WO2023077068A1

WO2023077068A1 - Novel binding agents related to glycoprotein-a

Info

Publication number: WO2023077068A1
Application number: PCT/US2022/078874
Authority: WO
Inventors: Neil Goldstein; Karla FRIETZE
Original assignee: Skunkworx Bio, Inc.
Priority date: 2021-10-28
Filing date: 2022-10-28
Publication date: 2023-05-04

Abstract

The disclosure is directed to compositions that specifically bind to glycoprotein-A repetitions predominant (GARR) and/or integrin subunit alpha V gene (ITGA V), compositions that bind to a protein of the extracelluar matrix, and related methods of use. Where a composition which specifically binds to glycoprotein-A repetitions predominant (GARR), comprises: (a) a single-chain antibody, or fragment thereof, comprising a CDR1, CDR2, and CDR3.

Description

GENERATION AND CHARACTERIZATION OF NOVEL BINDING AGENTS RELATED TO GLYCOPROTEIN-A REPETITIONS PREDOMINANT (GARP) AND THE INTEGRIN SUBUNIT ALPHA V GENE (ITGAV) FIELD OF THE DISCLOSURE The disclosure is directed to compositions that specifically bind to glycoprotein-A repetitions predominant (GARP), or integrin subunit alpha V gene (ITGAV), and/or compositions that bind to a protein of the extracelluar matrix, and related methods. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/272,962, filed on October 28, 2021, the entire content of which is hereby incorporated herein by reference in its entirety. DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY This application contains a Sequence Listing in XML format submitted electronically herewith via EFS-Web. Said XML copy, created on October 18, 2022, is named SKX- 003PC_SequenceListing and is 56,332 bytes in size. The Sequence Listing is incorporated herein by reference in its entirety. BACKGROUND Regulatory T cells (Tregs) are physiologically engaged in the maintenance of immunological self-tolerance and homeostasis, and these cells have a critical role for the control of anti-tumor immune responses. In malignant tumors, Tregs promote tumor progression by suppressing effective anti-tumor immunity. Higher infiltration of Tregs is frequently observed in tumor tissues, and increases in the number of Tregs is correlated with poor prognosis in various types of human cancers. Glycoprotein-A Repetitions Predominant (GARP) is, inter alia, a protein with a single- pass transmembrane structure, and this protein is expressed on the cell surface of activated Tregs and forms a complex with latent TGF-β (a precursor of TGF-β which is an important molecule for inducing immune tolerance). GARP is expressed by Tregs in tumor infiltrating T cells at tumor sites of cancer patients, and also by Tregs circulating in the peripheral blood of cancer patients. In addition, overexpression of the integrin subunit alpha V gene (ITGAV) has been observed in several human cancers, including prostate and breast cancer. Overexpression of the α₅β₃ integrin, which contains ITGAV, has been associated with cancer metastasis and tumor progression.

Existing anti-GARP antibodies are limited, as they show partial or incomplete inhibition of Tregs, and existing anti-ITGAV antibodies have incomplete clinical outcomes. Thus, there remains a significant need for the development of effective agents that modulate the activity of GARP and/or ITGAV to treat various cancers and other diseases.

SUMMARY

Accordingly, In aspects, the present disclosure relates to compositions which specifically bind to GARP. Further, In aspects, the present disclosure relates to methods of using compositions which specifically bind to GARP and/or ITGAV, e.g. for the treatment or prevention of various cancers or autoimmune diseases.

In aspects, disclosed herein is a composition which specifically binds to glycoprotein- A repetitions predominant (GARP), wherein the composition comprises: (a) a single-chain antibody, or fragment thereof, comprising a CDR1, CDR2, and CDR3, wherein: CDR1 is LFYWMKMFDW (SEQ ID NO: 1) or EWYSHMATWW (SEQ ID NO: 4), or MDTSFVANS (SEQ ID NO: 11), or NTREYKVPPS (SEQ ID NO: 12), or HDDHWVQTVR (SEQ ID NO: 13), or a variant thereof, CDR2 is GRFIWKSPKQ (SEQ ID NO: 2) or VYQWWWWGDK (SEQ ID NO: 5), or GFHAYYRHRH (SEQ ID NO: 14), or EVYTHHHYYK (SEQ ID NO: 15), or EKLERREVYE (SEQ ID NO: 16), or a variant thereof, and CDR3 is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or NWYWRFNWNYVEWVVQHM (SEQ ID NO: 3), or TGPMLNLFHMKVSQGQFT (SEQ ID NO: 6), or TAGLGAWLMHIIHGMTHE (SEQ ID NO: 17), or NRTIPWVTGYYYAITDQV (SEQ ID NO: 18), or QMSMEIMARPRERRFQHL (SEQ ID NO: 19), or a variant thereof; or (b) a peptide, and the peptide is selected from: WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof.

In embodiments, CDR1 is EWYSHMATWW (SEQ ID NO: 4), or a variant thereof, CDR2 is VYQWWWWGDK (SEQ ID NO: 5), or a variant thereof, and CDR3 is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions. In embodiments, CDR1 is LFYWMKMFDW (SEQ ID NO: 1), or a variant thereof, CDR2 is GRFIWKSPKQ (SEQ ID NO: 2), or a variant thereof, and CDR3 is NWYWRFNWNYVEWVVQHM (SEQ ID NO: 3), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is EWYSHMATWW (SEQ ID NO: 4), or a variant thereof, CDR2 is VYQWWWWGDK (SEQ ID NO: 5), or a variant thereof, and CDR3 is TGPMLNLFHMKVSQGQFT (SEQ ID NO: 6), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is MDTSFVANS (SEQ ID NO: 11), or a variant thereof, CDR2 is GFHAYYRHRH (SEQ ID NO: 14), or a variant thereof, and CDR3 is TAGLGAWLMHIIHGMTHE (SEQ ID NO: 17), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is NTREYKVPPS (SEQ ID NO: 12), or a variant thereof, CDR2 is EVYTHHHYYK (SEQ ID NO: 15), or a variant thereof, and CDR3 is NRTIPWVTGYYYAITDQV (SEQ ID NO: 18), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is HDDHWVQTVR (SEQ ID NO: 13), or a variant thereof, CDR2 is EKLERREVYE (SEQ ID NO: 16), or a variant thereof, and CDR3 is QMSMEIMARPRERRFQHL (SEQ ID NO: 19), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the single-chain antibody, or fragment thereof, further comprises variable region framework (FW) sequences juxtaposed between the CDRs according to the formula (FW1)-(CDR1)-(FW2)-(CDR2)-(FW3)-(CDR3)-(FW4), wherein the variable region FW sequences in the heavy chain variable region are heavy chain variable region FW sequences, and wherein the variable region FW sequences in the light chain variable region are light chain variable region FW sequences. In embodiments, the variable region FW sequences are human. In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASEWYSHMATWWWFRQAPGKEREFVAVYQW WWWGDKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWLYRSLSMV KDYYYYLTLPYWGQGTLVTVSSGPGGQ (SEQ ID NO: 41), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 41.

In embodiments, the single-chain antibody comprises an amino acid sequence of: MQVQLVESGGGVVQPGRSLRLSCAASLFYWMKMFDWWFRQAPGKEREFVAGRFI WKSPKQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNWYWRFNWN YVEWVVQHMWGQGTLVTVSSGPGGQ (SEQ ID NO: 8), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 8.

In embodiments, the single-chain antibody comprises an amino acid sequence of: MQVQLVESGGGVVQPGRSLRLSCAASEWYSHMATWWWFRQAPGKEREFVAVYQ WWWWGDKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGPMLNLF HMKVSQGQFTWGQGTLVTVSSGPGGQ (SEQ ID NO: 9), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 9.

In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASSMDTSFVANSWFRQAPGKEREFVAGFHAYYR HRHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTAGLGAWLMHIIHG MTHEWGQGTLVTVSSGPGGQ (SEQ ID NO: 20), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 20.

In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASNTREYKVPPSWFRQAPGKEREFVAEVYTHHH YYKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNRTIPWVTGYYYAI TDQVWGQGTLVTVSSGPGGQ (SEQ ID NO: 21), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 21.

In embodiments, the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASHDDHWVQTVRWFRQAPGKEREFVAEKLERR EVYEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMSMEIMARPRER RFQHLWGQGTLVTVSSGPGGQ (SEQ ID NO: 22), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 22.

In embodiments, the peptide is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the peptide further comprises an additional peptide. In embodiments, the composition comprises a dimer of peptides. In embodiments, composition comprises a trimer of peptides. In embodiments, the peptides are joined with a linker which is substantially comprised of glycine and serine residues. In embodiments, the linker is (GGS)n, wherein _nis 1, or 2, or 3, or 4, or 5. In embodiments, the linker is GGSGGSGGSG (SEQ ID NO: 10), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the composition comprises a hinge-CH₂-CH₃ Fc domain. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgG, IgA, IgD, or IgE. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgG, IgA, IgD, or IgE. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgG4. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgG4.

In embodiments, the hinge-CH₂-CH₃ Fc domain comprises an amino acid sequence of GPGGPEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 43), or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% thereof.

In embodiments, the human IgG Fc domain comprises one or more mutations that reduce or eliminate the effector function of the Fc domains. In embodiments, the mutations are L234A and L235A (LALA) substitutions in human IgGl, or an equivalent thereof. In embodiments, the human IgG Fc domain comprises one or mutations that stabilize a hinge region in the Fc domain. In embodiments, the mutation is S228P in human IgG4, or an equivalent thereof.

In any of the preceding embodiments, the composition comprises a targeting moiety. In embodiments, the targeting moiety is directed to a tumor cell. In embodiments, the targeting moiety is directed to a tumor-associated antigen (TAA). In embodiments, the TAA is selected from HER2, PSA, TRP-2, EpCAM, GPC3, mesothelin (MSLN), and EGFR.

In embodiments, the disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding the single-chain antibody, or a fragment thereof, or peptide of any one of the preceding embodiments. In embodiments, the disclosed herein is a vector comprising the polynucleotide. In embodiments, the disclosed herein is a host cell comprising the vector. In embodiments, the disclosed herein is a pharmaceutical composition comprising the composition of any one of the preceding embodiments, and a pharmaceutically acceptable excipient or carrier.

In aspects, disclosed herein is a composition which specifically binds to integrin subunit alpha-V (ITGAV), wherein the composition comprises: (a) a single-chain antibody, or fragment thereof, comprising a CDR1, CDR2, and CDR3, wherein: CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO: 31), or SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 32), or FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof; or (b) a peptide, and the peptide is selected from: QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or a variant thereof, TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof, ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof,

VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or a variant thereof, FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO: 31), or a variant thereof,

SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 32), or a variant thereof, or

FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof.

In embodiments, CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO: 31), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is: SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 32), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is: FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the single-chain antibody, or fragment thereof, further comprises variable region framework (FW) sequences juxtaposed between the CDRs according to the formula (FW1)-(CDR1)-(FW2)-(CDR2)-(FW3)-(CDR3)-(FW4), wherein the variable region FW sequences in the heavy chain variable region are heavy chain variable region FW sequences, and wherein the variable region FW sequences in the light chain variable region are light chain variable region FW sequences. In embodiments, the variable region FW sequences are human.

In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQDQTWFGNRWW DLWMQPWWGQGTLVTVSSGPGGQ (SEQ ID NO: 34), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 34.

In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSSGFTRWIKFCD SKCSLSAFTQGYGVKYFVARLARMWGGWGQGTLVTVSSGPGGQ (SEQ ID NO: 35), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 35.

In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALMAYVREMGQ QVVNWTRERVFFRVGSANVRKVACHGECNWGQGTLVTVSSGPGGQ (SEQ ID NO: 36), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 36.

In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSLFRIPAGAWQV VKTYFKYAVYGFGGFGSQCRGHEGESCWGQGTLVTVSSGPGGQ (SEQ ID NO: 37), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 37.

In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFWLFNLGRCENA FWMDGWRVFKSLNNTVEDMGFVMSHLLKWGQGTLVTVSSGPGGQ (SEQ ID NO: 38), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 38.

In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFNHGGSTWSGLS GAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQWGQGTLVTV SSGPGGQ (SEQ ID NO: 39), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 39.

In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFNHGGSTWSGLS GAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 40), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 40.

In embodiments, the peptide is selected from QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or a variant thereof,

TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof, ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof, VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or a variant thereof, FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO: 31), or a variant thereof,

FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the peptide further comprises an additional peptide. In embodiments, the composition comprises a dimer of peptides. In embodiments, the composition comprises a trimer of peptides. In embodiments, the peptides are joined with a linker which is substantially comprised of glycine and serine residues. In embodiments, the linker is (GGS)n, wherein nis 1, or 2, or 3, or 4, or 5. In embodiments, the linker is GGSGGSGGSG (SEQ ID NO: 10), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the composition comprises a hinge- CH2-CH3 Fc domain. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgG, IgA, IgD, or IgE. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgG, IgA, IgD, or IgE. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In embodiments, the hinge-CH2-CH3 Fc domain is from or is derived from human IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from IgG4. In embodiments, hinge-CH₂-CH₃ Fc domain is from or is derived from human IgG4.

In embodiments, the hinge-CH₂-CH₃ Fc domain comprises an amino acid sequence of GPGGPEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 43), or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% thereof. In embodiments, the human IgG Fc domain comprises one or more mutations that reduce or eliminate the effector function of the Fc domains. In embodiments, the mutations are L234A and L235A (LALA) substitutions in human IgGl, or an equivalent thereof. In embodiments, the human IgG Fc domain comprises one or mutations that stabilize a hinge region in the Fc domain. In embodiments, the mutation is S228P in human IgG4, or an equivalent thereof.

In embodiments, the composition prevents or reduces tumor growth kinetics of the dissociation of the ECM. In embodiments, composition binds to an integrin. In embodiments, the composition binds to one or more one or more of the α₅β₁, α₅β₃, and α₅β₅ integrins. In embodiments, the composition prevents or reduces tumor growth kinetics of tumorigenesis. In embodiments, the composition prevents or reduces tumor growth kinetics of breast cancer metastasis or a primary breast tumor. In embodiments, the composition prevents or reduces tumor growth kinetics of glioblastoma. In embodiments, the composition prevents or reduces tumor growth kinetics of bladder cancer. In embodiments, the composition prevents or reduces tumor growth kinetics of colorectal cancer. In embodiments, the composition prevents or reduces tumor growth kinetics of prostate cancer. In embodiments, the composition prevents or reduces tumor growth kinetics of lung cancer.

In embodiments, the composition further comprises a targeting moiety. In embodiments, the targeting moiety is directed to a tumor cell. In embodiments, the targeting moiety is directed to a tumor-associated antigen (TAA). In embodiments, the TAA is selected from HER2, PSA, TRP-2, EpCAM, GPC3, mesothelin (MSLN), and EGFR.

In aspects, disclosed herein is a bispecific molecule comprising a first binding arm having the composition of any one of the preceding embodiments, and a second binding arm comprising the composition of any one of the preceding embodiments, wherein the bispecific molecule binds to a protein of the extracellular matrix (ECM). In embodiments, the bispecific molecule comprises a GARP single chain antibody comprising an amino acid sequence of SEQ ID NO: 41, and a ITGAV single chain antibody comprising an amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% thereto.

In embodiments, the single chain antibodies are joined with a linker which is substantially comprised of glycine and serine residues. In embodiments, the linker is (GGS)n, wherein n is 1, or 2, or 3, or 4, or 5. In embodiments, the linker is GGSGGSGGSG (SEQ ID NO: 10), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the GARP-ITGAV bispecific molecule comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASEWYSHMATWWWFRQAPGKEREFVAVYQW WWWGDKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWLYRSLSMV KDYYYYLTLPYWGQGTLVTVSSGPGGQGGGGSGGGGSQVQLVESGGGVVQPGRSL RLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMWYDKNYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCARQDQTWFGNRWWDLWMQPWWGGWGQGTL VTVSSGPGGQ (SEQ ID NO: 42), or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% thereto.

In embodiments, the composition comprises a hinge-CH₂-CH₃ Fc domain. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgG, IgA, IgD, or IgE. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgG, IgA, IgD, or IgE. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from IgG4. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgG4.

In embodiments, the bispecific molecule prevents or reduces tumor growth kinetics of the dissociation of the ECM. In embodiments, the bispecific molecule binds to an integrin. In embodiments, the bispecific molecule binds to one or more one or more of the α₅β₁, α₅β₃, and oyfE integrins. In embodiments, the bispecific molecule prevents or reduces tumor growth kinetics of tumorigenesis. In embodiments, the bispecific molecule prevents or reduces tumor growth kinetics of breast cancer metastasis or a primary breast tumor. In embodiments, the bispecific molecule prevents or reduces tumor growth kinetics of glioblastoma. In embodiments, the bispecific molecule prevents or reduces tumor growth kinetics of bladder cancer. In embodiments, the bispecific molecule prevents or reduces tumor growth kinetics of colorectal cancer. In embodiments, the bispecific molecule prevents or reduces tumor growth kinetics of prostate cancer. In embodiments, the bispecific molecule prevents or reduces tumor growth kinetics of lung cancer.

In embodiments, disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding the single-chain antibody, or a fragment thereof, or peptide of any one of the preceding embodiments, and a nucleic acid sequence encoding the single-chain antibody, or a fragment thereof, or peptide of any one of the preceding embodiments.

In aspects, disclosed herein is a method for treating or preventing cancer, comprising administering an effective amount of the composition of any one of the preceding embodiments to a patient in need thereof. In embodiments, the cancer is a solid tumor. In embodiments, the cancer is a blood cancer. In embodiments, the cancer is selected form one or more of a cancer of a blood vessel, an eye tumor, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; primary breast cancer; metastatic breast cancer, colorectal cancer, cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi’s sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs’ syndrome.

In embodiments, the primary breast cancer expresses the integrin subunit alpha-V gene (ITGAV).

In embodiments, the disclosed herein is a method for treating or preventing an autoimmune disease or disorder, comprising administering an effective amount of the composition of any one of the preceding embodiments to a patient in need thereof. In embodiments, the autoimmune disease or disorder is selected from graft versus host disease, transplantation rejection (e.g., prevention of allograft rejection), multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Meniere's syndrome; pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and Grave's disease. In embodiments, the autoimmune disease or disorder is graft versus host disease.

The details of one or more examples of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings, detailed description of several examples, and also from the appended claims. The details of the disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, illustrative methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows microscopic images of a A549 wound healing assay.

FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D show images and graphs demonstrating a reduction in migration of adenocarcinomic human alveolar basal epithelial cells (“A549”) cells by the GARP peptide, “173 3” (FIG. 2A and FIG. 2B), and the GP3 peptide (FIG. 2C and FIG 2D). FIG. 3A shows a microscopic image of an anti-GARP antibody (“Ref Tool 1”), a second anti-GARP antibody (“Ref Tool 2”), and the GARP peptide, “173 3” in an A549 wound healing assay. FIG. 3B shows a graph of percentage wound closure for an anti-GARP antibody (“Ref Tool 1”), a second anti-GARP antibody (“Ref Tool 2”), and the GARP peptide, “173 3” in the A549 wound healing assay.

FIG. 4 is a graph showing the overexpression of the integrin subunit alpha V gene (“ITGAV”) in multiple cancers.

FIG. 5 are fluorescencent microscopic images showing the binding of the bispecific molecules against GARP and ITGAV to A549 cells.

FIG. 6 is a graph showing A6pep3 and A6pep3:480-l constructs inhibited TGFp/SMAD luciferase activity in U87 GARP overexpressed cell lines,

FIG. 7A - FIG. 7C show graphs and images demonstrating the results of alkaline phosphatase (AP) reporter assays performed to assess inhibition of various GARP constructs. FIG. 7 A shows a graph of the absorbance of AP activity of various treatments after 24 hours. FIG. 7B shows a graph of the absorbance of AP activity of various treatments after 48 hours. FIG. 7C shows representative microscopy images of cells treated with TGFpi, A6Spep3, and A6Spep3 + TGFpi after 48 hours.

FIG. 8 A - FIG. 8B show graphs demonstrating A6pep3 inhibited TGFpi activity at various dosages and time course. FIG. 8A shows a graph of the absorbance of AP activity of various dosages of A6Spep3 treatments after 24 hours. FIG. 8B shows a graph of the absorbance of AP activity of various dosages of A6Spep3 treatments after 48 hours.

FIG. 9A - FIG. 9B show an image and graph of A549-GARP overexpressed cells treated with A6Spep3 and A6Spep3:480-l. FIG. 9A shows microscopy images of A549-GARP overexpressed cells treated with A6Spep3 and A6Spep3:480-l. FIG. 9B shows a graph of wound healing assay of A549-GARP overexpressed cells treated with A6Spep3 and A6Spep3:480-l.

FIG. 10 shows a bar graph of the expression of key genes in the TGFP pathway. For orientation purposes, the bars on the left represent A549-GARP and the bars on the right represent A549-GARP 173 3. FIG. 11A - FIG. 11D show images and graphs of WT-G292 and G292-GARP overexpressed cells treated with 173 3 peptides. FIG. 11A shows microscopy images of WT- G292 treated with 1733 peptides. FIG. 1 IB shows a bar graph depicting the results of a wound healing assay comparing G292 cells treated with DMSO and 173 3 peptides. FIG. 11C shows microscopy images of G292-GARP overexpressed cells treated with 173 3 peptides. FIG. 1 ID shows a bar graph depicting the results of a wound healing assay comparing G292-GARP overexpressed cells treated with DMSO and 173 3 peptides.

DETAILED DESCRIPTION

The present invention is based, in part, on the surprising discovery of a composition that specifically binds glycoprotein- A repetitions predominant (GARP), and/or ITGAV. The composition disclosed herein can be a small protein, or a peptide, or an antibody (e.g., a singlechain antibody), that binds to GARP, or the composition disclosed herein can be a small protein, or a peptide, or an antibody (e.g., a single-chain antibody), that binds to ITGAV. In embodiments, the composition disclosed herein is a bispecific molecule comprising a first binding arm having the composition that binds to GARP, and comprising a second binding arm having the composition that binds to ITGAV, wherein the bispecific molecule binds to a protein of the extracellular matrix (ECM). The compositions disclosed herein are capable of functioning as agonists or antagonists.

In addition, the present disclosure describes peptide, small protein, and antibody compositions that are interchangeable. The peptide or small protein can be derived from the binding region of an antibody (e.g, a single-chain antibody), and target specific peptides and small proteins can be incorporated into the binding regions of the antibody (e.g., a single-chain antibody). The compositions disclosed herein may be used in a method of treating or preventing cancer (e.g., a solid tumor, or blood cancer), or an autoimmune disease (e.g., graft versus host disease).

In embodiments, disclosed herein is a composition that specifically binds to GARP.

In embodiments, disclosed herein is a composition that specifically binds to ITGAV.

In embodiments, disclosed herein is a bispecific molecule comprising a first binding arm having the composition that binds to GARP, and comprising a second binding arm having the composition that binds to ITGAV. In embodiments, disclosed herein is a composition which specifically binds to GARP on the surface of a cell. In embodiments, disclosed herein is a composition which specifically binds to GARP on the surface of blood cells. In embodiments, disclosed herein is a composition which specifically binds to GARP on the surface of activated Tregs. In embodiments, disclosed herein is a composition which specifically binds to GARP on the surface of placenta, lung, kidney, heart, liver, skeletal muscle, pancreas, and lymphoid tissues. In embodiments, disclosed herein is a composition which specifically binds to GARP on the surface of megakaryocytes, platelets, B lymphocytes, T lymphocytes, mesenchymal stromal cells (MSCs) and human umbilical vein endothelial cells. In embodiments, disclosed herein is a composition which specifically binds to GARP on the surface of cancer cells. In embodiments, disclosed herein is a composition which specifically binds to GARP in invasive, metastatic, or treatment-resistant cancer.

In embodiments, the composition is a bispecific molecule comprising a first binding arm having the composition that binds to GARP of any one of the embodiments above, and a second binding arm comprising a peptide or a single-chain antibody, wherein the bispecific molecule binds to a protein of the extracellular matrix (ECM). In embodiments, the bispecific molecule disclosed herein prevents or reduces tumor growth kinetics of the dissociation of the ECM. In embodiments, the bispecific molecule binds to an integrin (e.g, the α₅β₁ integrin, the aspis integrin, and/or the α₅β₅ integrin). In embodiments, the bispecific molecule binds to the α₅β₁ integrin. In embodiments, the bispecific molecule binds to the α₅β₃ integrin. In embodiments, the bispecific molecule binds to the α₅β₅ integrin. In embodiments, the bispecific molecule binds to one or more of α₅β₁, α₅β₃, and α₅β₅ integrins. In embodiments, the bispecific molecule binds to a subunit of ITGAV.

In embodiments, disclosed herein is a composition which specifically binds to human GARP.

In embodiments, disclosed herein is a composition which specifically binds to murine GARP.

In embodiments, disclosed herein is a composition which specifically binds to human ITGAV.

In embodiments, disclosed herein is a composition which specifically binds to murine

ITGAV. In embodiments, disclosed herein is a composition which specifically binds to both human GARP and human ITGAV.

In embodiments, disclosed herein is a composition which specifically binds to both murine GARP and murine ITGAV.

In embodiments, disclosed herein is a composition which specifically binds to a particular antigen, e.g, GARP, ITGAV, or both GARP and ITGAV, when it binds to that antigen in a sample, and does not recognize and bind, or recognizes and binds to a lesser extent, other molecules in the sample. In embodiments, the compositions of the present disclosure can selectively bind to an epitope with an affinity (Kd) equal to or less than, for example, about 1 x 10'⁶ M (e.g., equal to or less than about 1 x 10'⁹ M, equal to or less than about 1 x IO⁴⁰ M, equal to or less than about 1 x 10⁴¹ M, or equal to or less than about 1 x IO⁴² M) in phosphate buffered saline. The ability of a compositions of the present disclosure to specifically bind a protein epitope can be determined using any of the methods known in the art or those methods described herein (e.g., by Biacore/Surface Plasmon Resonance). This can include, for example, binding to e.g, GARP, ITGAV, or both GARP and ITGAV, on live cells as a method to stimulate caspase activation in live transformed cells, binding to an immobilized target substrate including human GARP, ITGAV, or both GARP and ITGAV fusion proteins as detected using an ELISA method, binding to GARP, ITGAV, or both GARP and ITGAV on live cells as detected by flow cytometry, or binding to an immobilized substrate by surface plasmon resonance (including ProteOn).

In embodiments, the composition disclosed herein is a peptide, a small protein, or an antibody (e.g., a single-chain antibody), that binds to GARP, and/or ITGAV.

In embodiments, a single-chain antibody is a single-chain variable fragment (scFv).

In embodiments, the composition disclosed herein is a single-chain antibody, small protein, or a single polypeptide that contains at least one variable binding domain (e.g, a variable domain of a mammalian heavy or light chain immunoglobulin, a camelid VHH, or a cartilaginous fish (e.g., shark) Ig-NAR domain) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies include single-domain antibodies.

As used herein, the term “single-domain antibody” refers to a polypeptide that contains one camelid VHH or at least one cartilaginous fish Ig-NAR domain that is capable of specifically binding to an antigen. Non-limiting examples of single-domain antibodies are described, for example, in U.S. Publication No. 2010/0092470. In embodiments, the small protein ranges in size from about 2 kDa to about 4 kDa. In embodiments, the small protein ranges in size from about 2 kDa to about 3.5 kDa, from about 2 kDa to about 3 kDa, or from about 2 kDa to about 2.5 kDa.

In embodiments, the peptide or small protein is an agonist. In embodiments, the peptide or small protein is an antagonist.

In embodiments, the antibody ranges in size from about 5 kDa to about 30 kDa, from about 5 kDa to about 25 kDa, from about 5 kDa to about 20 kDa, or from about 5 kDa to about 15 kDa. In embodiments, the antibody (e.g, a single-chain antibody) is a 12 kDa fragment.

In embodiments, the antibody retains the biological function of the parent, whole antibody. In embodiments, the small protein and antibody compositions are interchangeable. In embodiments, the peptide or small protein is derived from the binding region of the antibody. In embodiments, the target specific small protein is incorporated into the binding regions of the antibody (e.g., a single-chain antibody).

In embodiments, a single-chain antibody is a single-chain variable fragment (scFv). In embodiments, the composition disclosed herein is a single-chain antibody, small protein, or a single polypeptide that contains at least one variable binding domain (e.g, a variable domain of a mammalian heavy or light chain immunoglobulin, a camelid VHH, or a cartilaginous fish (e.g. , shark) Ig-NAR domain) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies include single-domain antibodies.

In embodiments, the composition comprises a single-chain antibody, or fragment thereof, having a heavy chain variable domain. The variable domain determines the specificity of the antibody. Each variable region comprises three hypervariable regions also known as complementarity determining regions (CDRs) flanked by four relatively conserved framework regions (FRs). The three CDRs, referred to as CDR1, CDR2, and CDR3, contribute to the antibody binding specificity. In embodiments, the single-chain antibody is a chimeric antibody. In embodiments, the single-chain antibody is a humanized antibody.

In embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.

“Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices.

As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

GARP Binding Agents

In embodiments, the single-chain antibody, or fragment thereof, comprises a CDR1, CDR2, and CDR3, wherein:

CDR1 is LFYWMKMFDW (SEQ ID NO: 1) or EWYSHMATWW (SEQ ID NO: 4), or MDTSFVANS (SEQ ID NO: 11), or NTREYKVPPS (SEQ ID NO: 12), or HDDHWVQTVR (SEQ ID NO: 13), or a variant thereof;

CDR2 is GRFIWKSPKQ (SEQ ID NO: 2) or VYQWWWWGDK (SEQ ID NO: 5), or GFHAYYRHRH (SEQ ID NO: 14), or EVYTHHHYYK (SEQ ID NO: 15), or EKLERREVYE (SEQ ID NO: 16), or a variant thereof, and

CDR3 is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or NWYWRFNWNYVEWVVQHM (SEQ ID NO: 3) or TGPMLNLFHMKVSQGQFT (SEQ ID NO: 6), or TAGLGAWLMHIIHGMTHE (SEQ ID NO: 17), or NRTIPWVTGYYYAITDQV (SEQ ID NO: 18), or QMSMEIMARPRERRFQHL (SEQ ID NO: 19), or a variant thereof.

In embodiments, the composition comprises a peptide, and the peptide is selected from: WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof.

In embodiments, the single-chain antibody, or fragment thereof, further comprises variable region framework (FW) sequences juxtaposed between the CDRs according to the formula (FW1)-(CDR1)-(FW2)-(CDR2)-(FW3)-(CDR3)-(FW4), wherein the variable region FW sequences in the heavy chain variable region are heavy chain variable region FW sequences, and wherein the variable region FW sequences in the light chain variable region are light chain variable region FW sequences.

In embodiments, the variable region FW sequences are human. In embodiments, the single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASEWYSHMATWWWFRQAPGKEREFVAVYQW WWWGDKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWLYRSLSMV KDYYYYLTLPYWGQGTLVTVSSGPGGQ (SEQ ID NO: 41), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 8.

In embodiments, the single-chain antibody comprises an amino acid sequence of:

In embodiments, the peptide is or comprises: WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the peptide further comprises an additional peptide. In embodiments, the composition comprises a dimer of peptides. In embodiments, the composition comprises a trimer of peptides.

In embodiments, the peptides are joined with a linker which is substantially comprised of glycine and serine residues. In embodiments, the linker is (GGS)n, wherein n is 1, or 2, or 3, or 4, or 5. In embodiments, the linker is GGSGGSGGSG (SEQ ID NO: 21), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the composition comprises a hinge- CH2-CH3 Fc domain. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgG, IgA, IgD, or IgE. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgG, IgA, IgD, or IgE. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from IgG4. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgG4.

In embodiments, the present compositions comprise a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the nucleic acid sequences of any one of SEQ ID NOs: 23, or 24, as shown below, or a codon-optimized version thereof: atgcaggtgcagctggtggagtctgggggaggcgtggtacagcctgggaggtccctgc gactctcctgtgcagcctctctgttctactggatgaaaatgttcgactggtggttccgccag gctccagggaaggagcgtgagttcgtggcaggtcgtttcatctggaaatctccgaaacag tacgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctg tatctgcaaatgaacagcctgcgtgccgaggacacggccgtgtattactgcgcgcgtaac tggtactggcgtttcaactggaactacgttgaatgggttgttcagcatatgtggggtcaagg taccctggtcaccgtctcctcaggaccgggaggacaa (SEQ ID NO: 23) atgcaggtgcagctggtggagtctgggggaggcgtggtacagcctgggaggtccctgc gactctcctgtgcagcctctgaatggtactctcatatggctacttggtggtggttccgccag gctccagggaaggagcgtgagttcgtggcagtttaccagtggtggtggtggggtgacaa atacgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgct gtatctgcaaatgaacagcctgcgtgccgaggacacggccgtgtattactgcgcgcgta ctggtccgatgctgaacctgttccatatgaaagtttctcagggtcagttcacttggggtcaa ggtaccctggtcaccgtctcctcaggaccgggaggacaa (SEQ ID NO: 24)

ITGA V Binding Agents

In embodiments, the composition specifically binds to ITGAV. In embodiments, the composition binds to a subunit of ITGAV (e.g., the a?P i integrin, the α₅β₃ integrin, and/or the 01^5 integrin). In embodiments, the composition binds to the a?P i integrin. In embodiments, the composition binds to the α₅β₃ integrin. In embodiments, the composition binds to the α₅β₅ integrin. In embodiments, the composition binds to one or more of α₅β₁, α₅β₃, and α₅β₅ integrins. In embodiments, the composition disclosed herein prevents or reduces tumor growth kinetics of the dissociation of the ECM. In embodiments, the composition comprises: (a) a single-chain antibody, or fragment thereof, comprising a CDR1, CDR2, and CDR3, wherein: CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO:

30), or FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO:

31), or SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ

(SEQ ID NO: 32), or FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof; or (b) a peptide, and the peptide is selected from: QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or a variant thereof, TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof, ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof,

FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof.

In embodiments, CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 32), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the variable region FW sequences are human.

TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof, ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof,

SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 32), or a variant thereof,

In embodiments, the peptide further comprises an additional peptide. In embodiments, the composition comprises a dimer of peptides. In embodiments, the composition comprises a trimer of peptides. In embodiments, the peptides are joined with a linker which is substantially comprised of glycine and serine residues. In embodiments, the linker is (GGS)_n, wherein _nis 1, or 2, or 3, or 4, or 5. In embodiments, linker is GGSGGSGGSG (SEQ ID NO: 10), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the composition comprises a hinge-CFh-CHs Fc domain. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from IgG, IgA, IgD, or IgE. In embodiments, the hinge-CH₂-CH₃ Fc domain is from or is derived from human IgG, IgA, IgD, or IgE. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from IgGl, IgG2, IgG3, IgGl, IgAl, or IgA2. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from human IgGl, IgG2, IgG3, IgGl, IgAl, or IgA2. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from IgG4. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from human IgG4.

In embodiments, the hinge- CH2-CH3 Fc domain comprises an amino acid sequence of GPGGPEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 43), or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% thereof.

In embodiments, the human IgG Fc domain comprises one or more mutations that reduce or eliminate the effector function of the Fc domains. In embodiments, the mutations are L234A and L235A (LALA) substitutions in human IgGl, or an equivalent thereof. In embodiments, the human IgG Fc domain comprises one or mutations that stabilize a hinge region in the Fc domain. In embodiments, the mutation is S228P in human IgG4, or an equivalent thereof. In embodiments, the composition prevents or reduces tumor growth kinetics of the dissociation of the ECM.

In embodiments, the composition binds to an integrin.

In embodiments, the composition binds to one or more one or more of the α₅β₁, α₅β₃, and 015P5 integrins.

In embodiments, the composition prevents or reduces tumor growth kinetics of tumorigenesis.

In embodiments, the composition prevents or reduces tumor growth kinetics of breast cancer metastasis or a primary breast tumor.

In embodiments, the composition prevents or reduces tumor growth kinetics of glioblastoma.

In embodiments, the composition prevents or reduces tumor growth kinetics of bladder cancer.

In embodiments, the composition prevents or reduces tumor growth kinetics of colorectal cancer.

In embodiments, the composition prevents or reduces tumor growth kinetics of prostate cancer.

In embodiments, the composition prevents or reduces tumor growth kinetics of lung cancer.

In embodiments, the composition decreases the gene expression of SMAD3 in the TGFP pathway. In embodiments, the composition decreases the gene expression of SMAD7 in the TGFP pathway. In embodiments, the composition decreases the gene expression of TGFP- 1 in the TGFP pathway. In embodiments, the composition decreases the gene expression of TGFP-2 in the TGFP pathway. In embodiments, the composition decreases the gene expression of TGFP receptor 2 in the TGFP pathway. In embodiments, the composition decreases the gene expression of LTBP1 in the TGFP pathway.

In embodiments, the composition increases the gene expression of SMAD5 in the TGFP pathway. In embodiments, the composition increases the gene expression of SMAD6 in TGFP pathway. Bispecific Molecule Binding Agents

In embodiments, the composition is a bispecific molecule comprising a first binding arm having the composition that binds to GARP of any one of the embodiments above, and a second binding arm comprising a peptide or a single-chain antibody, wherein the bispecific molecule binds to a protein of the extracellular matrix (ECM). In embodiments, the bispecific molecule disclosed herein prevents or reduces tumor growth kinetics of the dissociation of the ECM. In embodiments, the bispecific molecule binds to an integrin. In embodiments, the bispecific molecule binds to a subunit of ITGAV (e.g., the a?P i integrin, the α₅β₃ integrin, and/or the asfri integrin). In embodiments, the bispecific molecule binds to the α₅β₁ integrin. In embodiments, the bispecific molecule binds to the asfE integrin. In embodiments, the bispecific molecule binds to the asfri integrin. In embodiments, the bispecific molecule binds to one or more of α₅β₁, α₅β₃, and α₅β₅ integrins.

In embodiments, the bispecific molecule comprises: (a) the first arm of the bispecific molecule is a single chain antibody, or fragment thereof, comprising a CDR1, CDR2, and CDR3, wherein: CDR1 is LFYWMKMFDW (SEQ ID NO: 1) or EWYSHMATWW (SEQ ID NO: 4), or MDTSFVANS (SEQ ID NO: 11), or NTREYKVPPS (SEQ ID NO: 12), or HDDHWVQTVR (SEQ ID NO: 13), or a variant thereof, CDR2 is GRFIWKSPKQ (SEQ ID NO: 2) or VYQWWWWGDK (SEQ ID NO: 5), or GFHAYYRHRH (SEQ ID NO: 14), or EVYTHHHYYK (SEQ ID NO: 15), or EKLERREVYE (SEQ ID NO: 16), or a variant thereof, and CDR3 is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or

NWYWRFNWNYVEWVVQHM (SEQ ID NO: 3), or TGPMLNLFHMKVSQGQFT (SEQ ID NO: 6), or TAGLGAWLMHIIHGMTHE (SEQ ID NO: 17), or

NRTIPWVTGYYYAITDQV (SEQ ID NO: 18), or QMSMEIMARPRERRFQHL (SEQ ID NO: 19), or a variant thereof; and (b) the second arm of the bispecific molecule is a single chain antibody, or fragment thereof, comprising a CDR1, CDR2, and CDR3, wherein: CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO: 31), or SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 32), or FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof. In embodiments, the first arm of the bispecific molecule is a peptide, and the peptide is selected from: WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof. In embodiments, the second arm of the bispecific molecule is a peptide, and the peptide is selected from: QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or a variant thereof, TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof,

ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof, VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or a variant thereof,

FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO: 31), or a variant thereof,

SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 32), or a variant there, or

FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof.

In embodiments, the first arm of the bispecific molecule is a single chain antibody, or fragment thereof, comprising a CDR1, CDR2, and CDR3 wherein:

CDR3 is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), NWYWRFNWNYVEWVVQHM (SEQ ID NO: 3) or TGPMLNLFHMKVSQGQFT (SEQ ID NO: 6), or TAGLGAWLMHIIHGMTHE (SEQ ID NO: 17), or NRTIPWVTGYYYAITDQV (SEQ ID NO: 18), or QMSMEIMARPRERRFQHL (SEQ ID NO: 19), or a variant thereof.

In embodiments, the first arm of the bispecific molecule comprises a peptide, and the peptide is selected from: WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof.

In embodiments, CDR1 is EWYSHMATWW (SEQ ID NO: 4), or a variant thereof, CDR2 is VYQWWWWGDK (SEQ ID NO: 5), or a variant thereof, and CDR3 is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, CDR1 is LFYWMKMFDW (SEQ ID NO: 1), or a variant thereof, CDR2 is GRFIWKSPKQ (SEQ ID NO: 2), or a variant thereof, and CDR3 is NWYWRFNWNYVEWVVQHM (SEQ ID NO: 3), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the first arm of the bispecific molecule comprises a single-chain antibody comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASEWYSHMATWWWFRQAPGKEREFVAVYQW WWWGDKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWLYRSLSMV KDYYYYLTLPYWGQGTLVTVSSGPGGQ (SEQ ID NO: 41), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 8.

In embodiments, the first arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: MQVQLVESGGGVVQPGRSLRLSCAASLFYWMKMFDWWFRQAPGKEREFVAGRFI WKSPKQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNWYWRFNWN YVEWVVQHMWGQGTLVTVSSGPGGQ (SEQ ID NO: 8), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 8.

In embodiments, the first arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: MQVQLVESGGGVVQPGRSLRLSCAASEWYSHMATWWWFRQAPGKEREFVAVYQ WWWWGDKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGPMLNLF HMKVSQGQFTWGQGTLVTVSSGPGGQ (SEQ ID NO: 9), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 9.

In embodiments, the first arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASSMDTSFVANSWFRQAPGKEREFVAGFHAYYR HRHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTAGLGAWLMHIIHG MTHEWGQGTLVTVSSGPGGQ (SEQ ID NO: 20), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 20.

In embodiments, the first arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASNTREYKVPPSWFRQAPGKEREFVAEVYTHHH YYKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNRTIPWVTGYYYAI TDQVWGQGTLVTVSSGPGGQ (SEQ ID NO: 21), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 2E In embodiments, the first arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASHDDHWVQTVRWFRQAPGKEREFVAEKLERR EVYEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMSMEIMARPRER RFQHLWGQGTLVTVSSGPGGQ (SEQ ID NO: 22), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 22.

In embodiments, the second arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQDQTWFGNRWW DLWMQPWWGQGTLVTVSSGPGGQ (SEQ ID NO: 34), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 34.

In embodiments, the second arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSSGFTRWIKFCD SKCSLSAFTQGYGVKYFVARLARMWGGWGQGTLVTVSSGPGGQ (SEQ ID NO: 35), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 35.

In embodiments, the second arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALMAYVREMGQ QVVNWTRERVFFRVGSANVRKVACHGECNWGQGTLVTVSSGPGGQ (SEQ ID NO: 36), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 36.

In embodiments, the second arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSLFRIPAGAWQV VKTYFKYAVYGFGGFGSQCRGHEGESCWGQGTLVTVSSGPGGQ (SEQ ID NO: 37), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 37.

In embodiments, the second arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFWLFNLGRCENA FWMDGWRVFKSLNNTVEDMGFVMSHLLKWGQGTLVTVSSGPGGQ (SEQ ID NO: 38), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 38.

In embodiments, the second arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFNHGGSTWSGLS GAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQWGQGTLVTV SSGPGGQ (SEQ ID NO: 39), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 39.

In embodiments, the second arm of the bispecific molecule comprises a single-chain antibody comprising an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKKMW YDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFNHGGSTWSGLS GAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 40), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 40.

In embodiments, the second binding arm is a peptide selected from QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or a variant thereof, TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof, ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof,

FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the peptide further comprises an additional peptide. In embodiments, the composition comprises a dimer of peptides. In embodiments, the composition comprises a trimer of peptides. In embodiments, the peptides are joined with a linker which is substantially comprised of glycine and serine residues. In embodiments, the linker is (GGS)n, wherein _nis 1, or 2, or 3, or 4, or 5.

In embodiments, the linker is GGSGGSGGSG (SEQ ID NO: 10), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

In embodiments, the composition comprises a hinge-CH₂-CH₃ Fc domain. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from IgG, IgA, IgD, or IgE. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from human IgG, IgA, IgD, or IgE. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from IgGl, IgG2, IgG3, IgGl, IgAl, or IgA2. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from human IgGl, IgG2, IgG3, IgGl, IgAl, or IgA2. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from IgG4. In embodiments, the hinge- CH2-CH3 Fc domain is from or is derived from human IgG4.

In embodiments, the bispecific molecule binds to a protein of the extracellular matrix (ECM), and prevents or reduces tumor growth kinetics of tumorigenesis. In embodiments, the protein of the ECM is one or more of α₅β₁, α₅β₃, and α₅β₅ integrins.

In embodiments, the bispecific molecule binds to the ECM (e.g, one or more of α₅β₁, α₅β₃, and α₅β₅ integrins), and prevents or reduces tumor growth kinetics of breast cancer metastasis or a primary breast tumor. In embodiments, the bispecific molecule binds to ITGAV, and prevents or reduces tumor growth kinetics of breast cancer metastasis or a primary breast tumor.

In embodiments, the bispecific molecule binds to the ECM (e.g., one or more of α₅β₁, o^Ps. and α₅β₅ integrins), and prevents or reduces tumor growth kinetics of a glioblastoma. In embodiments, the bispecific molecule binds to ITGAV, and prevents or reduces tumor growth kinetics of a glioblastoma.

In embodiments, the bispecific molecule binds to the ECM (e.g, one or more of α₅β₁, o^Ps. and α₅β₅ integrins), and prevents or reduces tumor growth kinetics of bladder cancer. In embodiments, the bispecific molecule binds to ITGAV, and prevents or reduces tumor growth kinetics of bladder cancer.

In embodiments, the bispecific molecule binds to the ECM (e.g, one or more of α₅β₁, o^Ps. and α₅β₅ integrins), and prevents or reduces tumor growth kinetics of colorectal cancer. In embodiments, the bispecific molecule binds to ITGAV, and prevents or reduces tumor growth kinetics of colorectal cancer.

In embodiments, the bispecific molecule binds to the ECM (e.g., one or more of α₅β₁, o^Ps. and α₅β₅ integrins), and prevents or reduces tumor growth kinetics of prostate cancer. In embodiments, the bispecific molecule binds to ITGAV, and prevents or reduces tumor growth kinetics of prostate cancer.

In embodiments, the bispecific molecule binds to the ECM (e.g, one or more of α₅β₁, o^Ps. and α₅β₅ integrins), and prevents or reduces tumor growth kinetics of lung cancer. In embodiments, the bispecific molecule binds to ITGAV, and prevents or reduces tumor growth kinetics of lung cancer. In embodiments, the composition further comprises a targeting moiety. In embodiments, the targeting moiety is directed to a tumor cell. In embodiments, the targeting moiety is directed to a tumor-associated antigen (TAA). In embodiments, the TAA is selected from HER2, PSA, TRP-2, EpCAM, GPC3, mesothelin (MSLN), and EGFR.

In embodiments, the present disclosure provides a polynucleotide comprising a nucleic acid sequence encoding the single-chain antibody, or a fragment thereof, or peptide described in any of the preceding embodiments.

In embodiments, the polynucleotide is RNA or DNA. In embodiments, the RNA is a messenger RNA (mRNA) or a modified mRNA.

In embodiments, a vector comprising the polynucleotide is provided. In embodiments, the vector is an expression vectors (e.g, a recombinant expression vector containing a sequence encoding a polypeptide of interest). In embodiments, the expression vector comprises one or more additional sequences such as, but not limited to, regulatory sequences (e.g., promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide variety of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors.

In embodiments, a host cell comprising the vector is provided. Cells may be cultured in vitro or genetically engineered, for example. Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.

Cells suitable for use in the expression of the single-chain antibody, or fragment thereof, as described herein can be eukaryotic cells, more preferably cells of plant, rodent, or human origin, for example but not limited to NSO, CHO, CHOK1, perC.6, Tk-tsl3, BHK, HEK293 cells, COS-7, T98G, CV-l/EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2/0 myeloma cells, and BHK cell lines, among others. In addition, expression of a single-chain antibody may be accomplished using hybridoma cells. Methods for producing hybridomas are well established in the art.

In embodiments, the single-chain antibody, or fragment thereof, is chimeric. As used herein, the term “chimeric” refers to a single-chain antibody, or fragment thereof, having at least some portion of at least one variable domain derived from the antibody amino acid sequence of a non-human mammal, a rodent, or a reptile, while the remaining portions of the antibody, or antigen-binding fragment thereof, are derived from a human.

In embodiments, the single-chain antibody, or fragment thereof, is a humanized antibody. Humanized single-chain antibodies may be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigenbinding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized single-chain antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary -determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In general, the humanized single-chain antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized singlechain antibody may include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

In embodiments, the present disclosure provides a pharmaceutical composition comprising the composition of any of the embodiments disclosed herein, and a pharmaceutically acceptable excipient. A “pharmaceutically acceptable carrier” (also referred to as an “excipient” or a “carrier”) is a pharmaceutically acceptable solvent, suspending agent, stabilizing agent, or any other pharmacologically inert vehicle for delivering one or more therapeutic compounds to a subject (e.g, a mammal, such as a human, non-human primate, dog, cat, sheep, pig, horse, cow, mouse, rat, or rabbit), which is nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties, when combined with one or more of therapeutic compounds and any other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers that do not deleteriously react with amino acids include, by way of example and not limitation: water, saline solution, binding agents (e.g, polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate), lubricants (e.g., starch, polyethylene glycol, or sodium acetate), disintegrates (e.g., starch or sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate). Pharmaceutically acceptable carriers also include aqueous pH buffered solutions or liposomes (small vesicles composed of various types of lipids, phospholipids and/or surfactants which are useful for delivery of a drug to a mammal). Further examples of pharmaceutically acceptable carriers include buffers such as phosphate, citrate, and other organic acids, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, saltforming counterions such as sodium, and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

Pharmaceutical compositions can be formulated by mixing one or more active agents with one or more physiologically acceptable carriers, diluents, and/or adjuvants, and optionally other agents that are usually incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A pharmaceutical composition can be formulated, e.g., in lyophilized formulations, aqueous solutions, dispersions, or solid preparations, such as tablets, dragees or capsules. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences (18th ed, Mack Publishing Company, Easton, PA (1990)), particularly Chapter 87 by Block, Lawrence, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies as described herein, provided that the active agent in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See, also, Baldrick, Regul Toxicol Pharmacol 32:210-218, 2000; Wang, Ini J Pharm 203:1-60, 2000; Charman J Pharm Sci 89:967-978, 2000; and Powell et al. PDA J Pharm Sci Technol 52:238-311, 1998), and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

Pharmaceutical compositions include, without limitation, solutions, emulsions, aqueous suspensions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, for example, preformed liquids, selfemulsifying solids, and self-emulsifying semisolids. Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other; in general, emulsions are either of the water-in-oil (w/o) or oil-in-water (o/w) variety. Emulsion formulations have been widely used for oral delivery of therapeutics due to their ease of formulation and efficacy of solubilization, absorption, and bioavailability.

Compositions and formulations can contain sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers). Compositions additionally can contain other adjunct components conventionally found in pharmaceutical compositions. Thus, the compositions also can include compatible, pharmaceutically active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or additional materials useful in physically formulating various dosage forms of the compositions provided herein, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. Furthermore, the composition can be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings, and aromatic substances. When added, however, such materials should not unduly interfere with the biological activities of the polypeptide components within the compositions provided herein. The formulations can be sterilized if desired.

In embodiments, a composition containing a composition as provided herein can be in the form of a solution or powder with or without a diluent to make an injectable suspension. The composition may contain additional ingredients including, without limitation, pharmaceutically acceptable vehicles, such as saline, water, lactic acid, mannitol, or combinations thereof, for example.

Any appropriate method can be used to administer a composition as described herein to a mammal. Administration can be, for example, parenteral (e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous drip). Administration can be rapid (e.g, by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations). In embodiments, administration can be topical (e.g., transdermal, sublingual, ophthalmic, or intranasal), pulmonary (e.g., by inhalation or insufflation of powders or aerosols), or oral. In addition, a composition containing a composition as described herein can be administered prior to, after, or in lieu of surgical resection of a tumor.

In embodiments, the compositions disclosed herein further comprise a therapeutic agent, as necessary for the particular disorder being treated. In embodiments, the single-chain antibody, or fragment thereof, and the therapeutic agent have complementary activities that do not adversely affect each other. In embodiments, the further therapeutic agent is a chemotherapeutic agent. In embodiments, the further therapeutic agent is a biologic, e.g., an antibody, or e.g., a checkpoint inhibitor.

Methods of Treating or Preventing Diseases

In embodiments, the compositions of the disclosure find use in the treatment or prevention of various diseases or disorders.

In embodiments, the methods disclosed herein treat or prevent cancer by administering an effective amount of the composition that specifically binds to GARP and/or ITGAV. In embodiments, the methods disclosed herein prevent an onset or progression of cancer by administering an effective amount a composition disclosed herein (e.g, a single-chain antibody or peptide) to the subject.

As a non-limiting example, the prevention of an onset, the presence, and/or the evaluation of the progression of a cancer in a subject can be assessed according to the Tumor/Nodes/Metastases (TNM) system of classification (International Union Against Cancer, 6th edition, 2002), or the Whitmore- Jewett staging system (American Urological Association). Typically, cancers are staged using a combination of physical examination, blood tests, and medical imaging. If tumor tissue is obtained via biopsy or surgery, examination of the tissue under a microscope can also provide pathologic staging. In embodiments, the stage or grade of a cancer assists a practitioner in determining the prognosis for the cancer and in selecting the appropriate modulating therapy.

In embodiments, the prevention of an onset, or progression, of cancer is assessed using the overall stage grouping as a non-limiting example: Stage I cancers are localized to one part of the body, typically in a small area; Stage II cancers are locally advanced and have grown into nearby tissues or lymph nodes, as are Stage III cancers. Whether a cancer is designated as Stage II or Stage III can depend on the specific type of cancer. The specific criteria for Stages II and III can differ according to diagnosis. Stage IV cancers have often metastasized or spread to other organs or throughout the body. The onset or progression of cancer can be assessed using conventional methods available to one of skill in the art, such as a physical exam, blood tests, and imaging scans (e.g., X-rays, MRI, CT scans, ultrasound etc.).

As disclosed herein, administering, or administering a treatment/therapy, refers to a treatment/therapy from which a subject receives a beneficial effect, such as the reduction, decrease, attenuation, diminishment, stabilization, remission, suppression, inhibition or arrest of the development or progression of cancer, or a symptom thereof.

In embodiments, the treatment/therapy that a subject receives, or the prevention in the onset of cancer results in at least one or more of the following effects: (1) the reduction or amelioration of the severity of cancer and/or a genetic disease or disorder, and/or a symptom associated therewith; (2) the reduction in the duration of a symptom associated with cancer and/or a genetic disease or disorder; (3) the prevention in the recurrence of a symptom associated with cancer and/or a genetic disease or disorder; (4) the regression of cancer and/or a genetic disease or disorder, and/or a symptom associated therewith; (5) the reduction in hospitalization of a subject; (6) the reduction in hospitalization length; (7) the increase in the survival of a subject; (8) the inhibition of the progression of cancer and/or a genetic disease or disorder and/or a symptom associated therewith; (9) the enhancement or improvement the therapeutic effect of another therapy; (10) a reduction or elimination in the cancer cell population, and/or a cell population associated with a genetic disease or disorder; (11) a reduction in the growth of a tumor or neoplasm; (12) a decrease in tumor size; (13) a reduction in the formation of a tumor; (14) eradication, removal, or control of primary, regional and/or metastatic cancer; (15) a decrease in the number or size of metastases; (16) a reduction in mortality; (17) an increase in cancer-free survival rate of a subject; (18) an increase in relapse- free survival; (19) an increase in the number of subjects in remission; (20) a decrease in hospitalization rate; (21) the size of the tumor is maintained and does not increase in size or increases the size of the tumor by less 5% or 10% after administration of a therapy as measured by conventional methods available to one of skill in the art, e.g, X-rays, MRI, CAT scan, ultrasound etc.; (22) the prevention of the development or onset of cancer and/or a genetic disease or disorder, and/or a symptom associated therewith; (23) an increase in the length of remission for a subject; (24) the reduction in the number of symptoms associated with cancer and/or a genetic disease or disorder; (25) an increase in symptom-free survival of a cancer subject and/or a subject associated with a genetic disease or disorder; and/or (26) limitation of or reduction in metastasis. In embodiments, the treatment/therapy that a subject receives does not cure cancer, but prevents the progression or worsening of the disease. In certain embodiments, the treatment/therapy that a subject receives does not prevent the onset/development of cancer, but may prevent the onset of cancer symptoms.

In embodiments, “preventing” an onset or progression of cancer in a subject in need thereof, is inhibiting or blocking the cancer or disorder. In embodiments, the methods disclosed herein prevent, or inhibit, the cancer or disorder at any amount or level. In embodiments, the methods disclosed herein prevent or inhibit the cancer or genetic disease or disorder by at least or about a 10% inhibition (e.g., at least or about a 20% inhibition, at least or about a 30% inhibition, at least or about a 40% inhibition, at least or about a 50% inhibition, at least or about a 60% inhibition, at least or about a 70% inhibition, at least or about a 80% inhibition, at least or about a 90% inhibition, at least or about a 95% inhibition, at least or about a 98% inhibition, or at least or about a 100% inhibition).

In embodiments, disclosed herein is a method for treating or preventing cancer, comprising administering an effective amount of any one of the compositions described herein to a patient in need thereof. In embodiments, the cancer is a solid tumor. In embodiments, the cancer is a blood cancer. In embodiments, the cancer is selected form one or more of a cancer of a blood vessel, an eye tumor, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; primary breast cancer; metastatic breast cancer, colorectal cancer, cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi’s sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs’ syndrome.

In aspects, the present disclosure provides a method of increasing anti-tumor immunity in a subject having cancer comprising administering an effective amount of composition of any one of the present embodiments to a patient in need thereof or contacting cells with a composition of any one of the present embodiments (e.g. ex vivo, e.g. with allogeneic or autologous cells).

In embodiments, the anti-tumor immunity includes the processing and presentation of released antigens by antigen-presenting cells (APCs), interaction with T lymphocytes, subsequent immune/T-cell activation, trafficking of antigen-specific effector cells, and the engagement of the target tumor cell by the activated effector T cell. Anti -tumor immunity can be imposed by antigen-specific CD8+ T cells and tumoricidal macrophages. Antigens (Ag), typically foreign substances of environmental, viral, or bacterial origin, products of somatically altered proteins, or debris from dying (apoptotic) cells are processed and presented by major histocompatibility complex (MHC) on antigen presenting cells, including (but not limited to) dendritic cells, macrophages, and B cells. CD8+ T cells utilize T cell receptors (TCRs) to recognize WIC-presented peptides and subsequently mount an antigen-specific cytolytic attack. In particular, Ag-TCR engagement ultimately leads to activation and proliferation of CD8+ T cells that play a crucial role in autoimmunity, response to pathogens, and tumor suppression. Also, myeloid cells, including various subsets of monocytes, neutrophils, and macrophages, are implicated in T cell suppression.

In embodiments, increased anti-tumor immunity can be identified by measuring at least one of: increased CD8+ T cells, increased CD4+ T cells, increased CD4+ or CD8+ T cells secreting interferon gamma, and/or decreased CD4+ T cells secreting IL10 among tumorinfiltrating lymphocytes.

In embodiments, disclosed herein is a method for treating or preventing an autoimmune disease or disorder, comprising administering an effective amount of any one of the compositions described herein to a patient in need thereof. In embodiments, the autoimmune disease or disorder is selected from graft versus host disease, transplantation rejection (e.g, prevention of allograft rejection), multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Meniere's syndrome; pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and Grave's disease. In embodiments, the autoimmune disease or disorder is graft versus host disease.

In aspects, the present disclosure provides a method for inhibiting or reducing tumor growth and/or inducing or increasing immune tolerance comprising administering an effective amount of composition of any one of the present embodiments to a patient in need thereof or contacting cells with a composition of any one of the present embodiments (e.g. ex vivo, e.g. with allogeneic or autologous cells).

In embodiments, there is provided a method of antagonizing GARP, comprising contacting a cell expressing GARP, e.g. in vivo or ex vivo, with the present compositions.

In embodiments, there is provided a method of reducing or disrupting an interaction between GARP and TGF-pi, e.g. on the surface of Tregs and/or B cells, e.g. in vivo or ex vivo, with the present compositions. In embodiments, there is provided a method of reducing or disrupting an interaction between GARP and latent TGF-pi, e.g. on the surface of Tregs and/or B cells, e.g. in vivo or ex vivo, with the present compositions.

In embodiments, there is provided a method of modulating a Treg cell, comprising contacting the Treg, e.g. in vivo or ex vivo, with the present compositions.

In embodiments, there is provided a method of reducing an immunosuppressive activity of a cell, e.g. Tregs and/or B cells, and/or a tumor microenvironment with the present compositions. In embodiments, there is provided a method of, e.g. in vivo or ex vivo, depleting Tregs and/or attenuating their suppressive activity with the present compositions. In embodiments, there is provided a method of, e.g. in vivo or ex vivo, reducing T reg-mediated peripheral tolerance in a subject, by administering to the subject a therapeutically effective amount of a composition of the disclosure. In embodiments, the Tregs are Foxp3+ Tregs. In embodiments, the number of Tregs is decreased in the subject relative to prior to administration of the present compositions. The present invention also provides, in embodiments, methods for inhibiting the immune suppressive function of human Tregs in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of the disclosure.

In embodiments, the present disclosure provides a kit comprising at least one GARP- binding composition of the present disclosure. In embodiments, the kit comprises a package or a container comprising at least one reagent, e.g., at least one GARP-binding composition of the present disclosure, for specifically binding to GARP. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Furthermore, any or all of the kit reagents may be provided within containers that protect them from the external environment, such as in sealed containers. The kits may also contain a package insert describing the kit and methods for its use.

As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features. Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present technology, or embodiments thereof, may alternatively be described using more limiting terms such as “consisting of’ or “consisting essentially of’ the recited ingredients.

Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes.

This disclosure is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1: Identification of Peptides, Small Proteins and Antibodies that bind to GARP In the experiments of this example, single-chain antibodies, and peptides based on the CDR3 regions of antibodies, were developed that specifically bind to glycoprotein- A repetitions predominant (GARP). These experiments demonstrate, in part, how the synthesized peptides, and single-chain antibodies, in the presence of TGF-pi, reduce wound healing and migration of A549 cells, which mimics cancer cell migration in vivo.

FIG. 1 shows microscopic images of a A549 wound healing assay. Wounds in A549 cells were created by scraping a monolayer of A549 cells with a pipette tip. Cells were washed with PBS, and the media was replaced with DMEM containing only 1% FBS to limit cellular proliferation. Treatments of either the GARP peptide, “173 3” (SEQ ID NO: 7) (100 pg/ml), or DMSO, were added directly to wells of a 6 well dish. TGF-P peptide (5 ng/ml) was added to all wells. At both hour 0 and hour 16 after the creation of wounds, images were acquired, and wound healing was quantified. The experiments in this example show a reduction in migration of A549 cells by the GARP peptide, “173 3” in three replicates of this experiment.

FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D show images and graphs demonstrating a reduction in migration of A549 cells by the GARP peptide, “173 3” (SEQ ID NO: 7) (FIG. 2A and FIG. 2B), and the GP3 peptide (FIG. 2C and FIG 2D). FIG. 2A (GARP peptide, “173 3”) and FIG. 2C (GP3 peptide) show microscopic images of a wound healing assay. At both hour 0 and hour 16 after the creation of wounds, images were acquired (FIG. 2A, FIG. 2C), and the percentage of wound healing was quantified as shown in FIG. 2B (GARP peptide, “173 3” (SEQ ID NO: 7)), and FIG. 2D (GP3 peptide, a dimer of the GARP peptide). The data in FIG. 2B and FIG. 2D was measured, in part, using an unpaired t-test. In FIG. 2B, the p-value = 0.0059 and a 14.7% average difference was observed between the DMSO (left column), and the GARP peptide, “173 3” (right column). In FIG. 2D, the p-value = 0.0295, and a 8.7% average difference was observed between the DMSO (left column), and the GP3 peptide (right column). The experiments in this example show a reduction in migration of A549 cells by the GARP peptide, “173 3”, and the GP3 peptide.

FIG. 3A shows a microscopic image of an anti-GARP antibody (“Ref Tool 1”), a second anti-GARP antibody (“Ref Tool 2”), and the GARP peptide, “173 3” in an A549 wound healing assay. FIG. 3B shows a graph of percentage wound closure for an anti-GARP antibody (“Ref Tool 1”), a second anti-GARP antibody (“Ref Tool 2”), and the GARP peptide, “173 3” in the A549 wound healing assay. The experiments in this example show a significant reduction in migration of A549 cells by the GARP peptide, “173 3”, compared to the Ref Tool 1 antibody, and the Ref Tool 2. Together, these experiments demonstrate that, inter aha, peptides derived from the present CDR3 regions of the present single-chain antibodies that specifically bind to GARP, reduce wound healing and migration, thereby demonstrating how the synthesized peptides are capable of reducing cancer cell migration in vivo.

Example 2: Identification Of Bispecific Molecules That Bind To ITGAV

In the experiments of this example, bispecific molecules comprising a (1) first binding arm having a single-chain antibody, or a peptide based on the CDR3 region of an antibody, (2) and a second binding arm comprising a peptide or a single-chain antibody, were developed. These experiments demonstrate in part, how the synthesized bispecific molecules bind to a protein of the extracellular matrix, and can prevent or slow down the dissociation of the ECM.

FIG. 4 is a graph showing the overexpression of ITGAV in multiple cancers. This graph shows how ITGAV is overexpressed in multiple cancers and is involved in cell migration and metastasis.

FIG. 5 are fluorescencent microscopic images showing the binding of the bispecific molecules to A549 cells. In these experiments, A549 cells were grown to confluency in a 24 microtiter plate in D5 medium (DMEM-5% FBS-gentamycin). A 1000 pl pipet tip was used to score the plate, leaving a gap between two areas of cells. Control medium and bispecific molecules were then added (final concentration 50pg/ml) to a final volume of 0.5ml. Next, the plates were incubated at 37°C in 8% CO2 for 48 hours. The wells were then photographed under phase, medium was removed, and the cells were washed 2x with Hanks Balanced Salt Solution, and calcium was added (1 Opl in 10ml HBSS; Invitrogen; stock Img/ml). The plates were then incubated at 37°C for 30-45 minutes, and then the wells were washed 2x with HBSS and resuspended in 0.5ml HBSS. The plates were visualized using fluorescence microscopy and photographed (FIG. 5).

Amino acid sequences of ITGAV single chain antibodies in Fig. 5 are shown in table below.

Together, these experiments demonstrate that, inter aha, how the synthesized bispecific molecules bind to a protein of the extracellular matrix (e.g, ITGAV, an integrin of ITGAV, for example, the asfii integrin, the α₅β₃ integrin, and/or the α₅β₅ integrin), thereby demonstrating how the synthesized bispecific molecules prevent or slow down the dissociation of the ECM in vivo.

All of the features disclosed herein may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

Example 3: GARP -ITGAV Bispeciflc Construct Assessment

GARP overexpressed cell lines were developed from U87 (glioblastoma, astrocytoma cell line), G292 (osteosarcoma), and A549 (adenosarcoma) cell lines. All cell lines were transfected with TGFp/SMAD-LucReporter. GARP overexpressed U87 cells were treated with A6pep3 construct (SEQ ID NO: 41) (GARP single chain antibody) and A6pep3:480-l bispecific constructs (SEQ ID NO: 42) (GARP and ITGAV bispecific antibody) at various dosages. Results showed that both A6pep3 and A6pep3:480-l inhibited TGFp/SMAD luciferase activity in U87 GARP overexpressed cell lines (FIG. 6). Alkaline phosphatase (AP) reporter assays were performed to assess inhibition of various GARP constructs. 5K cells were seeded on a 96 well plate overnight. Treatments were added the following day in complete media. Supernatant of treated cells was used to measure AP activity using QUANTI-blue reagent. FIG. 7 A shows absorbance of AP activity of groups treated for 24 hours. FIG. 7B shows absorbance of AP activity of groups treated for 48 hours. FIG. 7C shows representative images of cells at 48 hours post treatment at 20x magnification. Results showed that GARP antibody, A6pep3, inhibited TGFpi activity in AP assay. FIG. 8A- 8B show additional data that A6pep3 inhibited TGFpi activity at various dosages and time course. GARP overexpressed A549 cells were also treated with A6pep3 construct and A6pep3:480-l bispecific construct. FIG. 9A shows GARP bispecific construct blocks migration in GARP overexpressed A549 cells. FIG. 9B shows that both A6pep3 and A6pep3:480-l treatments reduced wound closure compared to untreated cells in a wound healing assay. Statistical analysis was done using an unpaired t-test (p > .05 = *, p > .01 = **, p > .001 = ***, p > .0001 = ****).

TGFP pathway in A549-GARP overexpressed cells were characterized with 173 3 peptides. A549 cells were treated with 5 ngs/mL of TGFpi for 4 hours. For peptide treatments, 100 ugs/mL of peptide 173 3 was used to treat cells for 4 hours. TGFpi and peptide were added simultaneous to cells. Cells were collected and processed for RNA extraction. 173 3 peptide reduced key genes in TGFP pathway as shown in FIG. 10.

Wound healing assays were also performed in WT-G292 cells and G292-GARP overexpressed cells (FIG. 11A-11D). Confluent G292 cells and G292-GARP overexpressing (OE) cells were scratched using a p20 tip. Cells were washed and serum free media was added to cells. The cells were then treated with 5 ngs/mL of TGFpi. Additional treatment of either DMSO or 100 ugs/mL of peptide 173 3 was added to cells. 173 3 peptide inhibited wound healing in both WT-G292 cells and G292-GARP overexpressed cells. In G292-GARP overexpressed cells, 173 3 peptide displayed toxicity to the cells, which was not notable in WT cells.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims

What is claimed is:

1. A composition which specifically binds to glycoprotein-A repetitions predominant (GARP), wherein the composition comprises:

(a) a single-chain antibody, or fragment thereof, comprising a CDR1, CDR2, and CDR3, wherein:

CDR1 is LFYWMKMFDW (SEQ ID NO: 1) or EWYSHMATWW (SEQ ID NO:

4), or MDTSFVANS (SEQ ID NO: 11), or NTREYKVPPS (SEQ ID NO: 12), or HDDHWVQTVR (SEQ ID NO: 13), or a variant thereof,

CDR2 is GRFIWKSPKQ (SEQ ID NO: 2) or VYQWWWWGDK (SEQ ID NO:

5), or GFHAYYRHRH (SEQ ID NO: 14), or EVYTHHHYYK (SEQ ID NO: 15), or EKLERREVYE (SEQ ID NO: 16), or a variant thereof, and

CDR3 is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or

NWYWRFNWNYVEWVVQHM (SEQ ID NO: 3), or

TGPMLNLFHMKVSQGQFT (SEQ ID NO: 6), or

TAGLGAWLMHIIHGMTHE (SEQ ID NO: 17), or

NRTIPWVTGYYYAITDQV (SEQ ID NO: 18), or

QMSMEIMARPRERRFQHL (SEQ ID NO: 19), or a variant thereof; or

(b) a peptide, and the peptide is selected from:

WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof

2. The composition of claim 1, wherein:

CDR1 is EWYSHMATWW (SEQ ID NO: 4), or a variant thereof,

CDR2 is VYQWWWWGDK (SEQ ID NO: 5), or a variant thereof, and

CDR3 is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

3. The composition of claim 1, wherein:

CDR1 is LFYWMKMFDW (SEQ ID NO: 1), or a variant thereof,

CDR2 is GRFIWKSPKQ (SEQ ID NO: 2), or a variant thereof, and CDR3 is NWYWRFNWNYVEWVVQHM (SEQ ID NO: 3), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions. The composition of claim 1, wherein

CDR1 is EWYSHMATWW (SEQ ID NO: 4), or a variant thereof,

CDR2 is VYQWWWWGDK (SEQ ID NO: 5), or a variant thereof, and

CDR3 is TGPMLNLFHMKVSQGQFT (SEQ ID NO: 6), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions. The composition of claim 1, wherein:

CDR1 is MDTSFVANS (SEQ ID NO: 11), or a variant thereof,

CDR2 is GFHAYYRHRH (SEQ ID NO: 14), or a variant thereof, and

CDR3 is TAGLGAWLMHIIHGMTHE (SEQ ID NO: 17), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions. The composition of claim 1, wherein

CDR1 is NTREYKVPPS (SEQ ID NO: 12), or a variant thereof,

CDR2 is EVYTHHHYYK (SEQ ID NO: 15), or a variant thereof, and

CDR3 is NRTIPWVTGYYYAITDQV (SEQ ID NO: 18), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions. The composition of claim 1, wherein

CDR1 is HDDHWVQTVR (SEQ ID NO: 13), or a variant thereof,

CDR2 is EKLERREVYE (SEQ ID NO: 16), or a variant thereof, and

CDR3 is QMSMEIMARPRERRFQHL (SEQ ID NO: 19), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

8. The composition of claim 1, wherein the single-chain antibody, or fragment thereof, further comprises variable region framework (FW) sequences juxtaposed between the CDRs according to the formula (FW1)-(CDR1)-(FW2)-(CDR2)-(FW3)-(CDR3)-(FW4), wherein the variable region FW sequences in the heavy chain variable region are heavy chain variable region FW sequences, and wherein the variable region FW sequences in the light chain variable region are light chain variable region FW sequences.

9. The composition of claim 7, wherein the variable region FW sequences are human.

10. The composition of claim 1 or 2, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASEWYSHMATWWWFRQAPGKEREFVAVYQ WWWWGDI<YADSVI<GRFTISRDNSI<NTLYLQMNSLRAEDTAVYYCARW LYRSLSMVKDYYYYLTLPYWGQGTLVTVSSGPGGQ (SEQ ID NO: 41), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 41.

11. The composition of claim 1 or 3, wherein the single-chain antibody comprises an amino acid sequence of:

MQVQLVESGGGVVQPGRSLRLSCAASLFYWMKMFDWWFRQAPGKEREFVAGR FIWKSPKQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNW YWRFNWNYVEWVVQHMWGQGTLVTVSSGPGGQ (SEQ ID NO: 8), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 8.

12. The composition of claim 1 or 4, wherein the single-chain antibody comprises an amino acid sequence of:

MQVQLVESGGGVVQPGRSLRLSCAASEWYSHMATWWWFRQAPGKEREFVAV YQWWWWGDKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RTGPMLNLFHMKVSQGQFTWGQGTLVTVSSGPGGQ (SEQ ID NO: 9), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 9.

13. The composition of claim 1 or 5, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASSMDTSFVANSWFRQAPGKEREFVAGFHA YYRHRHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTAGL GAWLMHIIHGMTHEWGQGTLVTVSSGPGGQ (SEQ ID NO: 20), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 20.

14. The composition of claim 1 or 6, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASNTREYKVPPSWFRQAPGKEREFVAEVYTH HHYYKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNRTIP WVTGYYYAITDQVWGQGTLVTVSSGPGGQ (SEQ ID NO: 21), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 21.

15. The composition of claim 1 or 7, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASHDDHWVQTVRWFRQAPGKEREFVAEKLE RREVYEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMS MEIMARPRERRFQHLWGQGTLVTVSSGPGGQ (SEQ ID NO: 22), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 22.

16. The composition of claim 1, wherein the peptide is WLYRSLSMVKDYYYYLTLPY (SEQ ID NO: 7), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

17. The composition of any one of claims 1 or 15, wherein the peptide further comprises an additional peptide.

18. The composition of claim 16, wherein the composition comprises a dimer of peptides.

19. The composition of claim 16, wherein the composition comprises a trimer of peptides.

20. The composition of any one of claims 16-19, wherein the peptides are joined with a linker which is substantially comprised of glycine and serine residues.

21. The composition of claim 20, wherein the linker is (GGS)n, wherein nis 1, or 2, or 3, or 4, or 5.

22. The composition of claim 21, wherein the linker is GGSGGSGGSG (SEQ ID NO: 10), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

23. The composition of any one of claims 1-22 wherein the composition comprises a hinge- CH2-CH3 Fc domain.

24. The composition of any one of claims 1-23, wherein the hinge- CH2-CH3 Fc domain is from or is derived from IgG, IgA, IgD, or IgE.

25. The composition of any one of claims 1-24, wherein the hinge- CH2-CH3 Fc domain is from or is derived from human IgG, IgA, IgD, or IgE.

26. The composition of any one of claims 1-25, wherein the hinge- CH2-CH3 Fc domain is from or is derived from IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2.

27. The composition of any one of claims 1-26, wherein the hinge- CH2-CH3 Fc domain is from or is derived from human IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2.

28. The composition of any one of claims 26-27, wherein the hinge- CH2-CH3 Fc domain is from or is derived from IgG4.

29. The composition of any one of claims 26-28, wherein the hinge- CH2-CH3 Fc domain is from or is derived from human IgG4.

30. The composition of any one of claims 1-29, wherein the hinge- CH2-CH3 Fc domain comprises an amino acid sequence of

GPGGPEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<A LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPG K (SEQ ID NO: 43), or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% thereto.

31. The composition of any one of claims 23-30, wherein the human IgG Fc domain comprises one or mutations to reduce or eliminate the effector function of the Fc domains.

32. The composition of claim 31, wherein the mutations are L234A and L235A (LALA) substitutions in human IgGl.

33. The composition of any one of claims 24-32, wherein the human IgGFc domain comprises one or mutations to stabilize a hinge region in the Fc domain.

34. The composition of claim 33, wherein the mutation is S228P.

35. The composition of any one of the above claims, further comprising a targeting moiety.

36. The composition of claim 35, wherein the targeting moiety is directed to a tumor cell.

37. The composition of claim 35, wherein the targeting moiety is directed to a tumor-associated antigen (TAA).

38. The composition of claim 37, wherein the TAA is selected from HER2, PSA, TRP-2, EpCAM, GPC3, mesothelin (MSLN), and EGFR.

39. A polynucleotide comprising a nucleic acid sequence encoding the single-chain antibody, or a fragment thereof, or peptide of any one of the preceding claims.

40. A vector comprising the polynucleotide of claim 39.

41. A host cell comprising the vector of claim 40.

42. A pharmaceutical composition comprising the composition of any of claims 1-41, and a pharmaceutically acceptable excipient or carrier.

43. A composition which specifically binds to integrin subunit alpha-V (ITGAV), wherein the composition comprises:

(a) a single-chain antibody, or fragment thereof, comprising a CDR1, CDR2, and CDR3, wherein: CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof,

CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and

CDR3 is QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or

VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK

(SEQ ID NO: 31), or

SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSS GPGGQ (SEQ ID NO: 32), or FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof; or

(b) a peptide, and the peptide is selected from:

QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or a variant thereof,

TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof,

ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof,

VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or a variant thereof,

FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO: 31), or a variant thereof,

SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGG Q (SEQ ID NO: 32), or a variant thereof, or

FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof. The composition of claim 43, wherein:

CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof, CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and

CDR3 is QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

The composition of claim 43, wherein:

CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof,

CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and

CDR3 is TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

The composition of claim 43, wherein:

CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof,

CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and

CDR3 is ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

The composition of claim 43, wherein:

CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof,

CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and

CDR3 is VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

The composition of claim 43, wherein:

CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof,

CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and CDR3 is FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO: 31), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

49. The composition of claim 43, wherein:

CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof,

CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and

CDR3 is

SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGG Q (SEQ ID NO: 32), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

50. The composition of claim 43, wherein:

CDR1 is PMSPMDSESW (SEQ ID NO: 25), or a variant thereof,

CDR2 is KPKKMWYDKN (SEQ ID NO: 26), or a variant thereof, and

CDR3 is

51. The composition of claim 43, wherein the single-chain antibody, or fragment thereof, further comprises variable region framework (FW) sequences juxtaposed between the CDRs according to the formula (FW1)-(CDR1)-(FW2)-(CDR2)-(FW3)-(CDR3)-(FW4), wherein the variable region FW sequences in the heavy chain variable region are heavy chain variable region FW sequences, and wherein the variable region FW sequences in the light chain variable region are light chain variable region FW sequences.

52. The composition of claim 51, wherein the variable region FW sequences are human.

53. The composition of claim 43 or 44, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKK MWYDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQDQ TWFGNRWWDLWMQPWWGQGTLVTVSSGPGGQ (SEQ ID NO: 34), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 34.

54. The composition of claim 43 or 45, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKK MWYDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSS GFTRWnCFCDSKCSLSAFTQGYGVKYFVARLARMWGGWGQGTLVTVSS GPGGQ (SEQ ID NO: 35), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 35.

55. The composition of claim 43 or 46, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKK MWYDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAL MAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECNWGQGTLVT VSSGPGGQ (SEQ ID NO: 36), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 36.

56. The composition of claim 43 or 47, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKK MWYDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSL FRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESCWGQGTLVTVSS GPGGQ (SEQ ID NO: 37), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 37.

57. The composition of claim 43 or 48, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKK MWYDKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFWL FNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLKWGQGTLVTVS SGPGGQ (SEQ ID NO: 38), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 38.

58. The composition of claim 43 or 49, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKK MWYDI<NYADSVI<GRFTISRDNSI<NTLYLQMNSLRAEDTAVYYCARFNH GGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVS SGPGGQWGQGTLVTVSSGPGGQ (SEQ ID NO: 39), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 39.

59. The composition of claim 43 or 50, wherein the single-chain antibody comprises an amino acid sequence of:

QVQLVESGGGVVQPGRSLRLSCAASPMSPMDSESWWFRQAPGKEREFVAKPKK MWYDI<NYADSVI<GRFTISRDNSI<NTLYLQMNSLRAEDTAVYYCARFNH GGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVS SGPGGQ (SEQ ID NO: 40), or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 40.

60. The composition of claim 43, wherein the peptide is selected from QDQTWFGNRWWDLWMQPW (SEQ ID NO: 27), or a variant thereof, TSSGFTRWIKFCDSKCSLSAFTQGYGVKYFVARLARMWGG (SEQ ID NO: 28), or a variant thereof, ALMAYVREMGQQVVNWTRERVFFRVGSANVRKVACHGECN (SEQ ID NO: 29), or a variant thereof, VSLFRIPAGAWQVVKTYFKYAVYGFGGFGSQCRGHEGESC (SEQ ID NO: 30), or a variant thereof,

FWLFNLGRCENAFWMDGWRVFKSLNNTVEDMGFVMSHLLK (SEQ ID NO: 31), or a variant thereof,

SGLSGAGLTAFRGWGLMQASRQFREWLWAFLWWGQGTLVTVSSGPGGQ (SEQ ID NO: 32), or a variant thereof, FNHGGSTWSGLSGAGLTAFRGWGLMQASRQFREWLWAFLW (SEQ ID NO: 33), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

61. The composition of any one of claims 43 or 60, wherein the peptide further comprises an additional peptide.

62. The composition of claim 61, wherein the composition comprises a dimer of peptides.

63. The composition of claim 61, wherein the composition comprises a trimer of peptides.

64. The composition of any one of claims 61-63, wherein the peptides are joined with a linker which is substantially comprised of glycine and serine residues.

65. The composition of claim 64, wherein the linker is (GGS)n, wherein nis 1, or 2, or 3, or 4, or 5.

66. The composition of claim 65, wherein the linker is GGSGGSGGSG (SEQ ID NO: 10), or a variant thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

67. The composition of any one of claims 43-66, wherein the composition comprises a hinge- CH2-CH3 Fc domain.

68. The composition of any one of claims 43-67, wherein the hinge-CEh-CEE Fc domain is from or is derived from IgG, IgA, IgD, or IgE.

69. The composition of any one of claims 43-68, wherein the hinge-CEh-CEE Fc domain is from or is derived from human IgG, IgA, IgD, or IgE.

70. The composition of any one of claims 43-69, wherein the hinge-CHz-CEE Fc domain is from or is derived from IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2.

71. The composition of any one of claims 43-70, wherein the hinge-CFb-CHa Fc domain is from or is derived from human IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2.

72. The composition of any one of claims 70-71, wherein the hinge-CHz-CEE Fc domain is from or is derived from IgG4.

73. The composition of any one of claims 70-72, wherein the hinge-CEE-CEE Fc domain is from or is derived from human IgG4.

74. The composition of any one of claims 43-73, wherein the hinge-CEE-CEE Fc domain comprises an amino acid sequence of

75. The composition of any one of claims 68-74, wherein the human IgG Fc domain comprises one or mutations to reduce or eliminate the effector function of the Fc domains.

76. The composition of claim 75, wherein the mutations are L234A and L235A (LALA) substitutions in human IgGl.

77. The composition of any one of claims 68-76, wherein the human IgG Fc domain comprises one or mutations to stabilize a hinge region in the Fc domain.

78. The composition of claim 77, wherein the mutation is S228P.

79. The composition of any one of claims 43-78, wherein the composition prevents or reduces tumor growth kinetics of the dissociation of the ECM.

80. The composition of any one of claims 43-79, wherein the composition binds to an integrin.

81. The composition of claim 50, wherein the composition binds to one or more one or more of the α₅β₁, α₅β₃, and α₅β₅ integrins.

82. The composition of any one of claims 43-81, wherein the composition prevents or reduces tumor growth kinetics of tumorigenesis.

83. The composition of any one of claims 43-82, wherein the composition prevents or reduces tumor growth kinetics of breast cancer metastasis or a primary breast tumor.

84. The composition of any one of claims 43-82, wherein the composition prevents or reduces tumor growth kinetics of glioblastoma.

85. The composition of any one of claims 43-82, wherein the composition prevents or reduces tumor growth kinetics of bladder cancer.

86. The composition of any one of claims 43-82, wherein the composition prevents or reduces tumor growth kinetics of colorectal cancer.

87. The composition of any one of claims 43-82, wherein the composition prevents or reduces tumor growth kinetics of prostate cancer.

88. The composition of any one of claims 43-82, wherein the composition prevents or reduces tumor growth kinetics of lung cancer.

89. The composition of any one of claims 43-88, further comprising a targeting moiety.

90. The composition of claim 89, wherein the targeting moiety is directed to a tumor cell.

91. The composition of claim 89, wherein the targeting moiety is directed to a tumor-associated antigen (TAA).

92. The composition of claim 91, wherein the TAA is selected from HER2, PSA, TRP-2, EpCAM, GPC3, mesothelin (MSLN), and EGFR.

93. A polynucleotide comprising a nucleic acid sequence encoding the single-chain antibody, or a fragment thereof, or peptide of any one of claims 43 to 92.

94. A vector comprising the polynucleotide of claim 93.

95. A host cell comprising the vector of claim 94.

96. A pharmaceutical composition comprising the composition of any of claims 43 to 95, and a pharmaceutically acceptable excipient or carrier.

97. A bispecific molecule comprising a first binding arm having the composition of any one of claims 1 to 22, and a second binding arm comprising the composition of any one of claims 43 to 66, wherein the bispecific molecule binds to a protein of the extracellular matrix (ECM).

98. The composition of claim 97, wherein the bispecific molecule comprises a GARP single chain antibody comprising an amino acid sequence of SEQ ID NO: 41, or an amino acid sequence having at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% thereto, and a ITGAV single chain antibody comprising an amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% thereto.

99. The composition of any one of claims 97-98, wherein the single chain antibodies are joined with a linker which is substantially comprised of glycine and serine residues.

100. The composition of claim 99, wherein the linker is (GGS)n, wherein n is 1, or 2, or 3, or 4, or 5.

101. The composition of claim 100, wherein the linker is GGSGGSGGSG (SEQ ID NO: 10), or a variant thereof, wherein the variant comprising about 1, or about 2, or about 3, or about 4, or about 5 mutations, the mutations selected from substitutions or deletions.

102. The composition of any one of claims 97-101, wherein the GARP -ITGAV bispecific molecule comprises an amino acid sequence of: QVQLVESGGGVVQPGRSLRLSCAASEWYSHMATWWWFRQAPGKEREFVAVYQWWW WGDKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWLYRSLSMVKDYY YYLTLPYWGQGTLVTVSSGPGGQGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAA SPMSPMDSESWWFRQAPGKEREFVAKPKKMWYDKNYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCARQDQTWFGNRWWDLWMQPWWGGWGQGTLVTVSSGPGGQ (SEQ ID NO: 42), or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% thereto.

103. The composition of any one of claims 97-102 wherein the composition comprises a hinge- CH2-CH3 Fc domain.

104. The composition of any one of claims 97-103, wherein the hinge-CEE-CEE Fc domain is from or is derived from IgG, IgA, IgD, or IgE.

105. The composition of any one of claims 97-104, wherein the hinge-CEE-CEE Fc domain is from or is derived from human IgG, IgA, IgD, or IgE.

106. The composition of any one of claims 97-105, wherein the hinge-CEE-CEE Fc domain is from or is derived from IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2.

107. The composition of any one of claims 97-106, wherein the hinge-CEE-CEE Fc domain is from or is derived from human IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2.

108. The composition of any one of claims 106-107, wherein the hinge-CEE-CEE Fc domain is from or is derived from IgG4.

109. The composition of any one of claims 106-108, wherein the hinge-CEE-CEE Fc domain is from or is derived from human IgG4.

110. The composition of any one of claims 97-109, wherein the hinge-CEE-CEE Fc domain comprises an amino acid sequence of

111. The composition of any one of claims 104-110, wherein the human IgG Fc domain comprises one or mutations to reduce or eliminate the effector function of the Fc domains.

112. The composition of claim 111, wherein the mutations are L234A and L235A (LALA) substitutions in human IgGl.

113. The composition of any one of claims 104-112, wherein the human IgG Fc domain comprises one or mutations to stabilize a hinge region in the Fc domain.

114. The composition of claim 113, wherein the mutation is S228P.

115. The bispecific molecule of any one of claims 97-114, wherein the bispecific molecule prevents or reduces tumor growth kinetics of the dissociation of the ECM.

116. The bispecific molecule of claim any one of claims 97-114, wherein the bispecific molecule binds to an integrin.

117. The bispecific molecule of claim 116, wherein the bispecific molecule binds to one or more one or more of the α₅β₁, α₅β₃, and α₅β₅ integrins.

118. The bispecific molecule of any one of claims 97-114, wherein the bispecific molecule prevents or reduces tumor growth kinetics of tumorigenesis.

119. The bispecific molecule of any one of claims 97-114, wherein the bispecific molecule prevents or reduces tumor growth kinetics of breast cancer metastasis or a primary breast tumor.

120. The bispecific molecule of any one of claims 97-114, wherein the bispecific molecule prevents or reduces tumor growth kinetics of glioblastoma.

121. The bispecific molecule of any one of claims 97-114, wherein the bispecific molecule prevents or reduces tumor growth kinetics of bladder cancer.

122. The bispecific molecule of any one of claims 97-114, wherein the bispecific molecule prevents or reduces tumor growth kinetics of colorectal cancer.

123. The bispecific molecule of any one of claims 97-114, wherein the bispecific molecule prevents or reduces tumor growth kinetics of prostate cancer.

124. The bispecific molecule of any one of claims 97-114, wherein the bispecific molecule prevents or reduces tumor growth kinetics of lung cancer.

125. The composition of any one of claims 97-124, further comprising a targeting moiety.

126. The composition of claim 125, wherein the targeting moiety is directed to a tumor cell.

127. The composition of claim 125, wherein the targeting moiety is directed to a tumor- associated antigen (TAA).

128. The composition of claim 127, wherein the TAA is selected from HER2, PSA, TRP-2, EpCAM, GPC3, mesothelin (MSLN), and EGFR.

129. A polynucleotide comprising a nucleic acid sequence encoding the single-chain antibody, or a fragment thereof, or peptide of any one of claims 97 to 114.

130. A vector comprising the polynucleotide of claim 129.

131. A host cell comprising the vector of claim 130.

132. A pharmaceutical composition comprising the composition of any of claims 97 to 131, and a pharmaceutically acceptable excipient or carrier.

133. A method for treating or preventing cancer, comprising administering an effective amount of the composition of any one of claims 1 to 132 to a patient in need thereof.

134. The method of claim 133, wherein the cancer is a solid tumor.

135. The method of claim 133, wherein the cancer is a blood cancer.

136. The method of any one of claims 133-135, wherein the cancer is selected form one or more of a cancer of a blood vessel, an eye tumor, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; primary breast cancer; metastatic breast cancer, colorectal cancer, cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi’s sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs’ syndrome.

137. The method of claim 136, wherein the primary breast cancer expresses the integrin subunit alpha- V gene (ITGAV).

138. A method for treating or preventing an autoimmune disease or disorder, comprising administering an effective amount of the composition of any one of claims 1-132 to a patient in need thereof.

139. The method of claim 138, wherein the autoimmune disease or disorder is selected from graft versus host disease, transplantation rejection (e.g., prevention of allograft rejection), multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Meniere's syndrome; pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and Grave's disease.

140. The method of claim 138, wherein the autoimmune disease or disorder is graft versus host disease.