigen-binding construct sequences (SEQ ID NOs: 26, 41, 27, and 43). [Figure 47] FIG. 1 shows a sequence alignment of the heavy chains of sibrotuzumab (SEQ ID NO: 7) and muF19 (SEQ ID NO: 10) with 14 exemplary antigen-binding construct sequences. Highlighted residues indicate predicted mutations or backmutations based on frequency of occurrence in humans. [Figure 48] 1 shows a sequence alignment of the light chains of sibrotuzumab (SEQ ID NO: 187) and muF19 (SEQ ID NO: 10) with 14 exemplary antigen-binding construct sequences. Highlighted residues indicate predicted mutations or backmutations based on frequency of occurrence in humans. [Figure 49A] FIG. 1 shows some schematic embodiments of a minibody with bivalent binding to CD8. [Figure 49B] FIG. 1 illustrates an embodiment of a schematic portion of a minibody. [Figure 50A] FIG. 1 shows some schematic embodiments of cys-diabodies that exhibit bivalent binding to antigen. [Figure 50B] FIG. 1 shows a schematic of a cys-diabody that exhibits bivalent binding to an antigen. [Figure 51] 1A-1C illustrate an exemplary embodiment of a minibody. [Figure 52A] FIG. 1 shows an embodiment of the hinge region of the antigen-binding construct of SEQ ID NO: 188. [Figure 52B] FIG. 1 shows an embodiment of the hinge region of the antigen-binding construct of SEQ ID NO: 189. [Figure 52C] FIG. 1 shows an embodiment of the hinge region of the antigen-binding construct of SEQ ID NO: 190. [Fig. 52D] FIG. 1 shows an embodiment of the hinge region of the antigen-binding construct of SEQ ID NO: 191. [Figure 52E] FIG. 1 shows an embodiment of the hinge region of the antigen-binding construct of SEQ ID NO: 192. [Fig. 52F] FIG. 1 shows an embodiment of the hinge region of the antigen-binding construct of SEQ ID NO: 193. [Diagram 53] FIG. 1 shows an embodiment of the sequence of the His-tagged extracellular domain of human FAP of SEQ ID NO: 147. [Figure 54] FIG. 1 shows an embodiment of the sequence of His-tagged FAP alpha of SEQ ID NO:148. [Figure 55] FIG. 1 shows an alignment of the wild-type human FAP amino acid sequence (SEQ ID NO: 148) with the wild-type human DPP4 amino acid sequence (SEQ ID NO: 194). [Figure 56A] 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16M1-8, IAB16M2-9, IAB16M1-10, IAB16M1-12, and IAB16M2-13 to purified human FAP antigen using ELISA. [Figure 56B] 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16M1-30, IAB16M2-31, IAB16M1-32, and IAB16M2-33 to purified human FAP antigen using ELISA. [Figure 56C] FIG. 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16M1-36 and IAB16M2-37 to purified human FAP antigen using ELISA. [Figure 56D] FIG. 1 is a graph showing the binding kinetics of antigen-binding construct IAB16C1-38 to purified human FAP antigen using ELISA. [Figure 56E] FIG. 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16M2-29, IAB16C3-21, and IAB16C1-20 to purified human FAP antigen using ELISA. [Fig. 56F] 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16C1-24, IAB16C2-25, IAB16C3-26, and IAB16C4-27 to purified human FAP antigen using ELISA. [Figure 56G] 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16C2-16, IAB16C4-17, IAB16C2-18, and IAB16C4-19 to purified human FAP antigen using ELISA. [Figure 57A] FIG. 1 is a graph showing the cross-reactive binding kinetics of antigen-binding construct IAB16C3-26 to purified mouse and human FAP antigens using ELISA. [Figure 57B] 1 is a graph showing the cross-reactive binding kinetics of antigen-binding constructs IAB16M2-9, IAB16M1-10, and IAB16M2-37 to purified mouse FAP antigen and purified human FAP antigen using ELISA. [Figure 57C] 1 is a graph showing the cross-reactive binding kinetics of antigen-binding constructs IAB16M2-9, IAB16M1-10, and IAB16M1-12 to purified human FAP antigen and purified human DPP4 antigen using ELISA. [Figure 58A] 1 is a graph showing binding of antigen-binding constructs IAB16M1-8, IAB16M2-9, IAB16M1-10, IAB16M1-12, IAB16M2-13, IAB16M2-7, and IAB16M1-6 to MRC-5 fibroblasts using flow cytometry. [Figure 58B]FIG. 1 is a graph showing binding of antigen-binding constructs IAB16M1-8, IAB16M2-9, IAB16M1-10, IAB16M1-12, IAB16M2-13, IAB16M2-7, IAB16M1-6, and anti-FAP to U87-MG glioblastoma cells using flow cytometry. [Figure 58C] FIG. 1 is a graph showing binding of the antigen-binding construct IAB16M2-37 to MRC-5 fibroblasts and U87-MG glioblastoma cells using flow cytometry. [Figure 59A] Visualization of IAB16M2-9 internalization into HT1080 human FAP cells after 1 hour using a fluorescent microscope. White arrows point to cells showing particularly significant construct internalization. [Figure 59B] Visualization of IAB16M1-10 internalization into HT1080 human FAP cells after 1 hour using a fluorescent microscope. White arrows point to cells showing particularly significant construct internalization. [Figure 59C] Visualization of IAB16M2-37 internalization into HT1080 human FAP cells after 1 hour using a fluorescent microscope. White arrows point to cells showing particularly significant construct internalization. [Figure 60] FIG. 2 shows embodiments of the heavy chain (H) and light chain (L) CDR sequences of SEQ ID NOs: 81 to 86, 104 to 109, and 134 to 139. [Figure 61] FIG. 2 shows embodiments of heavy chain (VH) sequences and light chain (VL) sequences of SEQ ID NOs: 87 to 96, 110 to 115, 122 to 125, and 140 to 143. [Figure 62] FIG. 1 shows an embodiment of the antigen-binding construct 9A5 amino acid sequence of SEQ ID NO:97. [Figure 63] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-42 amino acid sequence of SEQ ID NO:98. [Figure 64] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-43 amino acid sequence of SEQ ID NO:99. [Figure 65]FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-48 amino acid sequence of SEQ ID NO: 100. [Figure 66] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-51(HC-N76S) amino acid sequence of SEQ ID NO: 101. [Figure 67] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-52 amino acid sequence of SEQ ID NO: 102. [Figure 68] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-53 amino acid sequence of SEQ ID NO: 103. [Figure 69] FIG. 1 shows an embodiment of the antigen-binding construct 9A2 amino acid sequence of SEQ ID NO:116. [Figure 70] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-44 amino acid sequence of SEQ ID NO: 117. [Figure 71] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-45 amino acid sequence of SEQ ID NO: 118. [Figure 72] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-46 amino acid sequence of SEQ ID NO: 119. [Figure 73] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-47 amino acid sequence of SEQ ID NO: 120. [Figure 74] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-56 amino acid sequence of SEQ ID NO: 126. [Figure 75] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-59 amino acid sequence of SEQ ID NO: 127. [Figure 76] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-60 amino acid sequence of SEQ ID NO: 128. [Figure 77] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-61 amino acid sequence of SEQ ID NO: 129. [Figure 78] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-54 amino acid sequence of SEQ ID NO: 130. [Figure 79]FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-55 amino acid sequence of SEQ ID NO: 131. [Figure 80] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-57 amino acid sequence of SEQ ID NO: 132. [Figure 81] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-58 amino acid sequence of SEQ ID NO: 133. [Figure 82] FIG. 1 shows an embodiment of the antigen-binding construct 3A9 amino acid sequence of SEQ ID NO: 144. [Figure 83] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M1-49 amino acid sequence of SEQ ID NO: 145. [Figure 84] FIG. 1 shows an embodiment of the antigen-binding construct IAB16M2-50 amino acid sequence of SEQ ID NO: 146. [Figure 85] FIG. 1 shows a sequence alignment of the heavy chain of the murine 9A5 construct (SEQ ID NO: 87) with five exemplary antigen-binding construct sequences (SEQ ID NOs: 90, 92, 94, 96, and 123). The underlined residues represent the CDR regions. Highlighted residues indicate humanized germline differences. [Figure 86] FIG. 1 shows a sequence alignment of the light chain of the murine 9A5 construct (SEQ ID NO: 87) with five exemplary antigen-binding construct sequences (SEQ ID NOs: 89, 91, 93, 95, and 122). The underlined residues represent the CDR regions. Highlighted residues indicate humanized germline differences. [Figure 87] FIG. 1 shows a sequence alignment of the heavy chain of the murine 9A2 construct (SEQ ID NO: 110) with five exemplary antigen-binding construct sequences (SEQ ID NOs: 112, 91, 114, 95, and 124). The underlined residues represent the CDR regions. Highlighted residues indicate humanized germline differences. [Figure 88] 1 shows a sequence alignment of the light chain of the mouse 9A2 construct (SEQ ID NO: 110) with five exemplary antigen-binding construct sequences (SEQ ID NOs: 113, 92, 115, 96, and 125). The underlined residues represent the CDR regions. Highlighted residues indicate humanized germline differences. [Figure 89] 1 shows a sequence alignment of the heavy chain of the mouse 3A9 construct (SEQ ID NO: 140) with four exemplary antigen-binding construct sequences (SEQ ID NOs: 142, 91, 122, and 143). The underlined residues represent the CDR regions. Highlighted residues indicate humanized germline differences. [Figure 90] 1 shows a sequence alignment of the light chain of the mouse 3A9 construct (SEQ ID NO: 140) with three exemplary antigen-binding construct sequences (SEQ ID NOs: 90, 125, and 94). The underlined residues represent the CDR regions. Highlighted residues indicate differences in the humanized germline. [Figure 91A] FIG. 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16M1-54, IAB16M2-55, and 9A2 to purified human FAP antigen using ELISA. [Figure 91B] FIG. 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16M1-42 and IAB16M2-43 to purified human FAP antigen using ELISA. [Fig. 91C] FIG. 1 is a graph showing the binding kinetics of antigen-binding construct 9G5 to purified human FAP antigen using ELISA. [Fig. 91D] FIG. 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16M2-48 and IAB16M2-51 to purified human FAP antigen using ELISA. [Fig. 91E] FIG. 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16M2-52 and IAB16M2-53 to purified human FAP antigen using ELISA. [Fig. 91F] FIG. 1 is a graph showing the binding kinetics of antigen-binding constructs IAB16M1-49 and IAB16M2-50 to purified human FAP antigen using ELISA. [Figure 92] FIG. 1 is a graph showing the cross-reactive binding kinetics of antigen-binding construct IAB16M2-43 to purified mouse FAP antigen, cynomolgus monkey FAP, human FAP antigen, and human DPP4 antigen using ELISA. [Figure 93]FIG. 1 is a graph showing binding of antigen-binding constructs 9A2, 3A9, and 9A5 to HT1080 human FAP cells using flow cytometry. [Figure 94A] Fluorescence microscopy visualization of IAB16M2-43 internalization into HT1080 human FAP cells. White arrows point to cells showing particularly significant construct internalization. [Figure 94B] Figure 1: Visualization of IAB16M2-43 internalization into MRC5 cells using fluorescence microscopy. White arrows point to cells showing particularly significant construct internalization. [Fig. 94C] Figure 1: Visualization of IAB16M2-43 internalization into U87-MG cells using fluorescence microscopy. White arrows point to cells showing particularly significant construct internalization. [Figure 95] Graph showing uptake of radiolabeled minibodies IAB16M2-77 and IAB16M2-78 in the liver, kidney and spleen. [Figure 96] Graph showing uptake of radiolabeled minibody IAB16M2-37 conjugated with LicorIRDye800 in liver, kidney and spleen. [Figure 97] Graph showing uptake of radiolabeled minibody IAB16M2-56 conjugated with LicorIRDye800 and the metal chelator DTPA in the liver, kidney, and spleen. [Figure 98] FIG. 13 is a graph showing the biodistribution of radiolabeled minibody IAB16M2-56 conjugated to the metal chelator DTPA at different chelator to minibody ratios (CMR) in the liver, kidney, and spleen. [Figure 99] FIG. 13 is a graph showing the biodistribution of radiolabeled minibody IAB16C3-26 conjugated with the metal chelator DTPA in the liver, kidney and spleen. [Figure 100] FIG. 1 shows an embodiment of the radiolabeled minibody IAB16M2-77 of SEQ ID NO: 149. [Figure 101]FIG. 1 shows an embodiment of the radiolabeled minibody IAB16M2-78 of SEQ ID NO: 150. [Figure 102] FIG. 1 shows an embodiment of framework 2 of the germline sequences of antibodies represented by SEQ ID NOs: 151 to 186. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Antibodies that bind to FAP are known in the art. An antibody designated F19 that specifically binds to FAP is described in US Patent No. 5,059,523. F19 can be obtained from hybridoma cell line ATCC Accession No. HB8269. A minibody that targets FAP, designated MO36, was developed by phage display and is disclosed in Brocks et al. (2001) Phage Display Selection of FAP-Specific scFv. Molec. Med, vol. 7(no. 7) pp. 461-469. An antibody called sibrotuzumab specifically binds to FAPα and is disclosed in US Patent No. 6,455,677. Antibodies 4B9 and 28H1, which also bind to FAP, are disclosed in US Patent Publication No. 20120128591.

[0044] Described herein are antigen-binding constructs, including minibody and cys-diabody formats thereof. In some embodiments, the antigen-binding constructs bind to FAPs. In some embodiments, the antigen-binding constructs can be used to target FAPs for diagnostic and / or therapeutic purposes. In some embodiments, the antigen-binding constructs can be at least one of an antibody, a minibody, and a cys-diabody. In some embodiments, the antigen-binding constructs can be combined with at least one of a payload, a chelator, a label (such as a detectable marker), a small molecule, and / or a therapeutic agent. In some embodiments, targeting a FAP provides a health benefit to a subject in need thereof.

[0045] definition As used herein, "a" or "an" can mean one or more than one.

[0046] As used herein, the terms "about" or "approximately" have their ordinary meaning as understood by one of ordinary skill in the art, and thus indicate that a numerical value includes the inherent variation of error for the method used to determine the numerical value or the variation that exists between multiple determinations.

[0047] Throughout this specification, unless the context requires otherwise, the terms "comprise", "comprises", and "comprising" will be understood to mean the inclusion of the stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means inclusive and limited to everything that follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and no other elements may be present. "Consisting essentially of" means including any elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that other elements are optional and may or may not be present depending on whether they materially affect the activity or action of the recited elements.

[0048] As used herein, "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, an optionally substituted group means that the group is either unsubstituted or substituted.

[0049] The terms "function" and "functional" as used herein have their plain and ordinary meaning as understood in the context of this specification and refer to biological, enzymatic, or therapeutic functions.

[0050] The term "inhibit" as used herein has its plain and ordinary meaning as understood in light of the present specification and can refer to a reduction or prevention of biological activity. The reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, about, at least, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount within a range defined by any two of the above values. The term "delay" as used herein has its plain and ordinary meaning as understood in light of this specification, and refers to the slowing, postponement, or postponement of a biological event to a later time than would otherwise be expected. The delay may be 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, about, at least, at least about 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or less, or about 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the above values. A non-limiting example of inhibition as used herein can refer to a reduction in FAP activity upon contact with an antigen-binding construct, e.g., a reduction in dipeptidyl peptidase activity, endopeptidase activity, and / or proteolytic activity. In some embodiments, inhibition is measured in an enzyme assay, which quantifies the decrease in activity of the enzyme of interest with increasing inhibitor concentration. For example, the amount of fluorescent substrate cleaved by the FAP is measured in the presence of a potential FAP inhibitor, such as BR03354. Inhibition values ​​are typically expressed as IC50, where half of the activity of the enzyme is inhibited at that given inhibitor concentration. In some embodiments, FAP inhibitors have IC50 values ​​of 10-20 nM. The terms inhibition and delay do not necessarily indicate 100% inhibition or delay.Partial inhibition or retardation may be achieved.

[0051] The term "yield" of any given substance, compound, or material, as used herein, has its plain and ordinary meaning as understood in light of the present specification, and refers to the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material may be about, at least, or at least about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount. , or 100%, 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or less, or about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, including all decimal points therebetween. Yields can be affected by the efficiency of the reaction or process, undesired side reactions, decomposition, quality of the input substances, compounds, or materials, or loss of desired substances, compounds, or materials during any step of production.

[0052] As used herein, the term "isolated" has its plain and ordinary meaning as understood in light of the present specification and refers to a substance and / or entity that (1) has been separated from at least a portion of the components that were associated with it when initially produced (whether in nature and / or in an experimental setting) and / or (2) has been produced, prepared, and / or manufactured by the hand of man. An isolated substance and / or entity is one that is about, at least, at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or equal to 100% of the other components with which it was initially associated. It may be separated from 0%, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or less than 100%, from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or less than 100% (or from a range including and / or spanning the recited values). In some embodiments, an isolated agent is about, at least, at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure, or is 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, About 96%, about 97%, about 98%, about 99%, substantially 100%, or less than 100% pure, or about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or less than 100% pure (or ranges including and / or spanning the above values). As used herein, an "isolated" material may be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" may refer to a cell that is not contained in a multicellular organism or tissue.

[0053] As used herein, "in vivo" is to be given its plain and ordinary meaning as understood in the context of this specification and refers to the performance of a method in a living organism, usually animals, mammals, including humans, and plants, or in the living cells that make up such a living organism, as opposed to a tissue extract or a dead organism.

[0054] As used herein, "ex vivo" is given its plain and ordinary meaning as understood in the context of this specification and refers to carrying out the method outside a living organism with minimal alteration from natural conditions.

[0055] As used herein, "in vitro" is given its plain and ordinary meaning as understood in the context of this specification and refers to performing a method outside biological conditions, e.g., in a petri dish or test tube.

[0056] As used herein, "nucleic acid", "nucleic acid molecule", or "nucleotide" refers to polynucleotides or oligonucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by either ligation, cleavage, endonuclease action, exonuclease action, and synthetic production. Nucleic acid molecules may be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have alterations in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar may be functionalized as an ether or ester. Additionally, the entire sugar moiety may be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. The nucleic acid monomers may be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, and phosphoramidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain naturally occurring or modified nucleobases attached to a polyamide backbone. Nucleic acids may be single-stranded or double-stranded.

[0057] The terms "peptide", "polypeptide" and "protein" as used herein have their plain and ordinary meaning as understood in the context of this specification and refer to a polymer composed of amino acids linked by peptide bonds. The functions of peptides, polypeptides and proteins are numerous and known in the art, including but not limited to enzymatic, structural, transport, defensive, hormonal or signal transduction. Peptides, polypeptides and proteins are often, but not always, produced biologically by ribosomal complexes using nucleic acid templates, although chemical synthesis is also available. By manipulating the nucleic acid template, peptide, polypeptide and protein mutations can be performed, such as substitutions, deletions, truncations, additions, duplications or fusions of more than one peptide, polypeptide or protein.Such fusions of more than one peptide, polypeptide, or protein may be contiguously joined in the same molecule, or may be joined via extra amino acids in between, such as a linker, repeat, epitope, or tag, or any other sequence, which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, about, at least, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 , 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 30 The length of the polypeptide may be 0 bases or less, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases or less, or any length in the range defined by any two of the above lengths. The term "downstream" of a polypeptide, as used herein, has its plain and ordinary meaning as understood in the context of this specification, and refers to a sequence that is located after the C-terminus of a preceding sequence. The term "upstream" of a polypeptide, as used herein, has its plain and ordinary meaning as understood in the context of this specification, and refers to a sequence that is located before the N-terminus of a following sequence.

[0058] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0059] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that code for identical or essentially identical amino acid sequences, or essentially identical sequences when the nucleic acid does not code for an amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that codes for a polypeptide also describes every possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon of a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, every silent variation of a nucleic acid that codes for a polypeptide is implicit in each described sequence.

[0060] With respect to amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small percentage of the amino acids in the encoded sequence are "conservatively modified variants" in which the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the constructs provided herein.

[0061] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0062] "Percentage of sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (e.g., a polypeptide of a construct provided herein) that does not contain additions or deletions in order to optimally align the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to calculate the percentage of sequence identity.

[0063] The term "identical" or percent "identity", in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same sequence. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a specified region or, if not specified, over the entire sequence of the reference sequence) when compared and aligned for maximum matching over a comparison window or over a specified region, as measured using one of the following sequence comparison algorithms or manual alignment and visual inspection. Some embodiments provided herein provide polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides exemplified herein, respectively. Optionally, identity exists over a region that is at least about 15, 25, or 50 nucleotides in length, or more preferably over a region that is 100-500 or 1000 or more nucleotides in length, or over the entire length of the reference sequence. With respect to amino acid sequences, identity or substantial identity may exist over a region that is at least 5, 10, 15, or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75, or 100 amino acids in length, optionally at least about 150, 200, or 250 amino acids in length, or over the entire length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, in some embodiments substantial identity exists when one or two amino acid residues are conservatively substituted according to the conservative substitutions defined herein.

[0064] In sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or embodiment parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0065] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, in some embodiments, a polypeptide is typically substantially identical to a second polypeptide, for example, where the only difference between the two peptides is a conservative substitution. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0066] The term "gene" as used herein has its plain and ordinary meaning as understood in the context of this specification and generally refers to a portion of a nucleic acid or functional RNA that codes for a protein, although the term may optionally include regulatory sequences. Those skilled in the art will appreciate that the term "gene" may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences. It will be further understood that the definition of a gene includes reference to nucleic acids that do not code for proteins, but rather code for functional RNA molecules such as tRNAs and miRNAs. In some cases, a gene includes regulatory sequences involved in transcription or message production or composition. In other embodiments, a gene includes a transcribed sequence that codes for a protein, polypeptide, or peptide. According to the terminology described herein, an "isolated gene" may include a transcribed nucleic acid, regulatory sequence, or coding sequence, etc., that is substantially isolated from other naturally occurring genes, regulatory sequences, other such sequences, such as polypeptide or peptide coding sequences. In this regard, the term "gene" is used in shorthand to refer to a nucleic acid that includes a transcribed nucleotide sequence and its complement. As will be understood by those skilled in the art, the functional term "gene" includes any smaller engineered nucleic acid segment, including genomic sequences, RNA or cDNA sequences, or nucleic acid segments of non-transcribed portions of a gene, including but not limited to non-transcribed promoter or enhancer regions of a gene. Smaller engineered gene nucleic acid segments can express or can be configured to express proteins, polypeptides, domains, peptides, fusion proteins, and / or mutants, etc., using nucleic acid engineering techniques.

[0067] As used herein, an "expression vector" or "vector" is a nucleic acid molecule that encodes a gene to be expressed in a host cell. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be "operably linked" to the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

[0068] As used herein, a "ligand" refers to a small molecule, peptide, or protein that can form a complex with another molecule or biomolecule for a biological purpose, such as signal induction. Binding can occur through intermolecular forces, such as ionic bonds, hydrogen bonds, van der Waals interactions, etc. Ligand binding to a receptor protein can change its three-dimensional structure and determine its functional state.

[0069] By way of example and not limitation, ligands can include substrates, proteins, small molecules, inhibitors, activators, and neurotransmitters. The binding strength of a ligand is referred to as binding affinity and can be determined by direct interactions and solvent effects. A ligand can be bound by a "ligand-binding domain." A ligand-binding domain refers to a conserved sequence in a structure that can bind to a particular ligand. Without limitation, a ligand-binding domain can be a particular protein domain that is specific for one or more ligands.

[0070] "Specific" or "specificity" can refer to characteristics of a ligand for a binding partner or alternatively a binding partner for a ligand, and can include complementary shape, charge, and hydrophobicity specificities for binding. Specificity of binding can include stereospecificity, regioselectivity, and chemoselectivity.

[0071] "Label," "detectable label," or "detectable marker" are used interchangeably herein to refer to a detectable compound or composition that is directly conjugated to or indirectly associated with an antibody to produce a "labeled" antibody. The label may itself be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0072] The term "payload" refers to an atom or molecule or other entity associated (covalently or otherwise) with an antigen-binding construct. The term "payload" includes labels or markers in the case of diagnostic aspects, as well as toxins, cytotoxic agents, chemotherapeutic agents in the case of various therapeutics. In some embodiments, the payload includes a chelator to attach the antigen-binding construct to a molecule or atom to be delivered or co-localized via the antigen-binding construct.

[0073] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. This term includes any substance that inhibits or prevents cellular function and / or causes cell death or destruction. 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 53 , Bi 212 , P 32 , Pb 212, and radioisotopes of Lu), chemotherapeutic agents (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents), toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including enzymes such as nucleases and fragments thereof, antibiotics, and fragments and / or variants thereof, toxins, growth inhibitory agents, drug moieties, and various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. Tumoricidal agents cause the destruction of tumor cells.

[0074] A "toxin" is any substance capable of detrimentally affecting the growth or proliferation of cells.

[0075] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and CYTOXAN™ cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL™); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analog topotecan (HYCAMTIN™), CPT-11 (irinotecan, CAMPTO SAR™), acetylcamptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and biceresin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); ereutherobiin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Enediyne antibiotics (e.g., the calicheamicins, especially calicheamicin gamma II and calicheamicin omegall (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); the dynemicins, including dynemicin A; the esperamicins; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), the aclacinomysins, actinomycins, autramycins, azaserine, bleomycins, cactinomycins, carabicins, carminomycins, carpinomycins, Dinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, noga Antibiotics such as ramycin, olivomycin, peplomycin, porfiromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; metabolic inhibitors such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; fludarabine, 6-mercaptopurine, Purine analogues such as thiamiprine and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid sources such as frolinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine;Bestravsil;bisantrene;edatraxate;defofamine;demecolcine;diaziquone;elfornithine;elliptinium acetate;epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidainine;maytansinoids such as maytansine and ansamitocin;mitoguazone;mitoxantrone;mopidanmol;nitraerine;pentostatin;phenamet;pirarubicin;rosoxantrone;2-ethylhydrazide;procarbazine;PSK(R) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine (ELDISINE™, FILDESIN™); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), a cremophor albumin-free engineered nanoparticle formulation of paclitaxel, ABRAXANE™ (American Pharmaceutical docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN™); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN™); oxaliplatin; leucovovin;Vinorelbine (NAVELBINE™); Novantrone; Edatrexate; Daunomycin; Aminopterin; Ibandronate; The topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid; Capecitabine (XELODA™); Pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for the treatment regimen with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

[0076] "Radiotherapy" refers to treatment using radiation or radioisotopes for therapeutic purposes. Radiotherapy includes radiotherapy intended to exhibit an abscopal effect as described in Yang Liu, Yinping Dong, Li Kong, Fang Shi, Hui Zhu & Jinming Yu; "Abscopal effect of radiotherapy combined with immune checkpoint inhibitors"; Journal of Hematology & Oncology, Volume 11, Article No: 104 (2018); and Melek Tugce Yilmaz, Aysenur Elmali, and Gozde Yazici; "Abscopal Effect, From Myth to Reality: From Radiation Oncologists' Perspective"; Cureus, January 2019; Volume 11 (Issue 1).

[0077] The terms "immune checkpoint inhibitors" (sometimes referred to as "ICIs") or "checkpoint inhibitors" (sometimes referred to as "CPIs") or "immune checkpoint blockade inhibitors" and all similar terms refer to a subclass of immunotherapy. Examples include molecules that block certain proteins made by some types of immune system cells, such as T cells, and some cancer cells. These proteins can help keep the immune response in check and prevent T cells from killing cancer cells. When these proteins are blocked, the immune system is free to act and T cells can kill cancer cells. Some embodiments include multispecific agents, including, but not limited to, anti-PD1 and anti-PD-L1 binding agents, anti-CTLA4 agents, and anti-CTLA-4 / B7-1 / B7-2. Additional immunotherapies include checkpoint inhibitors such as ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). IOTs also include tremelimumab and pidilizumab, and small molecule ICIs are in development including BMS-1001, BMS-1116, CA-170, CA-327, imiquimod, resiquimod, 852A, VTX-2337, ADU-S100, MK-1454, ibrutinib, 3AC, idelalisib, IPI-549, epacadostat, AT-38, CPI-444, vipadenant, preladenant, PBF, AZD4635, galuniseritib, OTX015 / MK-8628, CPI-0610 (see Kerr and Chisolm (2019) The Journal of Immunology, 2019, 202:11-19).

[0078] IOTs are not CPIs, but also include other modalities that activate the host immune system against cancer or render tumors vulnerable to CPI therapy. Such embodiment IOTs include, but are not limited to, T cell immunomodulators such as cytokines IL-2, IL-7, IL-15, IL-21, IL-12, GM-CSF, and IFNα (THOR-707 from Synthorx Therapeutics and NKTR-214 bempegaldesleukin from Nektar Therapeutics); various other interferons and interleukins; TGFβ1 inhibitors (such as SRK-181, which is being developed by Scholar Rock); oncolytic therapy (including oncolytic virus therapy); adoptive cell therapy such as T cell therapy (including CAR-T cell therapy); and cancer vaccines (both preventive and therapeutic). Immunotherapies also include strategies to increase the neo-antigen load of tumor cells, including targeted therapy to cause tumor cells to express or present tumor-associated antigens. (See Galon and Bruni (2019) Nature Reviews Drug Discovery, vol. 18, pp. 197-218.) Additional IOTs include TLR9 ligands (Checkmate Pharmaceuticals), A2A / A2B dual antagonists (Arcus Biosciences), and vaccination peptides against endogenous enzymes such as IDO-1 and arginase (IO Biotech). IOTs include HS-110, HS-130, PTX-35 (Heat Biologics).

[0079] Those skilled in the art will recognize that immunotherapies can be used in combination with each other, and can also be used before, after, or in combination with other therapies for the disease, including, in the case of cancer, radiation therapy, chemotherapy of any type (including the cytotoxic, chemotherapeutic, antihormonal, growth inhibitory agents referenced above), and surgical resection.

[0080] The term "antigen-binding construct" includes any type of antibody, including binding fragments thereof. It further includes constructs comprising one, two, three, four, five, and / or six CDRs. In some embodiments, such CDRs may be distributed among their appropriate framework regions in a conventional antibody. In some embodiments, the CDRs may be comprised within the heavy and / or light chain variable regions. In some embodiments, the CDRs may be comprised within the heavy and / or light chain. In some embodiments, the CDRs may be within a single peptide chain. In some embodiments, the CDRs may be within two or more peptides that are covalently linked to each other. In some embodiments, the peptides may be covalently linked by disulfide bonds. In some embodiments, the peptides may be linked via a linking molecule or moiety. In some embodiments, the antigen-binding protein is non-covalent, such as diabodies and monovalent scFVs. Unless otherwise stated herein, the antigen-binding constructs described herein bind to the indicated target molecule. The term also includes minibodies and cys-diabodies.

[0081] The term "antibody" includes, but is not limited to, genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, etc.). The term "antibody" includes cys-diabodies and minibodies. Thus, any and all embodiments provided herein with respect to "antibody" are also envisioned as cys-diabody and / or minibody embodiments, unless otherwise specified. The term "antibody" includes polypeptides of the immunoglobulin family, or polypeptides including fragments of immunoglobulins capable of non-covalently, reversibly, and specifically binding to a corresponding antigen. Exemplary antibody structural units include tetramers. In some embodiments, a full-length antibody may be composed of two identical pairs of polypeptide chains, each pair having one "light" chain and one "heavy" chain, connected via disulfide bonds. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. For full-length chains, light chains are classified as either kappa or lambda. For full-length chains, heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of the light and heavy chains, respectively. As used in this application, "antibody" encompasses all variations of antibodies and fragments thereof. Thus, within this concept are full-length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (scFV), Fab, Fab', and multimeric forms of such fragments having the same binding specificity (e.g., F(ab')2). In some embodiments, the antibody specifically binds to a desired target.

[0082] The term "sibrotuzumab" or "BIBH1", as used herein, is given its plain and ordinary meaning as understood in the context of this specification and refers to a known humanized monoclonal antibody that binds to FAP. It was developed by Boehringer Ingelheim Pharma KG for the treatment of cancer. However, in 2003, it failed a Phase II clinical trial for metastatic colorectal cancer.

[0083] "Complementarity determining domain" or "complementarity determining region ("CDR")" refers synonymously to the hypervariable regions of VL and VH. CDRs are the target protein binding sites of antibody chains that harbor the specificity for such target protein. In some embodiments, each VL and / or VH has three CDRs (CDR1-3, numbered consecutively from the N-terminus) that constitute about 15-20% of the variable domain. The CDRs are structurally complementary to the epitope of the target protein and are therefore directly responsible for the binding specificity. The remaining parts of the VL or VH, the so-called framework regions (FR), show less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4, WH Freeman & Co., New York, 2000).

[0084] The locations of the CDRs and framework regions may be determined according to various definitions well known in the art, e.g., Kabat (Wu, TT, EA Kabat, 1970. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132:211-250; Kabat, EA, Wu, TT, Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, Md.), Chothia (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997), ImMunoGeneTics Database (IMGT) (World Wide Web, imgt.org / ) Giudicelli, V., Duroux, P., Ginestoux, C., Folch, G., Jabado-Michaloud, J., Chaume, D., and Lefranc, M.-P. IMGT / LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences Nucl. Acids Res., 34, D781-D784 (2006), PMID: 16381979; Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V., and Lefranc, G., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains Dev. Comp. Immunol., vol. 27, pp. 55-77 (2003). PMID: 12477501; Brochet, X., Lefranc, M.-P., and Giudicelli, V. IMGT / V-QUEST: the highly customized and integrated system for IG and TR standardized VJ and VDJ sequence analysis Nucl. Acids Res, vol. 36, W503-508 (2008); AbM (Martin et al., Proc. Natl. Acad. Sci. USA, vol. 86:9268-9272 (1989)), North (North B., Lehmann A., Dunbrack RL, A new clustering of antibody CDRs loop conformations, J. Mol. Biol. (2011) 406(2):228-256), AHo (Honegger A., ​​Pluckthun, Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J. Mol. Biol. (2001) 309, 657-670); contact definitions (MacCallum et al., J. Mol. Biol., 262:732-745 (1996)), and / or automated modeling and analysis tools, Honegger A, Pluckthun A.(World Wide Web, bioc dot uzh dot ch / antibody / Numbering / index dot html). In some embodiments, any one or more of the CDRs in any of the sequences provided herein may be defined by any of these CDR definitions.

[0085] "Antibody variable light chain" or "antibody variable heavy chain" as used herein refers to a polypeptide comprising a VL or VH, respectively. An endogenous VL is encoded by gene segments V (variable) and J (junction), and an endogenous VH is encoded by V, D (diversity) and J. Each of the VL or VH comprises CDRs and framework regions. In the present application, the antibody variable light chain and / or the antibody variable heavy chain may sometimes be collectively referred to as "antibody chains". Such terms encompass antibody chains that contain mutations that do not disrupt the basic structure of the VL or VH, as would be readily recognized by one of skill in the art. In some embodiments, full-length heavy and / or light chains are contemplated. In some embodiments, the presence of only the variable regions of the heavy and / or light chains is contemplated.

[0086] Antibodies may exist as intact immunoglobulins or as a number of fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide linkage in the hinge region to produce F(ab)'2, a Fab' dimer, which is itself a light chain (VL-CL) joined to VH-CH1 by a disulfide linkage. F(ab)'2 is reduced under mild conditions to cleave the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. A Fab' monomer is a Fab with a portion of the hinge region. (Paul, Fundamental Immunology 3rd Edition (1993)). Similarly, Fab fragments can be derived by enzymatic digestion of full-length antibodies with papain, which cleaves the antibody at the upper hinge region. Although various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill in the art will appreciate that such fragments can be synthesized de novo, either chemically or by use of recombinant DNA methodology. Thus, the term "antibody," as used herein, also includes antibody fragments produced by the modification of whole antibodies, or antibody fragments synthesized de novo using recombinant DNA methodologies (e.g., single chain Fvs), or antibody fragments identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0087] For the preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, for example, Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96. Alan R. Liss, Inc. 1985; Advances in the production of human monoclonal antibodies Shixia Wang, Antibody Technology Journal 2011:1:1-4; J Cell Biochem. 2005 Oct. 1; 96(2):305-13; Recombinant polyclonal antibodies for cancer therapy; Sharon J, Liebman MA, Williams BR; and Drug Discov Today. 2006 (See Haurum JS, Recombinant polyclonal antibodies: the next generation of antibody therapeutics?, July 11(13-14):655-60). Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies against polypeptides. Also, transgenic mice, or other organisms such as other mammals, can be used to express fully human monoclonal antibodies. Alternatively, phage display technology can be used to identify high affinity binders to a selected antigen (see, e.g., McCafferty et al., supra; Marks et al., Biotechnology, 10:779-783 (1992)).B cell cloning can be used to identify fully human antibodies directly from a human subject (Wardemann H., Busse E., Expression Cloning of Antibodies from Single Human B Cells, Methods Mol. Biol. (2019) 1956:105-125).

[0088] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, in this method, an antibody or antibody fragment derived from a non-human source has one or more amino acid residues introduced into it from a human (or primate, in the case of primatization) source. In some embodiments, a non-human antibody has one or more amino acid residues introduced into it from a source that is human. Such human amino acid residues are often referred to as import residues, and are typically taken from an import variable domain. In some embodiments, the terms "donor" and "acceptor" sequences can be used. Humanization can be performed essentially according to the method of Winter et al. (see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)) by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Such humanized antibodies (described, for example, in U.S. Pat. No. 4,816,567) thus have substantially less intact non-human variable domain replaced by the corresponding sequences of human origin. In practice, humanized antibodies are typically rodent or other mammalian antibodies in which some complementarity determining region ("CDR") residues and possibly some framework ("FR") residues are substituted by residues from analogous sites in human antibodies.

[0089] Antibodies further comprise one or more immunoglobulin chains that are chemically conjugated to other proteins or expressed as fusion proteins with other proteins. In some embodiments, the antigen-binding construct may be a monovalent scFV construct. In some embodiments, the antigen-binding construct may be a bispecific construct. Bispecific or bifunctional antibodies are artificial hybrid antibodies that have two different heavy / light chain pairs and two different binding sites. Other antigen-binding fragments or antibody portions include bivalent scFVs (diabodies), bispecific scFV antibodies in which the antibody molecule recognizes two different epitopes, single binding domains (sdAbs or nanobodies), and minibodies.

[0090] The term "antibody fragment" includes, but is not limited to, one or more antigen-binding fragments of an antibody alone or in combination with other molecules, including, but not limited to, Fab', F(ab')2, Fab, Fv, rIgG (reduced IgG), scFV fragments (monovalent, trivalent, etc.), single domain fragments (nanobodies), peptibodies, minibodies, diabodies, and cys-diabodies. The term "scFV" refers to a single chain Fv ("variable fragment") antibody in which the variable domains of the heavy and light chains of a traditional two-chain antibody are combined to form one chain.

[0091] A "single-chain variable fragment" or scFV is a fusion protein comprising the variable regions of the immunoglobulin heavy (VH) and light (VL) chains connected by a short linker peptide. Without limitation, the linker may contain glycine for flexibility and hydrophilic amino acids such as serine or threonine for solubility. The linker may connect 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.

[0092] Minibodies are antibody formats that have a smaller molecular weight than full-length antibodies while maintaining bivalent binding properties to antigens. In some embodiments, minibodies are 50-100 kDa, most preferably about 80 kDa. In some embodiments, Fab2 is about 100 kDa. In some embodiments, full-length antibodies are about 150 kDa. In some embodiments, FAP diabodies are about 50 kDa. In some embodiments, cys-diabodies are about 50 kDa. In some embodiments, scFVs are about 25 kDa. In some embodiments, VHHs are about 12 kDa. Minibodies' small size allows for more rapid clearance from the system and enhanced penetration in targeting tumor tissue. Minibodies have strong and selective targeting capabilities combined with rapid clearance, making them advantageous for diagnostic imaging and delivery of cytotoxic / radioactive payloads that may have adverse side effects due to their extended circulation time. The "minibodies" described herein include human IgG1 C, human IgG2 C, human IgG3 C, human IgG4 C, human IgG5 C, human IgG6 C, human IgG7 C, human IgG8 C, human IgG1 C, human IgG1 C, human IgG1 C, human IgG1 C, human IgG2 ... H In some embodiments, the CH3 sequence is a homodimer that is a single chain variable fragment (scFV) linked to three domains. In some embodiments, the hinge sequence is a human IgG1 or IgG2 hinge sequence. In some embodiments, the CH3 sequence is a human IgG1 C H 3 sequences or IgG2 C H Contains 3 sequences.

[0093] In some embodiments, the hinge sequence is an artificial hinge sequence. In some embodiments, the hinge sequence may be an IgG hinge from any one or more of the four classes. The artificial hinge sequence may include a portion of a human IgG1 or IgG2 hinge and a GlySer linker (also known as an "extension" when this section is distinguished from the common linker sequence that connects the Vh and Vl regions) sequence. Artificial hinge sequences and linker sequences that can be usefully used with the present invention are described in WO2017027325A1.

[0094] scFV is V H- V LOrientation may be L- V H In some embodiments, the V H and V L are linked together by an amino acid linker sequence. The amino acid linker may be a linker described herein. In some embodiments, the linker is GlySer-rich and is approximately 15-20 amino acids in length. In another embodiment, the linker is GlySer-rich and is 18 amino acids in length. In some embodiments, the linker length varies from about 1-50 amino acids, e.g., 2-30, 3-20, 4-15, or 5 amino acids to 8 amino acids (and inclusive). In some embodiments, the minibody scFv has a sequence that is at least about 80%, e.g., at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, or 99% identical to the scFvs of the cys-diabodies described herein. The scFvs are H -V L Orientation may be L -V H The film may have an orientation.

[0095] In some embodiments, each monomer of a minibody comprises, from N-terminus to C-terminus, the following elements: L V linked to domain H (b) a hinge extension domain comprising a human IgG1 hinge region; and (c) a human IgG C H In some embodiments, each monomer of a minibody comprises, from N-terminus to C-terminus, the following elements: (a) V L V linked to domain H In some embodiments, each monomer of the minibody comprises an IgG2, IgG3, or IgG4 C domain and an scFV sequence that binds to a target molecule, (b) a hinge extension domain comprising an IgG2 hinge region as described herein, and (c) a human IgG CH3 sequence. HIn some embodiments, each monomer of a minibody may comprise an IgA or IgD CH3 domain and / or an IgM and / or IgE CH4 domain. In some embodiments, the minibody is encoded by a nucleic acid that can be expressed by a cell, cell line, or other suitable expression system described herein. Thus, a signal sequence can be fused to the N-terminus of the scFv to allow for secretion of the minibody when expressed in a cell or cell line.

[0096] The term "diabody" refers to a heavy chain (V H ) domain and the light chain variable (V L ) domains, each heavy chain domain being connected to a light chain domain via a linker.

[0097] The term "linker" refers to H Domain and V L The linker refers to a peptide sequence that connects the domains. H and V L Domain, V L -V H Orientation or V H -V L The linker can be oriented in either V or V orientation via the peptide backbone. H V L Connect to.

[0098] The term "extension sequence" refers to, for example, in a diabody, the first V H Domain to the second V H domain or the first V L The second V LThe term "extension sequence" refers to a region that connects the domains. The extension sequence can connect the domains via the C-terminus of each domain. In some embodiments, the extension sequence connects the domains by a covalent bond. In some embodiments, the extension sequence will contain one or more cysteines, allowing one or more disulfide bonds to form between two such extension sequences. Examples of extension sequences applied to the C-terminus of a cys-diabody are disclosed in WO2018147960A1. An example of a pair of extension sequences is shown as a line with two cysteines connecting either two heavy chain domains or two light chain domains. The extension sequence will be toward the C-terminus of the construct, but need not be the very last amino acid of the variable domain. That is, the linker may be positioned slightly N-terminal to the C-terminus. For example, the extension sequence may be located within 10 amino acids of the C-terminus. Similarly, additional sequences may be located between the natural C-terminus and where the extension sequence begins. The extension sequence may be linked to the V domain via a disulfide bond. H and V H To or V L and V L and can be connected.

[0099] A cys-diabody refers to a modified protein with an added C-terminal cys sequence that can result in a construct that is a disulfide-bonded dimer. In some embodiments, the cys-diabody is monospecific. In some embodiments, the cys-diabody is bispecific.

[0100] As will be appreciated by those skilled in the art, although the disclosure generally refers to antigen-binding constructs, certain cys-diabody embodiment configurations can be used in various embodiments to obtain certain advantages. In some embodiments, the cysteines are cross-linked to one another. In some embodiments, the cysteines are reduced, so that such tail-forming cysteines do not form disulfide bonds with one another. In some embodiments, one or more of the "tail-forming" cysteines form covalent bonds with one or more detectable markers, such as fluorescent probes. In some embodiments, one or more of the "tail-forming" cysteines may be covalently conjugated to half-life extending moieties, such as polyethylene glycol (PEG) of different molecular weights. In some embodiments, any covalently modifiable moiety can be used in place of one or more of the cysteines. For example, this can include GlySer linkers, GlyLeu linkers, and / or inserted cysteines after a short tag. In some embodiments, the connection can be established via a coiled coil or leucine zipper. In some embodiments, the "tail" itself may contain functional groups at its termini so that it can be selectively attached to desired residues and / or locations at each end of the polypeptide, instead of the disulfide bond itself. In some embodiments, rather than a tail providing space between the two polypeptide chains, a covalently modifiable moiety may be attached directly to the end of a heavy or light chain polypeptide, although the two covalently modifiable moieties may be connected by a linker. In such embodiments, the construct may still contain cysteines in the tail, but simply not be cross-linked. In other embodiments, the construct need not have cysteines in the tail, or need not have a tail at all.

[0101] The phrases "specifically bind" or "selectively bind", when used in the context of describing the interaction between an antigen, e.g., a protein, and an antibody or antibody-derived binding agent, refer to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biological products, e.g., in a biological sample, e.g., blood, serum, plasma, or tissue sample. Thus, under specified immunoassay conditions, in some embodiments, an antibody or binding agent with a particular binding specificity binds to a particular antigen at least twice background and does not bind substantially in significant amounts to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or agent be selected for its specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal that is at least twice background, more typically at least 10-100 times background.

[0102] The term "equilibrium dissociation constant (KD, M)" refers to the dissociation rate constant (kd, time -1 ) as the binding rate constant (ka, time -1 , M -1 The equilibrium dissociation constant can be measured using any method known in the art. The antibodies provided herein have an equilibrium dissociation constant of about 10 -7 or 10 -8 Less than m, e.g., about 10 -9 M or 10 -10 In some embodiments, less than about 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M or 10-15 The KD can have an equilibrium dissociation constant less than M. In some embodiments, the KD is determined with the antibody dissolved in a buffer. In some embodiments, the buffer is a phosphate buffer.

[0103] "PET" is a diagnostic technique that can be used to observe the function and metabolism of human organs and tissues at the molecular level. In PET, positron-emitting drugs (e.g., 18 F-FDG) may be injected into the human body. When FDG is used, the metabolism of fludoxyglucose (FDG) is similar to that of glucose, so FDG will accumulate in cells that digest glucose. 18 The positron released by the decay of F and the electron in the tissue undergo an annihilation reaction, generating two gamma photons of the same energy in opposite directions. A detector array surrounding the human body can detect the two photons using coincidence techniques to determine the positional information of the positron. This positional information can then be processed using image reconstruction software to construct a cross-sectional image of the positrons within the human body. In some circumstances, immunoPET can be used, in which the antigen-binding construct contains a label (e.g., 18 F) is attached or associated therewith. In such embodiments, the distribution of the antigen-binding construct can be monitored, which will depend on the binding and distribution characteristics of the antigen-binding construct. For example, when a CD8-directed minibody is used, PET can be used to monitor the distribution of CD8 molecules in the host system. PET systems are known in the art, for example, U.S. Patent Publication Nos. 20170357015, 20170153337, 20150196266, 20150087974, 20120318988, and 20090159804. The contents of each of these documents are incorporated herein by reference for their description of PET and its uses.

[0104] In some embodiments, imaging is performed by PET scan, PET / CT scan, or SPECT scan. In some embodiments, imaging is performed by photoacoustics, optical probes, MR imaging, magnetic nanoparticles for imaging, spectroscopy, and / or any other standard imaging method. In some embodiments, at least one optical probe is coupled together with at least one metal chelator. In some embodiments, optical imaging is used for surgical assistance. In some embodiments, photodynamic therapy is used. In some embodiments, photodynamic therapy is used for surgical assistance. In some embodiments, photodynamic therapy is used for theranostics.

[0105] In some embodiments, the diagnosis is performed in vivo. In some embodiments, the diagnosis comprises imaging using the antigen-binding constructs and / or formulations described herein. In some embodiments, the imaging is used for one or more of cancer diagnosis, determining subject stratification for therapy, monitoring subject response to therapy, assisting in surgery, and / or informing therapeutic decision-making.

[0106] In some embodiments, the antigen-binding constructs and / or compositions are used as part of a theranostic approach. The term "theranostics" as used herein has its ordinary meaning as understood by those skilled in the art, and thus refers to a combination of therapy and diagnosis. In some embodiments, theranostics involves a combination of first identifying a disease using a first compound (diagnosis) and then delivering a second compound to treat the disease (therapy). In some embodiments, the second compound is the same as the first compound. In some embodiments, the first and second compounds are derivatives of each other. In some embodiments, the first and second compounds are different from each other. In some embodiments, the first compound is conjugated to a detectable marker, such as a fluorescent marker, a chemically reactive marker, a luminescent marker, or a radioactive marker. In some embodiments, the detectable marker is monitored by imaging. In some embodiments, the second compound is conjugated to a medicamentously effective therapeutic small molecule or payload. In some embodiments, the second compound is conjugated to a toxic radiolabel, a cytotoxic agent, or other molecule capable of producing deleterious effects in a target cell, tissue, organ, or organ system. In some embodiments, the adverse effect is one or more of cell cycle arrest, apoptosis, proliferation arrest, stress induction, cytotoxicity, DNA repair inhibition, necrosis, oxidative stress, nitrosative stress, free radical stress, enhanced targeting by the subject's immune system, protein degradation, enhanced protein turnover, inhibition of protein turnover, metabolic arrest, organelle arrest, transcription arrest, DNA replication arrest, translation arrest, or any combination thereof.

[0107] Some embodiments described herein relate to pharmaceutical compositions or nutritional supplements comprising, consisting essentially of, or consisting of an effective amount of any one or more of the therapeutic agents described herein. Such pharmaceutical compositions and nutritional supplements are suitable for human and / or veterinary applications.

[0108] The terms "individual," "subject," "host," or "patient," as used herein, have their ordinary meaning as understood by one of ordinary skill in the art, and thus include a human or non-human mammal. The term "mammal" is used in its ordinary biological sense. Thus, the term specifically includes, but is not limited to, a primate, including a monkey (chimpanzee, ape, monkey), a human, a cow, a horse, a sheep, a goat, a pig, a rabbit, a dog, a cat, a rodent, a rat, a mouse, or a guinea pig.

[0109] As used herein, "treatment" or "therapy" of a disease or condition refers to reducing the severity, frequency, or occurrence of at least one symptom of the disease or condition compared to a similar but untreated patient. Treatment can also refer to halting, slowing, or reversing the progression of a disease or condition compared to a similar but untreated patient. Treatment may further include addressing the underlying cause of the disease and / or one or more symptoms. The term "prevent" does not require that the disorder or disease be completely halted.

[0110] The term "effective amount" or "effective dose" as used herein has its ordinary meaning as understood by those skilled in the art and refers to the amount of a described composition or compound that produces an observable biological effect. The actual dosage level of the active ingredients in the active compositions of the presently disclosed subject matter may vary to administer an effective amount of the active composition or compound to achieve the desired response for a particular subject and / or application. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the composition, the formulation, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and previous medical history of the subject being treated. In some embodiments, a minimum dose is administered, and the dose is increased to the minimum effective amount in the absence of dose-limiting toxicity. Determination and adjustment of the effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0111] A "therapeutically effective amount" or "therapeutically effective dose" is an amount that produces a desired therapeutic effect in a subject, such as prevention, treatment, delay of onset of a target condition, disorder and / or symptom, and / or alleviation of symptoms associated with the condition. This amount will vary depending on a variety of factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of drug), the nature of one or more pharmacologic carriers in the formulation, and / or the route of administration. Those skilled in the clinical and pharmacological arts, given the present disclosure, will be able to determine a therapeutically effective amount by routine experimentation, for example, by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st ed., Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0112] The term "biopharmaceutical" refers to biological products such as proteins (including fusion proteins), vaccines, blood and blood components, allergens, somatic cells, gene therapy components, tissues, and recombinant therapeutic proteins. Biologics may contain carbohydrates, proteins, or nucleic acids, or complex combinations thereof, and may be living entities such as cells and tissues. Biologics can be isolated from a variety of natural sources, such as humans, animals, or microorganisms, and can be produced by a variety of methods, including the use of recombinant DNA.

[0113] As used herein, "pharmacologically acceptable" has its plain and ordinary meaning as understood in the context of the present specification and refers to a carrier, excipient, and / or stabilizer that is non-toxic or has an acceptable level of toxicity to cells or mammals exposed thereto at the dosages and concentrations used. "Pharmaceutically acceptable", "diluent", "excipient", and / or "carrier" as used herein has its plain and ordinary meaning as understood in the context of the present specification and is intended to include any and all solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic or absorption delaying agents that are compatible with administration to a human, feline, canine, or other vertebrate host. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are those that are approved by federal, state, or other regulatory agencies for use in animals, including humans, and non-human mammals, such as cats and dogs, or are listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias. The terms diluent, excipient, and / or "carrier" can refer to a diluent, adjuvant, excipient, or vehicle administered with a pharmaceutical composition. Such pharmaceutical diluents, excipients, and / or carriers that can be incorporated into any one or more of the compositions described herein include sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline solution, or aqueous dextrose and glycerol solutions can be used as liquid diluents, excipients, and / or carriers. Suitable pharmaceutical diluents and / or excipients that can be incorporated into any one or more of the compositions described herein also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, or ethanol.The physiologically acceptable carrier may also include one or more of the following: antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, carbohydrates such as amino acids, glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol and sorbitol, salt-forming counterions such as sodium, non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), PLURONICS®, or preservatives such as essential oils, methylparaben, propylparaben, or sodium salts of parabens. Preferably, the preservative is bronidiol. The composition may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, if necessary. Such compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. The formulation should be suitable for the method of administration.

[0114] Additional excipients having desirable properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be present in residual amounts or may be contaminant substances from the manufacturing process, including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth media components, or any combination thereof.The amount of excipient may be 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, at least about 0.1%, at least about 0.2%, at least about 0.4%, at least about 0.6%, at least about 0.8%, at least about 0.9 ... , 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 1 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100w / w%, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0. 5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or less, or about 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8 %, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% weight / weight or less, or any weight percentage in a range defined by any two of the aforementioned numeric values.

[0115] As used herein, "carrier" has its plain and ordinary meaning as understood in light of this specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or uptake of a compound into cells, tissues, and / or body organs.

[0116] As used herein, "diluent" has its plain and ordinary meaning as understood in light of the present specification, and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity, but may be pharmacologic necessary or desirable. For example, a diluent can be used to increase the bulk of a potent drug that is too small in mass to manufacture and / or administer. A diluent may also be a liquid for dissolving a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate buffered saline, which mimics the composition of human blood.

[0117] Administered "in combination," as used herein, means delivering two (or more) different compositions to a subject during the course of the subject's illness, e.g., delivering two or more compositions after the subject has been diagnosed or selected as having a disorder, but before the disorder is cured or eliminated. In some embodiments, the subject is selected to receive any one or more of the compositions described herein by diagnostic analysis or clinical evaluation, or both. For example, in some embodiments, the subject is screened to determine whether the subject lacks one or more beneficial bacteria or has a reduced amount of the one or more beneficial bacteria before the subject receives any one or more of the compositions described herein. In some embodiments, there is an overlap because the delivery of one therapy is still occurring when the delivery of the second therapy begins. This may be referred to herein as "concurrent" or "simultaneous" or "co-delivery." In other embodiments, the delivery of one therapy ends before the delivery of the other therapy begins. This may be referred to herein as "sequential" or "sequential delivery." In either case, the embodiments, the therapies are more effective due to the combined administration. For example, the second therapy is more effective, e.g., a lower amount of the second therapy is seen to have the same effect, or the second therapy reduces symptoms to a greater extent than would be seen if the second therapy were administered in the absence of the first therapy, or a similar situation would be seen with the first therapy. In some embodiments, the delivery is such that the reduction in symptoms or other parameters of the disorder is greater than would be observed if one therapy were delivered in the absence of the other. The effects of the two therapies may be partially additive, fully additive, or greater than additive (e.g., synergistic). The delivery may be such that the effect of the first therapy delivered is still detectable when the second therapy is delivered.

[0118] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as referred to herein. The term "neoplasm" encompasses the term tumor.

[0119] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, bone cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma, brain and head and neck cancer, and associated metastases. The term cancer includes adult and pediatric solid cancers. In some embodiments, the cancer may be a solid tumor. In some embodiments, the cancer is a highly fibrotic tumor or cancer. In some embodiments, the cancer is desmoplastic.

[0120] Non-limiting examples of novel antigen-binding construct sequences and their properties FAP has a molecular weight of 170 kDa and is composed of two 97 kDa glycoprotein subunits (Figure 2 and Figure 55). A non-limiting example of the FAP sequence is shown in Figures 53-54. Based on their amino acid homology, FAP is most similar to DPP4 (Figure 55). The main difference between these two enzymes is that FAP has Ala657, while DPP4 contains Asp663 in their respective active sites.

[0121] Expression of FAP varies among organisms and tissue types. FAP appears to be conserved among chordates, and shows high homology, especially in mammals. FAP is not normally present in normal adult tissues. A soluble, enzymatically active FAP, antiplasmin cleaving enzyme (APCE), circulates in human plasma. FAP is expressed in reactive stromal fibroblasts in tumor tissues and wound healing, and in synovial cells in rheumatoid arthritis. FAP expression is weak in cervical and uterine stroma, but expression reaches its highest level during the proliferation phase. FAP is also present in multipotent bone marrow stromal cells (BM-MSCs) in both mice and humans. FAP has also been detected in human placenta and, in some cases, in perifollicular dermal fibroblasts. Furthermore, FAP expression can be altered under various pathological conditions, including liver cirrhosis, rheumatoid arthritis, tissue remodeling, tumor formation, and tumor-associated stromal cells.

[0122] Disclosed herein are novel antigen-binding constructs targeting FAP. Non-limiting examples of amino acid sequences of novel antigen-binding constructs are shown in Figures 6-39, 62-84. Non-limiting examples of nucleic acid sequences of novel antigen-binding constructs are shown in Figure 40. Non-limiting examples of CDR, VH, and VL sequences are shown in Figures 3-4, 60-61. It will be understood that any of the CDR, VH, and VL regions can be used alone or in any combination. In some alternatives, the constructs are expressed in a compatible cell with or without additional expression sequences such as constitutive promoters, inducible promoters, detectable markers, splicing factors, resistance genes, and / or cleavable sequences (Figure 5). It will be understood that any cell, cell line, tissue, organ, or organ system capable of expressing the antigen-binding construct can be used, preferably mammalian cells or cell lines, most preferably human or rodent cells or cell lines.

[0123] As disclosed herein, the novel antigen-binding constructs are derived from parent constructs. In some alternatives, the novel antigen-binding constructs are minibodies. In some alternatives, the novel antigen-binding constructs are cys-diabodies. Non-limiting examples of the novel antigen-binding constructs referenced herein, their parent antibody constructs, and their corresponding SEQ ID NOs are shown in Figure 41. Examples of alignments of the novel antigen-binding constructs with the parent antigen-binding constructs are shown in Figures 47-48. Examples of alignments of the novel antigen-binding constructs with known antibody sequences are shown in Figures 42-46 and 85-90.

[0124] In some alternatives, the novel antigen-binding constructs comprise at least one VH domain, VL domain, hinge domain, linker domain, or any combination thereof (Figures 47-51). In some alternatives, the novel antigen-binding constructs further comprise at least one signal peptide, CH3 domain, or any combination thereof. Non-limiting examples of hinge sequences are shown in Figures 52A-52F. In some alternatives, the hinge sequence comprises one or more of an upper hinge region, a core hinge region, and / or a lower hinge region. It will be understood that any of the hinge sequences of the present disclosure can be used in combination with any of the CDR sequences, VH sequences, and / or VL sequences provided herein. Furthermore, any of the CDR sequences, VH sequences, and / or VL sequences provided herein can be used in any of the constructs / methods disclosed herein with or without any of the hinge sequences provided herein.

[0125] As disclosed herein, the antigen-binding construct is designed with a specific sequence. In some embodiments, the isolated antigen-binding construct comprises an HCDR1 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:1 (EYTIH), an HCDR2 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:2 (GINPNNGIPNYNQKFKG), an HCDR3 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:3 (RRIAYGYDEGHAMDY), an LCDR1 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:4 (KSSQSLLYSRNQKNYLA), an LCDR2 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:5 (WASTRES), and an LCDR3 comprising amino acids having the amino acid sequence of SEQ ID NO:6 (QQYYSYPLT). In some embodiments, the isolated antigen-binding construct comprises a heavy chain having at least 80% identity with the amino acid sequence of SEQ ID NO:7. In some embodiments, the isolated antigen-binding construct comprises a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO:7. In some embodiments, the isolated antigen-binding construct comprises a heavy chain having at least 95% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 95% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the isolated antigen-binding construct comprises a light chain having at least 95% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the isolated antigen-binding construct comprises at least one VH framework residue selected from the group consisting of an alanine at position 24 of the sequence of SEQ ID NO:7 or a glycine at position 26 of the sequence of SEQ ID NO:7.In some embodiments, the isolated antigen-binding construct comprises at least one VL framework residue selected from the group consisting of a serine at position 73 of sequence SEQ ID NO:8, an arginine at position 83 of sequence SEQ ID NO:8, a glutamic acid at position 85 of sequence SEQ ID NO:8, a proline at position 86 of sequence SEQ ID NO:8, a phenylalanine at position 89 of sequence SEQ ID NO:8, or a tyrosine at position 98 of sequence SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises an alanine at position 24 and a glycine at position 26 of the heavy chain sequence of SEQ ID NO:7. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 and a serine at position 73 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 and a tyrosine at position 98 of the sequence of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the sequence of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the sequence of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the sequence of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a variable heavy domain (VH) of SEQ ID NO: 7 and a variable light domain (VL) of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a variable heavy domain (VH) of SEQ ID NO: 7 and a variable light domain (VL) of SEQ ID NO: 9.

[0126] Some embodiments of the present disclosure relate to an isolated antigen-binding construct specific for FAP alpha, comprising a CDR3 comprising amino acids having at least 90% identity with the amino acid sequence of SEQ ID NO:6 (QQYYSYPLT). Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a light chain CDR3 comprising amino acids having at least 100% identity with the amino acid sequence of SEQ ID NO:6. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising an LCDR1 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:4 (KSSQSLLYSRNQKNYLA), an LCDR2 comprising amino acids having at least 80% identity with the amino acid sequence of SEQ ID NO:5 (WASTRES), and an LCDR3 comprising amino acids having the amino acid sequence of SEQ ID NO:6 (QQYYSYPLT). Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a heavy chain comprising at least 99% identity with the amino acid sequence of SEQ ID NO:7. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO:7, comprising at least one VH framework residue selected from the group consisting of an alanine at position 24 of sequence SEQ ID NO:7 and a glycine at position 26 of sequence SEQ ID NO:7. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:8, comprising at least one VL framework residue selected from the group consisting of a serine at position 73 of sequence SEQ ID NO:8, an arginine at position 83 of sequence SEQ ID NO:8, a glutamic acid at position 85 of sequence SEQ ID NO:8, a proline at position 86 of sequence SEQ ID NO:8, a phenylalanine at position 89 of sequence SEQ ID NO:8, and a tyrosine at position 98 of sequence SEQ ID NO:8. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a light chain comprising an amino acid at least 95% identity to the amino acid sequence of SEQ ID NO:9. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising a light chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:8.Some aspects of the present disclosure relate to an isolated antigen-binding construct comprising a variable heavy domain (VH) comprising amino acids having at least 95% identity to the amino acid sequence of SEQ ID NO: 7, and a variable light domain (VL) comprising amino acids having at least 95% identity to the amino acid sequence of SEQ ID NO: 8. Some aspects of the present disclosure relate to an isolated humanized antigen-binding construct comprising a variable heavy domain (VH) comprising amino acids having at least 95% identity to the amino acid sequence of SEQ ID NO: 7, and a variable light domain (VL) comprising amino acids having at least 89% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the isolated antigen-binding construct comprises at least one VH framework residue selected from the group consisting of an alanine at position 24 of the sequence of SEQ ID NO: 7 and a glycine at position 26 of the sequence of SEQ ID NO: 7. In some embodiments, the isolated antigen-binding construct comprises at least one VL framework residue selected from the group consisting of a serine at position 73 of sequence SEQ ID NO:8, an arginine at position 83 of sequence SEQ ID NO:8, a glutamic acid at position 85 of sequence SEQ ID NO:8, a proline at position 86 of sequence SEQ ID NO:8, a phenylalanine at position 89 of sequence SEQ ID NO:8, and a tyrosine at position 98 of sequence SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 and a serine at position 73 of the light chain sequence of SEQ ID NO:4. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89 and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO: 8.In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO: 8. In some embodiments, the isolated antigen-binding construct comprises a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO: 8.

[0127] Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising amino acids having at least 80% identity to an amino acid sequence selected from the group of SEQ ID NOs: 23 to 44. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising amino acids having at least 90% identity to an amino acid sequence selected from the group of SEQ ID NOs: 23 to 44. Some embodiments of the present disclosure relate to an isolated antigen-binding construct comprising amino acids having at least 100% identity to an amino acid sequence selected from the group of SEQ ID NOs: 23 to 44, 12 to 43, 12 to 45, 87 to 96, 110 to 115, 122 to 125, or 140 to 143.

[0128] As disclosed herein, antigen-binding constructs are designed with specific sequences. In some embodiments, such constructs bind to FAP. In some embodiments, antigen-binding constructs include: (1) HCDR1 comprising the amino acid sequence of SEQ ID NO: 81 (NYDIN), or a sequence having 3 or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 81; (2) HCDR2 comprising the amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN), or a sequence having 3 or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 82; (3) HCDR3 comprising the amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY), or a sequence having 3 or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 83; (4) the sequence (5) LCDR1 comprising the amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 84; (6) LCDR3 comprising the amino acid sequence of SEQ ID NO: 86 (QQHYSNPRT), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 86; (7) or any combination thereof.

[0129] In any of the embodiments provided herein, the % similarity or % identity may be 80, 85, 90, 95, 96, 97, 98, 99, or 100% to any one or more of the sequences described (e.g., one, two, three, four, five, six of the CDRs and / or VH and / or VL provided herein). In some embodiments, any one or more of the protein segments provided herein (e.g., CDRs, VH, and / or VL) may contain one, two, or three substitutions, which may, but need not be, conservative substitutions.

[0130] In some embodiments, the antigen-binding construct comprises one or more of: (1) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 81 (NYDIN), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 81; (2) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 82; (3) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 83; (4) or any combination thereof. Also disclosed herein are antigen-binding constructs comprising one or more of: (1) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 884; (2) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 (SASYRYT), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 85; (3) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 86 (QQHYSNPRT), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 86; (4) or any combination thereof.

[0131] In some embodiments, an isolated antigen-binding construct comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 81 (NYDIN), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 81, is specific for a FAP. In some embodiments, the CDR1 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP comprising a CDR2 comprising an amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 82. In some embodiments, the CDR2 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 83. In some embodiments, the CDR3 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for FAP, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 84. In some embodiments, the CDR1 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for FAP, comprising a CDR2 comprising an amino acid sequence of SEQ ID NO: 85 (SASYRYT), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 85. In some embodiments, the CDR2 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 86 (QQHYSNPRT), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 86. In some embodiments, the CDR3 is part of a light chain.

[0132] In some embodiments, the antigen-binding construct is specific for a FAP. In some embodiments, the antigen-binding construct is specific for a FAP alpha. In some embodiments, the antigen-binding construct does not bind to DPP4.

[0133] In some embodiments, the antigen-binding construct comprises at least one CDR region having less than four point mutations, less than three point mutations, less than two point mutations, or less than one point mutation from any one of SEQ ID NOs: 81-86.

[0134] In some embodiments, the antigen-binding construct comprises at least one CDR region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% similarity to any one of SEQ ID NOs: 81-86. In some embodiments, the antigen-binding construct comprises one CDR region having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 1-6. In some embodiments, the antigen-binding construct comprises two CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 81-86. In some embodiments, the antigen-binding construct comprises three CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 81-86. In some embodiments, the antigen-binding construct comprises four CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 81-86. In some embodiments, the antigen-binding construct comprises five CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 81-86. In some embodiments, the antigen-binding construct comprises six CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 81-86. In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 88.In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:87, and a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:88.

[0135] In some embodiments, the antigen-binding construct has any of the CDRs provided herein, and one or more of the FRs provided in any of the VH and / or VL sequences provided herein, including any one of FR1, FR2, FR3, and / or FR4, or any sequence at least 60, 70, 80, 90, or 95, 96, 97, 98, or 99% identical or similar thereto.

[0136] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:90. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:89, and a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:90.

[0137] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:91, and a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:92.

[0138] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:93, and a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:94.

[0139] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:95, and a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:96.

[0140] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 123. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 122, and a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 123.

[0141] Also disclosed herein is an antigen-binding construct comprising a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:87, and a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:88. Also disclosed herein is an antigen-binding construct comprising a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:89, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:90. Also disclosed herein is an antigen-binding construct comprising a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:91, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:92. Also disclosed herein is an antigen-binding construct comprising a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:93, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:94.Also disclosed herein is an antigen-binding construct comprising a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:95, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:96. Also disclosed herein is an antigen-binding construct comprising a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:122, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:123.

[0142] In some embodiments, the antigen-binding construct comprises a variable light domain and a variable heavy domain, and the order of the variable regions is from the N-terminus to the C-terminus of the polypeptide: V L , V H In some embodiments, the antigen-binding construct comprises a variable light domain and a variable heavy domain, and the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, V H , V L It is.

[0143] In some embodiments, the antigen-binding construct comprises any of the sequences provided in any one or more of the figures provided herein, including those shown in Figures 52A-52F for the hinge region, and Figures 60-61 and 62-90 for any CDR, VH, and / or VL sequences provided herein, including variants thereof having at least 60, 70, 80, 90, 95, 96, 97, 98, 99, or more percent identity thereto or percent similarity thereto.

[0144] Also disclosed herein is an isolated antigen-binding construct comprising amino acids having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, and / or 100% identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 97-103 and 126.

[0145] Also provided herein is an HCDR1 comprising: (1) the amino acid sequence of SEQ ID NO: 104 (SYVMH), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 104; (2) the amino acid sequence of SEQ ID NO: 105 (YINPHNDGTK), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 105; (3) the amino acid sequence of SEQ ID NO: 106 (ARWGIYYGYGAWFAY), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 106; (4) the amino acid sequence of SEQ ID NO: 107 (KASQNVG (5) LCDR1 comprising an amino acid sequence of SEQ ID NO: 108 (YSASNRYS) or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 108; (6) LCDR3 comprising an amino acid sequence of SEQ ID NO: 109 (QQYNTYPYT) or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 109; (7) or any combination thereof. Also disclosed herein are antigen-binding constructs comprising one or more of: (1) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 104 (SYVMH), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 104; (2) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 105 (YINPHNDGTK), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 105; (3) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 106 (ARWGIYYGYGAWFAY), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 106; (4) or any combination thereof.Also disclosed herein are antigen-binding constructs comprising one or more of: (1) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 107 (KASQNVGTYVA), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 107; (2) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 108 (YSASNRYS), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 108; (3) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 109 (QQYNTYPYT), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 109; or (4) any combination thereof.

[0146] Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 104 (SYVMH), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 104. In some embodiments, the CDR1 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR2 comprising an amino acid sequence of SEQ ID NO: 105 (YINPHNDGTK), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 105. In some embodiments, the CDR2 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 106 (ARWGIYYGYGAWFAY), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 106. In some embodiments, the CDR3 is part of a heavy chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 107 (KASQNVGTYVA), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 107. In some embodiments, the CDR1 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR2 comprising an amino acid sequence of SEQ ID NO: 108 (YSASNRYS), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 108. In some embodiments, the CDR2 is part of a light chain. Also disclosed herein is an isolated antigen-binding construct specific for a FAP, comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 109 (QQYNTYPYT), a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 109. In some embodiments, the CDR3 is part of a light chain.

[0147] In some embodiments, the antigen-binding construct is specific for a FAP. In some embodiments, the antigen-binding construct is specific for a FAP alpha. In some embodiments, the antigen-binding construct does not bind to DPP4. In some embodiments, the antigen-binding construct comprises at least one CDR region having less than four point mutations, less than three point mutations, less than two point mutations, or less than one point mutation from any one of SEQ ID NOs: 104-109. In some embodiments, the antigen-binding construct comprises at least one CDR region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% similarity to any one of SEQ ID NOs: 104-109. In some embodiments, the antigen-binding construct comprises one CDR region having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 104-109. In some embodiments, the antigen-binding construct comprises two CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 104-109. In some embodiments, the antigen-binding construct comprises three CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 104-109. In some embodiments, the antigen-binding construct comprises four CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 104-109. In some embodiments, the antigen-binding construct comprises five CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 104-109. In some embodiments, the antigen-binding construct comprises six CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 104-109.

[0148] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:111. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:110, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:111.

[0149] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:112, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:113.

[0150] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:95, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:96.

[0151] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:91, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:92.

[0152] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:114, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:115.

[0153] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:95, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:96.

[0154] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 125. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 124, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 125.

[0155] In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:110, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:111. Also disclosed herein is an antigen-binding construct comprising a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:112, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:113.

[0156] In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:91, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:92.

[0157] In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:95, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:96.

[0158] In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:114, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:115.

[0159] In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 124, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 125.

[0160] In some embodiments, the antigen-binding construct comprises a variable light domain and a variable heavy domain, and the order of the variable regions is from the N-terminus to the C-terminus of the polypeptide: V L , V H In some embodiments, the antigen-binding construct comprises a variable light domain and a variable heavy domain, and the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, V H , V L Also disclosed herein is an isolated antigen-binding construct comprising an amino acid sequence having at least 98% identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 116-120 and 127-133.

[0161] In some embodiments, the antigen-binding construct comprises: (1) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 134 (SYTMS), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 134; (2) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 135 (TISSGGSYTY), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 135; (3) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 136 (TRDQVGYAMDY), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 136; (4) an HCDR4 comprising the amino acid sequence of SEQ ID NO: 137 (TRDQVGYAMDY), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 137; (5) LCDR1 comprising the amino acid sequence of SEQ ID NO: 137 (TRDQVGYAMDY), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 137; (6) LCDR3 comprising the amino acid sequence of SEQ ID NO: 139 (QQYSRYPYT), or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 139; (7) or any combination thereof.

[0162] In some embodiments, the antigen-binding construct comprises one or more of: (1) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 134, or a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 134; (2) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 135, or a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 135; (3) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 136, or a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 136; (4) or any combination thereof.

[0163] In some embodiments, the antigen-binding construct comprises one or more of: (1) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 137; (2) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 138, or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 138; (3) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 139, or a sequence having three or less point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 139; or (4) any combination thereof.

[0164] In some embodiments, the isolated antigen-binding construct is specific for a FAP and comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 134, a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 134. In some embodiments, the CDR1 is part of a heavy chain. In some embodiments, the isolated antigen-binding construct is specific for a FAP and comprises a CDR2 comprising the amino acid sequence of SEQ ID NO: 135, a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 135. In some embodiments, the CDR2 is part of a heavy chain.

[0165] In some embodiments, an isolated antigen-binding construct specific for a FAP comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 136, a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 136. In some embodiments, the CDR3 is part of a heavy chain. In some embodiments, an isolated antigen-binding construct specific for a FAP comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 137, a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 137. In some embodiments, the CDR1 is part of a light chain. In some embodiments, an isolated antigen-binding construct specific for a FAP comprises a CDR2 comprising the amino acid sequence of SEQ ID NO: 138, a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 138. In some embodiments, the CDR2 is part of a light chain. In some embodiments, the isolated antigen-binding construct specific for FAP comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 139, a sequence having no more than three point mutations thereto, and / or a sequence having at least 80% similarity to SEQ ID NO: 139. In some embodiments, the CDR3 is part of a light chain.

[0166] In some embodiments, the antigen-binding construct is specific for a FAP. In some embodiments, the antigen-binding construct is specific for a FAP alpha. In some embodiments, the antigen-binding construct does not bind to DPP4. In some embodiments, the antigen-binding construct comprises at least one CDR region having less than four point mutations, less than three point mutations, less than two point mutations, or less than one point mutation from any one of SEQ ID NOs: 134-139. In some embodiments, the antigen-binding construct comprises at least one CDR region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% similarity to any one of SEQ ID NOs: 134-139. In some embodiments, the antigen-binding construct comprises one CDR region having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 134-139. In some embodiments, the antigen-binding construct comprises two CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 134-139. In some embodiments, the antigen-binding construct comprises three CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 134-139. In some embodiments, the antigen-binding construct comprises four CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 134-139. In some embodiments, the antigen-binding construct comprises five CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 134-139. In some embodiments, the antigen-binding construct comprises six CDR regions, each having up to three point mutations and / or at least about 80% similarity to any one of SEQ ID NOs: 134-139.

[0167] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 141. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 140, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 141.

[0168] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 142. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:90. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 142, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:90.

[0169] In some embodiments, the antigen-binding construct comprises a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the antigen-binding construct comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:94. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 143, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:94.

[0170] In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 140, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 141. In some embodiments, the antigen-binding construct comprises a variable heavy domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 142, and / or a variable light domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:90. In some embodiments, the antigen-binding construct comprises a variable heavy chain domain (VH) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO: 143, and / or a variable light chain domain (VL) having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and / or at least 100% identity to the amino acid sequence of SEQ ID NO:94.

[0171] In some embodiments, the antigen-binding construct comprises a variable light domain and a variable heavy domain, and the order of the variable regions is from the N-terminus to the C-terminus of the polypeptide: V L , V H In some embodiments, the antigen-binding construct comprises a variable light domain and a variable heavy domain, and the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, V H , V LIn some embodiments, the antigen-binding construct comprises amino acids having at least 98% identity to at least one amino acid sequence selected from the group of amino acid sequences of SEQ ID NOs: 144-146.

[0172] In some embodiments, the isolated antigen-binding construct of any one of the above embodiments is an antibody. In some embodiments, the isolated antigen-binding construct is a minibody. In some embodiments, the isolated antigen-binding construct is a cys-diabody.

[0173] Some embodiments of the present disclosure relate to minibody antigen-binding constructs that include amino acids having at least about 80%, at least about 90%, or at least about 99% identity to an amino acid sequence selected from the group of SEQ ID NOs: 11-22. Some embodiments of the present disclosure relate to cys-diabody antigen-binding constructs that include amino acids having at least about 80%, at least about 90%, or at least about 99% identity to an amino acid sequence selected from the group of SEQ ID NOs: 11-22.

[0174] As disclosed herein, the antigen-binding construct can be isolated using any conventional method known to one of skill in the art. The term "isolated antigen-binding construct" can refer to a purified construct, a construct in solution, a construct expressed on the surface of a cell, a construct expressed inside a cell, a construct expressed on the surface of a tissue, a construct expressed inside a tissue, a construct expressed in a system, and / or a construct expressed in an organism. In some embodiments, the antigen-binding construct is mammalian in origin. In some embodiments, the antigen-binding construct is murine in origin. In some embodiments, the antigen-binding construct is human in origin. In some embodiments, the antigen-binding construct is humanized. In some embodiments, the antigen-binding construct is expressed in a mammalian cell, a mammalian cell line, a mammalian tissue, a mammalian organ, a mammalian organ system, and / or a mammalian organism. In some embodiments, the antigen-binding construct is expressed in a mouse cell, a mouse cell line, a mouse tissue, a mouse organ, a mouse organ system, and / or a mouse organism. In some embodiments, the antigen-binding construct is expressed in a human cell, a human cell line, a human tissue, a human organ, a human organ system, and / or a human organism.

[0175] In some embodiments, the antigen-binding construct is an antibody. In some embodiments, the antigen-binding construct is an antibody fragment. In some embodiments, the antigen-binding construct is an scFv. In some embodiments, the antigen-binding construct is a Fab. In some embodiments, the antigen-binding construct is a Fab2. In some embodiments, the antigen-binding construct is a nanobody. In some embodiments, the antigen-binding construct is a minibody. In some embodiments, the antigen-binding construct is a diabody. In some embodiments, the antigen-binding construct is a cys-diabody. In some embodiments, the antigen-binding construct is a combination of any two or more of the above as a composition and / or bispecific configuration.

[0176] In some embodiments, a minibody antigen-binding construct comprises amino acids having at least about 80%, at least about 90%, or at least about 99% identity to at least one of the amino acid sequences selected from the group of SEQ ID NOs: 97-103, 116-120, 126-133, and 144-146. In some embodiments, a cys-diabody antigen-binding construct comprises amino acids having at least about 80%, at least about 90%, or at least about 99% identity to at least one of the amino acid sequences selected from the group of SEQ ID NOs: 97-103, 116-120, 126-133, and 144-146.

[0177] In some embodiments, the isolated antigen-binding construct is specific for a human FAP. In some embodiments, the antigen-binding construct is specific for FAP alpha. In some embodiments, the isolated antigen-binding construct of any one of the above embodiments is specific for FAP alpha (Figure 54). In some embodiments, the isolated antigen-binding construct does not bind to DPP4. In some embodiments, the antigen-binding construct does not bind to DPP4. In some embodiments, the antigen-binding construct has a higher binding affinity for a FAP and / or FAP alpha than for DPP4. In some embodiments, the isolated antigen-binding construct exhibits at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold greater expression in mammalian cells than expression of sibrotuzumab minibody. In some embodiments, the isolated antigen-binding construct has a KD of less than about 3 x 10^-9 M, less than about 2.5 x 10^-9 M, less than about 2 x 10^-9 M, less than about 1.5 x 10^-9 M, less than about 1 x 10^-9 M, or less than about 0.5 x 10^-9 M. In some embodiments, the isolated antigen-binding construct has an on-rate (kD) of greater than about 7.0 (1 / Ms), greater than about 8.0 (1 / Ms), greater than about 9.0 (1 / Ms), or greater than about 10.0 (1 / Ms). on In some embodiments, the isolated antigen-binding construct has an on-rate (k) greater than 9.0 (1 / Ms). onIn some embodiments, the isolated antigen-binding construct has an off rate (k) of less than about 3 x 10^-3 (1 / s), less than about 2.5 x 10^-3 (1 / s), less than about 2 x 10^-3 (1 / s), less than about 1.5 x 10^-3 (1 / s), or less than about 1.0 x 10^-3 (1 / s). off ).

[0178] In some embodiments, the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, V L , V H In some embodiments, the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, V H , V L In some embodiments, the isolated antigen-binding construct further comprises a payload. In some embodiments, the payload is fluorescent. In some embodiments, the payload is luminescent. In some embodiments, the payload is chromogenic. In some embodiments, the payload is radioactive. In some embodiments, the payload is non-radioactive. In some embodiments, the payload is an ADC. In some embodiments, the isolated antigen-binding construct is humanized. In some embodiments, the payload is chemically reactive. In some embodiments, the payload is a detectable marker.

[0179] Some aspects of the present disclosure include the amino acid sequence of any one of embodiments 1 to 66, 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc,99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th, and 225 The present disclosure relates to compositions further comprising at least one payload, such as a label or therapeutic ion, selected from at least one of the group consisting of: Ac, ...

[0180] In some embodiments, the amino acid sequence of the antigen-binding construct further comprises at least one metal binding site. In some embodiments, the amino acid sequence of the antigen-binding construct further comprises a histidine (His) tag sequence. In some embodiments, the amino acid sequence of the antigen-binding construct further comprises 99m It further comprises a Tc-carbonyl radiolabel.

[0181] Some aspects of the present disclosure relate to a method for introducing any amino acid sequence or expression vector of an antigen-binding construct and / or an expression vector encoding same into a host cell, comprising performing electroporation, viral infection, and / or at least one chemical method. Some aspects of the present disclosure relate to a host cell comprising the amino acid sequence or expression vector of any one of the above embodiments disclosed herein.

[0182] Some aspects of the present disclosure relate to a composition comprising any one of the amino acid sequences of the above embodiments disclosed herein and at least one chelating agent. In some embodiments, the at least one chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NOGADA, NETA, deferoxamine (DFO), porphyrin, polyamine, crown ether, bis-thiosemicarbazone, polyoxime, dimercaprol, penicillamine, trientine, zinc, deferasirox, deferiprone, deferoxamine, succimer, pyrophosphate, tripolyphosphate, citric acid, tartaric acid, glycine, DMPS, DMSA, NTA, calcium, sodium, desferryloxamine, dicobalt EDTA, dimercarpol, BAL, and demercaptosuccinic acid, or any combination thereof. In some embodiments, the composition further comprises a dual chelating agent. In some embodiments, the dual chelating agent is 64 Cu / 67 In some embodiments, the dual chelator is 89 Zr / 177Lu. In some embodiments, the at least one chelator is configured to capture the isotope. In some embodiments, the composition further comprises at least one payload. In some embodiments, the at least one payload is 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227Th, and 225 Ac, or any combination thereof.

[0183] Some aspects of the present disclosure relate to the use of the compositions disclosed herein as a medicament. Some aspects of the present disclosure relate to the use of the compositions disclosed herein in the manufacture of a medicament for administration to a subject. Some aspects of the present disclosure relate to the use of the compositions disclosed herein for imaging a cell, tissue, organ, and / or subject. Some aspects of the present disclosure relate to the use of the compositions disclosed herein for identifying a disease in a subject. In some embodiments, the disease is a cancer or tumor. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is fibrosis. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is the cardiovascular system.

[0184] Some aspects of the present disclosure relate to a method for identifying a disease in a subject, the method comprising administering to the subject at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies of any one of the embodiments disclosed herein; screening the binding of the at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to a FAP; and determining whether the subject has the disease based on the presence or absence of binding to the FAP. In some embodiments, the FAP is FAP alpha. In some embodiments, the disease is a cancer or tumor. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is fibrosis. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is cardiovascular. In some embodiments, the subject is a mammal or human. In some embodiments, the subject is a mouse or a rat.

[0185] In some embodiments, at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies binds to tumor stroma. In some embodiments, at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies binds to fibroblasts. In some embodiments, at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies binds to cancer-associated fibroblasts. In some embodiments, the disease is epithelial. In some embodiments, at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies further comprises a payload. In some embodiments, the payload (which may be a marker or a label) is: 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re,188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th, and 225 In some embodiments, the payload is used to determine binding of at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP. In some embodiments, the binding of at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP is determined by PET scan. In some embodiments, the binding of at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP is determined by in vitro testing of a tissue or cell sample removed from the subject. In some embodiments, the method further comprises identifying the subject as disease negative if there is no significant binding of at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP. In some embodiments, the method further comprises identifying the subject as disease-negative if there is significant binding of at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP.

[0186] Some aspects provide pharmaceutical compositions comprising an amount of at least one antibody, antigen-binding construct thereof, minibody, and / or cys-diabody of any one of the embodiments disclosed herein effective for treating a subject having cancer and / or a tumor; and a pharma- ceutically acceptable carrier. In some embodiments, the amount of the at least one antibody, antigen-binding construct, minibody, and / or cys-diabody is about 0.01 mg / kg to about 25 mg / kg. In some embodiments, the amount of the at least one antibody, antigen-binding construct, minibody, and / or cys-diabody is about 1 mg / kg to about 20 mg / kg.

[0187] In some embodiments, the pharmaceutical composition further comprises an eluent, diluent, carrier, and / or other molecules to aid in the delivery and efficacy of the composition. In some embodiments, the pharmaceutical composition is formulated for oral delivery. In some embodiments, the pharmaceutical composition is formulated for nasal delivery. In some embodiments, the pharmaceutical composition is formulated for intravenous delivery. In some embodiments, the pharmaceutical composition is formulated for single dose delivery. In some embodiments, the pharmaceutical composition is formulated for multiple dose delivery. In some embodiments, the pharmaceutical composition further comprises at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating a disease. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating a disease is an antibody. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating a disease is used as part of a chemotherapy. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating a disease is an immuno-oncology drug. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating a disease is an immuno-oncology drug. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating the disease is a DNA repair inhibitor. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating the disease is used as part of a photodynamic therapy. In some embodiments, the at least one known small molecule, therapeutic agent, or antigen-binding construct effective in treating cancer and / or tumors is selected from the group consisting of alkylating agents, metabolic inhibitors, radiosensitizers, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, nitrosoureas, corticosteroids, antiangiogenic agents, apoptosis inducers, antimicrotubule agents, vinca alkaloids, taxanes, anthracyclines, antiandrogens, VEGF pathway inhibitors, VEGF pathway inhibitors, MAPK / Ras / Raf pathway inhibitors, and EGFR pathway inhibitors. In some embodiments, the disease is fibrosis. In some embodiments, the disease is cancer or tumor. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is an autoimmune disease.In some embodiments, the disease is cardiovascular.

[0188] Some aspects of the present disclosure relate to a method for treating, inhibiting, or ameliorating a disease in a subject, comprising administering a pharmaceutical composition of any one of the embodiments disclosed herein to a subject in need thereof. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the disease is fibrosis. In some embodiments, the disease is cancer or a tumor. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is cardiovascular. In some embodiments, the method further comprises imaging the disease using a composition of any of the embodiments disclosed herein. In some embodiments, the method further comprises imaging the disease using any of the antigen-binding constructs disclosed herein.

[0189] Some aspects of the present disclosure relate to a method for targeting a FAP protein on fibroblasts in a subject, comprising administering to the subject a pharmaceutical composition of any one of the embodiments disclosed herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the fibroblasts are cancer-associated fibroblasts. In some embodiments, the cancer is in a solid tumor. In some embodiments, following targeting of the fibroblasts, the cancer cells and / or tumor-associated macrophages are damaged or killed.

[0190] Some aspects of the present disclosure relate to a method for inhibiting, ameliorating, impairing, or inducing apoptosis in cancer or tumor associated macrophages in a subject. In some embodiments, the method comprises administering to a subject in need thereof a pharmaceutical composition of any one of the embodiments disclosed herein.

[0191] In some embodiments, the subject is a mammal or a human.

[0192] In some embodiments, the method further comprises administering radiation therapy, photodynamic therapy, and / or chemotherapy to the subject.

[0193] In some embodiments, the disease is a solid tumor. In some embodiments, the disease is an epithelial cancer. In some embodiments, the cancer or tumor is selected from the group consisting of bone cancer, osteosarcoma, breast cancer, triple-negative breast cancer, carcinoid, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, epithelial ovarian cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, glioma, glioblastoma, brain cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, medullary thyroid cancer, melanoma, non-small cell lung cancer, small cell lung cancer, osteosarcoma, oral squamous cell carcinoma, oral cancer, ovarian cancer, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, anal cancer, skin cancer, gastric cancer, testicular cancer, thyroid cancer, anaplastic thyroid cancer, and urothelial cancer.

[0194] Therapeutic Agents and Compositions In some embodiments, therapy involves administration of a composition of at least one therapeutic agent, such as any of the suitable antigen-binding constructs provided herein, optionally with other excipients.

[0195] The proper formulation of the treatment may depend on the route of administration selected. Techniques for formulating and administering the compounds described herein are known to those skilled in the art. There are multiple techniques for administering the compounds in the art, including, but not limited to, parenteral delivery, including enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, intramuscular, subcutaneous, intraarterial, intravenous, continuous infusion, intraportal, intraarticular, intradermal, peritoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injection, subcutaneous, intracranial injection, surgically created resection cavity (SCRC) injection, injection through Ommaya reservoir, injection through Rickham reservoir. Preparations suitable for parenteral administration include, for example, aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Preparations may be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type previously described.

[0196] In some embodiments, unit dosage formulations contain a daily dose or unit, a daily partial dose, or a suitable fraction thereof, of drug.However, it will be understood that the specific dose level for any particular patient will depend on various factors, including the activity of the specific compound used; the age, weight, general health, sex, and diet of the individual being treated; the time and route of administration; excretion rate; other drugs previously administered; and the severity of the specific disease being treated, as well understood by those skilled in the art.

[0197] Alternatively, the formulations can be provided in a form suitable for daily, weekly, or monthly administration, for example, an insoluble salt of the active compound can be configured to provide a preparation for intramuscular injection. The pharmaceutical formulations described herein may be administered to a patient on their own, or in the case of combination therapy, in a pharmaceutical formulation mixed with other active ingredients or suitable pharma- ceutical acceptable carriers or excipients. Techniques for formulating and administering the compounds of the present application can be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pa., 18th Edition, 1990.

[0198] Pharmaceutical preparations for parenteral administration, for example by bolus injection or continuous infusion, include aqueous solutions of the active compound in water-soluble form. In addition, suspensions of the active compound can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or other organic oils such as soybean oil, grapefruit oil, or almond oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of highly concentrated solutions. Injectable preparations can be provided in unit dosage form, for example in ampoules or multi-dose containers, with the addition of preservatives. The preparations can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In some embodiments, the formulation includes at least one agent that acts to reduce radiolysis (also known as a "radioprotectant").Non-limiting examples of radiolysis reducing agents include: acetylcholine, AET, ACE inhibitors, acteoside, alpha-tocopherol acetate, amifostine, ascorbic acid, aspirin, atorvastatin, beta-carotene, Bowman-Birk protease inhibitors, caffeic acid, captopril, carbaminoylcholine, carvacrol, celecoxib, coenzyme Q10, COX2 inhibitors / NSAIDs, curcumin, cysteine, cysteamine, cystamine, dendrodine analogs, dithiolthione, dopamine, enalapril, epigallocatechin-3-gallate, epinephrine, 17-β-estradiol, GANRA-5, genistein, green tea extract, growth factors, guanine nucleotides, halofuginone, Hmg-CoA reductase inhibitors (statins), helo In, histamine, ibuprofen, inapoyl-E-glucoside, isoflavone, isoflavin, kukoamine A, lactoferrin, amifostine, lipoic acid, lovastatin, luteolin-7-O-(2-apiosyl)-glucoside, 2-mercaptoethylguanidine, melatonin, methacholine, morphine, N-acetylcysteine, oltipraz, palifermin, phenethyl esther esters, polyphenols, pravastatin, protease inhibitors, quercetin-3-O-rhamnoside-7-O-glucoside, quercetin-3-O-rhamnoside, ramipril, resveratrol, rutin, serotonin, simvastatin, sodium ascorbate, superoxide dismutase, TGF-signaling inhibitors, tocopherols, vitamin C, vitamin E, watermelon juice, black grape juice, and thiols such as glutathione.

[0199] The dosing regimen using the compound of the present embodiment is selected according to various factors, including the type, race, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound used. A physician or veterinarian of ordinary skill can easily determine and prescribe the effective amount of drug required to prevent, counter, or stop the progression of the condition. Achieving with optimal precision the drug concentration within the range that provides efficacy without toxicity requires a regimen based on the kinetics of the availability of the pharmaceutical formulation to the target site. This includes considering the distribution, equilibration, and elimination of the compound. Advantageously, the compound of the present embodiment may be administered, for example, in a single daily dose, or the total daily dosage may be administered in divided doses, two, three, or four times a day.

[0200] In some embodiments, the pharmaceutical formulations detailed herein are typically administered in accordance with conventional pharmaceutical practice. Furthermore, suitable pharma- ceutically acceptable carriers can also be incorporated into the mixture, if desired or necessary.

[0201] The dosage of the product may vary over a wide range. An effective amount of the compound is usually provided at a dosage level of about 0.01 mg / kg body weight to about 25 mg / kg body weight per dose. In some embodiments, the dosage is administered daily. In some embodiments, the dosage is administered in a single dosage. In some embodiments, the dosage is administered as divided dosages repeated at intervals of about once every 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, or about 10 weeks.

[0202] The embodiments of the present disclosure provided herein are illustrated by the following example numbering scheme. 1. HCDR1 comprising amino acids having at least 80% identity to the amino acid sequence of SEQ ID NO:1 (EYTIH), HCDR2 comprising amino acids having at least 80% identity to the amino acid sequence of SEQ ID NO: 2 (GINPNNGIPNYNQKFKG); an HCDR3 comprising amino acids having at least 80% identity to the amino acid sequence of SEQ ID NO: 3 (RRIAYGYDEGHAMDY); LCDR1 comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4 (KSSQSLLYSRNQKNYLA), an LCDR2 comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:5 (WASTRES); and LCDR3 comprising amino acids having the amino acid sequence of SEQ ID NO:6 (QQYYSYPLT) and an isolated antigen-binding construct thereof comprising: 2. The isolated antigen-binding construct of configuration 1, comprising a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO:7. 3. The isolated antigen-binding construct of configuration 1, comprising a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:7. 4. The isolated antigen-binding construct of configuration 1, comprising a heavy chain having at least 95% identity to the amino acid sequence of SEQ ID NO:7. 5. The isolated antigen-binding construct of any one of configurations 1-4, comprising a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:8. 6. The isolated antigen-binding construct of any one of configurations 1-4, comprising a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:8. 7. The isolated antigen-binding construct of any one of configurations 1-4, comprising a light chain having at least 95% identity to the amino acid sequence of SEQ ID NO:8. 8. The isolated antigen-binding construct of any one of configurations 1-4, comprising a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:9. 9. The isolated antigen-binding construct of any one of configurations 1-4, comprising a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:9. 10. The isolated antigen-binding construct of any one of configurations 1-4, comprising a light chain having at least 95% identity to the amino acid sequence of SEQ ID NO:9. 11. an alanine at position 24 of sequence SEQ ID NO:7, or Glycine at position 26 of sequence SEQ ID NO:7 11. The isolated antigen-binding construct of any one of configurations 1-10, comprising at least one VH framework residue selected from the group consisting of: 12. serine at position 73 of sequence SEQ ID NO:8, an arginine at position 83 of sequence SEQ ID NO:8, glutamic acid at position 85 of sequence SEQ ID NO:8, a proline at position 86 of sequence SEQ ID NO:8, a phenylalanine at position 89 of sequence SEQ ID NO:8, or Tyrosine at position 98 of sequence SEQ ID NO:8 12. The isolated antigen-binding construct of any one of configurations 1-11, comprising at least one VL framework residue selected from the group consisting of: 13. The isolated antigen-binding construct of any one of configurations 1-12, comprising a phenylalanine at position 89 of the light chain sequence of SEQ ID NO:8. 14. The isolated antigen-binding construct of any one of configurations 1-13, comprising an alanine at position 24 and a glycine at position 26 of the heavy chain sequence of SEQ ID NO:7. 15. The isolated antigen-binding construct of any one of configurations 1 to 14, comprising a phenylalanine at position 89 and a serine at position 73 of the light chain sequence of SEQ ID NO:8. 16. The isolated antigen-binding construct of any one of configurations 1 to 15, comprising a phenylalanine at position 89, a serine at position 73, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. 17. The isolated antigen-binding construct of any one of configurations 1 to 16, comprising a phenylalanine at position 89 and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. 18. The isolated antigen-binding construct of any one of configurations 1-17, comprising a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. 19. The isolated antigen-binding construct of any one of configurations 1-18, comprising a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO:8. 20. The isolated antigen-binding construct of any one of configurations 1-19, comprising a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. 21. The isolated antigen-binding construct of any one of configurations 1-20, comprising a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO:8. twenty two. a variable heavy domain (VH) of SEQ ID NO: 7, and A variable light domain (VL) of SEQ ID NO: 8 22. The antigen-binding construct of any one of claims 1 to 21, comprising: twenty three. a variable heavy domain (VH) of SEQ ID NO: 7, and A variable light domain (VL) of SEQ ID NO: 9 22. The antigen-binding construct of any one of claims 1 to 21, comprising: 24. An isolated antigen-binding construct specific for FAP alpha, comprising a CDR3 comprising amino acids having at least 90% identity to the amino acid sequence of SEQ ID NO: 6 (QQYYSYPLT). 25. An isolated antigen-binding construct comprising a light chain CDR3 comprising an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO:6. 26. LCDR1 comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4 (KSSQSLLYSRNQKNYLA), an LCDR2 comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:5 (WASTRES); and LCDR3 comprising amino acids having the amino acid sequence of SEQ ID NO:6 (QQYYSYPLT) and an isolated antigen-binding construct thereof comprising: 27. An isolated antigen-binding construct comprising a heavy chain having at least 99% identity to the amino acid sequence of SEQ ID NO:7. 28. A heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO:7, an alanine at position 24 of sequence SEQ ID NO:7, Glycine at position 26 of sequence SEQ ID NO:7 1. An isolated antigen-binding construct comprising at least one VH framework residue selected from the group consisting of: 29. A light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:8, serine at position 73 of sequence SEQ ID NO:8, an arginine at position 83 of sequence SEQ ID NO:8, glutamic acid at position 85 of sequence SEQ ID NO:8, a proline at position 86 of sequence SEQ ID NO:8, a phenylalanine at position 89 of sequence SEQ ID NO:8, Tyrosine at position 98 of sequence SEQ ID NO:8 1. An isolated antigen-binding construct comprising at least one VL framework residue selected from the group consisting of: 30. An isolated antigen-binding construct comprising a light chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:9. 31. An isolated antigen-binding construct comprising a light chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:8. 32. a variable heavy domain (VH) comprising amino acids having at least 95% identity to the amino acid sequence of SEQ ID NO:7, and a variable light domain (VL) comprising amino acids having at least 95% identity to the amino acid sequence of SEQ ID NO:8; 1. An isolated antigen-binding construct comprising: 33. a variable heavy domain (VH) comprising amino acids having at least 95% identity to the amino acid sequence of SEQ ID NO:7, and a variable light domain (VL) comprising amino acids having at least 89% identity to the amino acid sequence of SEQ ID NO:9; 1. An isolated humanized antigen-binding construct comprising: 34. an alanine at position 24 of sequence SEQ ID NO:7, Glycine at position 26 of sequence SEQ ID NO:7 34. The isolated antigen-binding construct of any one of configurations 27, 32, or 33, comprising at least one VH framework residue selected from the group consisting of: 35. serine at position 73 of sequence SEQ ID NO:8, an arginine at position 83 of sequence SEQ ID NO:8, glutamic acid at position 85 of sequence SEQ ID NO:8, a proline at position 86 of sequence SEQ ID NO:8, a phenylalanine at position 89 of sequence SEQ ID NO:8, Tyrosine at position 98 of sequence SEQ ID NO:8 33. The isolated antigen-binding construct of any one of configurations 29, 31, or 32, comprising at least one VL framework residue selected from the group consisting of: 36. The isolated antigen-binding construct of any one of configurations 29, 31, or 32, comprising a phenylalanine at position 89 of the light chain sequence of SEQ ID NO:8. 37. The isolated antigen-binding construct of any one of configurations 29, 31, or 32, comprising a phenylalanine at position 89 and a serine at position 73 of the light chain sequence of SEQ ID NO:4. 38. The isolated antigen-binding construct of any one of configurations 29, 31, or 32, comprising a phenylalanine at position 89, a serine at position 73, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. 39. The isolated antigen-binding construct of any one of configurations 29, 31, or 32, comprising a phenylalanine at position 89 and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. 40. The isolated antigen-binding construct of any one of configurations 29, 31, or 32, comprising a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. 41. The isolated antigen-binding construct of any one of configurations 29, 31, or 32, comprising a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO:8. 42. The isolated antigen-binding construct of any one of configurations 29, 31, or 32, comprising a phenylalanine at position 89, a serine at position 73, an arginine at position 83, a glutamic acid at position 85, a proline at position 86, and a tyrosine at position 98 of the light chain sequence of SEQ ID NO:8. 43. The isolated antigen-binding construct of any one of configurations 29, 31, or 32, comprising a phenylalanine at position 89, an arginine at position 83, a glutamic acid at position 85, and a proline at position 86 of the light chain sequence of SEQ ID NO:8. 44. An isolated antigen-binding construct comprising an amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-44. 45. An isolated antigen-binding construct comprising an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-44. 46. ​​An isolated antigen-binding construct comprising an amino acid sequence having at least 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-44, 12-43, 12-45, 87-96, 110-115, 122-125, or 140-143. 47. HCDR1 comprising the amino acid sequence of SEQ ID NO: 81 (NYDIN) or a sequence having three or fewer point mutations thereto, HCDR2 comprising the amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN) or a sequence having three or fewer point mutations thereto; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY) or a sequence having three or fewer point mutations thereto; LCDR1 comprising the amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA) or a sequence having three or fewer point mutations thereto; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 85 (SASYRYT) or a sequence having three or fewer point mutations thereto, and / or An LCDR3 comprising the amino acid sequence of SEQ ID NO: 86 (QQHYSNPRT) or a sequence having three or fewer point mutations thereto. and an isolated antigen-binding construct thereof comprising: 48. HCDR1 comprising the amino acid sequence of SEQ ID NO: 81 (NYDIN) or a sequence having three or fewer point mutations thereto, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN) or a sequence having three or fewer point mutations thereto, and / or HCDR3 comprising the amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY) or a sequence having three or fewer point mutations thereto and an isolated antigen-binding construct thereof comprising: 49. LCDR1 comprising the amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA) or a sequence having three or fewer point mutations thereto; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 85 (SASYRYT) or a sequence having three or fewer point mutations thereto, and / or An LCDR3 comprising the amino acid sequence of SEQ ID NO: 86 (QQHYSNPRT) or a sequence having three or fewer point mutations thereto. and an isolated antigen-binding construct thereof comprising: 50. An isolated antigen-binding construct specific for FAP, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 81 (NYDIN), or a sequence having three or fewer point mutations thereto. 51. The isolated antigen-binding construct of embodiment 50, wherein CDR1 is part of a heavy chain. 52. An isolated antigen-binding construct specific for FAP, comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN), or a sequence having three or fewer point mutations thereto. 53. The isolated antigen-binding construct of embodiment 52, wherein CDR2 is part of a heavy chain. 54. An isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY), or a sequence having three or fewer point mutations thereto. 55. The isolated antigen-binding construct of embodiment 54, wherein CDR3 is part of a heavy chain. 56. An isolated antigen-binding construct specific for FAP, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA), or a sequence having three or fewer point mutations thereto. 57. The isolated antigen-binding construct of embodiment 56, wherein CDR1 is part of a light chain. 58. An isolated antigen-binding construct specific for FAP, comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 85 (SASYRYT), or a sequence having three or fewer point mutations thereto. 59. The isolated antigen-binding construct of embodiment 58, wherein CDR2 is part of a light chain. 60. An isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 86 (QQHYSNPRT), or a sequence having three or fewer point mutations thereto. 61. The isolated antigen-binding construct of embodiment 60, wherein CDR3 is part of a light chain. 62. The isolated antigen-binding construct of any one of configurations 47-61, wherein no more than two point mutations are present. 63. The isolated antigen-binding construct of any one of configurations 47-61, wherein no more than one point mutation is present. 64. The isolated antigen-binding construct of any one of configurations 47-61, wherein there are no point mutations. 65. The isolated antigen-binding construct of any one of configurations 47-64, further comprising a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO:87. 66. The isolated antigen-binding construct of any one of configurations 47-65, further comprising a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:88. 67. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 87, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 88 67. The antigen-binding construct of any one of claims 47 to 66, further comprising: 68. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 87, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 88 and an isolated antigen-binding construct thereof comprising: 69. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 89, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 90 67. The antigen-binding construct of any one of claims 47 to 66, further comprising: 70. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 89, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 90 and an isolated antigen-binding construct thereof comprising: 71. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 91, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 92 67. The antigen-binding construct of any one of claims 47 to 66, further comprising: 72. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 91, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 92 and an isolated antigen-binding construct thereof comprising: 73. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 93, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO:94 67. The antigen-binding construct of any one of claims 47 to 66, further comprising: 74. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 93, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO:94 and an isolated antigen-binding construct thereof comprising: 75. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 95, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 96 67. The antigen-binding construct of any one of claims 47 to 66, further comprising: 76. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 95, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 96 and an isolated antigen-binding construct thereof comprising: 77. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 122, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 123 67. The antigen-binding construct of any one of claims 47 to 66, further comprising: 78. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 122, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 23 and an isolated antigen-binding construct thereof comprising: 79. HCDR1 comprising the amino acid sequence of SEQ ID NO: 104 (SYVMH) or a sequence having three or fewer point mutations thereto; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 105 (YINPHNDGTK) or a sequence having three or fewer point mutations thereto; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 106 (ARWGIYYGYGAWFAY) or a sequence having three or fewer point mutations thereto; LCDR1 comprising the amino acid sequence of SEQ ID NO: 107 (KASQNVGTYVA) or a sequence having three or fewer point mutations thereto; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 108 (YSASNRYS) or a sequence having three or fewer point mutations thereto, and / or LCDR3 comprising the amino acid sequence of SEQ ID NO: 109 (QQYNTYPYT) or a sequence having three or fewer point mutations thereto and an isolated antigen-binding construct thereof comprising: 80. HCDR1 comprising the amino acid sequence of SEQ ID NO: 104 (SYVMH) or a sequence having three or fewer point mutations thereto; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 105 (YINPHNDGTK) or a sequence having three or fewer point mutations thereto, and / or HCDR3 comprising the amino acid sequence of SEQ ID NO: 106 (ARWGIYYGYGAWFAY) or a sequence having three or fewer point mutations thereto and an isolated antigen-binding construct thereof comprising: 81. LCDR1 comprising the amino acid sequence of SEQ ID NO: 107 (KASQNVGTYVA) or a sequence having three or fewer point mutations thereto; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 108 (YSASNRYS) or a sequence having three or fewer point mutations thereto, and / or LCDR3 comprising the amino acid sequence of SEQ ID NO: 109 (QQYNTYPYT) or a sequence having three or fewer point mutations thereto and an isolated antigen-binding construct thereof comprising: 82. An isolated antigen-binding construct specific for FAP, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 104 (SYVMH), or a sequence having three or fewer point mutations thereto. 83. The isolated antigen-binding construct of embodiment 82, wherein CDR1 is part of a heavy chain. 84. An isolated antigen-binding construct specific for FAP, comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 105 (YINPHNDGTK), or a sequence having three or fewer point mutations thereto. 85. The isolated antigen-binding construct of embodiment 84, wherein CDR2 is part of a heavy chain. 86. An isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 106 (ARWGIYYGYGAWFAY), or a sequence having three or fewer point mutations thereto. 87. The isolated antigen-binding construct of embodiment 86, wherein CDR3 is part of a heavy chain. 88. An isolated antigen-binding construct specific for FAP, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 107 (KASQNVGTYVA), or a sequence having three or fewer point mutations thereto. 89. The isolated antigen-binding construct of embodiment 88, wherein CDR1 is part of a light chain. 90. An isolated antigen-binding construct specific for FAP, comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 108 (YSASNRYS), or a sequence having three or fewer point mutations thereto. 91. The isolated antigen-binding construct of embodiment 90, wherein CDR2 is part of a light chain. 92. An isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 109 (QQYNTYPYT), or a sequence having three or fewer point mutations thereto. 93. The isolated antigen-binding construct of embodiment 92, wherein CDR3 is part of a light chain. 94. The isolated antigen-binding construct of any one of configurations 79-93, wherein no more than two point mutations are present. 95. The isolated antigen-binding construct of any one of configurations 79-93, wherein no more than one point mutation is present. 96. The isolated antigen-binding construct of any one of configurations 79 to 93, wherein there are no point mutations. 97. The isolated antigen-binding construct of any one of configurations 79 to 96, further comprising a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO:110. 98. The isolated antigen-binding construct of any one of configurations 79 to 97, further comprising a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:111. 99. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 110, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 111 99. The antigen-binding construct of any one of embodiments 79 to 98, further comprising: 100. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 110, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 111 and an isolated antigen-binding construct thereof comprising: 101. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 112, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 113 99. The antigen-binding construct of any one of embodiments 79 to 98, further comprising: 102. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 112, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 113 and an isolated antigen-binding construct thereof comprising: 103. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 91, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 92 99. The antigen-binding construct of any one of embodiments 79 to 98, further comprising: 104. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 114, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 115 99. The antigen-binding construct of any one of embodiments 79 to 98, further comprising: 105. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 114, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 115 and an isolated antigen-binding construct thereof comprising: 106. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 95, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 96 99. The antigen-binding construct of any one of embodiments 79 to 98, further comprising: 107. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 124, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 125 99. The antigen-binding construct of any one of embodiments 79 to 98, further comprising: 108. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 124, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 125 and an isolated antigen-binding construct thereof comprising: 109. HCDR1 comprising the amino acid sequence of SEQ ID NO: 134 (SYTMS) or a sequence having three or fewer point mutations thereto; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 135 (TISSGGSYTY) or a sequence having three or fewer point mutations thereto; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 136 (TRDQVGYAMDY) or a sequence having three or fewer point mutations thereto; LCDR1 comprising the amino acid sequence of SEQ ID NO: 137 (TRDQVGYAMDY) or a sequence having three or fewer point mutations thereto; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 138 (YWASTRHT) or a sequence having three or fewer point mutations thereto, and / or LCDR3 comprising the amino acid sequence of SEQ ID NO: 139 (QQYSRYPYT) or a sequence having three or fewer point mutations thereto and an isolated antigen-binding construct thereof comprising: 110. HCDR1 comprising the amino acid sequence of SEQ ID NO: 134 (SYTMS) or a sequence having three or fewer point mutations thereto; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 135 (TISSGGSYTY) or a sequence having three or fewer point mutations thereto; HCDR3 comprising the amino acid sequence of SEQ ID NO: 136 (TRDQVGYAMDY) or a sequence having three or fewer point mutations thereto and an isolated antigen-binding construct thereof comprising: 111. LCDR1 comprising the amino acid sequence of SEQ ID NO: 137 (TRDQVGYAMDY) or a sequence having three or fewer point mutations thereto; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 138 (YWASTRHT) or a sequence having three or fewer point mutations thereto, and / or LCDR3 comprising the amino acid sequence of SEQ ID NO: 139 (QQYSRYPYT) or a sequence having three or fewer point mutations thereto and an isolated antigen-binding construct thereof comprising: 112. An isolated antigen-binding construct specific for FAP, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 134 (SYTMS), or a sequence having three or fewer point mutations thereto. 113. The isolated antigen-binding construct of embodiment 112, wherein CDR1 is part of a heavy chain. 114. An isolated antigen-binding construct specific for FAP, comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 135 (TISSGGSYTY), or a sequence having three or fewer point mutations thereto. 115. The isolated antigen-binding construct of embodiment 114, wherein CDR2 is part of a heavy chain. 116. An isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 136 (TRDQVGYAMDY), or a sequence having three or fewer point mutations thereto. 117. The isolated antigen-binding construct of embodiment 116, wherein the CDR3 is part of a heavy chain. 118. An isolated antigen-binding construct specific for FAP, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 137 (TRDQVGYAMDY), or a sequence having three or fewer point mutations thereto. 119. The isolated antigen-binding construct of embodiment 118, wherein CDR1 is part of a light chain. 120. An isolated antigen-binding construct specific for FAP, comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 138 (YWASTRHT), or a sequence having three or fewer point mutations thereto. 121. The isolated antigen-binding construct of embodiment 120, wherein CDR2 is part of a light chain. 122. An isolated antigen-binding construct specific for FAP, comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 139 (QQYSRYPYT), or a sequence having three or fewer point mutations thereto. 123. The isolated antigen-binding construct of embodiment 122, wherein CDR3 is part of a light chain. 124. The isolated antigen-binding construct of any one of configurations 109 to 123, wherein there are no more than two point mutations thereto. 125. The isolated antigen-binding construct of any one of configurations 109 to 123, wherein no more than one point mutation is present thereto. 126. The isolated antigen-binding construct of any one of configurations 109 to 123, wherein there are no point mutations. 127. The isolated antigen-binding construct of any one of configurations 109 to 126, further comprising a heavy chain having at least 80% identity to the amino acid sequence of SEQ ID NO:140. 128. The isolated antigen-binding construct of any one of configurations 109 to 127, further comprising a light chain having at least 80% identity to the amino acid sequence of SEQ ID NO:11. 129. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 140, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 141 129. The antigen-binding construct of any one of claims 109 to 128, further comprising: 130. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 140, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 141 and an isolated antigen-binding construct thereof comprising: 131. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 142, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 90 129. The antigen-binding construct of any one of claims 109 to 128, further comprising: 132. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 142, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO: 90 and an isolated antigen-binding construct thereof comprising: 133. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 143, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO:94 129. The antigen-binding construct of any one of claims 109 to 128, further comprising: 134. a variable heavy domain (VH) having at least 80% identity to the amino acid sequence of SEQ ID NO: 143, and / or A variable light domain (VL) chain having at least 80% identity to the amino acid sequence of SEQ ID NO:94 and an isolated antigen-binding construct thereof comprising: 135. The order of variable regions is, from the N-terminus to the C-terminus of the polypeptide, V L , V H135. The isolated antigen-binding construct of any one of embodiments 47 to 134, 136. The order of variable regions is, from the N-terminus to the C-terminus of the polypeptide, V H , V L 135. The isolated antigen-binding construct of any one of embodiments 47 to 134, 137. An isolated antigen-binding construct comprising an amino acid sequence having at least 98% identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 97-103 and 126. 138. An isolated antigen-binding construct comprising an amino acid sequence having at least 98% identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 116-120 and 127-133. 139. An isolated antigen-binding construct comprising an amino acid sequence having at least 98% identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 144-146. 140. The isolated antigen-binding construct of any one of configurations 47-139, which is mammalian. 141. The isolated antigen-binding construct of configuration 140, which is murine. 142. The isolated antigen-binding construct of embodiment 140, which is human. 143. The isolated antigen-binding construct of any one of configurations 1-139, which is an antibody. 144. The isolated antigen-binding construct of any one of configurations 1 to 139, wherein the humanized antigen-binding construct is selected from the group consisting of an scFV, a Fab, a Fab2, a nanobody, a minibody, a cys-diabody, or any combination thereof. 145. The isolated antigen-binding construct of any one of configurations 1 to 144, wherein the humanized antigen-binding construct is a minibody. 146. The isolated antigen-binding construct of any one of configurations 1 to 144, wherein the humanized antigen-binding construct is a cys-diabody. 147. A minibody antigen-binding construct comprising an amino acid sequence having at least about 80%, at least about 90%, or at least about 99% identity to an amino acid sequence selected from the group of SEQ ID NOs: 11-22. 148. A cys-diabody antigen-binding construct comprising an amino acid sequence having at least about 80%, at least about 90%, or at least about 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-22. 149. A minibody antigen-binding construct comprising an amino acid sequence having at least about 80%, at least about 90%, or at least about 99% identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 97-103, 116-120, 126-133, and 144-146. 150. A cys-diabody antigen-binding construct comprising an amino acid sequence having at least about 80%, at least about 90%, or at least about 99% identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 97-103, 116-120, 126-133, and 144-146. 151. An isolated antigen-binding construct of any one of constructs 47-146 or 149-150, which is specific for human FAP. 152. An isolated antigen-binding construct according to any one of configurations 1 to 150, which is specific for FAP alpha. 153. An isolated antigen-binding construct of any one of configurations 1-150, which does not bind to DPP4. 154. An isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-153, which exhibits expression in mammalian cells that is at least two-fold greater than the expression of sibrotuzumab minibodies (IAB16M1-12 and IAB16M2-13). 155. An isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-154, which exhibits expression in mammalian cells that is at least 6-fold greater than the expression of sibrotuzumab minibodies (IAB16M1-12 and IAB16M2-13). 156. An isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-155, having a KD of less than 2×10^-9M. 157. An isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-156, having a KD of less than 1x10^-9M. 158. An isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-157, having an on rate (kon) greater than 8.0 (1 / Ms). 159. An isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-158, having an on rate (kon) greater than 9.0 (1 / Ms). 160. An isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-159, having an off rate (koff) of less than 2.5 x 10^-3 (1 / s). 161. An isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-160, having an off rate (koff) of less than 1.5 x 10^-3 (1 / s). 162. The isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-161, wherein the order of the variable regions is, from the N-terminus to the C-terminus of the polypeptide, VL, VH. 163. The isolated antigen-binding construct of any one of configurations 1-46, 147-148, or 152-161, wherein the order of the variable regions, from N-terminus to C-terminus of the polypeptide, is VH, VL. 164. The isolated antigen-binding construct of any one of configurations 1 to 163, further comprising a payload. 165. The isolated antigen-binding construct of embodiment 164, wherein the payload is fluorescent. 166. The isolated antigen-binding construct of embodiment 164, wherein the payload is luminescent. 167. The isolated antigen-binding construct of embodiment 164, wherein the payload is chromogenic. 168. The isolated antigen-binding construct of embodiment 164, wherein the payload is radioactive. 169. The isolated antigen-binding construct of embodiment 164, wherein the payload is non-radioactive. 170. The isolated antigen-binding construct of claim 164, wherein the payload is an ADC. 171. The isolated antigen-binding construct of embodiment 164, wherein the payload is chemically reactive. 172. The isolated antigen-binding construct of any one of configurations 1 to 114, wherein the payload is a detectable marker. 173. The isolated antigen-binding construct of any one of configurations 1 to 172, which is humanized. 174. 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu,177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th, and 225 A composition comprising an amino acid sequence as defined in any one of configurations 1 to 173, further comprising at least one payload selected from the group consisting of: A, B, C, D, E, Et, G, Gt ... 175. The isolated antigen-binding construct of any one of configurations 47-146, 149-153, or 164-174, wherein the amino acid sequence further comprises at least one metal binding site. 176. The isolated antigen-binding construct of any one of configurations 47-146, 149-153, or 164-175, wherein the amino acid sequence further comprises a histidine (His) tag sequence. 177. The isolated antigen-binding construct of any one of configurations 47-146, 149-153, or 164-176, wherein the amino acid sequence further comprises a 99mTc-carbonyl radiolabel. 178. An expression vector configured to express a sequence specified in any one of constructs 1 to 177. 179. An expression vector capable of expressing a sequence specified in any one of constructs 1 to 177. 180. An expression vector according to construct 179, which is a vector for transfection in mammalian cells. 181. The expression vector of construct 178 or 179, which is a viral vector selected from a lentiviral vector or an adenoviral vector. 182. An expression vector according to any one of constructs 178 to 181, comprising a sequence encoding a cleavable signal peptide having at least 99% identity to the amino acid sequence of SEQ ID NO: 46 and 121 (METDTLLLWVLLLWVPGSTG). 183. A method for introducing an amino acid sequence or an expression vector as defined or described in any one of configurations 1 to 182 into a host cell, the method comprising the steps of performing electroporation, viral infection, and / or at least one chemical method. 184. A host cell comprising an amino acid sequence or an expression vector as defined or set forth in any one of configurations 1 to 182. 185. A method for using an amino acid sequence or an expression vector as defined or described in any one of configurations 1 to 184 as a pretargeting modality, comprising: adding a non-radioactive sequence, vector, or antigen-binding construct to the system; and adding a rapid clearance radiolabeled product that recognizes the protein product of the sequence or vector; The method includes: 186. The method of claim 185, wherein the rapid clearance radiolabeled product comprises a small molecule and / or a peptide. 187. A host cell comprising an amino acid sequence or an expression vector as defined or set forth in any one of configurations 1 to 182. 188. A composition comprising an amino acid sequence as defined in any one of compositions 1 to 177 and at least one chelating agent. 189. The composition of embodiment 188, wherein the at least one chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NOGADA, NETA, deferoxamine (DfO), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, dimercaprol, penicillamine, trientine, zinc, deferasirox, deferiprone, deferoxamine, succimer, pyrophosphate, tripolyphosphate, citric acid, tartaric acid, glycine, DMPS, DMSA, NTA, calcium, sodium, desferryloxamine, dicobalt EDTA, dimercarpol, BAL, and demercaptosuccinic acid, or any combination thereof. 190. The composition of embodiment 188 or 189, further comprising a double or triple chelating agent. 191. The composition of claim 190, wherein the double or triple chelator is 64Cu / 67Cu, 89Zr / 177Lu, or 89Zr / 227Th. 192. The composition of any one of aspects 188-191, wherein at least one chelating agent is configured to capture an isotope. 193. The composition of any one of configurations 188-192, further comprising an optical probe. 194. The composition of any one of embodiments 188-193, further comprising at least one payload. 195. At least one payload is 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th, and 225 The composition of embodiment 194, wherein the composition is selected from the group consisting of: 196. Use of a composition according to any one of claims 188 to 195 as a medicine. 197. Use of a composition according to any one of claims 188 to 195 in the manufacture of a medicament for administration to a subject. 198. Use of a composition described in any one of configurations 188 to 195 for imaging a cell, tissue, organ, and / or subject. 199. Use of a composition described in any one of configurations 188 to 195 for identifying a disease in a subject. 200. The use according to claim 199, wherein the disease is cancer or a tumor. 201. The use according to claim 199, wherein the disease is a solid tumor. 202. The use according to claim 199, wherein the disease is fibrosis. 203. The use according to claim 199, wherein the disease is an autoimmune disease. 204. The use according to claim 199, wherein the disease is cardiovascular. 205. A method for identifying a disease in a subject, comprising: administering to a subject at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies defined or described in any one of configurations 1-118; screening the binding of at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP; and determining whether the subject has a disease based on the presence or absence of binding to FAP The method includes: 206. The method of claim 205, wherein the disease is cancer or a tumor. 207. The method of claim 205, wherein the disease is a solid tumor. 208. The method of claim 205, wherein the disease is fibrosis. 209. The method of claim 205, wherein the disease is an autoimmune disease. 210. The method according to claim 205, wherein the disease is cardiovascular. 211. The method of any one of aspects 205 to 210, wherein the subject is a mammal or a human. 212. The method of any one of configurations 205-211, wherein at least one of the antibody, antigen-binding construct, minibody, and / or cys-diabody binds to the tumor stroma. 213. The method of any one of aspects 205-212, wherein at least one of the antibody, antigen-binding construct, minibody, and / or cys-diabody binds to fibroblasts. 214. The method of any one of configurations 205-213, wherein at least one of the antibody, antigen-binding construct, minibody, and / or cys-diabody binds to cancer-associated fibroblasts. 215. A method according to any one of configurations 205 to 214, wherein the disease is epithelial. 216. The method of any one of configurations 205-215, wherein at least one of the antibodies, antigen-binding constructs, minibodies, and / or cys-diabodies further comprises a payload. 217. The payload is 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc,94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227Th , and 225 The method of claim 216, wherein the at least one of the groups consisting of: Ac. 218. The method of any one of configurations 216 or 217, wherein the payload is used to determine binding of at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP. 219. The method of claim 218, wherein binding of at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP is determined by PET scanning. 220. The method of embodiment 218, wherein binding of at least one antibody, antigen-binding construct, minibody, and / or cys-diabody to the FAP is determined by at least one of the group consisting of MR imaging, optical probes, magnetic nanoparticles, spectrosc...

Claims

1. An HCDR1 comprising an amino acid having at least 80% identity with the amino acid sequence of SEQ ID NO: 1 (EYTIH), an HCDR2 comprising an amino acid having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 (GINPNNGIPNYNQKFKG), an HCDR3 comprising an amino acid having at least 80% identity with the amino acid sequence of SEQ ID NO: 3 (RRIAYGYDEGHAMDY), an LCDR1 comprising an amino acid having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 (KSSQSLLYSRNQKNYLA), an LCDR2 comprising an amino acid having at least 80% identity with the amino acid sequence of SEQ ID NO: 5 (WASTRES), and an LCDR3 comprising an amino acid having the amino acid sequence of SEQ ID NO: 6 (QQYYSYPLT) comprising an isolated antigen-binding construct.

2. The isolated antigen-binding construct according to claim 1, comprising a heavy chain having at least 80% identity with the amino acid sequence of SEQ ID NO:

7.

3. The heavy chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 7 is alanine at position 24 of the sequence of SEQ ID NO: 7, or glycine at position 26 of the sequence of SEQ ID NO: 7 The isolated antigen-binding construct according to claim 2, comprising at least one VH framework residue selected from the group consisting of

4. The isolated antigen-binding construct according to claim 1, comprising a light chain having at least 80% identity with the amino acid sequence of SEQ ID NO:

8.

5. The light chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 is serine at position 73 of the sequence of SEQ ID NO: 8, arginine at position 83 of the sequence of SEQ ID NO: 8, glutamic acid at position 85 of the sequence of SEQ ID NO: 8, proline at position 86 of the sequence of SEQ ID NO: 8, or phenylalanine at position 89 of the sequence of SEQ ID NO: 8 The isolated antigen-binding construct according to claim 4, comprising at least one VL framework residue selected from the group consisting of

6. The variable heavy chain domain (VH) of SEQ ID NO: 7, and The variable light chain domain (VL) of SEQ ID NO: 9 The isolated antigen-binding construct according to claim 1, comprising

7. An LCDR1 comprising an amino acid having the amino acid sequence of SEQ ID NO: 4 (KSSQSLLYSRNQKNYLA), an LCDR2 comprising an amino acid having the amino acid sequence of SEQ ID NO: 5 (WASTRES), and an LCDR3 comprising an amino acid having the amino acid sequence of SEQ ID NO: 6 (QQYYSYPLT) An isolated antigen-binding construct comprising **Claim 8** An isolated antigen-binding construct comprising an amino acid having 100% identity with any one of the amino acid sequences listed in SEQ ID NOs: 23-44, 12-43, 12-45, 87-96, 110-115, 122-125, or 140-143. **Claim 9** An HCDR1 comprising the amino acid sequence of SEQ ID NO: 81 (NYDIN) or a sequence having no more than 3 point mutations relative thereto, An HCDR2 comprising the amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN) or a sequence having no more than 3 point mutations relative thereto, An HCDR3 comprising the amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY) or a sequence having no more than 3 point mutations relative thereto, An LCDR1 comprising the amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA) or a sequence having no more than 3 point mutations relative thereto, An LCDR2 comprising the amino acid sequence of SEQ ID NO: 85 (SASYRYT) or a sequence having no more than 3 point mutations relative thereto, and / or An LCDR3 comprising the amino acid sequence of SEQ ID NO: 86 (QQHYSNPRT) or a sequence having no more than 3 point mutations relative thereto comprising an isolated antigen-binding construct thereof. **Claim 10** An HCDR1 comprising the amino acid sequence of SEQ ID NO: 81 (NYDIN) or a sequence having no more than 3 point mutations relative thereto, An HCDR2 comprising the amino acid sequence of SEQ ID NO: 82 (LIWTGGGTN) or a sequence having no more than 3 point mutations relative thereto, An HCDR3 comprising the amino acid sequence of SEQ ID NO: 83 (GGPLVWYALDY) or a sequence having no more than 3 point mutations relative thereto, comprising an isolated antigen-binding construct thereof. **Claim 11** An LCDR1 comprising the amino acid sequence of SEQ ID NO: 84 (KASQDVSTAVA) or a sequence having no more than 3 point mutations relative thereto, An LCDR2 comprising the amino acid sequence of SEQ ID NO: 85 (SASYRYT) or a sequence having no more than 3 point mutations relative thereto, and / or An LCDR3 comprising the amino acid sequence of SEQ ID NO: 86 (QQHYSNPRT) or a sequence having no more than 3 point mutations relative thereto comprising an isolated antigen-binding construct thereof. **Claim 12** The isolated antigen-binding construct according to any one of claims 9 to 11, wherein there are no more than 2 point mutations. **Claim 13** An isolated antigen-binding construct according to any one of claims 9 to 11, further comprising a heavy chain having the amino acid sequence of SEQ ID NO:

87.

14. An isolated antigen-binding construct according to any one of claims 9 to 11, further comprising a light chain having the amino acid sequence of SEQ ID NO:

88.

15. An isolated antigen-binding construct according to any one of claims 1 and 7 to 11, which is an antibody, scFv, Fab, Fab2, nanobody, minibody or cys-diabody.

16. Further comprising a payload, optionally, said payload is selected from the group consisting of fluorescent, luminescent, chromogenic, radioactive, non-radioactive, chemotherapeutic agents, chemically reactive agents, and detectable markers. An isolated antigen-binding construct according to any one of claims 1 and 7 to 11.

17. 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th, and 225 Ac, or at least one payload selected from the group consisting of combinations thereof, a composition comprising the isolated antigen-binding construct according to any one of claims 1 and 7-11.

18. An expression vector configured to express the antigen-binding construct according to any one of claims 1 and 7 to 11, or capable of expressing said antigen-binding construct.

19. A host cell comprising the antigen-binding construct according to any one of claims 1 and 7 to 11 or the expression vector according to claim 18.

20. A medicament for treating or diagnosing the human or animal body, comprising the isolated antigen-binding construct according to any one of claims 1 and 7 to 11 or the composition according to claim 17.

21. A pharmaceutical composition for treating a subject having cancer and / or a tumor, comprising the isolated antigen-binding construct according to any one of claims 1 and 7 to 11 and a pharmaceutically acceptable carrier.

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