Integrin beta 6 (ITGB6) targeting trispecific protein for treatment of cancer

Abstract

Provided herein are ITGB6 binding proteins, pharmaceutical compositions comprising such proteins or fragments thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such ITGB6 binding proteins. Also disclosed are methods of using the disclosed ITGB6 binding proteins in the prevention, and / or treatment diseases, conditions and disorders associated with ITGB6.

Description

INTEGRIN BETA 6 (ITGB6) TARGETING TRISPECIFIC PROTEIN FOR TREATMENTOF CANCERCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 387,007, filed December 12, 2022, and U.S. Provisional Application No. 63 / 496,182, filed on April 14, 2023, each of which is incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BACKGROUND OF THE DISCLOSURE

[0003] Integrins are a0 heterodimeric glycoproteins which span the cell’s membrane, integrating the outside and inside of the cell. Integrins bind to specific extracellular proteins in the extracellular matrix or on other cells and subsequently transduce signals intracellularly to affect cell behavior. Integrin beta 6 (encoded by the ITGB6 gene in humans) is the 06 subunit of the integrin av06. 06 associates exclusively with the av subunit. Integrin av06 is found on epithelial cells. In most resting normal cells, little ITGB6 is produced, however the highest levels are found in the stomach, gall bladder and lung. ITGB6 levels increase in cells remodeling tissues so av06 expression is increased in development, wound healing, fibrosis and cancer.

[0004] The principal function of av06 is the activation of cytokine transforming growth factor-bl (TGF-01). Latent-TGF-01 is bound to the extracellular matrix, covered by its pro-peptide latency associated peptide (LAP). av06 binds LAP, and releases TGF-01 through cytoskeletal force. TGF- 01 regulates multiple processes including cell proliferation, differentiation, angiogenesis, epithelial- mesenchymal-transition (EMT) and immune suppression. These processes combine to heal wounds but when uncontrolled can promote tissue pathologies. As av06 expression is increased in response to tissue damage and is a principal activator of TGF-0 (a major driver of fibroblast activation), it is therefore a potential drug target in treating fibrosis. Increased av06 expression occurs in up to one third of solid tumors including breast cancer, lung cancer and pancreatic cancer. Integrin av06 promotes tumor progression in multiple ways. When av06 is over-expressed in cancers it often correlates with poorer overall survival. Because it is not found on most normal cells, it is a potential therapeutic and imaging target in cancer research.

[0005] There is a need for a greater choice of treatment options which allows physicians to select the therapeutic with the best side effect profile for an individual patient. The present disclosure provides novel polypeptides and protein therapeutics useful in methods of treatment, particularly for treatment of conditions associated with abnormal expression of Integrin beta 6 (ITGB6).SUMMARY OF THE DISCLOSURE

[0006] Provided herein is an ITGB6 binding domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149- 222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296. In some embodiments, the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296. In some embodiments, an amino acid sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74. In some embodiments, the ITGB6 binding domain is part of a multispecific protein. In some embodiments, the multispecific protein further comprises a CD3 binding domain. In some embodiments, the multispecific protein comprises an active drug format. In some embodiments, the multispecific protein further comprises a bulk serum protein binding domain. In some embodiments, the bulk serum protein comprises a serum albumin protein. In some embodiments, the serum albumin protein comprises a human serum albumin protein. In some embodiments, the bulk serum protein binding domain comprises a sequence that is at least 75% identical the sequence as set forth in SEQ ID NO: 654 or 728. In some embodiments, the CD3 binding domain comprises a sequence that is at least 75% identical the sequence as set forth in SEQ ID NO: 655. In some embodiments, the multispecific protein comprises a sequence that is at least about 75% identical to the sequence as set forth in SEQ ID NOS: 353-357. In some embodiments, the bulk serum protein binding domain is a binding moiety comprising a linker and a masking moiety, wherein the masking moiety masks or is capable of masking the binding of the ITGB6 binding domain or the CD3 binding domain, to their respective targets. In some embodiments, the multispecific protein comprises a non-cleavable prodrug format. In some embodiments, the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOS: 801-845, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 801-845. In some embodiments, the linker comprises a sequence selected from the group consisting of SEQ ID NOS: 659-661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 718-727, 731-772, and 846-856, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 659-661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 718-727, 731-772, and 846-856. In some embodiments, the bulk serum protein binding domain comprises a sequence that is at least 75% identical the sequence selected from the group consisting of SEQ ID NO: 728. In some embodiments, wherein the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO: 655. In some embodiments, wherein the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 345-357. In some embodiments, the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 345-352. In some embodiments, the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the active drug comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 297-344.

[0007] In some embodiments, the ITGB6 binding domain is part of a chimeric antigen receptor (a CAR) or a conditionally activatable chimeric antigen receptor (a ProCAR), wherein the CAR further comprises at least one of a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In some embodiments, the ITGB6 binding domain is part of the ProCAR and the ProCAR further comprises (a) a binding moiety comprising a non-CDR loop and a cleavable linker; (b) a transmembrane domain; and (c) an intracellular signaling domain; wherein the binding moiety is capable of masking the binding of the ITGB6 binding domain to its target. In some embodiments, the binding moiety further comprises one or more complementarity determining regions (CDRs). In some embodiments, the CDR loop provides a binding site specific for a bulk serum protein. In some embodiments, the bulk serum protein comprises at least one of: a serum albumin, a transferrin, an IgGl, an IgG2, an IgG4, an IgG3, an IgA monomer, a Factor XIII, a fibrinogen, a pentameric IgM. In some embodiments, the bulk serum protein comprises the serum albumin. In some embodiments, the serum albumin is a human serum albumin. In some embodiments, the ProCAR further comprising a costimulatory domain, wherein the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDl la / CD18), ICOS (CD278), and 4-lBB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.

[0008] In some embodiments, the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the encoded T-cell receptor fusion protein. In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some embodiments, the intracellular signaling domain is derived from CD3 epsilon, CD3 gamma, CD3 delta, CD3 alpha, CD3 beta, or a combination thereof. In some embodiments, the intracellular signaling domain is derived from CD3 epsilon.

[0009] Provided herein is a method for the treatment or amelioration of a proliferative disease, or a tumorous disease, comprising the administration of an ITGB6 binding domain according to descriptions herein, or a pharmaceutical composition comprising the same, to a subject in need thereof. In some embodiments, the subject is human.

[0010] Provided here in is a conditionally active chimeric antigen receptor (ProCAR) that comprises a single polypeptide chain, comprising (a) a binding moiety comprising a non-CDR loop and a cleavable linker; (b) an ITGB6 binding domain, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296; (c) a transmembrane domain; and (d) an intracellular signaling domain; wherein the binding moiety is capable of masking the binding of the ITGB6 binding domain to its target.

[0011] In some embodiments, the ITGB6 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74.

[0012] Provided herein is a conditionally active ITGB6 binding protein comprising a binding moiety (M) which comprises a non-CDR loop, a cleavable linker (L), a first target antigen binding domain (Tl), and a second target antigen binding domain (T2), wherein at least one of the first target antigen binding domain (Tl) and the second target antigen binding domain (T2) comprisesan ITGB6 binding domain, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296, wherein the non-CDR loop is capable of binding to the ITGB6 binding domain or the second target antigen binding domain, and wherein the binding moiety masks or is capable of masking the binding of the ITGB6 binding domain or the second target antigen binding domain to its target. In some embodiments, the binding moiety comprises a masking moiety and wherein the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOS: 801-845, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOS: 801-845. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854. In some embodiments, the binding moiety comprises a sequence that is at least 75% identical the sequence selected from the group consisting of SEQ ID NO: 728. In some embodiments, the second target antigen binding domain (T2) comprises a CD3 binding domain. In some embodiments, the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO: 655.

[0013] Provided herein is a method for the treatment or amelioration of a proliferative disease, or a tumorous disease, comprising the administration of a conditionally active chimeric antigen receptor according to descriptions herein, or a pharmaceutical composition comprising the same, to a subject in need thereof. In some embodiments, the subject is human.

[0014] Provided herein is a method for the treatment or amelioration of a proliferative disease, or a tumorous disease, comprising the administration of a conditionally active ITGB6 binding protein according to descriptions herein, or a pharmaceutical composition comprising the same, to a subject in need thereof. In some embodiments, the subject is human.

[0015] In some embodiments, the binding domain is a humanized antibody or an antigen binding fragment thereof. In some embodiments, the binding domain is a single domain antibody, a VHHdomain, a scFv, a VH domain, a VL domain, a Fab, a Fab’, a non-Ig domain, a ligand, a knotin, or a small molecule entity. In some embodiments, binding domain comprises the single domain antibody. In some embodiments, the binding domain binds to ITGB6 with a binding affinity (Kd) of about 0.001 nM to about 500 nM. In some embodiments, the binding domain binds to human ITGB6, mouse ITGB6, cynomolgus ITGB6, or a combination thereof.

[0016] Provided herein is a multispecific protein comprising an ITGB6 binding domain, wherein the ITGB6 binding domain is according to descriptions herein. In some embodiments, the ITGB6 binding domain according to descriptions herein (anti-ITGB6 domain), and a CD3 binding domain (anti-CD3 domain). In some embodiments, the anti-ITGB6 domain and the anti-CD3 domain are in an anti-ITGB6:anti-CD3 orientation. In some embodiments, the anti-ITGB6 domain and the anti- CD3 domain are in an anti-CD3: anti-ITGB6 orientation. In some embodiments, the ITGB6 binding domain according to descnptions herein (anti-ITGB6 domain), the CD3 binding domain (anti-CD3 domain), and an albumin binding domain (anti-ALB domain). In some embodiments, the anti-CD3 domain comprises an amino acid as set forth in SEQ ID NO: 655. In some embodiments, the anti- ALB domain comprises an amino acid sequence as set forth in SEQ ID NO: 728. In some embodiments, the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3: anti-ALB: anti-ITGB6 orientation. In some embodiments, the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ITGB6: anti-ALB: anti-CD3 orientation. In some embodiments, the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB: anti-ITGB6: anti-CD3 orientation. In some embodiments, the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3: anti-ITGB6: anti-ALB orientation. In some embodiments, the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB: anti-CD3: anti-ITGB6 orientation. In some embodiments, the anti- ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ITGB6: anti-CD3: anti-ALB orientation.

[0017] Provided here in is a multivalent protein comprising a sequence as set forth in any one of SEQ ID NOS: 345-357.

[0018] Provided herein is an active drug comprising a sequence as set forth in any one of SEQ ID NOS: 297-337.

[0019] Provided herein is an active drug comprising a sequence as set forth in any one of SEQ ID NOS: 338-344.

[0020] Provided herein is a pharmaceutical composition compnsing (i)(a) an ITGB6 binding domain according to descnptions herein; (i)(b) a conditionally active chimeric antigen receptor according to descriptions herein; (i)(c) a conditionally active ITGB6 binding protein according todescriptions herein; (i)(d) a multispecific protein according to descriptions herein; (i)(e) a multivalent protein according to descriptions herein; or (i)(f) an active drug according to descriptions herein, and (ii) a pharmaceutically acceptable carrier.

[0021] Provided herein is a process producing an ITGB6 binding domain according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the ITGB6 binding domain according to descriptions herein, under conditions allowing the expression of the ITGB6 binding domain and recovering and purifying the produced protein from the culture.

[0022] Provided herein is a process for producing a multispecific protein according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the multispecific ITGB6 binding protein according to descriptions herein under conditions allowing the expression of the multispecific protein and recovering and purifying the produced protein from the culture.

[0023] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering an ITGB6 binding domain according to descriptions herein, or a pharmaceutical composition according to descriptions herein, to the subject.

[0024] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering a multispecific protein according to descriptions herein, a multivalent protein according to descriptions herein, an active drug according to descriptions herein, or a pharmaceutical composition according to descriptions herein, to the subject. In some embodiments, the subject is human. In some embodiments, the method further comprises administration of an agent in combination with an ITGB6 binding domain according to descnptions herein, a multispecific protein according to descriptions herein, a multivalent protein according to descriptions herein, an active drug according to descriptions herein, or a pharmaceutical composition according to descriptions herein. In some embodiments, the ITGB6 binding domain selectively binds to tumor cells expressing ITGB6. In some embodiments, the tumorous disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. In some embodiments, the tumorous disease comprises at least one of: a colorectal cancer, a prostate cancer, a neuroendocrine cancer, a thyroid cancer, a nonsmall cell lung cancer, a small cell lung cancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, a biliary track cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, or a combination thereof. In some embodiments, the method further comprises administration of an agent in combination with conditionally active chimericantigen receptor according to descriptions herein, conditionally active ITGB6 binding protein according to any one of descriptions herein, or a pharmaceutical composition comprising the same. In some embodiments, the ITGB6 binding domain selectively binds to tumor cells expressing ITGB6. In some embodiments, the tumorous disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. In some embodiments, the tumorous disease is at least one of: a colorectal cancer, a prostate cancer, a neuroendocrine cancer, a thyroid cancer, a non-small cell lung cancer, a small cell lung cancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, a biliary track cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, or any combination thereof.

[0025] Provided herein is a process for the production of a conditionally active chimeric antigen receptor according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the conditionally active chimeric antigen receptor according to descriptions herein, under conditions allowing the expression of the conditionally active chimeric antigen receptor and recovering and purifying the produced protein from the culture.

[0026] Provided herein is a process for the production of a conditionally active ITGB6 binding protein according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the conditionally active ITGB6 binding protein according to descriptions herein, under conditions allowing the expression of the conditionally active ITGB6 binding protein and recovering and purifying the produced protein from the culture.

[0027] Provided herein is a process for producing a multivalent protein according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the multivalent protein according to descriptions herein, under conditions allowing the expression of the multivalent protein and recovering and purifying the produced protein from the culture.

[0028] Provided herein is a process for producing an active drug according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the active drug according to descriptions herein, under conditions allowing the expression of the active drug and recovering and purifying the produced drug from the culture.

[0029] Provided herein is a cell comprising a CAR according to descriptions herein. Provided herein is a cell comprising a ProCAR according to descriptions herein. In some embodiments, the cell is a T cell or a natural killer (NK) cell. Provided herein is a method comprising transfecting acell according to descriptions herein, with a vector or an RNA comprising a nucleotide sequence encoding the CAR or the ProCAR.

[0030] Provided herein is a conditionally active chimeric antigen receptor, wherein the conditionally active chimeric antigen receptor has a greater therapeutic index compared to a chimeric antigen receptor (CAR) that does not comprise the (a) binding moiety but is otherwise identical to the conditionally active chimeric antigen receptor. In some embodiments, the conditionally active chimeric antigen receptor has a therapeutic index that is at least about 5-fold to about 100-fold greater than that of a chimeric antigen receptor (CAR) that does not comprise the (a) binding moiety but is otherwise identical to the conditionally active chimeric antigen receptor. In some embodiments, the protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, wherein the protein comprises a sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, wherein the protein comprises a sequence that is at least about 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, wherein the conditionally active ITGB6 binding protein has a greater therapeutic index compared to an ITGB6 binding protein that does not comprise the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active ITGB6 binding protein. In some embodiments, the conditionally active ITGB6 binding protein has a therapeutic index that is at least about 5-fold to about 100-fold greater than that of an ITGB6 binding protein that does not comprise the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active ITGB6 binding protein.

[0031] Provided herein is a pharmaceutical composition comprising: (i)(a) a conditionally active chimeric antigen receptor according to descriptions herein; or (i)(b) a conditionally active ITGB6 binding protein according to descriptions herein, and (ii) a pharmaceutically acceptable carrier.

[0032] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering the conditionally active chimeric antigen receptor according to descriptions herein, or the pharmaceutical composition according to descriptions herein, to the subject.

[0033] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering the conditionally active ITGB6 binding protein according to descriptions herein, or the pharmaceutical composition according to descriptions herein, to the subject. In some embodiments, the subject is human. In some embodiments, the tumorous disease comprises at least one of: a colorectal cancer, a prostate cancer, a neuroendocrine cancer, a thyroid cancer, a non-small cell lung cancer, a small cell lungcancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, a biliary track cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, or any combination thereof.

[0034] Provided herein is a method of increasing a therapeutic index of an ITGB6 binding domain, the method comprising conjugating the ITGB6 binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein the non-CDR loop comprises a binding site specific for the ITGB6 binding domain, wherein the ITGB6 binding domain is masked from binding its target by the binding moiety, wherein the ITGB6 binding domain is able to bind its target upon cleavage of the cleavable linker. In some embodiments, the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296. In some embodiments, the ITGB6 binding domain conjugated to the binding moiety is part of a conditionally active multispecific protein, wherein the multispecific protein further comprises a CD3 binding domain. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 728. In some embodiments, the CD3 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 655. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854. In some embodiments, the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to asequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the ITGB6 binding domain conjugated to the binding moiety is part of a conditionally active chimeric antigen receptor, wherein the conditionally active chimeric antigen receptor further comprises at least one of: a transmembrane domain, an intracellular signaling domain, and a costimulatory domain. In some embodiments, the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 728. In some embodiments, the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74. In some embodiments, the ITGB6 binding domain comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74.

[0035] Provided herein is a method of increasing a therapeutic index of an ITGB6 binding protein comprising a first target antigen binding domain and a second target antigen binding domain, wherein at least one of the first and the second target antigen binding domain comprises an ITGB6 binding domain, the method comprising conjugating the first or the second target antigen binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein the non- CDR loop comprises a binding site specific for the first or the second target antigen binding domain, wherein at least one of the first or the second target antigen binding domain is masked from binding its target by the binding moiety, and wherein the first or the second target antigen binding domain that is masked, is able to bind its target upon cleavage of the cleavable linker. In some embodiments, the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149- 222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS:223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296. In some embodiments, the non-CDR loop comprises a binding site specific for the ITGB6 binding domain. In some embodiments, at least one of the first or the second target antigen binding domain comprises a CD3 binding domain. In some embodiments, the non-CDR loop comprises a binding site specific for the CD3 binding domain. In some embodiments, the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 655. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 728. In some embodiments, the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846- 854, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

[0036] Provided herein is a method of increasing a therapeutic index of an ITGB6 binding protein comprising an ITGB6 binding domain and a CD3 binding domain, the method comprising conjugating CD3 binding domain to a binding moiety comprising a cleavable linker and a non- CDR loop, wherein the non-CDR loop comprises a binding site specific for CD3 binding domain. In some embodiments, the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149- 222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296. In some embodiments, the CD3 binding domain comprises asequence that is at least about 75% identical to SEQ ID NO: 655. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 728. In some embodiments, the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

[0037] Provided herein is an ITGB6 targeting conditionally active multispecific protein, comprising: an ITGB6 binding domain, a CD3 binding domain, an albumin binding domain, wherein the albumin binding domain comprises a non-CDR loops that comprises a binding site specific for the CD3 binding domain and a cleavable linker, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 39-76; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296. In some embodiments, the albumin binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 728. In some embodiments, the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668,670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

[0038] Provided herein is a pharmaceutical composition compnsing an ITGB6 targeting conditionally active multispecific protein of descriptions herein. In some embodiments, further comprising a pharmaceutically acceptable earner.

[0039] Provided herein is a process for the production of ITGB6 targeting conditionally active multispecific protein of descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the ITGB6 targeting conditionally active multispecific protein of descriptions herein, under conditions allowing the expression of the ITGB6 targeting conditionally active multispecific protein and recovering and purifying the produced protein from the culture.

[0040] Provided herein is a method for the treatment or amelioration of a proliferative disease, or a tumorous disease, comprising the administration of an of ITGB6 targeting conditionally active multispecific protein of descriptions herein, or a pharmaceutical composition according to descriptions herein, to a subject in need thereof. In some embodiments, the tumorous disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. In some embodiments, the tumorous disease comprises at least one of: a colorectal cancer, a prostate cancer, a neuroendocrine cancer, a thyroid cancer, a non-small cell lung cancer, a small cell lung cancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, a biliary track cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0042] FIG. 1 provides results from a TDCC assay with CAPAN2 cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequences I6L61, 16L190, 16L272, 16L265, and I6L19.

[0043] FIG. 2 provides results from a TDCC assay with CAPAN2 cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequences I6L241, I6L243, 16L260, I6L280, and I6L211.

[0044] FIG. 3 provides results from a TDCC assay with CAPAN2 cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequences I6L262, 16L254, 16L17, 16L164, and I6L133.

[0045] FIG. 4 provides results from a TDCC assay with CAPAN2 cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequences I6L292, 16L290, 16L41, 16L32, and I6L29.

[0046] FIG. 5 provides results from a TDCC assay with CAPAN2 cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequences I6L25, 16L294, 16L70, and I6L28.

[0047] FIG. 6 provides results from a TDCC assay with CAPAN2 cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequences I6L152, 16L21, 16L83, 16L69, and I6L160.

[0048] FIG. 7 provides results from a TDCC assay with CAPAN2 cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequences I6L33, 16L18, and negative control GFP.

[0049] FIG. 8 provides results from a TDCC assay with CAPAN2 cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequence I6L251.

[0050] FIG. 9 provides results from a TDCC assay with CAPAN2 cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequence I6L285.

[0051] FIG. 10 provides results from a TDCC assay with SW780 Cells and anti-CD3 / anti-ITGB6 fusion proteins containing llama anti-ITGB6 sequence I6L18, 16L32, 16L41, 16L61, and I6L211.

[0052] FIG. 11 provides results from a TDCC assay with SW780 Cells and anti-CD3 / anti-ITGB6 fusion proteins containing llama anti-ITGB6 sequence I6L251, 16L265, and I6L285.

[0053] FIG. 12 provides results from a TDCC assay with HPAFII Cells and anti-CD3 / anti- ITGB6 fusion proteins containing llama anti-ITGB6 sequence I6L18, 16L32, 16L41, 16L61, I6L211, 16L265, and I6L285.

[0054] FIG. 13 provides results from a TDCC assay with HPAFII Cells and anti-CD3 / anti- ITGB6 fusion proteins containing humanized anti-ITGB6 sequence I6H18C, I6H32A, I6H41C, I6H61A, I6H211B, I6H265A, and I6H285A.

[0055] FIG. 14 provides results from a SDS-PAGE Gel of anti-CD3 / anti-ITGB6 non-cleavable fusion proteins consisting of llama anti-ITGB6 sequence I6L18, 16L32, 16L41, 16L61, 16L21L I6L251, 16L265, and I6L285.

[0056] FIG. 15 provides results from a flow cytometry assay of the binding to cells as histogram overlays of anti-CD3 / anti-ITGB6 Non-cleavable fusion proteins containing llama anti-ITGB6 sequence I6L18, 16L32, 16L41, 16L61, 16L211, 16L265, and I6L285 to SW780, HCC70, and HPAF-II cells.

[0057] FIG. 16 shows results from a SDS-PAGE of humanized anti-ITGB6 ProTriTAC linker 040 fusion proteins I6H41C, I6H61A, I6H265A, and I6H285A.

[0058] FIG. 17 shows results of a flow cytometry assay of humanized anti-CD3 / anti-ITGB6 ProTriTAC Linker 040 fusion proteins on human primary alveolar cells.

[0059] FIG. 18 shows results of a flow cytometry assay of humanized anti-CD3 / anti-ITGB6 ProTriTAC linker 040 fusion proteins on cynomolgus monkey (Cyno) primary alveolar cells.

[0060] FIG. 19 shows results of a TDCC assay with HCC70 cells and anti-CD3 / anti-ITGB6 fusion proteins containing humanized anti-ITGB6 sequence I6H18C and I6H41C.

[0061] FIG. 20 shows results of a TDCC assay with SW780 cells and anti-CD3 / anti-ITGB6 fusion proteins containing humanized anti-ITGB6 sequence I6H18C and I6H41C

[0062] FIG. 21 shows results of a TDCC assay with HT29 cells and anti-CD3 / anti-ITGB6 fusion proteins containing humanized anti-ITGB6 sequence I6H18C and I6H41C.

[0063] FIG. 22 shows results of a TDCC assay with CAL27 cells and anti-CD3 / anti-ITGB6 fusion proteins containing humanized anti-ITGB6 sequence I6H18C and I6H41C.

[0064] FIG. 23 shows results of a cell-based assay with HCC70 cells and anti-CD3 / anti-ITGB6 ProTriTAC Linker 040 fusion proteins containing humanized anti-ITGB6 sequence I6H18C and I6H41C.

[0065] FIG. 24 shows results of a cell-based assay with SW780 Cells and anti-CD3 / anti-ITGB6 ProTriTAC Linker 040 fusion proteins containing humanized anti-ITGB6 sequence I6H18C and I6H41C.

[0066] FIG. 25 shows results of a cell-based assay with HT29 Cells and anti-CD3 / anti-ITGB6 ProTriTAC Linker 040 fusion proteins containing humanized anti-ITGB6 sequence I6H18C and I6H41C.

[0067] FIG. 26 shows results of a cell-based assay with CAL27 Cells and anti-CD3 / anti-ITGB6 ProTriTAC Linker 040 fusion proteins containing humanized anti-ITGB6 sequence I6H18C and I6H41C.

[0068] FIGS. 27A-27B demonstrate ProTriTAC has substantially reduced pre-cleaved active drug in CHO productions with L276 versus L040 cleavable linker in 4-20% TRIS glycine with 16 pl CM loaded (FIG. 27A) or 10-20% TRIS glycine with 10 pl CM loaded (FIG. 27B).

[0069] FIG. 28 provides protease substrates by sequence and selectivity to metalloproteinases.

[0070] FIGS. 29A-29F illustrate exemplary ProCAR constructs. FIG. 29A illustrates construct C2483 which includes an anti-human serum albumin sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 662). FIG. 29B illustrates construct C3544 which includes an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 663). FIG. 29C illustrates construct C3546 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 4 (SEQ ID NO: 664), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 665). FIG. 29D illustrates construct C3548 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an antihuman EpCAM sdAb, a protease cleavable linker 5 (SEQ ID NO: 666), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 667). FIG. 29E illustrates construct C3549 which includes an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a protease cleavable linker 6 (SEQ ID NO: 668), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 669). FIG. 29F illustrates construct C3550 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 7 (SEQ ID NO: 670), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 671).

[0071] FIGS. 30A-30D illustrate that pan-MMP substrates are not stable when expressed on T cells in ProCAR format. CAR-T cells made from the constructs of FIG. 29 were prepared. Shown are dot plots of M2 staining (x-axis; BV421 channel) and EpCAM-Fc staining (y-axis; Al exaFluor 647 channel) of CAR-T cells. FIG 30A provides results for constructs C2483, C3546, and C3544. FIG. 30B provides results for constructs C2483, C3548, and C3544. FIG. 30C provides results for constructs C2483, C3549, and C3544. FIG. 30D provides results for constructs C2483, C3550, and C3544.

[0072] FIG. 31 illustrates that MMP2 and MMP7 are expressed at very low levels in T cells compared to MMP9.

[0073] FIGS. 32A-32E illustrate exemplary ProCAR constructs. FIG. 32A illustrates construct C2483 which includes an anti-human serum albumin sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 662). FIG. 32B illustrates construct C3269 includes an anti-human serum albuminsdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- 1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 672). FIG. 32C illustrates construct C3440 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 8 (SEQ ID NO: 673), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 674). FIG. 32D illustrates construct C3444 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 9 (SEQ ID NO: 675), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 689). FIG. 32E illustrates construct C3445 which includes an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a protease cleavable linker 10 (SEQ ID NO: 676), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 690).

[0074] FIGS. 33A-33C illustrate that MMP2-selective substrates show increased stability when expressed on T cells in ProCAR format. CAR-T cells made from the constructs of FIG. 32 were prepared. Shown are dot plots of M2 staining (x-axis; BV421 channel) and EpCAM-Fc staining (y- axis; AlexaFluor 647 channel) of CAR-T cells. FIG 33A provides results for constructs C3440 and C3269 for MMP2-selective substrate 1. FIG. 33B provides results for constructs C3444 and C3269 for MMP2-selective substrate 2. FIG. 33C provides results for constructs C3445 and C3269 for MMP2-selective substrate 3.

[0075] FIG. 34A-34E illustrate exemplary ProCAR constructs. FIG. 34A illustrates construct C2483 which includes an anti-human serum albumin sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 662). FIG. 34B illustrates construct C3543 includes an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- 1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 677). FIG. 34C illustrates construct C3558 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 11 (SEQ ID NO: 678), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 679). FIG. 34D illustrates construct C3559 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 12 (SEQ ID NO: 680), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 681). FIG. 34E illustrates construct C3560 which includes an anti-human serumalbumin sdAb, an anti-human EpCAM sdAb, a protease cleavable linker 13 (SEQ ID NO: 682), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 683).

[0076] FIGS. 35A-35C illustrate that MMP-7-selective substrates show increased stability when expressed on T cells in ProCAR format. CAR-T cells made from the constructs of FIG. 34 were prepared. Shown are dot plots of M2 staining (x-axis; BV421 channel) and EpCAM-Fc staining (y- axis; AlexaFluor 647 channel) of CAR-T cells. FIG. 35A provides results for constructs C2483, C3558, and C3543 for MMP7-selective substrate 1. FIG. 35B provides results for constructs C2483, C3559, and C3543 for MMP7-selective substrate 2. FIG. 35C provides results for constructs C2483, C3560, and C3543 for MMP7-selective substrate 3.

[0077] FIGS. 36A-36C illustrate that MMP7-selective substrates are cleavable with recombinant MMP7. CAR-T cells made from the constructs of FIG. 34 were prepared. Shown are dot plots of M2 staining (x-axis; BV421 channel) and EpCAM-Fc staining (y-axis; AlexaFluor 647 channel) of CAR-T cells. FIG. 36A provides results for constructs C3558 and C3558+MMP7 for MMP7- selective substrate 1. FIG. 36B provides results for constructs C3559 and C3559+MMP7 for MMP7-selective substrate 2. FIG. 36C provides results for constructs C3560 and C3560+MMP7 for MMP7-selective substrate 3.

[0078] FIGS. 37A-37E illustrate exemplary ProCAR constructs. FIG. 37A illustrates construct C2446 which includes an anti-human serine albumin sdAb, a protease cleavage site 3, an antihuman EGFR sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 684). FIG. 37B illustrates construct C2510 which includes an anti -human serine albumin sdAb, a protease cleavage site, an anti -human EGFR sdAb, a FLAG epitope, a dimerization-deficient CD8a hinge / transmembrane domain, a 4- 1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 685). FIG. 37C illustrates construct C2513 which includes an anti -human serine albumin sdAb, a protease cleavage site 3, an anti-human EGFR sdAb, a FLAG epitope, a dimerization-deficient CD8a hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 686). FIG. 37D illustrates construct C2514 which includes an anti -human serine albumin sdAb, an anti-human EGFR sdAb, a FLAG epitope, a dimerization-deficient CD8a hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 687). FIG. 37E illustrates construct C2448, which includes an anti-human EGFR sdAb, a FLAG epitope, a dimerization-deficient CD8a hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 688).

[0079] FIGS. 38A-38B illustrate that the level of protease site-dependent cell killing activity ofthe ProCAR constructs. The level of protease site-dependent cell killing activity of the ProCAR constructs was reduced by eliminating the dimerization activity of the CD8a transmembrane domain (FIG. 38B) compared to wild-type (FIG. 38A).

[0080] FIG. 39 illustrates that dimerization-deficient EGFR CAR has similar cell killing activity compared to wild-type.

[0081] FIG. 40 shows all EpCAM targeting ProTriTAC molecules comprising a cleavable linker were able to reduce tumor growth at both 0. 1 mg. / kg and 0.3 mg / kg dose.

[0082] FIG. 41 shows human PBMC cross-reactivity of eight anti-CD3 / anti-ITGB6 non- cleavable fusion proteins with llama anti-ITGB6 sequences.

[0083] FIG. 42 shows mouse splenocytes cross -reactivity of eight anti-CD3 / anti-ITGB6 non- cleavable fusion proteins with llama anti-ITGB6 sequences.

[0084] FIG. 43 shows human alveolar epithelial cells cross-reactivity of eight anti-CD3 / anti- ITGB6 non-cleavable fusion proteins with llama anti-ITGB6 sequences.

[0085] FIG. 44 shows cyno alveolar epithelial cells cross-reactivity of eight anti-CD3 / anti- ITGB6 non-cleavable fusion proteins with llama anti-ITGB6 sequences.

[0086] FIG. 45 shows mouse alveolar epithelial cells cross-reactivity of eight anti-CD3 / anti- ITGB6 non-cleavable fusion proteins with llama anti-ITGB6 sequences.

[0087] FIG. 46 shows human kidney epithelial cells cross-reactivity of eight anti-CD3 / anti- ITGB6 non-cleavable fusion proteins with llama anti-ITGB6 sequences.

[0088] FIG. 47 shows cyno kidney epithelial cells cross-reactivity of eight anti-CD3 / anti-ITGB6 non-cleavable fusion proteins with llama anti-ITGB6 sequences.

[0089] FIG. 48 shows mouse kidney epithelial cells cross-reactivity of eight anti-CD3 / anti- ITGB6 non-cleavable fusion proteins with llama anti-ITGB6 sequences.

[0090] FIG. 49 illustrates xenograft human HPAF-II tumor cells growth curve with treatment of anti-ITGB6 I6H41C ProTriTAC.

[0091] FIG. 50 illustrates xenograft human SW780 tumor cells growth curve with treatment of anti-ITGB6 I6H41C ProTriTAC.

[0092] FIG. 51 illustrates xenograft human HCC70 tumor cells growth curve with treatment of anti-ITGB6 I6H41C ProTriTAC.

[0093] FIG. 52 illustrates xenograft human CAL27 tumor cells growth curve with treatment of anti-ITGB6 I6H41C ProTriTAC.

[0094] FIG. 53 shows results of a TDCC assay with human alveolar epithelial cells and activated form of an ITGB6 ProTriTAC with binder I6H41C.

[0095] FIG. 54 shows results of a TDCC assay with cyno alveolar epithelial cells and activatedform of an ITGB6 ProTriTAC with binder I6H41C.

[0096] FIG. 55 shows results of a TDCC assay with human kidney epithelial cells and activated form of an ITGB6 ProTriTAC with binder I6H41C.

[0097] FIG. 56 shows results of a TDCC assay with cyno kidney epithelial cells and activated form of an ITGB6 ProTriTAC with binder I6H41C.

[0098] FIG. 57 shows results of a TDCC assay with human bladder epithelial cells and activated form of an ITGB6 ProTriTAC with binder I6H41C.

[0099] FIG. 58 shows results of a TDCC assay with cyno bladder epithelial cells and activated form of an ITGB6 ProTriTAC with binder I6H41C.

[0100] FIG. 59 shows results of a TDCC assay with human stomach epithelial cells and activated form of an ITGB6 ProTriTAC with binder I6H41C.

[0101] FIG. 60 shows results of a TDCC assay with cyno stomach epithelial cells and activated form of an ITGB6 ProTriTAC with binder I6H41C.

[0102] FIG. 61 shows results of a TDCC assay with human mammary epithelial cells and activated form of an ITGB6 ProTriTAC with binder I6H41C.

[0103] FIG. 62 shows results of a TDCC assay with cyno mammary epithelial cells and activated form of an ITGB6 ProTriTAC with binder I6H41C.

[0104] FIG. 63 shows results of a TDCC assay with SW780 parental cells and anti-CD3 / anti- ITGB6 fusion proteins containing humanized anti-ITGB6 sequence I6H41C.

[0105] FIG. 64 shows results of a TDCC assay with SW780 ITGB6 knockout cells and anti- CD3 / anti-ITGB6 fusion proteins containing humanized anti-ITGB6 sequence I6H41C.

[0106] FIG. 65 shows experimental design for the weekly 3-fold intra-animal dose-escalation.

[0107] FIG. 66 demonstrates that the ITGB6 ProTnTAC shows favorable pharmacokinetics, half-life and systemic accumulation in the intra-animal dose-escalation experiment.

[0108] FIG. 67 shows that comparable anti-tumor activity is achieved by molar equivalents of prodrug and active drug.DETAILED DESCRIPTION OF THE DISCLOSURE

[0109] Described herein are trispecific proteins that target ITGB6, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such proteins thereof. Also provided are methods of using the disclosed ITGB6 targeting trispecific proteins in the prevention, and / or treatment of diseases, conditions and disorders. The ITGB6 targeting trispecific proteins are capable of specifically binding to ITGB6 as well as CD3 and have a half-life extension domain, such as a domain binding to human albumin (ALB).ITGB6 binding proteins

[0110] Described herein are proteins that bind ITGB6, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such proteins thereof Also provided are methods of using the disclosed ITGB6 binding proteins in the prevention, and / or treatment of diseases, conditions and disorders. In some embodiments, the ITGB6 binding proteins are part of multispecific (e.g., trispecific) proteins that comprise an ITGB6 binding domain as described herein.

[0111] Human ITGB6 gene encodes the protein integrin beta 6, which is the 06 subunit of the integrin av06. Integrins are membrane-spanning a0 heterodimeric glycoproteins. An integrin binds to a specific extracellular protein in the extracellular matrix or on other cells, and subsequently transduces signals intracellularly. Each integrin consists of one a and one 0 subunit, which associate non-covalently to form 24 unique integnns in humans (R.O. Hynes, Integrins: bidirectional, allosteric signaling machines, cell, 110 (6) (2002), pp. 673-687). While some 0 integrin subunits partner with multiple a subunits, integrin beta 6 associates exclusively with the av subunit. Thus, the function of ITGB6 is associated with integrin av06. Integrin av06 is involved in wound healing and the pathogenesis of diseases including fibrosis and cancer. Therefore, it is of great clinical interest to develop ITGB6 binding domains and multispecific proteins containing the ITGB6 binding domains which have reduced side effects and / or efficacy.

[0112] av06 expression is exclusively on epithelial cells but is absent or expressed at low levels in normal healthy adult tissue. av06 expression increases during development, wound healing, cancer and fibrosis. av06 binds to proteins containing the RGD (Arg-Gly-Asp) motif, including fibronectin and the Transforming Growth Factor Beta (TGF-0) pro-peptide, Latency Associated Peptide (LAP). TGF-01 is post-translationally cleaved to release LAP, but LAP is anchored as a homodimer to the extracellular matrix (ECM) via latent TGF0 binding protein 1, remaining directly associated with the cytokine. The whole LAP-TGF-01 complex remains inert as latent TGF-01. av06’s binding to LAP physically moves LAP and exposes TGF-01 to receptors on adjacent cells, allowing an actin-myosin dependent activation of TGF-01. The cytoplasmic tail of integrin 06 also has binding domains that recruit signaling and structural partners including HAX-1, Fyn, ERK2, and Kindlin-1. Extracellular ligand binding to av06 is able to effect a conformational change in the cytoplasmic tail and thus initiate downstream signaling (outside-in signaling). Through these signaling pathways, av06 regulates some basic cell functions such as proliferation, migration and ECM degradation Expression of av06 is found in up to one third of all solid tumors. Expression of av06 is associated with a poorer prognosis and increased invasiveness. av06 expression is associated with the progression of pulmonary fibrosis through its activation of TGF- 1. SinceITGB6 is the exclusive binding partner of av, ITGB6 availability determines the cellular expression of av06. ITGB6’s transcription is regulated by transcription factors C / EBPa (CCAAT-enhancer- binding protein a), signal transducer and activator transcription 3 (STAT3), Ets-1, and a positive feedback loop involving TGF-01. ITGB6 is also subjected to post-transcriptional regulation.

[0113] ITGB6 levels increase in cells remodeling tissues so av 6 expression is increased in development. Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling and wound healing. Expression of integrin alphavbeta6 and TGF-beta in scarless vs scar-forming wound healing, but also in fibrosis. Expression of integrin alphavbeta6 and TGF-beta in scarless vs scar-forming wound healing and cancer.

[0114] Fibrosis occurs in response to chronic tissue insult and results in the deposit of excess collagen by activated fibroblasts in the matrix resulting in hardening of tissue. Fibroblasts are mesenchymal cells in all tissues that maintain the normal tissue matrix. When they become activated, as occurs in wound healing, they secrete extra matrix proteins and cytokines to promote wound repair. Chronic activation of fibroblasts can result in diseases such as pulmonary fibrosis, where the hardening and thickening of the lung tissue makes it difficult for patients to breathe. A major driver of fibroblast activation is TGF-0 and as av06 expression is increased in response to tissue damage, and is a principal activator of TGF-0, it is therefore a potential drug target in treating fibrosis. av06 can promote fibrosis in kidney, lung and skin, despite av06 being almost absent in their healthy equivalents.

[0115] otv06 expression is increased in up to one third of solid tumors, including breast cancer, lung cancer and pancreatic cancer. av06 over-expression in cancers is often correlated with poorer overall survival. Integrin av06 promotes tumor progression in multiple ways. Through its cytoplasmic tail integrin av06 promotes cancer cell migration, increased secretion of matrix metalloproteinases (MMPs) that can degrade the ECM, leading to increased invasion. Intracellular signals generated by av06 increase pErk and pAkt that increase cell proliferation and survival, respectively. Through its extracellular domain integrin av06 activates TGF-01 which increases processes that aid cancer progression including angiogenesis, activation of cancer associated fibroblasts, immune suppression, and epithelial-to-mesenchymal transition (EMT). EMT is the process by which epithelial cells adopt a mesenchymal phenotype, breaking away from neighboring epithelial cells and becoming more migratory, a crucial stage in the development of cancer. In cancer, this promotes invasion of the local healthy tissue and ultimately spread to other parts of the body. av06 can be found in cells which are undergoing EMT. Because av06 is not found on most normal cells, it is a potential therapeutic and imaging target in cancer research.

[0116] ITGB6 is also known by the following alternate names: Integrin subunit beta 6, integrin beta 6, integrin beta-6 and AI1H. In some embodiments, the ITGB6 binding proteins of this disclosure binds to an ITGB6 sequence provided in UniProtKB ID Nos. P18564 (SEQ ID NO: 857). In some embodiments, the ITGB6 binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 857. In some embodiments, the ITGB6 binding domain binds to a protein comprising the sequence of SEQ ID NO: 857.

[0117] In some embodiments, the ITGB6 binding domains disclosed herein recognize full-length ITGB6. In certain instances, the ITGB6 binding domains disclosed herein recognize an epitope within ITGB6, such as, in some cases the ITGB6 binding proteins interact with one or more amino acids found within a domain of human ITGB6. The epitope to which the antibodies bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within a domain of ITGB6. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within a domain of ITGB6.

[0118] In some embodiments, the ITGB6 binding proteins of this disclosure binds to the full length ITGB6 protein or to a fragment thereof, such as epitope containing fragments within the full length ITGB6 protein, as described above. In some cases, the epitope containing fragment comprises antigenic or immunogenic fragments and derivatives thereof of the ITGB6 protein. Epitope containing fragments, including antigenic or immunogenic fragments, are, in some embodiments, 12 amino acids or more, e.g., 20 amino acids or more, 50 or 100 amino acids or more. The ITGB6 fragments, in some embodiments, comprises 95% or more of the length of the full protein, 90% or more, 75% or 50% or 25% or 10% or more of the length of the full protein. In some embodiments, the epitope-containing fragments of ITGB6 including antigenic or immunogenic fragments are capable of eliciting a relevant immune response in a patient.Derivatives of ITGB6 include, in some embodiments, variants on the sequence in which one or more (e.g., 1-20 such as 15 amino acids, or up to 20% such as up to 10% or 5% or 1% by number of amino acids based on the total length of the protein) deletions, insertions or substitutions have been made to the ITGB6 sequence provided in SEQ ID NO: 857.

[0119] In some embodiments, substitutions comprise conservative substitutions. Derivatives and variants of, in some examples, have essentially the same biological function as the protein from which they are derived. For instance, derivatives and variants of ITGB6 are, in some cases, comparably antigenic or immunogenic to the protein from which they are derived, have either the ligand-binding activity, or the active receptor-complex forming ability, or preferably both, of the protein from which they are derived, and have the same tissue distribution as ITGB6.

[0120] In some embodiments, the ITGB6 binding protein specifically binds ITGB6 with equivalent or better affinity as that of a reference ITGB6 binding protein, and the ITGB6 binding protein in such embodiments comprises an affinity matured ITGB6 binding molecule, and is derived from the ITGB6 binding parental molecule, comprising one or more amino acid mutations (e.g., a stabilizing mutation, a destabilizing mutation) with respect to the ITGB6 binding parental molecule. In some embodiments, the affinity matured ITGB6 binding molecule has superior stability with respect to selected destabilizing agents, as that of a reference ITGB6 binding parental molecule. In some embodiments, the affinity matured ITGB6 binding molecule is identified in a process comprising panning of one or more pre-candidate ITGB6 binding molecules derived from one or more ITGB6 binding parental molecule, expressed in a phage display library, against an ITGB6 protein, such as a human ITGB6 protein. The pre-candidate ITGB6 binding molecule comprises, in some embodiments, amino acid substitutions in the variable regions, CDRs, or framework residues, relative to a parental molecule.

[0121] As used herein, “Phage display” refers to a technique by which variant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of phage, e.g., filamentous phage, particles. A utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and efficiently selected for those sequences that bind to a target molecule with high affinity. Display of peptide and protein libraries on phage has been used for screening millions of polypeptides for ones with specific binding properties. Polyvalent phage display methods have been used for displaying small random peptides and small proteins through fusions to either gene HI or gene VIII of filamentous phage. In monovalent phage display, a protein or peptide library is fused to a gene III, or a portion thereof, and expressed at low levels in the presence of wild type gene III protein so that phage particles display one copy or none of the fusion proteins. Avidity effects are reduced relative to poly valent phage so that selection is on the basis of intrinsic ligand affinity, and phagemid vectors are used, which simplify DNA manipulations.

[0122] In some embodiments, the panning comprises using varying binding times and concentrations to identify ITGB6 binding molecules with increased or decreased on-rates, from precandidate ITGB6 binding molecules. In some embodiments, the panning comprises using varying wash times to identify ITGB6 binding molecules with increased or decreased off-rates, from precandidate ITGB6 molecules. In some embodiments, the panning comprises using both varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of the affinity matured ITGB6 binding molecule, for example, by shuffling to create a second-stage combinatorial library from such mutants and conducting a second round of panning followed by a binding selection.

[0123] In some embodiments, the affinity matured ITGB6 binding molecule comprises an equivalent or better affinity to a ITGB6 protein (such as human ITGB6 protein) as that of a parental molecule, but that has reduced cross reactivity, or in some embodiments, increased cross reactivity, with selected substances, such as ligands, proteins, antigens, or the like, other than the ITGB6 epitope for which the ITGB6 binding parental molecule is specific, or is designed to be specific for. In regard to the latter, an affinity matured ITGB6 binding molecule, in some embodiments, is more successfully tested in animal models if the affinity matured binding molecule is reacted with both human ITGB6 and the corresponding target of the animal model, e.g. , mouse ITGB6 or cynomolgus monkey (cyno) ITGB6. In some embodiments, the parental ITGB6 binding molecule binds to human ITGB6 with an affinity of about 20000 nM or less, 15000 nM or less, 10000 nM or less, 5000 nM or less, 1000 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, and to cynomolgus ITGB6 with an affinity of about 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 15 nM or less, or 10 nM or less. In some embodiments, the affinity matured ITGB6 binding molecule, identified after one round of panning, binds to human ITGB6 with an affinity of about 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, such as 1 nM or less, and to cynomolgus ITGB6 with an affinity of about 7.5 nM or less, such as 1 nM or less. In some embodiments, the affinity matured ITGB6 binding molecule, identified after two rounds of panning, binds to human ITGB6 with an affinity of about 2.5 nM or less, and to cynomolgus ITGB6 with an affinity of about 3.5 nM or less.

[0124] In some embodiments, the ITGB6 binding protein comprises an antigen-specific binding domain polypeptide that specifically bind to targets, such as targets on diseased cells, or targets on other cells that support the diseased state, such as targets on stromal cells that support tumor growth or targets on immune cells that support disease-mediated immunosuppression. In some examples, the antigen-specific binding domain includes antibodies, single chain antibodies, Fabs, Fv, T-cell receptor binding domains, ligand binding domains, receptor binding domains, domain antibodies, single domain antibodies, minibodies, nanobodies, peptibodies, or various other antibody mimics (such as AFFIMERS®, affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, fynomers, darpins, affibodies, affilins, DARPINS®, monobodies and armadillo repeat protein-based proteins).

[0125] In some embodiments, the ITGB6 binding domain is an anti-ITGB6 antibody or an antigen binding fragment thereof, or an antibody variant of the ITGB6 binding domain or an antigen binding fragment thereof. As used herein, the term “antibody variant” refers to variants and derivatives of an antibody or an antigen binding fragment as described herein. In certainembodiments, amino acid sequence variants of the anti-ITGB6 antibodies or antigen binding fragments thereof, as described herein, are contemplated. For example, in certain embodiments amino acid sequence variants of anti- ITGB6 antibodies or antigen binding fragments thereof, as described herein, are contemplated to improve the binding affinity and / or other biological properties of the same. Exemplary method for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or antigen binding fragment thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody or antigen binding fragments thereof.

[0126] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigenbinding. In certain embodiments, variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into an antibody or antigen binding fragments thereof of interest and the products screened for a desired activity, e.g , retained / improved antigen binding, decreased immunogenicity, altered Antibody dependent cellular cytotoxicity (ADCC), or improved T-cell mediated cytotoxicity (TDCC). Both conservative and non-conservative amino acid substitutions are contemplated for preparing the antibody variants.

[0127] In another example of a substitution to create a variant anti-ITGB6 antibody or antigen binding fragments thereof, one or more hypervariable region residues of a parent antibody are substituted. In general, variants are then selected based on improvements in desired properties compared to a parent antibody or antigen binding fragments thereof, for example, increased affinity, reduced affinity, reduced immunogenicity, increased pH dependence of binding.

[0128] In some embodiments, the ITGB6 binding domain is a single domain antibody (sdAb) such as a heavy chain variable domain (VH), a variable domain (VHH) of a llama derived sdAb, a peptide, a ligand or a small molecule entity specific for ITGB6. In some embodiments, the ITGB6 binding domain described herein is any domain that binds to ITGB6 including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In certain embodiments, the ITGB6 binding domain is a singledomain antibody. In other embodiments, the ITGB6 binding domain is a peptide. In further embodiments, the ITGB6 binding domain is a small molecule.

[0129] Generally, it should be noted that the term single domain antibody as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation.Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4- chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. For example, in some embodiments, the single domain antibodies of the disclosure are obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by “camelization” of a naturally occurring VH domain from any animal species, and in particular from a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization” of a “domain antibody” or “Dab,” or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi -synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (7) by preparing a nucleic acid encoding a single domain antibody using techniques for nucleic acid synthesis known in the field, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0130] In one embodiment, a single domain antibody corresponds to the VHH domains of naturally occurring heavy chain antibodies directed against ITGB6. As further described herein, such VHH sequences can generally be generated or obtained by suitably immunizing a species of Llama with ITGB6, i.e., so as to raise an immune response and / or heavy chain antibodies directed against ITGB6), by obtaining a suitable biological sample from said Llama (such as a blood sample, serum sample or sample of B-cells), and by generating VHH sequences directed against ITGB6, starting from said sample, using any suitable technique known in the field.

[0131] In another embodiment, such naturally occurring VHH domains against ITGB6, are obtained from naive libraries of Camelid VHH sequences, for example by screening such a library using ITGB6, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known in the field. Such libraries and techniques are for example described in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, such as VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as for example described in WO 00 / 43507.

[0132] In a further embodiment, yet another technique for obtaining VHH sequences directed against ITGB6, involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e., so as to raise an immune response and / or heavy chain antibodies directed against ITGB6), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, serum sample or sample of B-cells), and then generating VHH sequences directed against ITGB6, starting from said sample, using any suitable technique known in the field. For example, for this purpose, the heavy chain antibody-expressing rats or mice and the further methods and techniques described in WO 02 / 085945 and in WO 04 / 049794 can be used.

[0133] In some embodiments, an anti-ITGB6 single domain antibody of this disclosure comprises a single domain antibody with an amino acid sequence that corresponds to the amino acid sequence of a non-human antibody and / or a naturally occurring VHH domain, e.g. , a llama anti-ITGB6 antibody, but that has been “humanized,” i.e., by replacing one or more amino acid residues in the amino acid sequence of said non-human anti-ITGB6 and / or the naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g. , as indicated above). This can be performed in a manner known in the field, which will be clear to the skilled person, for example on the basis of the further description herein. Again, it should be noted that such humanized anti-ITGB6 single domain antibodies of the disclosure are obtained in any suitable manner known per se (i.e., as indicated under points (l)-(8) above) and thus are not stnctly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material. In some additional embodiments, a single domain anti-ITGB6 antibody, as described herein, comprises a single domain antibody with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized” i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. Such “camelizing” substitutions are preferably inserted at amino acid positions that form and / or are present at the VH-VL interface, and / or at the so-called Camelidae hallmark residues. See, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence that is used as a starting material or starting point for generating or designing the camelized single domain is preferably a VH sequence from a mammal, more preferably the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized anti-ITGB6 single domain antibodies of the disclosure, in certain embodiments, are obtained in any suitable manner known in the field (i.e. , asindicated under points (1 )-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises anon-human anti-ITGB6 antibody and / or the naturally occurring VH domain as a starting material. For example, as further described herein, both “humanization” and “camelization” is performed by providing a nucleotide sequence that encodes a naturally occurring VHH domain or VH domain, respectively, and then changing, one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a “humanized” or “camelized” single domain antibody, respectively. This nucleic acid can then be expressed, so as to provide a desired anti-ITGB6 single domain antibody of the disclosure. Alternatively, in other embodiments, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized anti-ITGB6 single domain antibody of the disclosure, respectively, are designed and then synthesized de novo using known techniques for peptide synthesis. In some embodiments, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized anti- ITGB6 single domain antibody of the disclosure, respectively, is designed and then synthesized de novo using known techniques for nucleic acid synthesis, after which the nucleic acid thus obtained is expressed in using known expression techniques, so as to provide the desired anti-ITGB6 single domain antibody of the disclosure.

[0134] Other suitable methods and techniques for obtaining the anti-ITGB6 single domain antibody of the disclosure and / or nucleic acids encoding the same, starting from naturally occurring VH sequences or VHH sequences for example comprises combining one or more parts of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences, in a suitable manner, so as to provide an anti-ITGB6 single domain antibody of the disclosure or a nucleotide sequence or nucleic acid encoding the same.

[0135] In some embodiments, the ITGB6 binding domain is an anti-ITGB6 specific antibody comprising a heavy chain variable complementarity determining region CDR1, a heavy chain variable CDR2, a heavy chain variable CDR3, a light chain variable CDR1, a light chain variable CDR2, and a light chain variable CDR3. In some embodiments, the ITGB6 binding domain comprises any domain that binds to ITGB6 including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or antigen binding fragments such as single domain antibodies (sdAb), Fab, Fab', F(ab)2, and Fvfragments, fragments comprised of one or more CDRs, single-chain antibodies (e.g., single chain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain. In some embodiments, the ITGB6 binding domain is a single domain antibody. In some embodiments, the anti-ITGB6 single domain antibody comprises heavy chain variable complementarity determining regions (CDR), CDR1, CDR2, and CDR3.

[0136] In some embodiments, the ITGB6 binding domain is a polypeptide comprising an amino acid sequence that is comprised of four framework regions / sequences (fl -f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: fl -rl-f2-r2-f3-r3- f4, wherein rl, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and fl, f2, f3, and f4 are framework residues. The framework residues of the ITGB6 binding protein of the present disclosure comprise, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and the complementarity determining regions comprise, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the ITGB6 binding domain comprises an amino acid sequence selected from SEQ ID NOs: 1-74.

[0137] In some embodiments, the binding proteins described herein comprise a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1-74, subsequences thereof, and variants thereof. In some embodiments, the ITGB6 binding protein comprises at least 70%-95% or more homology to an amino acid sequence selected from SEQ ID NOs: 1-74, subsequences thereof, and variants thereof. In some embodiments, the ITGB6 binding protein comprises at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology to an amino acid sequence selected from SEQ ID NOs: 1-74, subsequences thereof, and variants thereof. In some embodiments, the ITGB6 binding protein comprises at least 70%-95% or more identity to an amino acid sequence selected from SEQ ID NOS: 1-74, subsequences thereof, and variants thereof. In some embodiments, the ITGB6 binding protein comprises at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from SEQ ID NOS: 1-74, subsequences thereof, and variants thereof.

[0138] In some embodiments, the CDR1 comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 75-148 or an amino acid sequence comprising one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOS: 75-148. In some embodiments, the CDR2 comprises an amino acid sequence as set forth in any one of SEQID NOS: 149-222 or an amino acid sequence comprising one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOS: 149-222. In some embodiments, the CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 223-296 an amino acid sequence comprising one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOS: 223-296.

[0139] In various embodiments, the ITGB6 binding domain of the present disclosure is at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOS: 1-74.

[0140] In various embodiments, a complementarity determining region of the ITGB6 binding domain of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NOS: 75-148.

[0141] In various embodiments, a complementarity determining region of the ITGB6 binding domain of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NOS: 149-222.

[0027] In various embodiments, a complementarity determining region of the ITGB6 binding domain of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NOS: 223-296.

[0142] In some embodiments, the ITGB6 binding domain is cross-reactive with human cynomolgus and mouse ITGB6. In some embodiments, the ITGB6 binding domain is specific for human ITGB6. In certain embodiments, the ITGB6 binding domains disclosed herein bind to human ITGB6 with a human Kd (hKd). In certain embodiments, the ITGB6 binding domainsdisclosed herein bind to cynomolgus ITGB6 with a cyno Kd (cKd). In certain embodiments, the ITGB6 binding domains disclosed herein bind to cynomolgus ITGB6 with a mouse Kd (mKd). In certain embodiments, the ITGB6 binding domains disclosed herein bind to both cynomolgus ITGB6 and a human ITGB6, with a cyno Kd (cKd) and a human Kd (hKd), respectively. In certain embodiments, the ITGB6 binding domains disclosed herein bind to cynomolgus ITGB6, mouse ITGB6, and a human ITGB6, with a cyno Kd (cKd), mouse Kd (mKd), and a human Kd (hKd), respectively. In some embodiments, the ITGB6 binding protein binds to human, mouse and cynomolgus ITGB6 with comparable binding affinities i.e., hKd, mKd and cKd values do not differ by more than ± 10%). In some embodiments, the hKd, mKd and the cKd range from about 0.001 nM to about 500 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.001 nM to about 450 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.001 nM to about 400 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.001 nM to about 350 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.001 nM to about 300 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.001 nM to about 250 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.001 nM to about 200 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.001 nM to about 150 nM. In some embodiments, the hKd, mKd and the cKd range from about 0,001 nM to about 100 nM. In some embodiments, the hKd, mKd and the cKd range from about 0. 1 nM to about 90 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.2 nM to about 80 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.3 nM to about 70 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.4 nM to about 50 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.5 nM to about 30 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.6 nM to about 10 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.7 nM to about 8 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.8 nM to about 6 nM. In some embodiments, the hKd, mKd and the cKd range from about 0.9 nM to about 4 nM. In some embodiments, the hKd, mKd and the cKd range from about 1 nM to about 2 nM.

[0143] In some embodiments, any of the foregoing ITGB6 binding domains (e.g, anti- ITGB6 single domain antibodies of SEQ ID NOS: 1-74) are affinity peptide tagged for ease of purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also referred to as 6X-His (SEQ ID NO: 658).

[0144] It is contemplated that in some embodiments the ITGB6 binding protein is fairly small and no more than 25 kDa, no more than 20 kDa, no more than 15 kDa, or no more than 10 kDa insome embodiments. In certain instances, the ITGB6 binding protein is 5 kDa or less if it is a peptide or small molecule entity.

[0145] In other embodiments, the ITGB6 binding proteins described herein comprise small molecule entity (SME) binders for ITGB6. SME binders are small molecules averaging about 500 to 2000 Da in size and are attached to the ITGB6 binding proteins by known methods, such as sortase ligation or conjugation. In these instances, the ITGB6 binding protein comprises a domain comprising a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 717). To attach a SME binder to ITGB6 binding protein comprising a sortase recognition sequence, the protein is incubated with a sortase and a SME binder whereby the sortase attaches the SME binder to the recognition sequence. In yet other embodiments, the ITGB6 binding proteins described herein comprise a knottin peptide for binding ITGB6. Knottins are disulfide-stabilized peptides with a cysteine knot scaffold and have average sizes about 3.5 kDa. Knottins have been contemplated for binding to certain tumor molecules such as ITGB6. In further embodiments, the ITGB6 binding proteins described herein comprise a natural ITGB6 ligand.

[0146] In some embodiments, the ITGB6 binding protein comprises more than one domain and are of a single-polypeptide design with flexible linkage of the domains. This allows for facile production and manufacturing of the ITGB6 binding proteins as they can be encoded by single cDNA molecule to be easily incorporated into a vector. Further, in some embodiments where the ITGB6 binding proteins described herein are a monomeric single polypeptide chain, there are no chain pairing issues or a requirement for dimerization. It is contemplated that, in such embodiments, the ITGB6 binding proteins described herein have a reduced tendency to aggregate.

[0147] In the ITGB6 binding proteins comprising more than one domain, the domains are linked by one or more internal linker. In certain embodiments, the internal linkers are “short,” i.e., consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, the internal linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linkers are “long,” i.e., consist of 15, 20 or 25 amino acid residues. In some embodiments, the internal linkers consist of about 3 to about 15, for example 8, 9 or 10 contiguous amino acid residues. Regarding the amino acid composition of the internal linkers, peptides are selected with properties that confer flexibility to the ITGB6 binding proteins, do not interfere with the binding domains as well as resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance.Examples of internal linkers suitable for linking the domains in the ITGB6 binding proteins include but are not limited to (GS)n(SEQ ID NO: 718), (GGS)n(SEQ ID NO: 719), (GGGS)n(SEQ ID NO: 720), (GGSG)n(SEQ ID NO: 721), (GGSGG)n(SEQ ID NO: 722), (GGGGS)n(SEQ ID NO:723), (GGGGG)n(SEQ ID NO: 724), or (GGG)n(SEQ ID NO: 725), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 726), (GGGGSGGGGSGGGGS) (SEQ ID NO: 727), or (GGGGSGGGS) (SEQ ID NO: 659).

[0148] In some cases, where the ITGB6 binding protein comprises more than one domain, the domains within the ITGB6 binding proteins are conjugated using an enzymatic site-specific conjugation method which involves the use of a mammalian or bacterial transglutaminase enzyme. Microbial transglutaminases (mTGs) are versatile tools in modem research and biotechnology. The availability of large quantities of relatively pure enzymes, ease of use, and lack of regulation by calcium and guanosine-5 ’-triphosphate (GTP) has propelled mTG to be the main cross-linking enzyme used in both the food industry and biotechnology. Currently, mTGs are used in many applications to attach proteins and peptides to small molecules, polymers, surfaces, DNA, as well as to other proteins. See, e.g., Pavel Strp, Veracity of microbial transglutaminase, Bioconjugate Chem. 25, 5, 855-862.

[0149] In some examples are provided ITGB6 binding proteins comprising more than one domain, wherein one of the domains comprises an acceptor glutamine in a constant region, which can then be conjugated to another domain via a lysine-based linker (e.g., any primary' amine chain which is a substrate for TGase, e.g. comprising an alkylamine, oxoamine) wherein the conjugation occurs exclusively on one or more acceptor glutamine residues present in the targeting moiety outside of the antigen combining site (e.g., outside a variable region, in a constant region). Conjugation thus does not occur on a glutamine, e.g., an at least partly surface exposed glutamine, within the variable region. The ITGB6 binding protein, in some examples, is formed by reacting one of the domains with a lysine-based linker in the presence of a TGase.

[0150] In some embodiments, where one or more domains within the ITGB6 binding proteins are directly j oined, a hybrid vector is made where the DNA encoding the directly j oined domains are themselves directly ligated to each other. In some embodiments, where linkers are used, a hybrid vector is made where the DNA encoding one domain is ligated to the DNA encoding one end of a linker moiety and the DNA encoding another domain is ligated to the other end of the linker moiety.

[0151] In some embodiments, the ITGB6 binding protein is a single chain variable fragments (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived single domain antibody. In other embodiments, the ITGB6 binding protein is a non-Ig binding domain, i.e., an antibody mimetic, such as anticalins, affilins, affibody molecules, AFFIMERS®, affitins, alphabodies, avimers, DARPINS®, fynomers, kumtz domain peptides, and monobodies. In further embodiments, theITGB6 binding protein is a ligand or peptide that binds to or associates with ITGB6. In yet further embodiments, the ITGB6 binding protein is a knottin. In yet further embodiments, the binding domain to ITGB6 is a small molecular entity.

[0152] In certain embodiments, the ITGB6 binding proteins according to the present disclosure may be incorporated into ITGB6 targeting trispecific proteins. In some embodiments, the trispecific proteins comprise a CD3 binding domain, a half-life extension domain, and an ITGB6 binding domain according to this disclosure. In some embodiments, the ITGB6 binding trispecific protein comprises a trispecific antibody.Multispecific ITGB6 targeting proteins, such as ITGB6 targeting trispecific proteins (also referred to herein as ITGB6 targeting TriTAC proteins or molecules)

[0153] In one aspect is described herein a multispecific or a multivalent protein comprising an ITGB6 binding protein according to the present disclosure. In some embodiments, the multispecific protein further comprises a domain which specifically binds to CD3. In some embodiments, the multispecific protein further comprises a domain which specifically binds to human CD3. In some embodiments, the multispecific protein further comprises a domain which specifically binds to CD3-gamma. In some embodiments, the multispecific protein further comprises a domain which specifically binds to CD3-delta. In some embodiments, the multispecific protein further comprises a domain which specifically binds to CD3-epsilon.

[0154] In additional embodiments, the multispecific protein further comprises a domain which specifically binds to the T cell receptor (TCR). In some embodiments, the multispecific protein further comprises a domain which specifically binds the alpha chain of the TCR. In some embodiments, the multispecific protein further comprises a domain which specifically binds the chain of the TCR.

[0155] In certain embodiments, the CD3 binding domain of the multispecific protein exhibits not only potent CD3 binding affinities with human CD3, but also shows excellent cross-reactivity with the respective cynomolgus monkey CD3 proteins. In some instances, the CD3 binding domain of the multispecific proteins are cross-reactive with CD3 from cynomolgus monkey. In certain instances, human: cynomolgous KD (hKd: cKd) ratios for CD3 binding are between 20: 1 and 1 :2.

[0156] In some embodiments, the CD3 binding domain of the multispecific protein is any domain that binds to CD3 including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or antigen binding fragments of the CD3 binding antibodies, such as single domain antibodies (sdAb), Fab, F(ab')2, and Fv fragments, fragments comprised of one or more CDRs, single-chain antibodies (e.g., single chain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies(e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain. In some instances, it is beneficial for the CD3 binding domain to be derived from the same species in which the multispecific protein comprising a single domain serum albumin binding protein described herein will ultimately be used in. For example, for use in humans, it may be beneficial for the CD3 binding domain of the multispecific protein comprising an ITGB6 binding protein described herein to comprise human or humanized residues from the antigen binding domain of an antibody or antibody fragment. Exemplary amino acid sequence for the CD3 binding domain of a multispecific (e.g., trispecific) ITGB6 targeting TriTAC protein of this disclosure is provided as SEQ ID NO: 655, or an amino acid sequence that is at least about 75% to 100% identical to SEQ ID NO: 655, such as at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 655.

[0157] In some embodiments, the serum albumin binding domain (also referred to herein as the half-life extension domain) of a multispecific protein comprising an ITGB6 binding protein as described herein can be any domain that binds to serum albumin including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the serum albumin binding domain is a single chain variable fragments (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived sdAb, or antigen binding fragments of the HSA binding antibodies, such as Fab, F(ab')2. and Fv fragments, fragments comprised of one or more CDRs, single-chain antibodies (e.g., single chain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain, peptide, ligand or small molecule entity specific for serum albumin. In certain embodiments, the HSA binding domain is a single-domain antibody. In other embodiments, the serum albumin binding domain is a peptide. In further embodiments, the serum albumin binding domain is a small molecule. It is contemplated that the serum albumin binding domain of the multispecific binding protein comprising a single chain variable fragment CD3 binding protein is fairly small and no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD in some embodiments. In certain instances, the serum albumin binding is 5 kD or less if it is a peptide or small molecule entity. Exemplary amino acid sequence for a serum albumin binding domain of a multispecific (e.g. , trispecific) ITGB6 targeting TriTAC protein of this disclosure is provided as SEQ ID NOs: 654, 728, or an amino acidsequence that is at least about 75% to 100% identical to SEQ ID NOs: 654, 728, such as at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NOs: 654, 728.

[0158] The half-life extension domain of a multispecific binding protein, as described herein, comprising a single chain variable fragment CD3 binding protein provides for altered pharmacodynamics and pharmacokinetics of the single chain variable fragment CD3 binding protein itself. As above, the half-life extension domain extends the elimination half-time. The half- life extension domain also alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the single chain variable fragment CD3 binding protein. In some embodiments, the half-life extension domain provides for improved tissue (including tumor) targeting, tissue distnbution, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension domain. In one embodiment, therapeutic methods effectively and efficiently utilize a reduced amount of the multispecific binding protein comprising a single chain variable fragment CD3 binding protein, resulting in reduced side effects, such as reduced off-target, such as non-tumor cell cytotoxicity.

[0159] Further, the binding affinity of the half-life extension domain, in some embodiments, is selected so as to target a specific elimination half-time in a particular multispecific binding protein comprising an ITGB6 binding protein as described herein. Thus, in some embodiments, the half- life extension domain has a high binding affinity. In other embodiments, the half-life extension domain has a medium binding affinity. In yet other embodiments, the half-life extension domain has a low or marginal binding affinity. Exemplary binding affinities include Ka of 10 nM or less (high), between 10 nM and 100 nM (medium), and greater than 100 nM (low). As above, binding affinities to serum albumin are determined by known methods such as Surface Plasmon Resonance (SPR).

[0160] An ITGB6 targeting multispecific protein of this disclosure, in certain embodiments, comprises (A) a first domain which binds to a CD3; (B) a second domain which is an half-life extension domain; and (C) a third domain which is an ITGB6 binding protein as described herein. In certain embodiments, the first domain comprises an scFv that specifically binds the CD3. The CD3, for instance, is a human CD3 protein. In certain embodiments, the second domain comprises an sdAb that specifically binds a bulk serum protein. In some instances, the bulk serum protein is albumin, such as, a serum albumin, such as, a human serum albumin. The domains (A), (B), and (C), are, in some embodiments, linked via linkers LI and L2, in any one of the following onentations: H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(C)-L1-(B)-L2-(A)-C00H, H2N-(C)-L1-(A)-L2-(B)-COOH, H2N-(A)-L1-(B)-(C)-L2-COOH, or H2N-(B)-(C)- (A)-COOH.

[0161] An ITGB6 targeting multispecific protein of this disclosure, in some embodiments, comprises an ammo acid sequence that is at least about 70% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 345-357. In some embodiments, an ITGB6 targeting multispecific protein of this disclosure comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 76%, at least about 77%, about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 345-357.Conditionally active multispecific ITGB6 targeting proteins, such as conditionally active ITGB6 targeting trispecific proteins (also referred to herein as ITGB6 targeting ProTriTAC or protrispecific proteins or molecules)

[0162] One embodiment of this disclosure provides a conditionally active multispecific protein comprising an ITGB6 binding domain as disclosed herein (for example, in some embodiment this disclosure provides an ITGB6 targeting protrispecific / ProTriTAC protein comprising an ITGB6 binding domain of this disclosure).

[0163] In some embodiments, the conditionally active multispecific protein further comprises a domain which specifically binds to a CD3 and a binding moiety which specifically binds to a bulk serum protein, such as a human serum albumin. In some embodiments, the binding moiety masks or is capable of masking the interaction of the ITGB6 binding domain or the CD3 binding domain, to their targets. In some embodiments, a binding moiety of this disclosure comprises a masking moiety and a cleavable linker, such as a protease cleavable linker. Exemplary sequences for masking moiety within a binding moiety are provided in SEQ ID NO: 728, or an amino acid sequence comprising one or more substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NOS: 728. In some embodiments, the binding moiety comprises a modified non-CDR loop sequence and a cleavable linker. In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713, 714, 715, and 716, or an amino acid sequence that comprises one or substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713,714, 715, and 716. In some embodiments, the masking moiety comprises a modified non-CDR loop sequence and a non-cleavable linker. In some embodiments the non-cleavable linker comprises an amino acid sequence as set forth in SEQ ID NO: 660, or an amino acid sequence comprising one or more substitutions relative to SEQ ID NO: 660. In some embodiments, a binding moiety comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 654 and 728. In some embodiments, a CD3 binding domain of an ITGB6 ProTriTAC of this disclosure comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO: 655.

[0164] In some embodiments, an ITGB6 targeting ProTriTAC of this disclosure, comprises, from N-terminal to C-terminal, comprise a binding moiety that is an anti-ALB domain comprising a non- CDR loop with a binding site for a CD3 binding domain (e.g., a CD3 binding domain having the sequence of SEQ ID NO: 655, or at least about 75% identity to the same), a cleavable linker, the CD3 binding domain, and on the C-terminal end the anti-ITGB6 binding domain. The ITGB6 binding domain of the ProTriTAC, in some embodiments, is at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOS: 1-74.

[0165] In some embodiments, an ITGB6 targeting ProTriTAC of this disclosure comprises an amino acid sequence that is at least about at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 345-357. In some embodiments, an ITGB6 targeting ProTriTAC of this discloses comprises an amino acid sequence as set forth in SEQ ID NOs: 353-357, a pharmaceutical composition comprising the same, and method of using the same for treating a disease, such as a tumorous disease as described herein.

[0166] In some embodiments, an ITGB6 targeting ProTriTAC of this disclosure, in a non- cleavable prodrug format, comprises an amino acid sequence that is at least about at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NOs: 345-352.

[0167] An exemplary sequence for an active ITGB6 targeting drug (CT), as described herein, is an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NOs: 345-357.

[0168] The binding moiety is capable of synergistically expanding a therapeutic window of a conditionally active ITGB6 targeting protri specific protein, by both steric masking and specific masking. In some embodiments, the binding moiety combines both steric masking (for example, via binding to a bulk serum albumin) and specific masking (for example, via non-CDR loops binding to the CDRs of an anti-ITGB6 domain or an anti-CD3 scFv domain). For example, the binding moiety masks binding of the ITGB6 binding domain (e g., hides the ITGB6 binding domain and / or prevents premature binding), until activation and the ability of a sequence to “mask” can be tested utilizing an assay such as described in Example 12 where activity with and without the masking sequence (e.g., a sequence selected from the group consisting of SEQ ID NOS: 801- 845, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 801-845) is compared. In some instances, the masking moiety is masking peptide / moiety, inserted into one or more non-CDR loops, such that the binding moiety binds to and inhibits the ITGB6 antigen binding domain until such time that the construct is delivered to a tumor microenvironment. In some cases, modifying the non-CDR loops within the binding moiety does not affect albumin binding. The protease cleavable linker, in some cases, enables activation of an ITGB6 targeting protrispecific protein in a single proteolytic event, thereby allowing more efficient conversion of the protrispecific molecule in tumor microenvironment. Further, tumor-associated proteolytic activation, in some cases, reveals active T cell engager with minimal off-tumor activity after activation. The present disclosure, in some embodiments, provides a half-life extended T cell engager format (ProTriTAC) comprising an ITGB6 binding moiety as described herein, which in some cases represents a new and improved approach to engineer conditionally active T cell engagers.

[0169] The half-life of the ITGB6 binding domain in a conditionally active protrispecific format is, in some embodiments, extended in systemic circulation by using the binding moiety as described above which acts as a safety switch that keeps the multispecific protein in the pro format in an inert state until it reaches the tumor microenvironment where it is conditionally activated by cleavage of the linker and is able to bind its target antigen(s). The safety switch, in certain instances, provides several advantages: some examples including (i) expanding the therapeutic window of theconditionally active ITGB6 targeting protein; (ii) reducing target-mediated drug disposition by maintaining the conditionally active ITGB6 targeting protein in systemic circulation; (iii) reducing the concentration of undesirable activated protein in systemic circulation, thereby minimizing the spread of chemistry, manufacturing, and controls related impurities, e.g., pre-activated drug product, endogenous viruses, host-cell proteins, DNA, leachables, anti-foam, antibiotics, toxins, solvents, heavy metals; (iv) reducing the concentration of undesirable activated proteins in systemic circulation, thereby minimizing the spread of product related impurities, aggregates, breakdown products, product variants due to: oxidation, deamidation, denaturation, loss of C-term Lys in MAbs; (v) preventing aberrant activation in circulation; (vi) reducing the toxicities associated with the leakage of activated species from diseased tissue or other pathophysiological conditions, e.g., tumors, autoimmune diseases, inflammations, viral infections, tissue remodeling events (such as myocardial infarction, skin wound healing), or external injury (such as X-ray, CT scan, UV exposure); and (vii) reducing non-specific binding of the conditionally active ITGB6 targeting protein. Furthermore, post-activation, or in other words post breaking of the safety switch, the conditionally active ITGB6 targeting protein is separated from the safety switch which provided extended half-life, and thus is cleared from circulation. For instance, if the drug is inadvertently activated outside of a tumor environment or if it leaks out of a tumor environment after activation, it is likely to be cleared rapidly and is less likely to cause damage to normal tissues, thus reducing toxicity.

[0170] In some embodiments, a conditionally active multispecific ITGB6 binding protein as described herein has an improved therapeutic index as compared to that of an ITGB6 binding protein that is not conditionally active but is, rather, constitutively active. For instance, an ITGB6 ProTriTAC, in some embodiments, has an increased therapeutic index than an ITGB6 TriTAC. The increase in therapeutic index, in some embodiments, is from at least about 2-fold to about 1000- fold, such as about 4-fold to about 800-fold, about 6-fold to about 800-fold, about 6-fold to about 600-fold, about 10-fold to about 400-fold, about 20-fold to about 200-fold, about 30-fold to about 150-fold, about 50-fold to about 100-fold. The increase in therapeutic index, in some embodiments, is attributed to the conjugation of the ITGB6 binding domain to a binding moiety as described above, with the non-CDR loop and the cleavable linker.

[0171] A “therapeutic index” (TI) (also referred to as “therapeutic window”) is, in some embodiments, a comparison of the minimum amount of a therapeutic agent (e.g. , an ITGB6 TriTAC, an ITGB6 ProTriTAC, an ITGB6 CAR, an ITGB6 ProCAR) that causes the therapeutic effect (e.g, improved survival of a patient with an ITGB6 expressing cancer, treated with a therapeutic agent as mentioned above) to minimum tolerated dose. In some instances, thetherapeutic index improvement is manifested in terms of an improved EC50 of an ITGB6 TriTAC compared to an ITGB6 ProTriTAC, in T cell mediated killing of cancer cells.

[0172] In some embodiments, the conditionally active ITGB6 targeting protein format gives the ITGB6 binding domain a significantly longer serum half-life and reduces the likelihood of its undesirable activation in circulation, thereby producing a “biobetter” version.

[0173] A binding moiety as described herein comprises at least one non-CDR loop. In some embodiments, a non-CDR loop provides a binding site for binding of the binding moiety to an ITGB6 binding domain of this disclosure. In some cases, the binding moiety masks or is capable of masking binding of the ITGB6 binding domain to its target antigen, e.g., via steric occlusion, via specific intermolecular interactions, or a combination of both.

[0174] In some embodiments, a binding moiety as described herein further comprises complementarity determining regions (CDRs), for instance, specific for binding a bulk serum protein (e.g. , a human serum albumin). In some instances, a binding moiety of this disclosure is a domain derived from an immunoglobulin molecule (Ig molecule). The Ig may be of any class or subclass (IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). A polypeptide chain of an Ig molecule folds into a senes of parallel beta strands linked by loops. In the variable region, three of the loops constitute the “complementarity determining regions” (CDRs) which determine the antigen binding specificity of the molecule. An IgG molecule comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) with are hypervariable in sequence and / or involved in antigen recognition and / or usually form structurally defined loops, interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0175] In some embodiments, a binding moiety of this disclosure is a heavy chain only antibody. In some embodiments, the variable domain of a heavy chain only antibody has several beta strands that are arranged in two sheets. The variable domain of a heavy chain only antibody contains three hypervariable loops, or complementarity-determining regions (CDRs) and framework regions FR1, FR2, FR3, and FR4. The three CDRs of the variable domain (CDR1, CDR2, CDR3) cluster at oneend of the beta barrel. The CDRs are the loops that connect beta strands B-C, C'-C", and F-G of the immunoglobulin fold, whereas the bottom loops that connect beta strands AB, CC, C" -D and E-F of the immunoglobulin fold, and the top loop that connects the D-E strands of the immunoglobulin fold are the non-CDR loops.

[0176] In some embodiments of this disclosure at least some, or all, of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the “non-CDR loop” of the binding moieties described herein, such as a binding moiety that is a heavy chain only antibody. In some embodiments of this disclosure, at least some amino acid residues of a constant domain, CHI, CH2, or CH3, are part of the “non-CDR loop” of the binding moieties described herein. Non-CDR loops comprise, in some embodiments, one or more of AB, CD, EF, and DE loops of a Cl-set domain of an Ig or an Ig-like molecule; AB, CC’, EF, FG, BC, and EC’ loops of a C2-set domain of an Ig or an Ig-like molecule; DE, BD, GF, A(A1A2)B, and EF loops of I(Intermediate)-set domain of an Ig or Ig-like molecule.

[0177] Within the variable domain, the CDRs are believed to be responsible for antigen recognition and binding, while the FR residues are considered a scaffold for the CDRs. However, in certain cases, some of the FR residues play an important role in antigen recognition and binding. Framework region residues that affect Ag binding are divided into two categones. The first are FR residues that contact the antigen, thus are part of the binding-site, and some of these residues are close in sequence to the CDRs. Other residues are those that are far from the CDRs in sequence but are in close proximity to it in the 3-D structure of the molecule, e.g., a loop in heavy chain.

[0178] In some embodiments, the non-CDR loop is modified to generate an antigen binding site specific for a bulk serum protein, such as albumin. In some embodiments, the non-CDR loop is modified to generate an antigen binding site specific for an ITGB6 binding domain as described herein. In some embodiments, the non-CDR loop is modified to generate an antigen binding site specific for a CD3 binding domain as described herein.

[0179] It is contemplated that various techniques can be used for modifying the non-CDR loop, e.g., site-directed mutagenesis, random mutagenesis, insertion of at least one amino acid that is foreign to the non-CDR loop amino acid sequence, amino acid substitution. An antigen peptide is inserted into a non-CDR loop, in some examples. In some examples, an antigenic peptide is substituted for the non-CDR loop. The modification, to generate an antigen binding site, is in some cases in only one non-CDR loop. In other instances, more than one non-CDR loop are modified. For instance, the modification is in any one of the non-CDR loops are AB, CC, C" D, EF, and D-E. In some cases, the modification is in the DE loop. In other cases, the modifications are in all four of AB, CC, CD, E-F loops.

[0180] In certain examples, the binding moieties described herein are bound to the ITGB6 binding domain via their AB, CC, C" D, or EF loop and are bound to a bulk-serum protein, such as albumin, via their B-C, C-C", or F-G loop. In certain examples, the binding moiety is bound to the ITGB6 binding domain via its AB, CC, C" D, and EF loop and is bound to a bulk-serum protein, such as albumin, via its BC, CC", and FG loop. In certain examples, the binding moiety is bound to the ITGB6 binding domain via one or more of AB, CC, C" D, and E-F loop and is bound to a bulkserum protein, such as albumin, via one or more of BC, CC", and FG loop. In certain examples, the binding moiety is bound to a bulk serum protein, such as albumin, via its AB, CC, C" D, or EF loop and is bound to the ITGB6 binding domain via its BC, CC", or FG loop. In certain examples, the binding moiety is bound to a bulk serum protein, such as albumin, via its AB, CC, C" D, and EF loop and is bound to the ITGB6 binding domain via its BC, CC", and FG loop. In certain examples, the binding moiety of the first embodiment is bound to a bulk serum protein, such as albumin, via one or more of AB, CC, C" D, and E-F loop and is bound to the ITGB6 binding protein, via one or more of BC, CC", and FG loop. In certain examples, the binding moieties described herein are bound to a CD3 binding domain via their AB, CC, C" D, or EF loop and are bound to a bulk-serum protein, such as albumin, via their B-C, C-C", or F-G loop. In certain examples, the binding moieties described herein are bound to a bulk serum protein, such as albumin, via their AB, CC, C" D, or EF loop and are bound to a CD3 binding domain, via their B- C, C'-C", or F-G loop. In certain examples, the binding moieties described herein are bound to a CD3 binding domain via their AB, CC, C" D, or EF loop and are bound to an ITGB6 binding domain, via their B-C, C-C", or F-G loop. In certain examples, the binding moieties described herein are bound to an ITGB6 binding domain via their AB, CC, C" D, or EF loop and are bound to a CD3 binding domain, via their B-C, C-C", or F-G loop.

[0181] The bulk serum protein comprises, for example, albumin, fibrinogen, or a globulin. In some embodiments, the binding moieties are engineered scaffolds. Engineered scaffolds comprise, for example, sdAb, a scFv, a Fab, a VHH, a fibronectin type III domain, immunoglobulin-like scaffold (as suggested in Halaby et al. , 1999. Prof Eng 12(7):563-571), DARPin, cystine knot peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin-binding module, or a DNA or RNA aptamer scaffold.

[0182] In some cases, the binding moieties comprise a binding site for the bulk serum protein. In some embodiments, the CDRs within the binding moieties provide a binding site for the bulk serum protein. The bulk serum protein is, in some examples, a globulin, albumin, transferrin, IgGl, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, Fibrinogen, IgE, or pentameric IgM. In some embodiments, the binding moieties comprise a binding site for an immunoglobulin light chain. Insome embodiments, the CDRs provide a binding site for the immunoglobulin light chain. The immunoglobulin light chain is, in some examples, an IgK free light chain or an IgX free light chain.

[0183] In additional embodiments, the binding moieties are any kinds of polypeptides. For example, in certain instances the binding moieties are natural peptides, synthetic peptides, or fibronectin scaffolds, or engineered bulk serum proteins. In some examples, the binding moieties comprise any type of binding domain, including but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the binding moiety is a single chain variable fragment (scFv), a soluble TCR fragment, a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody. In other embodiments, the binding moieties are non-Ig binding domains, i.e., antibody mimetic, such as anticalins, affilins, affibody molecules, AFFIMERS®, affitins, alphabodies, avimers, DARPINS®, fynomers, kunitz domain peptides, or monobodies.

[0184] It is contemplated herein that the binding moiety described herein comprises at least one cleavable linker. In one aspect, the cleavable linker comprises a polypeptide having an amino acid sequence recognized and cleaved in an amino acid sequence-specific manner. The binding moiety described herein, in some cases, comprises a protease cleavable linker recognized and cleaved in an amino acid sequence-specific manner. In some embodiments, the protease cleavable linker is recognized in an amino acid sequence-specific manner by a matrix metalloprotease (MMP), for example a MMP9. In some cases, the protease cleavable linker recognized by a MMP9 comprises a polypeptide having an amino acid sequence PR(S / T)(L / I)(S / T) (SEQ ID NO: 733). In some cases, the protease cleavable linker recognized by a MMP9 comprises a polypeptide having an amino acid sequence LEATA (SEQ ID NO: 734). In some cases, the protease cleavable linker is recognized in an amino acid sequence-specific manner by a MMP11.

[0185] Proteases are proteins that cleave proteins, in some cases, in an amino acid sequencespecific manner. Proteases include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpam, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14,urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin- ip converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26). In some cases, the protease cleavable linker is recognized in an amino acid sequence-specific manner by a polypeptide having an amino acid sequence of one of SEQ ID NOS: 731-772.Table 1: Exemplary proteases and protease recognition sequences

[0186] Proteases are known to be secreted by some diseased cells and tissues, for example tumor or cancer cells, creating a microenvironment that is rich in proteases or a protease-rich microenvironment. In some case, the blood of a subject is rich in proteases. In some cases, cells surrounding the tumor secrete proteases into the tumor microenvironment. Cells surrounding the tumor secreting proteases include but are not limited to the tumor stromal cells, myofibroblasts, blood cells, mast cells, B cells, NK cells, regulatory T cells, macrophages, cytotoxic T lymphocytes, dendritic cells, mesenchymal stem cells, polymorphonuclear cells, and other cells. In some cases, proteases are present in the blood of a subject, for example proteases that target amino acid sequences found in microbial peptides. This feature allows for targeted therapeutics such as antigen binding proteins to have additional specificity because T cells will not be bound by the antigen binding protein except in the protease rich microenvironment of the targeted cells or tissue. Other non-limiting examples of linkers that may be utilized in constructs described herein are provided in the Sequence Listing below.Integration into chimeric antigen receptors (CAR)

[0187] The ITGB6 binding proteins of the present disclosure can, in certain examples, be incorporated into a chimenc antigen receptor (CAR), or to a ProCAR. An engineered immune effector cell, e.g, a T cell or NK cell, can be used to express a CAR that includes an ITGB6 binding protein containing, for example, an anti-ITGB6 single domain antibody as described herein. In one embodiment, the CAR including the ITGB6 binding protein as described herein is connected to a transmembrane domain via a hinge region, and further a costimulatory domain, e.g., a functional signaling domain obtained from 0X40, CD27, CD28, CD5, ICAM-1, LFA-1 (CDl la / CD18), ICOS (CD278), or 4-1BB. In some embodiments, the CAR further comprises an amino acid sequence encoding an intracellular signaling domain, such as 4-1BB and / or CD3 zeta.

[0188] The conditionally active receptors described herein comprise at least one binding moiety comprising anon-CDR loop. In one aspect, the binding moiety masks or is capable of masking binding of the ITGB6 binding domain, until activation. The cleavable linker, for example, comprises a protease cleavage site or a pH dependent cleavage site. The cleavable linker, in certain instances, is cleaved only in a tumor microenvironment. Thus, the binding moiety, connected to the cleavable linker, and further bound to the ITGB6 binding domain, in some examples, maintains the ITGB6 binding domain in an inert state in circulation until the cleavable linker is cleaved off in a tumor microenvironment. In some embodiments, the binding moiety binds to the ITGB6 binding domain. In some embodiments, a non-CDR loop provides a binding site for binding of the moiety to the ITGB6 binding domain. In some embodiments, the binding moiety masks or is capable of masking binding of the ITGB6 binding domain to its target antigen, e.g., via steric occlusion, via specific intramolecular interactions, such as interactions within the different domains of the polypeptide comprising the binding moiety. In some embodiments, the binding moiety further comprises complimentary determining regions (CDRs).

[0189] In some instances, the binding moiety of a CAR or a proCAR as described herein is a domain derived from an immunoglobulin molecule (Ig molecule), as described above in the section corresponding to conditionally active multispecific ITGB6 targeting proteins of this disclosure.

[0190] In some embodiments, the conditionally active receptor comprises an ITGB6 binding domain (aTargetl), a cleavable linker, and a binding moiety (aTarget2). The ITGB6 binding domain has specificity for a first target (ITGB6), while the binding moiety has specificity for a second target. The binding moiety also has a modified non-CDR loop, which inhibits binding of the ITGB6 binding domain to its target. Once cleaved at the cleavable linker, the binding moiety can be released, enabling binding of the ITGB6 binding domain.

[0191] In some embodiments, the inactive receptor (Inactive ProCAR) comprises an ITGB6 binding domain (Anti-Tumor Target sdAb or scFv) connected to a binding moiety (Anti-Target 2 sdAb) via a linker comprising a protease cleavage site. The binding moiety comprises a masking peptide / moiety, inserted into one or more non-CDR loops, such that the binding moiety binds to and inhibits the ITGB6 antigen binding domain. In some embodiments, the binding moiety has specificity for a given target, as described further elsewhere herein. The receptor also comprises a transmembrane domain and an intracellular signaling domain. The receptor is provided in a T-cell (CAR-T). Upon exposure to a tumor environment, the protease cleavage site is cleaved by tumor- associated proteases, thereby activating the receptor, generating the active receptor which does not comprise the binding moiety. The receptor now comprises an active antigen-binding domain. As the receptor is internalized by the cell, new receptors are generated which comprise the binding moiety and are inactive.

[0192] The cleavable linker of the binding moiety, in some embodiments, comprises a protease cleavable site similar to what is described above with respect to the conditionally active multispecific proteins comprising an ITGB6 binding domain of this disclosure. For instance, the cleavable linker in some embodiments comprises an amino acid sequence selected from the linker sequences provided in the sequence table.Transmembrane domain

[0193] The conditionally active chimeric antigen receptors, T-cell receptor fusion proteins, and T-cell receptors of the present disclosure include a transmembrane domain for insertion into a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the ITGB6 binding domain and the intracellular domain. In some embodiments, the transmembrane domain is interposed between the ITGB6 binding domain and the costimulatory domain.

[0194] Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use. As one non-limiting example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 773) can be used. Additional non-limiting examples of suitable TM sequences include: a) CD8 beta derived: GLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO: 774); b) CD4 derived: ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO: 775); c) CD3 zeta derived: LCYLLDGILFIYGVILTALFLRV (SEQ ID NO: 776); d) CD28 derived: WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 777); e) CD134 (0X40) derived: AAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO: 778); and I) CD7 derived: ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO: 779).Hinge Region

[0195] In some cases, the conditionally active chimeric antigen receptors, T-cell receptor fusion proteins, and T-cell receptors of the present disclosure compnse a hinge region (also referred to herein as a “spacer”), where the hinge region is interposed between the ITGB6 binding domain and the transmembrane domain. In some cases, the hinge region is an immunoglobulin heavy chain hinge region. In some cases, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0196] The hinge region can have a length of from about 4 amino acids to about 50 amino acids (aa), e.g. , from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa.

[0197] Suitable spacers can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0198] Exemplary spacers include glycine polymers (G)n, glycine- serine polymers (including, for example, (GS)n (SEQ ID NO: 718), (GSGGS)n (SEQ ID NO: 780) and (GGGS)n (SEQ ID NO: 720), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary spacers comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 781), GGSGG (SEQ ID NO: 782), GSGSG (SEQ ID NO: 783), GSGGG (SEQ ID NO: 784), GGGSG (SEQ ID NO: 785), GSSSG (SEQ ID NO: 786), and the like.

[0199] Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res. 14: 1779. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 788); CPPC (SEQ ID NO: 789); CPEPKSCDTPPPCPR (SEQ ID NO: 790); see, e.g. , Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQ ID NO: 791); KSCDKTHTCP (SEQ ID NO: 792); KCCVDCP (SEQ ID NO: 793);KYGPPCP (SEQ ID NO: 794); EPKSCDKTHTCPPCP (SEQ ID NO: 795); human IgGl hinge); ERKCCVECPPCP (SEQ ID NO: 796); human IgG2 hinge); (SEQELKTPLGDTTHTCPRCP IDNO: 797); human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 798); human IgG4 hinge); and the like.

[0200] In some embodiments, the hinge region comprises an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. The hinge region can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally-occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 799); see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891.

[0201] In some embodiments, the hinge region comprises an amino acid sequence derived from human CD8; e.g., the hinge region comprises the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 800), or a variant thereof.Conditionally Active Chimeric Antigen Receptors

[0202] In one embodiment, the disclosure provides a conditionally active chimeric antigen receptor (CAR). A CAR generally comprises multiple domains, including a target antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The conditionally active CAR of the present disclosure comprises multiple domains, including a binding moiety, a target antigen binding domain which binds ITGB6, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a signaling domain of a protein including, but not limited to, ZAP70, CD3 zeta, and 4-1BB.

[0203] In some embodiments, the conditionally active chimeric antigen receptor further comprises a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain of a protein including, but not limited to, 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDl la / CD18), ICOS (CD278), and 4-1BB (CD137), and ammo acid sequences thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications thereto.

[0204] In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein including, but not limited to, a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto.

[0205] In one aspect, the disclosure provides a cell e.g., T-cell) engineered to express a CAR. In one aspect a cell is transformed with the CAR and the CAR is expressed on the cell surface. Insome embodiments, the cell (e.g., T-cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the CAR. In another embodiment, the cell (e.g. , T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, the cell may transiently express the CAR.Conditionally Active T-cell Receptor fusion proteins

[0206] In one embodiment, the disclosure provides a conditionally active T-cell receptor fusion protein. As used herein, a “T-cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T-cell.

[0207] The conditionally active TFP comprises a binding moiety, an ITGB6 binding domain, and a T-cell receptor subunit. In some embodiments, the T-cell receptor subunit further comprises at least a portion of a T-cell receptor extracellular domain, a transmembrane domain, and a T-cell receptor intracellular domain. In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein including, but not limited to, a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, or amino acid sequences thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications thereto.

[0208] In some embodiments, the T-cell receptor intracellular domain comprises a stimulatory domain. The stimulatory domain may be from T-cell receptor subunit, including but not limited to the beta subunit, alpha subunit, delta subunit, gamma subunit, epsilon subunit, or a combination thereof. In some embodiments, the stimulatory domain comprises an immunoreceptor tyrosinebased activation motif (IT AM) or portion thereof including, but not limited to, CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CDS, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications thereto.

[0209] In some embodiments, the conditionally active TFP further comprises a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain of aprotein including, but not limited to, 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDl la / CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications thereto.

[0210] In some embodiments, the ITGB6 binding domain is connected to the T-cell receptor extracellular domain by a linker sequence. In some instances, the encoded linker sequence comprises (G4S)n (SEQ ID NO: 723), wherein n=l to 4. In some instances, the encoded linker sequence comprises a long linker (LL) sequence. In some instances, the encoded long linker sequence comprises (G4S)n (SEQ ID NO: 723), wherein n=2 to 4. In some instances, the encoded linker sequence comprises a short linker (SL) sequence. In some instances, the encoded short linker sequence comprises (G4S)n (SEQ ID NO: 723), wherein n=l to 3.

[0211] In one aspect, the disclosure provides a cell (e.g, T-cell) engineered to express a conditionally active T-cell receptor fusion protein (TFP). In one aspect a cell is transformed with the conditionally active TFP and the conditionally active TFP is expressed on the cell surface. In some embodiments, the cell (e.g, T-cell) is transduced with a viral vector encoding a conditionally active TFP. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the conditionally active TFP. In another embodiment, the cell (e.g, T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a conditionally active TFP. In some such embodiments, the cell may transiently express the conditionally active TFP.Conditionally Active T-cell Receptors

[0212] In one embodiment, the disclosure provides a conditionally active T-cell receptor. A T- cell receptor generally comprises multiple subunits, including alpha, beta, delta, gamma, epsilon, and zeta subunits. The conditionally active T-cell receptor of the present disclosure comprises a binding moiety. In some embodiments, the binding moiety is attached to a T-cell receptor subunit including, but not limited to, the alpha subunit, beta subunit, or a combination thereof.

[0213] In some embodiments, the binding moiety masks or is capable of masking binding of the T-cell receptor to its target. In some embodiments, the binding moiety binds to T-cell receptor. In some embodiments, a non-CDR loop provides a binding site for binding of the moiety to the T-cell receptor. In some embodiments, the non-CDR loop provides a binding site specific for T-cell receptor alpha, T-cell receptor beta, or a combination thereof. In some embodiments, the binding moiety masks or is capable of masking binding of the T-cell receptor to its target, e.g., via stenc occlusion, via specific intermolecular interactions.

[0214] In one aspect, the disclosure provides a cell (e.g, T-cell) engineered to express a conditionally active T-cell receptor (TCR). In one aspect a cell is transformed with theconditionally active TCR and the conditionally active TCR is expressed on the cell surface. In some embodiments, the cell (e.g, T-cell) is transduced with a viral vector encoding a conditionally active TCR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the conditionally active TCR. In another embodiment, the cell (e.g., T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a conditionally active TCR. In some such embodiments, the cell may transiently express the conditionally active TCR.Cells

[0215] In one embodiment, the present disclosure provides a cell comprising the chimeric antigen receptor or the conditionally active chimeric antigen receptor, conditionally active T-cell receptor fusion protein, or conditionally active T-cell receptor of the present disclosure. The cell may be a mammalian cell.

[0216] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g, ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, HuT- 78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0217] In some instances, the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell or progenitor cell obtained from an individual.

[0218] In recent studies, CAR constructs have been used to direct natural killer (NK) cell activity, reviewed by Hermanson & Kaufman (2015, Front Immunol 6: 195) and Carlsten & Childs (2015, Front Immunol 6:266). Similar to T cells, NK cells can be transfected with CAR expression constructs and used to induce an immune response. Because NK cells do not require HLA matching, they can be used as allogeneic effector cells (Harmanson & Kaufman, 2015). Also, peripheral blood NK cells (PB-NK), of use for therapy, may be isolated from donors by a simple blood draw. The CAR constructs of use may contain similar elements to those used to make CAR-T cells.

[0219] As such, in some embodiments, the present disclosure provides a cell comprising an NK cell comprising the chimeric antigen receptor, the conditionally active chimeric antigen receptor, conditionally active T-cell receptor fusion protein, or conditionally active T-cell receptor of the present disclosure.

[0220] As discussed above in the context of a conditionally active ITGB6 binding protein (e.g, an ITGB6 ProTriTAC), in some embodiments, a conditionally active chimeric antigen receptor as described herein has an improved therapeutic index as compared to that of a chimeric antigen receptor comprising the same ITGB6 binding domain as the conditionally active variant but is constitutively active instead of being conditionally active. For instance, an ITGB6 ProCAR, in some embodiments, has an increased therapeutic index than an ITGB6 CAR. The increased, in some embodiments, is from at least about 2-fold to about 1000-fold, such as about 4-fold to about 800-fold, about 6-fold to about 800-fold, about 6-fold to about 600-fold, about 10-fold to about 400-fold, about 20-fold to about 200-fold, about 30-fold to about 150-fold, about 50-fold to about 100-fold. The increase in therapeutic index, in some embodiments, is attributed to the conjugation of the ITGB6 binding domain to a binding moiety as described above, with the non-CDR loop and the cleavable linker.Methods of Generating a Cell Comprising a Conditionally Active Receptor

[0221] The present disclosure provides a method of generating a cell comprising a conditionally active chimeric antigen receptor, T-cell receptor fusion protein, or T-cell receptor. The method generally involves genetically modifying a mammalian cell with an expression vector, or an RNA (e.g, in vitro transcribed RNA), comprising nucleotide sequences encoding a conditionally active chimeric antigen receptor, T-cell receptor fusion protein, or T-cell receptor of the present disclosure. The genetic modification can be carried out in vivo, in vitro, or ex vivo. The cell can be, for example, an immune cell (e.g, a T lymphocyte or NK cell), a stem cell, or a progenitor cell.

[0222] In some cases, the genetic modification is carried out ex vivo. For example, a T ly mphocyte, a stem cell, or an NK cell is obtained from an individual; and the cell obtained from the individual is genetically modified to express a conditionally active chimeric antigen receptor, T- cell receptor fusion protein, or T-cell receptor of the present disclosure.Sources of T-Cells

[0223] In some embodiments, a source of T-cells is obtained from a subject. The term “subject,” as used throughout this disclosure, is intended to include living organisms in which an immune response can be elicited (e.g, mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T-cells can be obtained from a number of sources, including, but not limited to, allogenic T cells (e.g, allogeneic donor-derived CAR T cells), natural killer cells(e.g., donor derived natural killer cells), peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T-cell lines available in the art, may be used. In certain embodiments of the present disclosure, T-cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T-cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis are washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the disclosure, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0224] In one embodiment, T-cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T-cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T-cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T-cells are isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3*28)-conjugated beads, such as DYNABEADS® M- 450 CD3 / CD28 T, for a time period sufficient for positive selection of the desired T-cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation time period is 24 hours. Longer incubation times may be used to isolate T-cells in any situation where there are few T-cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumortissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T-cells. Thus, by simply shortening or lengthening the time T-cells are allowed to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T-cells (as described further herein), subpopulations of T-cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T-cells can be preferentially selected for or against at culture initiation or at other desired time points. Multiple rounds of selection can also be used in the context of this disclosure. In certain embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

[0225] Enrichment of a T-cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T- cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar method of selection.

[0226] In one embodiment, a T-cell population can be selected that expresses one or more of IFN-y, TNFa, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g, other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No. WO 2013 / 126712.

[0227] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g, particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g. , increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet one embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T-cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T-cells that normally have weaker CD28 expression.

[0228] In another embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T-cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T-cells express higher levels of CD28 and are more efficiently captured than CD8+ T-cells in dilute concentrations. In one embodiment, the concentration of cells used is 5xl0e6 / ml. In other embodiments, the concentration used can be from about 1 x 105 / ml to 1 x 106 / ml, and any integer value in between.

[0229] In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10° C. or at room temperature.

[0230] T-cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C. or in liquid nitrogen.

[0231] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.

[0232] Also contemplated in the context of the disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as describedherein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary , and desired cells, such as T-cells, isolated and frozen for later use in T-cell therapy for any number of diseases or conditions that would benefit from T-cell therapy, such as those described herein. In one embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T-cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, my cophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.

[0233] In a further embodiment of the present disclosure, T-cells are obtained from a patient directly following treatment that leaves the subject with functional T-cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T-cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T-cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and / or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T- cells, B cells, dendritic cells, and other cells of the immune system.Activation and Expansion of T-Cells

[0234] T-cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0235] Generally, the T-cells of the disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T-cells. In particular, T-cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of an accessory' molecule on the surface of the T-cells, a ligand that binds the accessory molecule is used. For example, a population of T-cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T- cells. To stimulate proliferation of either CD4+ T-cells or CD8+ T-cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg etal., Transplant Proc. 30(8): 3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland etal., J. Immunol. Meth. 227(l-2):53-63, 1999).

[0236] In certain embodiments, the primary stimulatory signal and the costimulatory signal for the T-cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (z.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In one embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. US 20040101519 Al and US 20060034810 Al for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T-cells in the present disclosure.

[0237] In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen binding fragment thereof and the agent providing the costimulatory signal is an anti-CD28 antibody or antigen binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In oneembodiment, a 1 : 1 ratio of each antibody bound to the beads for CD4+ T-cell expansion and T-cell growth is used. In certain embodiments of the present disclosure, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T-cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1-fold to about 3-fold is observed as compared to the expansion observed using a ratio of 1: 1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1: 100 and all integer values there between. In one embodiment of the present disclosure, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the disclosure, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1. In one particular embodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1 :10 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used. In yet one embodiment, a 3: 1 CD3:CD28 ratio of antibody bound to the beads is used.

[0238] Ratios of particles to cells from 1:500 to 500: 1 and any integer values in between may be used to stimulate T-cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T-cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T-cells that result in T-cell stimulation can vary as noted above, however certain values include 1: 100, 1 :50, 1 :40, 1 :30, 1:20, 1: 10, 1 :9, 1:8, 1 :7, 1:6, 1:5, 1:4, 1:3, 1:2, 1 :1, 2: 1, 3: 1, 4:1, 5: 1, 6: 1, 7: 1, 8:1, 9: 1, 10: 1, and 15: 1 with one preferred ratio being at least 1 :1 particles per T-cell. In one embodiment, a ratio of particles to cells of 1:1 or less is used. In one particular embodiment, a particle: cell ratio is 1 :5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1 : 1 to 10: 1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1: 1 to 1 : 10 (based on cell counts on the day of addition). In one particular embodiment, the ratio of particles to cells is 1 :1 on the first day of stimulation and adjusted to 1 :5 on the third and fifth days of stimulation. In one embodiment, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 :5on the third and fifth days of stimulation. In one embodiment, the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1 : 10 on the third and fifth days of stimulation. In one embodiment, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 : 10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present disclosure. In particular, ratios will vary depending on particle size and on cell size and type.

[0239] In further embodiments of the present disclosure, the cells, such as T-cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0240] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T-cells. In one embodiment the cells (for example, 104to 109T-cells) and beads (for example, DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a ratio of 1:1) are combined in a buffer, for example PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present disclosure. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In one embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet one embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T-cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T-cells that normally have weaker CD28 expression.

[0241] In one embodiment of the present disclosure, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In one embodiment, the mixture may be cultured for 21 days. In one embodiment of the disclosure the beads and the T- cells are cultured together for about eight days. In one embodiment, the beads and T-cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T-cells can be 60 days or more. Conditions appropriate for T-cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFP, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1 40, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T-cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0242] T-cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T-cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T-cell population (TC, CD8+). Ex vivo expansion of T-cells by stimulating CD3 and CD28 receptors produces a population of T-cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T-cells comprises an increasingly greater population of TC cells.ITGB6 Binding Protein Modifications

[0243] The ITGB6 binding proteins described herein, including ITGB6 binding domains (e.g., an ITGB6 binding sdAb of this disclosure) and ITGB6 targeting multispecific proteins (e.g., an ITGB6 targeting trispecific or protrispecific protein as described herein) encompass derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue that is not one encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or an amino acid sequence for purification of the protein.

[0244] Typical modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0245] Modifications are made anywhere in the ITGB6 binding proteins described herein, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Certain common peptide modifications that are useful for modification of the ITGB6 binding proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blockage of the ammo or carboxyl group in a polypeptide, or both, by a covalent modification, and ADP-ribosylation.

[0246] In some embodiments, derivatives of the ITGB6 binding proteins as described herein comprise immunoreactive modulator derivatives and antigen binding molecules comprising one or more modifications.

[0247] In some embodiments, the ITGB6 binding proteins of the disclosure are monovalent or multivalent bivalent, trivalent, etc.). As used herein, the term “valency” refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds one target molecule or specific position or locus on a target molecule. When an antibody is monovalent, each binding site of the molecule will specifically bind to a single antigen position or epitope. When an antibody comprises more than one target binding site (multivalent), each target binding site may specifically bind the same or different molecules (e.g. , may bind to different ligands or different antigens, or different epitopes or positions on the same antigen).

[0248] In some embodiments, the ITGB6 binding proteins as set forth above are fused to an Fc region from any species, including but not limited to, human immunoglobulin, such as human IgGl, a human IgG2, a human IgG3, human IgG4, to generate Fc-fusion ITGB6 binding proteins. In some embodiments, the Fc-fusion ITGB6 binding proteins of this disclosure have extended halflife compared to an otherwise identical ITGB6 binding protein. In some embodiments, the Fc- fusion ITGB6 binding proteins of this disclosure contain inter alia one or more additional amino acid residue substitutions, mutations and / or modifications, e.g. , in the Fc region, which result in abinding protein with preferred characteristics including, but not limited to, altered pharmacokinetics, extended serum half-life, etc.

[0249] In some embodiments, such Fc-fused ITGB6 binding proteins provide extended half-lives in a mammal, such as in a human, of greater than 5 days, greater than 10 days, greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-life, in some cases, results in a higher serum titer which thus reduces the frequency of the administration of the ITGB6 binding proteins and / or reduces the concentration of the antibodies to be administered. Binding to human FcRn in vivo and serum half-life of human FcRn high affinity binding polypeptides is assayed, in some examples, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered.

[0250] The ITGB6 binding proteins, in some cases, are differentially modified during or after production, e.g, by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications are carried out by techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, try psin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0251] Various post-translational modifications of the ITGB6 binding proteins also encompassed by the disclosure include, for example, N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N- terminal methionine residue as a result of prokaryotic host cell expression. Moreover, the ITGB6 binding proteins are, in some cases, modified with a detectable label, such as an enzy matic, fluorescent, radioisotopic or affinity label to allow for detection and isolation of the modulator.Polynucleotides Encoding ITGB6 Binding Proteins

[0252] Also provided, in some embodiments, are polynucleotide molecules encoding ITGB6 binding proteins described herein. In some embodiments, the polynucleotide molecules are provided as a DNA construct. In other embodiments, the polynucleotide molecules are provided as a messenger RNA transcript.

[0253] The polynucleotide molecules are constructed by known methods such as by combining the genes encoding a single domain ITGB6 binding protein or gene encoding various domains of ITGB6 binding proteins comprising more than one domain. In some embodiments, the geneencoding the domains are either separated by peptide linkers or, in other embodiments, directly linked by a peptide bond, into a single genetic construct operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate expression system such as, for example CHO cells. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. The promoter is selected such that it drives the expression of the polynucleotide in the respective host cell.

[0254] In some embodiments, the polynucleotide coding for an ITGB6 binding protein as described herein is inserted into a vector, preferably an expression vector, which represents a further embodiment. This recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.

[0255] A variety of expression vector / host systems may be utilized to contain and express the polynucleotide encoding the polypeptide of the described ITGB6 binding protein. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.Thus, the ITGB6 binding proteins as described herein, in some embodiments, are produced by introducing a vector encoding the protein as described above into a host cell and culturing said host cell under conditions whereby the protein domains are expressed, may be isolated and, optionally, further purified.Pharmaceutical Compositions

[0256] Also provided, in some embodiments, are pharmaceutical compositions comprising an anti-ITGB6 binding protein described herein, a vector comprising the polynucleotide encoding the polypeptide of the ITGB6 binding proteins or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibactenal and antifungal agents.A further embodiment provides one or more of the above described ITGB6 binding proteins packaged in lyophilized form or packaged in an aqueous medium.

[0257] In some embodiments of the pharmaceutical compositions, the ITGB6 binding proteins described herein are encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, the ITGB6 binding protein is attached to liposomes. In some instances, the ITGB6 binding proteins are conjugated to the surface of liposomes. In some instances, the ITGB6 binding proteins are encapsulated within the shell of a liposome. In some instances, the liposome is a cationic liposome.

[0258] The ITGB6 binding proteins described herein are contemplated for use as a medicament. Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient’s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently. An “effective dose” refers to amounts of the active ingredient that are sufficient to affect the course and the seventy of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.

[0259] In some embodiments, the ITGB6 binding proteins of this disclosure are administered at a dosage of up to 10 mg / kg at a frequency of once a week. In some cases, the dosage ranges from about 1 ng / kg to about 10 mg / kg, for example about 1 ng / kg to about 70 ng / kg. In some embodiments, the dose is from about 1 ng / kg to about 10 ng / kg, about 5 ng / kg to about 15 ng / kg, about 12 ng / kg to about 20 ng / kg, about 18 ng / kg to about 30 ng / kg, about 25 ng / kg to about 50 ng / kg, about 35 ng / kg to about 60 ng / kg, about 45 ng / kg to about 70 ng / kg, about 65 ng / kg to about 85 ng / kg, about 80 ng / kg to about 1 pg / kg, about 0.5 ig / kg to about 5 pg / kg. about 2 pg / kg to about 10 pg / kg, about 7 pg / kg to about 15 pg / kg, about 12 pg / kg to about 25 pg / kg, about 20 pg / kg to about 50 pg / kg, about 20 pg / kg to about 60 pg / kg, about 20 pg / kg to 180 pg / kg. about 20 pg / kg to 540 pg / kg, about 35 pg / kg to about 70 pg / kg, about 45 pg / kg to about 80 pg / kg, about 60 pg / kg to 180 pg / kg, about 60 pg / kg to 540 pg / kg, about 65 pg / kg to about 90 pg / kg, about 85 pg / kg to about 0.1 mg / kg, about 0.095 mg / kg to about 10 mg / kg or about 180 pg / kg to 540 pg / kg. In some cases, the dosage is about 0.1 mg / kg to about 0.2 mg / kg; about 0.25 mg / kg to about 0.5 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 3 mg / kg, about 2.5 mg / kg to about 4mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 6 mg / kg, about 5.5 mg / kg to about 7 mg / kg, about 6.5 mg / kg to about 8 mg / kg, about 7.5 mg / kg to about 9 mg / kg, or about 8.5 mg / kg to about 10 mg / kg. In some embodiments, the ITGB6 binding proteins of this disclosure are administered at a dosage of about 20pg / kg, about 60pg / kg, about 180pg / kg or about 540pg / kg. In some embodiments, the ITGB6 binding proteins of this disclosure are administered at a dosage of 1 ng / kg, 2 ng / kg, 5ng / kg, lOng / kg, 20ng / kg, 30 ng / kg, 40ng / kg, 50 ng / kg, 60 ng / kg, 60 ng / kg, 70 ng / kg, 80 ng / kg, 90 ng / kg, 100 ng / kg, 200 ng / kg, 300 ng / kg, 400 ng / kg, 500 ng / kg, 600 ng / kg, 700 ng / kg, 800 ng / kg, 900ng / kg, 1 pg / kg, 2 pg / kg, 5 pg / kg, 10 pg / kg, 12 pg / kg, 15 pg / kg, 20 pg / kg, 22.5 pg / kg, 25 pg / kg, 30 pg / kg, 40 pg / kg, 50 pg / kg, 60 pg / kg, 70 pg / kg, 80 pg / kg, 90 pg / kg, 100 pg / kg, 130 pg / kg, 150 pg / kg, 180 pg / kg, 200 pg / kg, 225 pg / kg, 250 pg / kg, 280 pg / kg, 300 pg / kg, 350 pg / kg, 370 pg / kg, 400 pg / kg, 430 pg / kg, 460 pg / kg, 500 pg / kg, 540 pg / kg, 590 pg / kg, 600 pg / kg, 630 pg / kg, 670 pg / kg, 700 pg / kg, 730 pg / kg, 780 pg / kg, 800 pg / kg, 840 pg / kg, 900 pg / kg, 950 pg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, or 10 mg / kg. The frequency of administration, in some embodiments, is about less than daily, every other day, less than once a day, twice a week, weekly, once in 7 days, once in two weeks, once in three weeks, once in four weeks, or once a month. In some cases, the frequency of administration is weekly. In some cases, the frequency of administration is weekly and the dosage is up to 10 mg / kg. In some cases, duration of administration is from about 1 day to about 4 weeks or longer.Methods of treatments and Tumor growth reduction properties

[0260] Also provided in certain embodiments are methods of treating a condition associated with malignant cells expressing ITGB6 in a subject comprising administering to a subject in need thereof an effective amount of an ITGB6 binding domains or multispecific proteins (including conditionally active multispecific proteins) comprising an ITGB6 binding domain of this disclosure, or a CAR or a ProCAR comprising an ITGB6 binding protein as described herein, or a pharmaceutical composition comprising the same. In some embodiments, the condition is a cancer.

[0261] In another aspect, the disclosure provides a method of inhibiting tumor growth or progression in a subject who has malignant cells expressing ITGB6, comprising administering to the subject in need thereof an effective amount of an ITGB6 binding domains or multispecific proteins comprising an ITGB6 binding domain of this disclosure, or a CAR comprising an ITGB6 binding protein as described herein, or a pharmaceutical composition comprising the same. In another aspect, the disclosure provides a method of inhibiting metastasis of malignant cells expressing ITGB6 in a subject, comprising administering to the subject in need thereof an effectiveamount of an ITGB6 binding domains or multispecific proteins comprising an ITGB6 binding domain of this disclosure, or a pharmaceutical composition comprising the same. In another aspect, the disclosure provides a method of inducing tumor regression in a subject who has malignant cells expressing ITGB6, comprising administering to the subject in need thereof an effective amount of an ITGB6 binding domains or multispecific proteins comprising an ITGB6 binding domain of this disclosure, or a pharmaceutical composition comprising the same. In some embodiments, the methods as described herein further comprise administering an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is a biotherapeutic agent, for example, an antibody. In some embodiments, the second therapeutic agent is a cytokine, TNFa (Tumor Necrosis Factor alpha), a PAP (phosphatidic acid phosphatase) inhibitor, an oncolytic virus, a kinase inhibitor, an IDO (Indoleamine-pyrrole 2,3-dioxygenase) inhibitor, a glutaminase GLS1 inhibitor, a CAR (Chimeric Antigen Receptor)-T cell or T cell therapy, a TLR (Toll-Like Receptor) Agonist (e.g , TLR3, TLR4, TLR5, TLR7, TLR9), or a tumor vaccine.

[0262] In certain embodiments, the ITGB6 binding proteins of the disclosure reduce the growth of tumor cells in vivo when administered to a subject who has tumor cells that express ITGB6. Measurement of the reduction of the growth of tumor cells can be determined by multiple different methodologies well known in the art. Nonlimiting examples include direct measurement of tumor dimension, measurement of excised tumor mass and comparison to control subjects, measurement via imaging techniques (e.g. , CT or MRI) that may or may not use isotopes or luminescent molecules (e.g., luciferase) for enhanced analysis, and the like. In specific embodiments, administration of the ITGB6 binding proteins of the disclosure results in a reduction of in vivo growth of tumor cells as compared to a control antigen binding agent by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, with an about 100% reduction in tumor growth indicating a complete response and disappearance of the tumor. In further embodiments, administration of the ITGB6 binding proteins of the disclosure results in a reduction of in vivo growth of tumor cells as compared to a control antigen binding agent by about 50-100%, about 75- 100% or about 90-100%. In further embodiments, administration of the ITGB6 binding proteins of the disclosure results in a reduction of in vivo growth of tumor cells as compared to a control antigen binding agent by about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90- 100%.

[0263] In some embodiments, the ITGB6 binding proteins of the present disclosure are administered to treat a neoplastic condition. Neoplastic conditions, in some embodiments, are benign or malignant; solid tumors or other blood neoplasia; and, in some embodiments, are selected from the group including, but not limited to: adrenal gland tumors, AIDS-associated cancers,alveolar soft part sarcoma, astrocytic tumors, autonomic ganglia tumors, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), blastocoelic disorders, bone cancer (adamantinoma, aneurismal bone cysts, osteochondroma, osteosarcoma), brain and spinal cord cancers, metastatic brain tumors, breast cancer including triple negative breast cancer, carotid body tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytomas, desmoplastic small round cell tumors, ependymomas, epithelial disorders, Ewing's tumors, extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder and bile duct cancers, gastric cancer, gastrointestinal, gestational trophoblastic disease, germ cell tumors, glandular disorders, head and neck cancers, hypothalamic, intestinal cancer, islet cell tumors, Kaposi's Sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemias, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphomas, lung cancers (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, efc.), macrophagal disorders, medulloblastoma, melanoma, meningiomas, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancers, papillary' thyroid carcinomas, parathyroid tumors, pediatric cancers, peripheral nerve sheath tumors, phaeochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, rare hematologic disorders, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcomas, skin cancer, soft-tissue sarcomas, squamous cell cancer, stomach cancer, stromal disorders, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid metastatic cancer, and uterine cancers (carcinoma of the cervix, endometrial carcinoma, and leiomyoma).

[0264] In certain embodiments the ITGB6 binding proteins of the present disclosure are used as a front-line therapy and administered to subjects who have not previously been treated for the cancerous condition. In other embodiments the ITGB6 binding proteins of the present disclosure are used to treat subjects that have previously been treated (with an ITGB6 binding protein of this disclosure or with other anti-cancer agent) and have relapsed or determined to be refractory to the previous treatment. In some embodiments the ITGB6 binding proteins of the present disclosure are used to treat subjects that have recurrent tumors.

[0265] In some embodiments, an ITGB6 binding protein as described herein, including a multispecific protein, a CAR or a ProCAR as described herein, is administered to treat a cancer with widespread ITGB6 expression and prevalence, including, but not limited to, colorectal, prostate, neuroendocrine, thyroid, lung (both non-small cell lung and small cell lung cancers),gastric, ovarian, endometrial, pancreatic, biliary tract and gallbladder cancer, esophageal, breast, an adenocarcinoma, or any combination thereof.

[0266] In some aspects, the ITGB6 binding proteins of the present disclosure are administered to treat a proliferative disorder comprising a solid tumor including, but not limited to, adrenal, liver, kidney, bladder, breast, gastric, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinomas, sarcomas, glioblastomas, various head and neck tumors, or any combination thereof.

[0267] In some embodiments, the ITGB6 binding proteins of the present disclosure are administered to a subject suffering from melanoma. In some embodiments, the ITGB6 binding proteins of the present disclosure are used to diagnose, monitor, treat or prevent melanoma. The term “melanoma,” as used herein, includes all types of melanoma including, but not limited to, primary melanoma, malignant melanoma, cutaneous melanoma, extracutaneous melanoma, superficial spreading melanoma, polypoid melanoma, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, nodular malignant melanoma, lentigo maligna melanoma, lentiginous melanoma, lentiginous malignant melanoma, mucosal lentiginous melanoma, mucosal melanoma, acral lentiginous melanoma, soft tissue melanoma, ocular melanoma, invasive melanoma, familial atypical mole and melanoma (FAM-M) syndrome, desmoplastic malignant melanoma, uveal melanoma, or any combination thereof.

[0268] In some embodiments, possible indications for administration of the ITGB6 binding proteins of this disclosure or pharmaceutical compositions comprising the same are tumorous diseases especially epithelial cancers / car cinomas such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, cancers of the genito-urinary tract, e.g., ovarian cancer, endometrial cancer, cervix cancer and kidney cancer, lung cancer, gastric cancer, cancer of the small intestine, liver cancer, pancreas cancer, gall bladder cancer, cancers of the bile duct, esophagus cancer, cancer of the salivatory glands and cancer of the thyroid gland. In some embodiments, the administration of the ITGB6 binding proteins of this disclosure or pharmaceutical compositions comprising the same is indicated for minimal residual disease, such as early solid tumor, advanced solid tumor or metastatic solid tumor, which is characterized by the local and non-local reoccurrence of the tumor caused by the survival of single cells, or any combination thereof.

[0269] In selected aspects an ITGB6 binding proteins of the disclosure is incorporated into a chimeric antigen receptors (CAR) and the ITGB6 CAR is administered in a CAR based therapy effective at treating a cancer, such as: epithelial cancers / carcinomas such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, cancers of the genito-urinary tract, e.g.,ovarian cancer, endometrial cancer, cervix cancer and kidney cancer, lung cancer, gastric cancer, cancer of the small intestine, liver cancer, pancreas cancer, gall bladder cancer, cancers of the bile duct, esophagus cancer, cancer of the salivatory glands and cancer of the thyroid gland, small cell lung cancer, non-small cell lung cancer (e.g., squamous cell non-small cell lung cancer or squamous cell small cell lung cancer), large cell neuroendocrine carcinoma (LCNEC), or any combination thereof.

[0270] A chimeric antigen receptor is generally an artificially constructed hybrid protein or polypeptide containing or comprising an antigen binding domain of an antibody linked to a signaling domain (e.g., T-cell signaling or T-cell activation domains). In some embodiments, CARs comprising the ITGB6 binding protein of the present disclosure have the ability to redirect the specificity and reactivity of sensitized lymphocytes (e.g., T-cells) toward ITGB6 positive target cells in a non-MHC-restricted manner by exploiting the antigen-binding properties of antibodies or antigen binding fragments thereof. The non-MHC-restricted antigen recognition gives T-cells expressing ITGB6 CARs the ability to recognize tumorigenic ITGB6 independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed in T- cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

[0271] In some embodiments the disclosed ITGB6 binding proteins are administered to refractory patients (i.e., those whose disease recurs during or shortly after completing a course of initial therapy); sensitive patients (i.e., those whose relapse is longer than 2-3 months after primary therapy); or patients exhibiting resistance to a platinum based agent (e.g. , carboplatin, cisplatin, oxaliplatin) and / or a taxane (e.g., docetaxel, paclitaxel, larotaxel or cabazitaxel). In another embodiment the disclosed ITGB6 CAR treatments are effective at treating ovarian cancer, including ovarian-serous carcinoma and ovarian-papillary serous carcinoma.

[0272] In another embodiment the ITGB6 binding proteins of the disclosure, the ITGB6 CAR, or the ITGB6 sensitized lymphocytes, or any combination thereof are used in maintenance therapy to reduce or eliminate the chance of tumor recurrence following the initial presentation of the disease. In some cases, the disorder has been treated and the initial tumor mass eliminated, reduced or otherwise ameliorated so the patient is asymptomatic or in remission. At such time the subject is administered pharmaceutically effective amounts of the disclosed the ITGB6 binding proteins of the disclosure, the ITGB6 CAR, or the ITGB6 sensitized lymphocytes, or any combination thereof one or more times regardless of if there is little or no indication of disease using standard diagnostic procedures. In some embodiments, the ITGB6 binding proteins of the disclosure, the ITGB6 CAR, or the ITGB6 sensitized lymphocytes, or any combination thereof is administered on a regularschedule over a period of time, such as weekly, every two weeks, monthly, every six weeks, every two months, every three months every six months or annually, for example, to reduce the potential of disease recurrence. Moreover, such treatments are in some embodiments continued for a period of weeks, months, years or even indefinitely depending on the patient response and clinical and diagnostic parameters.

[0273] In yet another embodiment, the ITGB6 binding proteins of the disclosure, the ITGB6 CAR, or the ITGB6 sensitized lymphocytes, or any combination thereof are used to prophy tactically or as an adjuvant therapy to prevent or reduce the possibility of tumor metastasis following a debulking procedure. As used in the present disclosure a “debulking procedure,” is means any procedure, technique or method that eliminates, reduces, treats or ameliorates a tumor or tumor proliferation. Exemplary debulking procedures include, but are not limited to, surgery, radiation treatments (z.e. , beam radiation), chemotherapy, immunotherapy or ablation. In some embodiments, at appropriate times, the ITGB6 binding proteins of the disclosure, the ITGB6 CAR, or the ITGB6 sensitized lymphocytes, or any combination thereof are administered as suggested by clinical, diagnostic or theranostic procedures to reduce tumor metastasis. In some embodiments, the dosing regimen is accompanied by appropriate diagnostic or monitoring techniques that allow it to be modified.

[0274] Yet other embodiments of the disclosure comprise administering the ITGB6 binding protein of the disclosure, the ITGB6 CAR, or the ITGB6 sensitized lymphocytes, or any combination thereof to subjects that are asymptomatic but at risk of developing a proliferative disorder. That is, in some embodiments, the ITGB6 binding protein of the disclosure, the ITGB6 CAR, or the ITGB6 sensitized lymphocytes, or any combination thereof are used in preventative sense and given to patients that have been examined or tested and have one or more noted risk factors (e.g. , genomic indications, family history, in vivo or in vitro test results, etc.) but have not developed neoplasia. In such cases those skilled in the art would be able to determine an effective dosing regimen through empirical observation or through accepted clinical practices.

[0275] In some embodiments of the methods described herein, the ITGB6 binding proteins, or compositions as described herein are administered in combination with an agent for treatment of the particular disease, disorder or condition, also referred to herein as an additional therapeutic agent. Such agents include but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies (y-rays, X- rays, and / or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g, antisense, retroviral therapy and the like) and other immunotherapies. In some embodiments, an ITGB6 binding protein as described herein is administered in combination withanti-diarrheal agents, anti-emetic agents, analgesics, opioids and / or non-steroidal anti-inflammatory agents. In some embodiments, an ITGB6 binding protein as described herein is administered in combination with anti-cancer agents. Nonlimiting examples of anti-cancer agents that can be used in the various embodiments of the disclosure, including pharmaceutical compositions and dosage forms and kits of the disclosure, include: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefmgol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochlonde; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-nl interferon alpha-n3; interferon beta-la; interferon gammalb; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochlonde; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safmgol; safmgol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochlonde; temoporfm; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa;tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinzolidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. Other examples of anti-cancer drugs include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrme; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis- porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophy cin 8; cryptophy cin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodi demnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9~; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfemmex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-I receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear poly amine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitor (such as but not limited to, Lovastatin, Pravastatin, Fluvastatin, Statin, Simvastatin, and Atorvastatin); loxoribine; lurtotecan; lutetium texaphyrin; ly sofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; punne nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelhptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin;- 11 -SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenyl acetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; spl enopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfm; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichlonde; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; Vitaxin®; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Additional anti-cancer drugs are 5- fluorouracil and leucovorin. These two agents are particularly useful when used in methods employing thalidomide and a topoisomerase inhibitor. In some embodiments, the ITGB6 binding protein of the present disclosure is used in combination with gemcitabine. In some embodiments, the ITGB6 binding protein as described herein is administered before, during, or after surgery.Methods of detection of ITGB6 expression and diagnosis of ITGB6 associated cancer

[0276] According to another embodiment of the disclosure, kits for detecting expression of ITGB6 in vitro or in vivo are provided. The kits include the foregoing ITGB6 binding protein (e.g., an ITGB6 binding protein containing a labeled anti- ITGB6 single domain antibody or antigen binding fragments thereof), and one or more compounds for detecting the label. In some embodiments, the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

[0277] In some cases, ITGB6 expression is detected in a biological sample. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum,plasma, sputum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate.

[0278] In one embodiment, provided is a method of determining if a subject has cancer by contacting a sample from the subject with an anti- ITGB6 single domain antibody as disclosed herein; and detecting binding of the single domain antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample identifies the subject as having cancer.

[0279] In another embodiment, provided is a method of confirming a diagnosis of cancer in a subject by contacting a sample from a subject diagnosed with cancer with an anti- ITGB6 single domain antibody as disclosed herein; and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample confirms the diagnosis of cancer in the subject.

[0280] In some examples of the disclosed methods, the ITGB6 single domain antibody is directly labeled. In some examples, the methods further include contacting a second antibody that specifically binds the anti- ITGB6 single domain antibody with the sample; and detecting the binding of the second antibody. An increase in binding of the second antibody to the sample as compared to binding of the second antibody to a control sample detects cancer in the subject or confirms the diagnosis of cancer in the subject. In some cases, the cancer is a neuroendocrine cancer, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer or ovanan cancer (such as epithelial ovarian carcinoma), or any other type of cancer that expresses ITGB6. In some examples, the control sample is a sample from a subject without cancer. In particular examples, the sample is a blood or tissue sample.

[0281] In some cases, the antibody that binds (for example specifically binds) ITGB6 is directly labeled with a detectable label. In another embodiment, the antibody that binds (for example, specifically binds) ITGB6 (the first antibody) is unlabeled and a second antibody or other molecule that can bind the antibody that specifically binds ITGB6 is labeled. A second antibody is chosen such that it is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a llama IgG, then the secondary antibody may be an anti-llama-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially. Suitable labels for the antibody or secondary antibody are described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferon, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include1251,131I,35S or3H.

[0282] In an alternative embodiment, ITGB6 can be assayed in a biological sample by a competition immunoassay utilizing ITGB6 standards labeled with a detectable substance and an unlabeled antibody that specifically binds ITGB6. In this assay, the biological sample, the labeled ITGB6 standards and the antibody that specifically bind ITGB6 are combined and the amount of labeled ITGB6 standard bound to the unlabeled antibody is determined. The amount of ITGB6 in the biological sample is inversely proportional to the amount of labeled ITGB6 standard bound to the antibody that specifically binds ITGB6.

[0283] The immunoassays and method disclosed herein can be used for a number of purposes. In one embodiment, the antibody that specifically binds ITGB6 may be used to detect the production of ITGB6 in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of ITGB6 in a biological sample, such as a tissue sample, or a blood or serum sample. In some examples, the ITGB6 is cell-surface ITGB6. In other examples, the ITGB6 is soluble ITGB6 (e.g., ITGB6 in a cell culture supernatant or soluble ITGB6 in a body fluid sample, such as a blood or serum sample).

[0284] In one embodiment, a kit is provided for detecting ITGB6 in a biological sample, such as a blood sample or tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be obtained to detect the presence of soluble ITGB6 protein or fragment. Kits for detecting a polypeptide will typically comprise a single domain antibody, according to the present disclosure, that specifically binds ITGB6. In some embodiments, an antibody fragment, such as an scFv fragment, a VH domain, or a Fab is included in the kit. In a further embodiment, the antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label).

[0285] In one embodiment, a kit includes instructional materials disclosing means of use of an antibody that binds ITGB6. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk), may be visual (such as video files), or provided through an electronic network, for example, over the internet, World Wide Web, an intranet, or other network. The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

[0286] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method of detecting ITGB6 in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to an ITGB6 polypeptide. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0287] Methods of determining the presence or absence of a cell surface marker are well known in the art. For example, the antibodies can be conjugated to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metal compounds, radioactive compounds or drugs. The antibodies can also be utilized in immunoassays such as but not limited to radioimmunoassays (RIAs), ELISA, or immunohistochemical assays. The antibodies can also be used for fluorescence activated cell sorting (FACS). FACS employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells. See U.S. Patent No. 5, 061,620). Any of the single domain antibodies that bind ITGB6, as disclosed herein, can be used in these assays. Thus, the antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.Certain definitions

[0288] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “wi th”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0289] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value.Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.

[0290] The terms “individual,” “patient,” or “subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker e.g., a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker).

[0291] An “antibody” typically refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Human light chains comprise a variable domain (VL) and a constant domain (CL) wherein the constant domain may be readily classified as kappa or lambda based on amino acid sequence and gene loci. Each heavy chain comprises one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD, comprises three domains termed CHI, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In IgG, IgA, and IgD classes the CHI and CH2 domains are separated by a flexible hinge region, which is a proline and cysteine rich segment of variable length (generally from about 10 to about 60 amino acids in IgG). The variable domains in both the light and heavy chains are joined to the constant domains by a “J” region of about 12 or more amino acids and the heavy chain also has a “D” region of about 10 additional amino acids. Each class of antibody further comprises inter-chain and intra-chain disulfide bonds formed bypaired cysteine residues. There are two types of native disulfide bridges or bonds in immunoglobulin molecules: interchain and intrachain disulfide bonds. The location and number of interchain disulfide bonds vary- according to the immunoglobulin class and species. Interchain disulfide bonds are located on the surface of the immunoglobulin, are accessible to solvent and are usually relatively easily reduced. In the human IgGl isotype there are four interchain disulfide bonds, one from each heavy chain to the light chain and two between the heavy- chains. The interchain disulfide bonds are not required for chain association. As is well known the cysteine rich IgGl hinge region of the heavy chain has generally been held to consist of three parts: an upper hinge, a core hinge, and a lower hinge. Those skilled in the art will appreciate that that the IgGl hinge region contain the cysteines in the heavy chain that comprise the interchain disulfide bonds (two heavy / heavy, two heavy / light), which provide structural flexibility' that facilitates Fab movements. The interchain disulfide bond between the light and heavy chain of IgGl are formed between C214 of the kappa or lambda light chain and C220 in the upper hinge region of the heavy chain. The interchain disulfide bonds between the heavy chains are at positions C226 and C229 (all numbered per the EU index according to Kabat, et al., infra.')

[0292] As used herein the term “antibody” includes polyclonal antibodies, multiclonal antibodies, monoclonal antibodies, chimeric antibodies, deimmunized, humanized and primatized antibodies, CDR grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies (e.g., a monovalent IgG), multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof, immunospecific antibody fragments such as: hdgG, a V-NAR, Fv, Fd, Fab, F(ab')2, F(ab'), Fab2, Fab3 fragments, single-chain fragments (e.g., di-scFv, scFv, scFvFc, scFv- zipper, scFab), disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as nanobodies or single variable domain antibodies comprising merely one variable domain such as sdAb (VH, VL, or VHH domains), “r IgG” (“half antibody”), diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, “minibodies” are in some instances exemplified by a structure which is as follows: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2, multibodies such as triabodies or tetrabodies; and derivatives thereof including Fc fusions and other modifications, and any other immunoreactive molecule so long as it comprises a domain having a binding site for preferential association or binding with an ITGB6-protein. Moreover, unless dictated otherw ise by contextual constraints the term further comprises all classes of antibodies (IgA, IgD, IgE, IgG, and IgM) and all subclasses (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2). Heavy -chain constant domains that correspond to the different classes of antibodies are typically denoted by the corresponding lower-case Greek letter alpha, delta, epsilon, gamma, and mu, respectively. Light chains of the antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ), based on the amino acid sequences of their constant domains. In some embodiments, the ITGB6 binding proteins comprise a heavy chain only antibody, such as a VH or a VHH domain. In some cases, the ITGB6 binding proteins comprise a heavy chain only antibody that is an engineered human VH domain. In some examples, the engineered human VH domain is produced by panning of phage display libraries. In some embodiments, the ITGB6 binding proteins comprise a VHH. The term “VHH,” as used herein, refers to single chain antibody binding domain devoid of light chain. In some cases, a VHH is derived from an antibody of the type that can be found in Camelidae or cartilaginous fish which are naturally devoid of light chains or to a synthetic and non-immunized VHH which can be constructed accordingly. Each heavy chain comprises a variable region encoded by V-, D- and J exons. A VHH, in some cases, is a natural VHH, such as a Camelid-derived VHH, or a recombinant protein comprising a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from the group consisting of camels, llamas, vicunas, guanacos, andcartilaginous fish (such as, but not limited to, sharks). In another embodiment, the VHH is derived from an alpaca (such as, but not limited to, a Huacaya Alpaca or a Suri alpaca).

[0293] As used herein, “Vanable region” or “variable domain” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity -determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain vanable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a P-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody -dependent cellular toxicity. The assignment of amino acids to each domain, framework region and CDR is, in some embodiments, in accordance with one of the numbering schemes provided by Kabat etal. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept, of Health and Human Services, PHS, NIH, NIH Publication no. 91- 3242; Chothia et al. , 1987, PMID: 3681981; Chothia et al. , 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily -V CH Verlag GmbH and Co or AbM (Oxford Molecular / MSI Pharmacopeia) unless otherwise noted. In some embodiments of this disclosure, the ITGB6 binding proteins comprise heavy chain only antibodies, such as VH or VHH domains, and comprise three CDRs. Such heavy chain only antibodies, in some embodiments, bind ITGB6 as a monomer with no dependency on dimerization with a VL (light chain variable) region for optimal binding affinity.

[0294] “Variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat etal., Sequences of Proteins of Immunological Interest, Sth Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a singleamino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. It is not intended that CDRs of the present disclosure necessarily correspond to the Kabat numbering convention.

[0295] The term “Framework” or “FR” residues (or regions) refer to variable domain residues other than the CDR or hypervariable region residues as herein defined. A “human consensus framework” is a framework which represents the most commonly occurring amino acid residue in a selection of human immunoglobulin VL or VH framework sequences.

[0296] The term “epitope,” as used herein, refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0297] As used herein, the term “percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent ammo acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0298] As used herein, “elimination half-time” is used in its ordinary sense, as is described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination. The elimination of mostdrugs is exponential (i.e., follows first-order kinetics), since drug concentrations usually do not approach those required for saturation of the elimination process. The rate of an exponential process may be expressed by its rate constant, k, which expresses the fractional change per unit of time, or by its half-time, ti / 2 the time required for 50% completion of the process. The units of these two constants are time-1 and time, respectively. A first-order rate constant and the half-time of the reaction are simply related (kxt 1 / 2=0.693) and may be interchanged accordingly. Since first-order elimination kinetics dictates that a constant fraction of drug is lost per unit time, a plot of the log of drug concentration versus time is linear at all times following the initial distribution phase i.e., after drug absorption and distribution are complete). The half-time for drug elimination can be accurately determined from such a graph.

[0299] As used herein, the term "binding affinity” refers to the affinity of the proteins described in the disclosure to their binding targets and is expressed numerically using “Kd” values. If two or more proteins are indicated to have comparable binding affinities towards their binding targets, then the Kd values for binding of the respective proteins towards their binding targets, are within ±2 -fold of each other. If two or more proteins are indicated to have comparable binding affinities towards single binding target, then the Kd values for binding of the respective proteins towards said single binding target, are within ±2-fold of each other. If a protein is indicated to bind two or more targets with comparable binding affinities, then the Kd values for binding of said protein to the two or more targets are within ±2-fold of each other. In general, a higher Kd value corresponds to a weaker binding. In some embodiments, the “Kd” is measured by a radiolabeled antigen binding assay (RIA) or surface plasmon resonance assays using a BIACORE™-2000 or a BIACORE™- 3000 (BIAcore, Inc., Piscataway, N.J.). In certain embodiments, an “on-rate” or “rate of association” or “association rate” or “kon” and an “off-rate” or “rate of dissociation” or “dissociation rate” or “koff” are also determined with the surface plasmon resonance technique using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N. J.). In additional embodiments, the “Kd”, “kon”, and “koff” are measured using the OCTET® Systems (Pall Life Sciences). In an exemplary method for measuring binding affinity using the OCTET® Systems, the ligand, e.g., biotinylated human or cynomolgus ITGB6, is immobilized on the OCTET® streptavidin capillary sensor tip surface which streptavidin tips are then activated according to manufacturer's instructions using about 20-50 pg / ml human or cynomolgus ITGB6 protein. A solution of PBS / Casein is also introduced as a blocking agent. For association kinetic measurements, ITGB6 binding protein variants are introduced at a concentration ranging from about 10 ng / mL to about 100 pg / mL, about 50 ng / mL to about 5 pg / mL, or about 2 ng / mL to about 20 pg / mL. In some embodiments, the ITGB6 binding single domain proteins are used at aconcentration ranging from about 2 ng / mL to about 20 pg / mL. Complete dissociation is observed in case of the negative control, assay buffer without the binding proteins. The kinetic parameters of the binding reactions are then determined using an appropriate tool, e.g, ForteBio software.

[0300] As used herein, in some embodiments, “treatment” or “treating” or “treated” refers to therapeutic treatment wherein the object is to slow (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (z.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. In other embodiments, “treatment” or “treating” or “treated” refers to prophylactic measures, wherein the object is to delay onset of or reduce severity of an undesired physiological condition, disorder or disease, such as, for example is a person who is predisposed to a disease (e.g. , an individual who carries a genetic marker for a disease such as breast cancer).

[0301] A “TriTAC,” an “ITGB6 targeting TriTAC,” or an “ITGB6 targeting trispecific protein,” as used herein refers to a trispecific binding protein that is not conditionally activated, and comprises a binding moiety that is specific for a bulk serum protein, a first target antigen binding domain, and a second target antigen binding domain, wherein at least one of the first target antigen binding domain and the second target antigen binding domain comprises an ITGB6 binding protein as described herein, and at least one of the first target antigen binding domain and the second target antigen binding domain comprises a domain that binds a CD3, such as a human CD3.

[0302] A “ProTnTAC,” or an “ITGB6 targeting protrispecific protein,” as used herein refers to a trispecific binding protein that is conditionally activated, and comprises (i) a cleavable linker (e.g., comprising an amino acid sequence as set forth in SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, and 731-772), (ii) a binding moiety that is specific for a bulk serum protein and also comprises a masking moiety (e.g., comprising an amino acid sequence as set forth in SEQ ID NO: 728) which prohibits the binding of a first target antigen binding domain or a second target antigen binding domain to its target, wherein at least one of the first target antigen binding domain and the second target antigen binding domain comprises an ITGB6 binding protein as described herein. The ProTriTAC proteins of this disclosure are, in some cases, activated from amasked state to an active state by cleavage of the cleavable linker, for example, in a protease rich environment, such as in a tumor microenvironment, to form an active drug. An active drug, as provided herein, in some instances, comprises an ITGB6 binding domain of the disclosure and a CD3 binding domain of the disclosure. An example of an active drug is provided in SEQ ID NOS: 353-357, or an amino acid sequence that is at least about 75% to 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 353-357 such as about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 353-357.

[0303] A “non-cleavable prodrug,” as used herein refers to a ProTnTAC as described above where the cleavable linker is replaced by a non-cleavable linker (e.g., a linker as in SEQ ID NO: 660). An example of an active drug is provided in SEQ ID NOS: 345-352, or an amino acid sequence that is at least about 75% to 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 345-352, such as about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 345-352.

[0304] In a non-beta-sandwich scaffold (e.g, a DARPin, an affimer, an affibody), the “non-CDR loops” refer to an area that is (1) amenable for sequence randomization to allow engineered specificities to a second antigen, and (2) distal to the primary specificity determining region(s) typically used on the scaffold to allow simultaneous engagement of the scaffold to both antigens without steric interference. For this purpose, the primary specificity determining region(s) can be defined using the framework established in the Skrlec 2015 publication (Trends in Biotechnol, 33:408-418). An excerpt of the framework is listed below in Table 2:

[0305] “Chimeric antigen receptor” or “CAR” or “CARs”, as used herein, refers to engineered receptors which provide antigen specificity to cells (for example T cells). CARs comprise multiple domains, for example, at least one target antigen binding domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. Each domain may be connected by a linker. A “ProCAR,” as used herein, refers to a conditionally activatable CAR, comprising an ITGB6 binding domain of this disclosure.EXAMPLES

[0306] The application may be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following examples are presented in order to more fully illustrate embodiments and should in no way be construed, however, as limiting the broad scope of the application.Example 1: Screening of Phage Display Library for Identification of ITGAV / ITGB6 Binding Domains

[0307] Llamas were immunized with purified ITGAV / ITGB6 dimer protein expressed in 293 cells. A phage display library for expression of heavy variable antibody domains was constructed from circulating B cells isolated from the immunized llamas (van der Linden, de Geus, Stok, Bos, van Wassenaar, Verrips, and Frenken. 2000. J Immunol Methods 240: 185-195). Phage clones were screened for binding to ITGAV / ITGB6 dimer by expressing llama anti-ITGAV / ITGB6 proteins in E. colt, preparing periplasmic extracts, and performing colorimetric ELISAs. 67 unique heavy chain only sequences were identified (SEQ ID NOS 1 to 67) that produced a signal in the ELISA screening relative to the control with human ITGAV / ITGB6 protein (Table 3). The CDR1, CDR2, and CDR3 sequences for these heavy variable domains are, respectively, SEQ ID NOS 75 to 141, 149 to 215, and 223 to 289.Table 3: Binding of Llama Anti -Human ITGB6 Antibodies to Human ITGAV / ITGB6 in an ELISA AssayExample 2: Incorporation of ITGB6 Binding Heavy Chain Only Single Domain Antibodies into fusion proteins and T Cell Dependent Cellular Cytotoxicity Assays

[0308] The anti-ITGB6 antibody sequences were cloned into DNA constructs for expression of recombinant fusion proteins (SEQ IDS 297 to 337). The coding sequences of the fusion proteins contained a signal peptide for secreted cell expression, a stub to mimic the cleaved version of a conditionally-active T cell engager (SEQ ID 656), humanized anti-CD3 antibody scFv fragment (SEQ ID NO 655), one of the anti-ITGB6 antibody variable domains (SEQ ID NOS 1 to 67), and a repeat of six histidine sequences (SEQ ID NO 658). A linker sequence was inserted at the junctions between antibody domains (SEQ ID NO 659). These anti-CD3 / anti-ITGB6 fusion protein constructs were transfected into Expi293 cells (Life Technologies). The amount fusion protein in the conditioned media from the transfected Expi293 cells was quantitated by using an Octet instrument with Protein A tips using a fusion protein of similar molecular weight to the anti- CD3 / anti-ITGB6 proteins as a standard.

[0309] The conditioned media were tested in a T-cell dependent cellular cytotoxicity assay (TDCC) (Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27). In this assay, luciferase labelled CAPAN2 cells, which express ITGB6,were combined with purified human T cells and a titration of anti-CD3 / anti-ITGB6 fusion protein. If a fusion protein directs T cells to kill the CAPAN2 cells, the signal in a luciferase assay performed at 48 hours after starting the experiment should decrease. Figures 1 to 9 have graphs of TDCC viability results. ECso values from the TDCC assay are listed in Table 4. The most potent molecule had an EC50 value of 0. 171 pM. A negative control for the TDCC assays was an anti- GFP / anti-albumin / anti-CD3 protein, and this protein did not direct the T cells to kill the CAPAN2 cells except for slight activity at the highest concentration tested (e.g., Figure 7). Binders I6L251 and I6L285 were tested in separate experiments from the other binders.Table 4: EC50 Values for Redirected T Cell Killing of CAPAN2 cells by anti-CD3 / anti-ITGB6 fusion proteins containing llama Anti-ITGB6 Sequences.n / a insufficient activity to calculate an EC50 using the protein concentrations tested, n.d. no data with this binder.

[0310] Specificity of the anti-ITGB6 antibodies for ITGB6 was determined by measuring the affinity of the antibodies in conditioned medium for either a dimer of ITGAV and ITGB6, or a dimer of ITGAV and either ITGB1, ITGB5, or ITGB8 by using an Octet instrument with streptavidin tips loaded at a single concentration with the appropriate biotinylated ITGAV / ITGB dimer. Only I6L72 showed measurable affinity to all four ITGAV / ITGB dimers. The remaining binders are specific for the ITGAV / ITGB6 dimer. The affinities of these binders for the ITGAV / ITGB6 dimer are reported in Table 5.Table 5: Affinities of anti-CD3 / anti-ITGB6 fusion proteins Containing llama anti-ITGB6 sequences for Human ITGB6.

[0311] A subset of ITGB6 binders were tested in a TDCC assay using an additional luciferase labeled ITGB6-expressing cell line, SW780. Figures 10 and 11 have graphs of TDCC viability results. ECso values are reported in Table 6. Results were similar to those obtained with CAPAN2 cells.Table 6: EC50 Values for Redirected T Cell Killing of SW780 Cells by anti-CD3 / anti-ITGB6 fusion proteins containing llama Anti-ITGB6 Sequences.

[0312] The affinities of the anti-ITGB6 antibody in this subset of anti-CD3 / anti-ITGB6 fusion proteins were determined as before only using three concentrations of biotinylated ITGAV / ITGB6 dimer protein. The affinities are reported in Table 7.Table 7: Affinities of anti-CD3 / anti-ITGB6 fusion proteins containing llama anti-ITGB6 sequences for Human ITGB6Example 3: Humanization of ITGB6 Binding Antibodies and T Cell Dependent CellularCytotoxicity Assays

[0313] Seven of the llama anti-ITGB6 antibody sequences were humanized by grafting their CDR sequences onto human germline sequences, while retaining some llama framework sequences to ensure the antibodies did not lose activity (SEQ IDS 68 to 74). These sequences were cloned into expression constructs for expression as anti-CD3 / anti-ITGB6 fusion proteins (SEQ IDS 338 to 344) in Expi293 cells, along with their parental constructs, as before, except using a different N-terminal stub (SEQ ID NO 657). The amount of anti-CD3 / anti-ITGB6 fusion proteins in the conditioned medium was quantitated as before. Conditioned medium containing the anti-CD3 / anti-ITGB6 fusion proteins was used in a TDCC assay as before except using the ITGB6-expressing, luciferase labeled cell line HPAFII. The results of the TDCC assay are plotted in Figures 12 and 13, and the ECso values for directed T cell killing are listed in Table 8. Potent directed T cell killing was observed with both the llama and humanized ITGB6 antibodies.Table 8: EC50 Values for Redirected T Cell Killing of HPAFII Cells by anti-CD3 / anti-ITGB6 fusion proteins containing llama or Humanized Anti-IFGB6 Sequences.Example 4: Purification of anti-CD3 / anti-ITGB6 non-cleavable fusion proteins

[0314] Eight anti-CD3 / anti-ITGB6 non-cleavable fusion proteins containing llama anti-ITGB6 sequences (SEQ ID Nos 345 to 352) were purified in a small-scale production system using 35mL of sterile-filtered conditioned medium. MabSelect PrismA resin was used to bind the anti-CD3 / anti- ITGB6 fusion proteins. Resin containing the bound protein was transferred to a unifilter microplate, where it was washed then eluted with 0. IM Glycine Buffer pH 3. Following the elution step, the purified protein was neutralized using IM Tris pH 9 and quantitated. The results of the purification are illustrated in Figure 14. The purified proteins had no visible impurities as analyzed except for I6L285, which showed pre-cleavage.Example 5: Flow cytometry assessment of anti-CD3 / anti-ITGB6 non-cleavable fusion proteins on targeting cell lines

[0315] Eight anti-CD3 / anti-ITGB6 non-cleavable fusion proteins containing llama anti-ITGB6 sequences were assessed by Flow Cytometry on three targeting cell lines: SW780, HCC70, and HPAF-II, all of which expresses ITGB6 based on Cancer Cell Line Encyclopedia (CCLE) RNAseq data (Broad Institute). Cell binding was acquired using the BD FACS Diva and analyzed on FlowJo. The results are shown in Figure 15, and the MFI values for each cell line are reported in Table 9.Table 9: Values Represent the Median Fluorescence Fold Change Over the Isotype Control for the Binding of anti-CD3 / anti-ITGB6 Non-cleavable fusion proteins containing llama anti-ITGB6 sequences to SW780, HCC70, and HPAF-II CellsExample 6: Human, cynomolgus monkey, and mouse cells cross-reactivity of anti-CD3 / anti- ITGB6 non-cleavable fusion proteins

[0317] Human, cynomolgus monkey (cyno), and mouse cross-reactivity of eight anti-CD3 / anti- ITGB6 non-cleavable fusion proteins with llama anti-ITGB6 sequences were evaluated by Flow Cytometry on primary lung and kidney epithelial cells. PBMCs and splenocytes were tested as negative controls. The non-cleavable ITGB6 fusion proteins were tested using an eight point dilution series method, starting at a concentration of 200nM to 0.09nM. An 8G1 antibody, that was conjugated to AlexaFlour 647, was used to detect the cell binding of the molecules as it recognized the anti-albumin domain. The non-cleavable ITGB6 fusion proteins exhibited binding to both human and cyno lung and kidney primary cells over the negative cell types. I6L18 was the only one that showed cross-reactivity to mouse primary kidney cells. The data are plotted and Figures. 41 to 48, and results of EC50 values calculated from these data are reported in Table 10.Table 10: EC 50 Values of anti-CD3 / anti-ITGB6 Non-Cleavable fusion proteins containing llama anti-ITGB6 sequences on PBMCs, Splenocytes, and Primary CellsExample 7: Purification of anti-CD3 / anti-ITGB6 ProTriTAC cleavable fusion proteins

[0319] Four humanized anti-CD3 / anti-ITGB6 ProTriTAC Linker 040 fusion proteins were purified as a small-scale production. MabSelect PrismA resin was used to bind the anti-CD3 / anti- ITGB6 fusion proteins. Bound protein was eluted and quantitated as in Example 4. The purification results are displayed in Figure 16. Some of fusion proteins contained a faint active fragment band.Example 8: Cell Binding to and TDCC of Human and Cyno Alveolar Cells Comprising of anti-CD3 / anti-ITGB6 ProTriTAC cleavable fusion proteins

[0320] Five humanized anti-CD3 / anti-ITGB6 ProTriTAC Linker 040 fusion proteins were assessed by Flow Cytometry on human and cyno primary alveolar epithelial cells as in Example 6. All binders demonstrated binding to both human and cyno alveolar cells, but I6H18C only bound tohuman alveolar cells. The EC50 values for cell binding are listed in Table 11. The FACS results are plotted in Figures 17 and 18.

[0321] Table 11: EC50 Values for Cell Binding to Human and Cyno Alveolar Cells Comprising of anti-CD3 / anti-ITGB6 ProTriTAC Linker 040 fusion proteins containing humanized Anti-ITGB6 Sequences.

[0322] nb = no binding.

[0323] A TDCC assay was also used to assess if the I6H41C binder recognizes both human and cynomolgus ITGB6. Co-cultures of purified human T cell and human or cyno alveola, kidney, bladder, stomach, or mammary epithelial cells, were treated with a titration of the activated form of an ITGB6 ProTriTAC with binder I6H41C or GFP TriTAC (Figures. 53 to 62). Compared to untreated cells (co-culture) or cells treated with GFP TriTAC, increased and comparable cell killing was observed with the activated ProTriTAC molecule with both the human and cyno cells, indicating the I6H41C binds to both human and cynomolgus monkey ITGB6.Example 9; T Cell Dependent Cellular Cytotoxicity Assays and cell binding of Anti-CD3 / anti- ITGB6 ProTriTAC Linker 040 fusion proteins

[0324] A subset of two partially purified ITGB6 binders were tested in a TDCC assay using four luciferase labeled ITGB6-expressing cell lines, HCC70, SW780, HT29, and CAL27. The TDCC viability results are shown in Figures 19-22. The EC50 values are reported in Table 12. 16H18C had complete killing only on HCC70 and had partial killing activity for the other three remaining cell lines. To further demonstrate that TDCC killing is dependent on TDCC expression, a separate TDCC assay was performed with SW780 cell where the ITGB6 gene was deleted using CRISPR gene editing (SW780 ITGB6 knockout). No killing was observed in co-cultures of T cells and SW780 ITGB6 knockout cells when cells were untreated (co-culture), treated with an anti-GFP TriTAC, or an activated form of an anti-ITGB6 ProTriTAC molecule (Fig. 64). In contrast, with parental SW780 cells, which still express ITGB6, directed T cell killing of the SW780 cells was observed with the activated for of an anti-ITGB6 ProTriTAC (Fig. 63).Table 12: EC50 Values for Redirected T Cell Killing of HCC70, SW780, HT29, and CAL27 Cells by anti-CD3 / anti-ITGB6 fusion proteins containing humanized Anti-ITGB6 Sequences

[0325] incomplete killing.

[0326] The two partially purified ITGB6 binders previously used were also tested by Flow Cytometry using the same four ITGB6-expressing cell lines HCC70, SW780, HT29, and CAL27 as stated above. The MFI results are plotted in Figures 23-26, and the ECso values are reported in Table 13. 16H18C and I6H41C ProTriTAC Linker 040 fusion proteins showed binding to all four cell lines, in which CAL27 cells had the lowest MFI signal.Table 13: ECso Values for Binding to HCC70, SW780, HT29, and CAL27 Cells by Flow Cytometry of anti-CD3 / anti-ITGB6 ProTriTAC Linker 040 fusion proteins Containing Human Anti-ITGB6 SequencesExample 10: Demonstration of improved tolerability in mouse, conferred by an exemplary anti-CD3 / anti-ITGB6 fusion proteins

[0327] All animal experiments were conducted according to the protocol approved by Institutional Animal Care and Use Committee of Harpoon Therapeutics (protocol number HAR- 001-2019). Animals were purchased from The Jackson Laboratory then housed in a pathogen free animal facility located at Harpoon Therapeutics in accordance with IACUC guidelines. All studies were performed in NSG™ (NOD-SCID IL2Rgammanull) female mice 6 or 11 weeks of age with n = 5-10 mice per group. For each experiment, mice were age matched.

[0328] An admixture of ITGB6-expressing human tumor cells, either HPAF-II (5E6), SW780 (5E6), HCC70 (10E6), or CAL27 (5E6) and activated and expanded human T cells (2.5E6 or 5E6) at an E:T ratio of 1:2 was implanted subcutaneously on the right flank of NSG™ mice (Day 0). For SW780 xenograft, treatment followed 1 day post implant (Day 1). For HPAF-II, HCC70, and CAL27 xenografts, treatment began once tumors were established (average of 127, 184, or 120 mm3, respectively) on Day 5, 7, or 4, respectively. Mice were administered a repeat intraperitoneal dose (qdxl4) of the negative control, non-ITGB6 targeting anti-GFP TriTAC, or anti-ITGB6 I6H41C (human binder) ProTriTAC Linker 040 (SEQ ID NO 354). Tumor growth was monitoredat least twice weekly as indicated. Average tumor volume shown was calculated from measurements taken on the final day of each xenograft model study. Plots of the tumor growth versus time are in Figures. 49 to 52. Statistics represent RM one-way ANOVA with Dunnett post- hoc test, all groups compared to the negative control, anti-GFP TriTAC. The statistics for each xenograft model study are reported in Table 14.Table 14: P Values from Multiple Rodent Xenograft Studies with anti-CD3 / anti-ITGB6 16H41CProTriTAC Linker 040 Fusion Protein

[0329] A: anti; E:T ; effector to target cell ratio; n / a: not applicable; P: probability value. In addition, the in vivo anti-tumor activity on HCC70 (breast carcinoma model) shows that comparable anti-tumor activity is achieved by molar equivalents of prodrug and active drug (Figure 67).Example 11: ProTriTACs with L276 have less pre-cleaved active drug and are more manufacturable than ProTriTACs with L040 when produced in CHO Cells

[0330] In this example, linker variants were evaluated in reducing the presence of pre-cleaved active drug and improving manufacturability. 2e6 CHOSource™ CHO-K1 GS null cells (Horizon) were nucleofected with 5 pg linearized expression vector plasmid DNA harboring ProTriTAC sequences comprising L040 (SEQ ID NO 661) or L276 (SEQ ID NO: 706) protease cleavable linkers. Cells were passaged in medium containing glutamine for 2 days then switched to selection medium lacking glutamine and supplemented with 50pM methionine sulfoximine (MSX) for up to threeweeks such that stable pools were allowed to recover until they reached > 95% viability with less than 24hr doubling time and then banked.

[0331] Stable pools were thawed, expanded in glutamine free media supplemented with 50pM MSX for two to three passages, and seeded into production media at 0.5e6 viable cells / ml in Optimum Growth™ Flasks (Thomson) shaken at 150 rpm, 5% CO2, 60% relative humidity, and 37°C. Fed- batch cultures were supplemented with feeds and glucose on days 3, 5, and 7 and viability and cell density determined by ViCell. Conditioned media (CM) was harvested on day 10 by centrifugation and sterile filtration. Titer was determined by biolayer interferometry using an OctetRed96 and Protein A tips with binding rates compared against a standard curve prepared from a ProTriTAC of known concentration as reference (Table 15). CM samples were heated in non-reducing sample buffer and separated on NuPage TRIS-Glycine gels, stained with SimplyBlue Safestain (Invitrogen), and imaged with an Azure c500 near infrared imaging system.

[0332] FIG. 27 demonstrates a ProTriTAC has substantially reduced pre-cleaved active drug in CHO productions with L276 versus L040 cleavable linker.Table 15: Comparison of lOd harvest % viability and ProA titer of ProTriTAC sequences comprising L040 or L276 protease cleavable linkers.Example 12: Constructs and Testing for MMP selectivity - Set 1

[0333] Constructs

[0334] The following ProCAR constructs were made. Construct C2483 includes an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 662; FIG. 29A). Construct C3544 includes an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 663; FIG. 29B). Construct C3546 includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 4 (SEQ ID NO: 664), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 665; FIG. 29C). Construct C3548 includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an antihuman EpCAM sdAb, a protease cleavable linker 5 (SEQ ID NO: 666), a FLAG epitope, a CD8hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 667; FIG. 29D). Construct C3549 includes an anti -human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 6 (SEQ ID NO: 668), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 669; FIG. 29E). Construct C3550 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 7 (SEQ ID NO: 670), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 671; FIG. 29F).

[0335] Pan-MMP substrates are not stable when expressed on T cells in ProCAR format.

[0336] T cells were infected with lentivirus made from the indicated constructs to generate CAR- T cells, which were subsequently stained with anti-FLAG antibodies and EpCAM-Fc along with fluorescently labeled secondary antibodies and analyzed by flow cytometry .

[0001] FIG. 30 shows dot plots of M2 staining (x-axis; BV421 channel) and EpCAM-Fc staining (y-axis; Al exaFluor 647 channel) of CAR-T cells. FIG 30A provides results for constructs C2483, C3546, and C3544. FIG. 30B provides results for constructs C2483, C3548, and C3544. FIG. 30C provides results for constructs C2483, C3549, and C3544. FIG. 30D provides results for constructs C2483, C3550, and C3544.

[0337] FIG. 31 illustrates that MMP2 and MMP7 are expressed at very low levels in T cells compared to MMP9.Example 13: Constructs and Testing for MMP selectivity - Set 2

[0338] Constructs

[0339] The following ProCAR constructs were made: Construct C2483 which includes an antihuman EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 662; FIG. 32A). Construct C3269 includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 672; FIG. 32B). Construct C3440 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an antihuman EpCAM sdAb, a protease cleavable linker 8 (SEQ ID NO: 673), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 674; FIG. 32C). Construct C3444 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 9 (SEQ ID NO: 675), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BBintracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 689; FIG. 32D). Construct C3445 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 10 (SEQ ID NO: 676), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 690; FIG. 32E).

[0340] MMP2-selective substrates show increased stability when expressed on T cells in ProCAR format

[0341] T cells were infected with lenti virus made from the indicated constructs to generate CAR- T cells, which were subsequently stained with anti-FLAG antibodies and EpCAM-Fc along with fluorescently labeled secondary antibodies and analyzed by flow cytometry. FIGS. 33A-33C show dot plots of M2 staining (x-axis; BV421 channel) and EpCAM-Fc staining (y-axis; AlexaFluor 647 channel) of CAR-T cells. FIG. 33A provides results for constructs C3440 and C3269 for MMP2- selective substrate 1. FIG. 33B provides results for constructs C3444 and C3269 for MMP2- selective substrate 2. FIG. 33C provides results for constructs C3445 and C3269 for MMP2- selective substrate 3. MMP2-selective substrates show increased stability when expressed on T cells in ProCAR format.Example 14: Constructs and Testing for MMP selectivity - Set 3

[0342] Constructs

[0343] The following ProCAR constructs were made: Construct C2483 which includes an antihuman EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 662; FIG. 34A). Construct C3543 includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 677; FIG. 34B). Construct C3558 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an antihuman EpCAM sdAb, a protease cleavable linker 11 (SEQ ID NO: 678), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 679; FIG. 34C). Construct C3559 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 12 (SEQ ID NO: 680), a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 681; FIG. 34D). Construct C3560 which includes an anti-human serum albumin sdAb that contains a mask in the CC' loop, an anti-human EpCAM sdAb, a protease cleavable linker 13 (SEQ ID NO: 682), a FLAG epitope, aCD8 hinge / transmembrane domain, a 4-1 BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 683; FIG. 34E).

[0344] MMP-7 -selective substrates show increased stability when expressed on T cells in ProCAR format

[0345] T cells were infected with lentivirus made from the indicated constructs to generate CAR- T cells, which were subsequently stained with anti-FLAG antibodies and EpCAM-Fc along with fluorescently labeled secondary antibodies and analyzed by flow cytometry . Shown above are dot plots of M2 staining (x-axis; BV421 channel) and EpCAM-Fc staining (y-axis; Al exaFluor 647 channel) of CAR-T cells. FIG 35A provides results for constructs C2483, C3558, and C3543 for MMP7-selective substrate 1. FIG. 35B provides results for constructs C2483, C3559, and C3543 for MMP7-selective substrate 2. FIG. 35C provides results for constructs C2483, C3560, and C3543 for MMP2-selective substrate 7.

[0346] MMP-7 -selective substrates show increased stability when expressed on T cells in ProCAR format andMMP7-selective substrates are cleavable withMMP7

[0347] T cells were infected with lentivirus made from the indicated constructs to generate CAR- T cells and either treated with recombinant MMP7 or buffer alone for 1 hour (hr) at 37 °C. The CAR-T cells were subsequently stained with anti-FLAG antibodies and EpCAM-Fc along with fluorescently labeled secondary antibodies and analyzed by flow cytometry . Shown above are dot plots of M2 staining (x-axis; BV421 channel) and EpCAM-Fc staining (y-axis; Al exaFluor 647 channel) of CAR-T cells. FIG. 36A provides results for constructs C3558 and C3558+MMP7 for MMP7-selective substrate 1. FIG. 36B provides results for constructs C3559 and C3559+MMP7 for MMP7-selective substrate 2. FIG. 36C provides results for constructs C3560 and C3560+MMP7 for MMP7-selective substrate 3.Example 15: Protease Site-Dependent Cell Killing Activity

[0348] Constructs

[0349] The following constructs were made. Construct C2446 includes an anti-human serine albumin sdAb, a protease cleavage site 3, an anti-human EGFR sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (FIG. 37A; SEQ ID NO: 684). Construct C2510 includes an anti-human serine albumin sdAb, a protease cleavage site, an anti-human EGFR sdAb, a FLAG epitope, a dimerization-deficient CD8a hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain (FIG. 37B; SEQ ID NO: 685). Construct C2513 includes an anti-human serine albumin sdAb, a protease cleavage site 3, an anti-human EGFR sdAb, a FLAG epitope, a dimerization-deficient CD8a hinge / transmembrane domain, a 4- IBB intracellular domain, and a CD3 zeta intracellular domain(FIG. 37C; SEQ ID NO: 686). Construct C2514 includes an anti-human serine albumin sdAb, an anti-human EGFR sdAb, a FLAG epitope, a dimerization-deficient CD8a hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (FIG. 37D; SEQ ID NO: 687). Construct C2448, includes an anti-human EGFR sdAb, a FLAG epitope, a dimerizationdeficient CD8a hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (FIG. 37E; SEQ ID NO: 688).

[0350] In vitro Protease Site-Dependent Cell Killing Activity

[0351] In a first assay, 300,000 primary human T cells isolated from healthy donors were infected with 1 mL lentiviral supernatant made from the indicated constructs to generate anti-EGFR CAR-T cells. Twenty-five thousand CAR-T cells were subsequently co-cultured under standard conditions at a 10:1 ratio (CAR-T:Target cells) with EGF-expressing cancer cells that stably express luciferase for 72 hours. Luciferase activity is determined as a proxy for cancer cell viability and normalized to the control CAR-T cells that do not contain scFv.

[0352] The data demonstrated that the level of protease side-dependent cell killing activity was reduced in ProCAR constructs that lacked the dimerization activity of the CD8a transmembrane domain (FIG. 38B) compared to wild-type (FIG. 38A; Constructs Cl 081, Cl 824, Cl 950, C2031, C2446; SEQ ID NOS 691, 692, 693, 694, 684). Cell killing activity occurs without adding protease. It is believed that the exogenous protease activity comes from the CAR-T cells themselves. The dimerization mutation reduces the amount of cell killing activity in the absence of exogenous protease (e.g., compare C2031 to C2510, or compare C2446 to C2513). Reducing the amount of activation of the ProCAR from T cell proteases may expand the therapeutic window of a given ProCAR.

[0353] In a second assay, 300,000 primary human T cells isolated from healthy donors were infected with 1 mL lentiviral supernatant made from the indicated constructs to generate anti-EGFR CAR-T cells, which were subsequently co-cultured at ratios of 10: 1, 5: 1, 2.5: 1, and 1: 1 (CAR- T:Target cells) with EGF-expressing cancer cells that stably express luciferase for 72 hours. Luciferase activity is determined as a proxy for cancer cell viability and normalized to the control CAR-T cells that do not contain scFv. The data demonstrates that dimerization-deficient EGFR CAR has similar killing activity compared to wild-type (FIG. 39; Constructs Cl 801, C1824, C2448; SEQ ID NOS 691, 692, 688).

[0354] In vivo Protease Site-Dependent Cell Killing Activity

[0355] Five NSG mice per group (The Jackson Laboratories, Bar Harbor, ME) were subcutaneously implanted with admixture of 5x106 HCT116 cancer cells and the indicated CAR-T cells (C1081, C1950, or C1824; SEQ ID NOS 691, 693, 692) at a ratio of 1: 1 and tumor volumeswere measured at the indicated days post-implantation. Significance was determined by using unpaired t test between groups. *** denotes p<0.0005.

[0356] These data provide an in vivo demonstration of masking of the anti- EGFR binding domain by the mask in the CC’ loop of the anti- ALB domain using non-cleavable ProCAR. Significance was determined by using unpaired t test between groups. *** denotes p<0.0005.Example 16: Testing efficacy of different cleavable linkers on reducing tumor growth

[0357] EpCAM targeting ProTriTAC molecules (EpCAM H90 Pro with protease cleavable linker L040, EpCAM H90 ProTriTAC with protease cleavable linker 11, EpCAM H90 ProTriTAC with protease cleavable linker 12, and EpCAM H90 ProTriTAC with protease cleavable linker 77) were made comprising different protease cleavable linkers (L040, SEQ ID NO: 661; protease cleavable linker 11, SEQ ID NO: 678; protease cleavable linker 12, SEQ ID NO: 680; and protease cleavable linker 77, SEQ ID NO: 707). All animal experiments were conducted according to the protocol approved by Institutional Animal Care and Use Committee of Harpoon Therapeutics (protocol number HAR-001-2019). Animals were purchased from The Jackson Laboratory then housed in a pathogen free animal facility located at Harpoon Therapeutics in accordance with IACUC guidelines. All studies were performed in NSG™ (NOD-SCID IL2Rgammanull) female mice 8 weeks of age with n = 5-10 mice per group. An admixture of HT-29 human colorectal adenocarcinoma tumor cells (5E6) and activated and expanded human T cells (2.5E6) was implanted subcutaneously on the nght flank of NSG™ mice, followed 5 days later by treatment. Mice with established tumors (average of 215 mm3) were administered a repeat intraperitoneal dose (q.d. x 14) of non-targeting GFP TriTAC (at 0.1 mg / kg) or EpCAM targeting ProTriTACs (at O.lmg / kg or at 0.3 mg / kg). Tumor growth was monitored at least twice weekly as indicated. All data shown as mean ± SEM. Statistics represent RM one-way ANOVA with Dunnett post-hoc test, all groups compared to control non-targeting GFP TriTAC (****, P0.0001), FIG. 40 shows all EpCAM targeting ProTriTAC molecules comprising a cleavable linker were able to reduce tumor growth at both doses.Example 17: One-Month Dose Escalation / Maximum Tolerated Dose Study

[0358] In this dose escalation / maximum tolerated dose study, ITGB6 ProTriTAC Linker 40 molecule (I6H41C) is used for intravenous (slow bolus) injection in Cynomolgus Monkeys.

[0359] The primary objectives of this Good Laboratory Practice (GLP) compliant, escalating repeat dose study were to evaluate the potential toxicity and systemic exposure of ITGB6 ProTriTAC, when administered by intravenous (iv) slow bolus injections on Days 1, 8, 15, and 22 at 20, 60, 180, and 540 pg / kg, respectively, to cynomolgus monkeys. FIG. 65 shows the experimental design. In addition, the toxicokinetic (TK) characteristics of ITGB6 ProTriTAC were determined. Study endpoints included: mortality, clinical observations (daily cage side, postdose, and weekly detailed),body weights, qualitative food consumption, clinical pathology parameters (hematology', coagulation, clinical chemistry, and urinalysis), bioanalytical and TK parameters, cytokine analysis, organ weights, and macroscopic and microscopic examinations.

[0360] ITGB6 ProTriTAC was well-tolerated up to 540 jxg / kg, the highest dose tested. PK was assessed by capturing with a biotinylated anti-idiotype Ab recognizing the aCD3 domain and detecting with a sulfotagged anti-idiotype antibody raised against the aALB domain. FIG. 66 demonstrates that the ITGB6 ProTriTAC shows favorable pharmacokinetics, half-life and systemic accumulation. The hematological changes consisted of minimally to moderately decreased neutrophils at all dose levels, as well as transient decreases in lymphocytes and basophils in week 3 of the study. There were no notable findings in plasma cytokines (IFNy, IL-10, IL-2, IL-6, IL-10, IL- 8, TNT -a) throughout the study, no notable findings from coagulation, clinical chemistry, or urinalysis parameters and no notable histopathological findings based on organ weight, gross and microscopic examination.SEQUENCES

[0361] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An ITGB6 binding domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149- 222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296.

2. The ITGB6 binding domain of claim 1, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296.

3. The ITGB6 binding domain of claim 1 or 2, comprising an amino acid sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1- 74.

4. The ITGB6 binding domain of any one of claims 1-3, wherein the ITGB6 binding domain is part of a multispecific protein.

5. The ITGB6 binding domain of claim 4, wherein the multispecific protein further comprises a CD3 binding domain.

6. The ITGB6 binding domain of claim 5, wherein the multispecific protein comprises an active drug format.

7. The ITGB6 binding domain of claim 4 or 5, wherein the multispecific protein further comprises a bulk serum protein binding domain.

8. The ITGB6 binding domain of claim 7, wherein the bulk serum protein comprises a serum albumin protein.

9. The ITGB6 binding domain of claim 8, wherein the serum albumin protein comprises a human serum albumin protein.

10. The ITGB6 binding domain of any one of claims 7-9, wherein the bulk serum protein binding domain comprises a sequence that is at least 75% identical the sequence as set forth in SEQ ID NO: 654 or 728.

11. The ITGB6 binding domain of any one of claims 4-9, wherein the CD3 binding domain comprises a sequence that is at least 75% identical the sequence as set forth in SEQ ID NO: 655.

12. The ITGB6 binding domain of any one of claims 4-9, wherein the multispecific protein comprises a sequence that is at least about 75% identical to the sequence as set forth in SEQ ID NOS: 353-357.

13. The ITGB6 binding domain of any one of claims 7-9, wherein the bulk serum protein binding domain is a binding moiety comprising a linker and a masking moiety, wherein the masking moiety masks binding of the ITGB6 binding domain or the CD3 binding domain, to their respective targets.

14. The ITGB6 binding domain of any one of claims 7-9 and 13, wherein the multispecific protein comprises a non-cleavable prodrug format.

15. The ITGB6 binding domain of claim 13 or 14, wherein the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOS: 801-845, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 801-845.

16. The ITGB6 binding domain of any one of claims 13-15, wherein the linker comprises a sequence selected from the group consisting of SEQ ID NOS: 659-661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 718-727, 731-772, and 846-856, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 659-661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 718-727, 731-772, and 846-856.

17. The ITGB6 binding domain of any one of claims 13-16, wherein the bulk serum protein binding domain comprises a sequence that is at least 75% identical the sequence selected from the group consisting of SEQ ID NO: 728.

18. The ITGB6 binding domain of any one of claims 5-9 and 13-17, wherein the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO: 655.

19. The ITGB6 binding domain of any one of claims 7-9 and 13-18, wherein the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 345-357.

20. The ITGB6 binding domain of any one of claims 7-9 and 13-19, wherein the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 345-352.

21. The ITGB6 binding domain of any one of claims 7-9 and 13-20, wherein the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

22. The ITGB6 binding domain of claim 6, wherein the active drug comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 297-344.

23. The ITGB6 binding domain of any one of claims 1-3, wherein the ITGB6 binding domain is part of a chimeric antigen receptor (a CAR) or a conditionally activatable chimeric antigen receptor (a ProCAR), wherein the CAR further comprises at least one of a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

24. The ITGB6 binding domain of claim 23, wherein the ITGB6 binding domain is part of the ProC AR and the ProCAR further comprises (a) a binding moiety comprising a non-CDR loop and a cleavable linker; (b) a transmembrane domain; and (c) an intracellular signaling domain; wherein the binding moiety masks the binding of the ITGB6 binding domain to its target.

25. The ITGB6 binding domain of claim 24, wherein the binding moiety further comprises one or more complementarity determining regions (CDRs).

26. The ITGB6 binding domain of claim 25, wherein the CDR loop provides a binding site specific for a bulk serum protein.

27. The ITGB6 binding domain of claim 26, wherein the bulk serum protein comprises at least one of: a serum albumin, a transferrin, an IgGl, an IgG2, an IgG4, an IgG3, an IgA monomer, a Factor XIII, a fibrinogen, or a pentameric IgM.

28. The ITGB6 binding domain of claim 27, wherein the bulk serum protein comprises the serum albumin.

29. The ITGB6 binding domain of claim 28, wherein the serum albumin is a human serum albumin.

30. The ITGB6 binding domain of any one of claims 23-29, wherein the ProCAR further comprising a costimulatory domain, wherein the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDl la / CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.

31. The ITGB6 binding domain of claim 30, wherein the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the encoded T-cell receptor fusion protein.

32. The ITGB6 binding domain of any one of claims 23-31, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, functional fragments of any one thereof, and amino acid sequences of any one thereof having at least one but not more than 20 modifications thereto.

33. The ITGB6 binding domain of any of claims 23-32, wherein the intracellular signaling domain is derived from CD3 epsilon, CD3 gamma, CD3 delta, CD3 alpha, CD3 beta, or a combination thereof.

34. The ITGB6 binding domain of any of claims 23-32, wherein the intracellular signaling domain is derived from CD3 epsilon.

35. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering an ITGB6 binding domain according to any one of claims 1-34, or a pharmaceutical composition comprising the same, to the subject.

36. The method of 35, wherein the subject is human.

37. A conditionally active chimeric antigen receptor (ProCAR) that comprises a single polypeptide chain, comprising:(a) a binding moiety comprising a non-CDR loop and a cleavable linker;(b) an ITGB6 binding domain, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296;(c) a transmembrane domain; and(d) an intracellular signaling domain; wherein the binding moiety masks binding of the ITGB6 binding domain to its target.

38. The conditionally active chimeric antigen receptor of claim 37, wherein the ITGB6 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74.

39. A conditionally active ITGB6 binding protein comprising a binding moiety (M) which comprises a non-CDR loop, a cleavable linker (L), a first target antigen binding domain (Tl), and a second target antigen binding domain (T2), wherein at least one of the first target antigen binding domain (Tl) and the second target antigen binding domain (T2) comprises an ITGB6 binding domain, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296, wherein the non-CDR loop is capable of binding to the ITGB6 binding domain or the second target antigen binding domain, and wherein the binding moiety is capable of masking the binding of the ITGB6 binding domain or the second target antigen binding domain to its target.

40. The conditionally active ITGB6 binding protein of claim 39, wherein the binding moiety comprises a masking moiety and wherein the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOS: 801-845, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOS: 801 - 845.

41. The conditionally active ITGB6 binding protein of claim 39 or 40, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854.

42. The conditionally active ITGB6 binding protein of any one of claims 39-41, wherein the binding moiety comprises a sequence that is at least 75% identical the sequence selected from the group consisting of SEQ ID NO: 728.

43. The conditionally active ITGB6 binding protein of any one of claims 39-42, wherein the second target antigen binding domain (T2) comprises a CD3 binding domain.

44. The conditionally active ITGB6 binding protein of claim 43, wherein the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO: 655.

45. A method for the treatment or amelioration of a proliferative disease, or a tumorous disease, comprising the administration of a conditionally active chimeric antigen receptor according to claim 37 or 38, or a pharmaceutical composition comprising the same, to a subject in need thereof.

46. The method of 45, wherein the subject is human.

47. A method for the treatment or amelioration of a proliferative disease, or a tumorous disease, compnsing the administration of a conditionally active ITGB6 binding protein according to any one of claims 39-44, or a pharmaceutical composition comprising the same, to a subject in need thereof.

48. The method of 47, wherein the subject is human.

49. The ITGB6 binding domain of any one of claims 1-3, wherein the binding domain is a humanized antibody or an antigen binding fragment thereof.

50. The ITGB6 binding domain of any one of claims 1-3 and 49, wherein the binding domain is a single domain antibody, a VHH domain, a scFv, a VH domain, a VL domain, a Fab, a Fab’, anon-Ig domain, a ligand, a knottin, or a small molecule entity.

51. The ITGB6 binding domain of claim 50, wherein the binding domain comprises the single domain antibody.

52. The ITGB6 binding domain of any one of claims 1-3 and 49-51, wherein the binding domain binds to ITGB6 with a binding affinity (Kd) of about 0.001 nM to about 500 nM.

53. The ITGB6 binding domain of any one of claims 1-3 and 49-52, wherein the binding domain binds to human ITGB6, mouse ITGB6, cynomolgus ITGB6, or a combination thereof.

54. A multispecific protein comprising an ITGB6 binding domain, wherein the ITGB6 binding domain is according to any one of claims 1-3 and 49-53.

55. The multispecific protein of claim 54, comprising the ITGB6 binding domain according to any one of claims 1-3 and 49-53 (anti-ITGB6 domain), and a CD3 binding domain (anti-CD3 domain).

56. The multispecific protein of claim 55, wherein the anti-ITGB6 domain and the anti-CD3 domain are in an anti-ITGB6:anti-CD3 orientation.

57. The multispecific protein of claim 55, wherein the anti-ITGB6 domain and the anti- CD3 domain are in an anti-CD3: anti-ITGB6 orientation.

58. The multispecific protein of any one of claims 54-56, comprising the ITGB6 binding domain according to any one of claims 1-3 and 49-53 (anti-ITGB6 domain), the CD3 binding domain (anti-CD3 domain), and an albumin binding domain (anti-ALB domain).

59. The multispecific protein of any one of claims 54-58, wherein the anti-CD3 domain comprises an amino acid as set forth in SEQ ID NO: 655.

60. The multispecific protein of any one of claims 58-59, wherein the anti-ALB domain comprises an amino acid sequence as set forth in SEQ ID NO: 728.

61. The multispecific protein of any one of claims 58-60, wherein the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3: anti-ALB: anti-ITGB6 orientation.

62. The multispecific protein of any one of claims 58-60, wherein the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ITGB6: anti-ALB: anti-CD3 orientation.

63. The multispecific protein of any one of claims 58-60, wherein the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB: anti-ITGB6: anti-CD3 orientation.

64. The multispecific protein of any one of claims 58-60, wherein the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3: anti-ITGB6: anti-ALB orientation.

65. The multispecific protein of any one of claims 58-60, wherein the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB: anti-CD3: anti-ITGB6 orientation.

66. The multispecific protein of any one of claims 58-60, wherein the anti-ITGB6 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ITGB6: anti-CD3: anti-ALB orientation.

67. A multivalent protein comprising a sequence as set forth in any one of SEQ ID NOS: 345-357.

68. An active drug comprising a sequence as set forth in any one of SEQ ID NOS: 297- 337.

69. An active drug comprising a sequence as set forth in any one of SEQ ID NOS: 338-344.

70. A pharmaceutical composition comprising (i)(a) an ITGB6 binding domain according to any one of claims 1-34 and 49-53; (i)(b) a conditionally active chimeric antigen receptor according to any one of claims 37-38; (i)(c) a conditionally active ITGB6 binding protein according to any one of claims 39-44; (i)(d) a multispecific protein according to any one of claims 54-66; (i)I a multivalent protein according to claim 67 or 69; or (i)(f) an active drug according to claim 68, and (ii) a pharmaceutically acceptable carrier.

71. A process for producing an ITGB6 binding domain, said process comprising culturing a host transformed or transfected with a vector compnsing a nucleic acid sequence encoding the ITGB6 binding domain according to any one of claims 1-3 and 49-53, under conditions allowing the expression of the ITGB6 binding domain and recovering and purifying the produced protein from the culture.

72. A process for producing a multispecific protein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the multispecific ITGB6 binding protein according to any one of claims 54-66 under conditions allowing the expression of the multispecific protein and recovering and purifying the produced protein from the culture.

73. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering an ITGB6 binding domain according to any one of claims 1-34 and 49-53, or a pharmaceutical composition according to claim 70, to the subject.

74. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering the multispecific protein according to any one of claims 54-66, a multivalent protein according to claim 67 or 69, an active drug according to claim 68, or a pharmaceutical composition according to claim 70, to the subject.

75. The method of claim 73 or 74, wherein the subject is human.

76. The method of claim 75, wherein the method further comprises administration of an agent in combination with an ITGB6 binding domain according to any one of claims 1-34 and 49- 53, a multispecific protein according to any one of claims 54-66, a multivalent protein according to claim 67 or 69, an active drug according to claim 68, or a pharmaceutical composition according to claim 70.

77. The method of any one of claims 73-76, wherein the ITGB6 binding domain selectively binds to tumor cells expressing ITGB6.

78. The method of any one of claims 73-77, wherein the tumorous disease comprises a solid tumor disease.

79. The method of claim 78, wherein the solid tumor disease is metastatic.

80. The method of any one of claims 73-79, wherein the tumorous disease comprises at least one of: a colorectal cancer, a prostate cancer, a neuroendocrine cancer, a thyroid cancer, a non-small cell lung cancer, a small cell lung cancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, a biliary track cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, or any combination thereof.

81. The method of any one of claims 45-48, further comprising administering an agent in combination with the conditionally active chimeric antigen receptor according to claim 37 or 38, the conditionally active ITGB6 binding protein according to any one of claims 39-44, or a pharmaceutical composition comprising the same.

82. The method of claim 81, wherein the ITGB6 binding domain selectively binds to tumor cells expressing ITGB6.

83. The method of claim 81 or 82, wherein the tumorous disease comprises a solid tumor disease.

84. The method of claim 83, wherein the solid tumor disease is metastatic.

85. The method of any one of claims 81-84, wherein the tumorous disease is at least one of: a colorectal cancer, a prostate cancer, a neuroendocrine cancer, a thyroid cancer, a non-small cell lung cancer, a small cell lung cancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, a biliary track cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, or any combination thereof.

86. A process for producing a conditionally active chimeric antigen receptor, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the conditionally active chimeric antigen receptor according to any one of claims 37-38, under conditions allowing the expression of the conditionally active chimeric antigen receptor and recovering and purifying the produced protein from the culture.

87. A process producing a conditionally active ITGB6 binding protein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the conditionally active ITGB6 binding protein according to any one of claims 39-44, under conditions allowing the expression of the conditionally active ITGB6 binding protein and recovering and purifying the produced protein from the culture.

88. A process for producing a multivalent protein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the multivalent protein according to claims 67 or 69, under conditionsallowing the expression of the multivalent protein and recovering and purifying the produced protein from the culture.

89. A process for producing an active drug, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the active drug according to claim 68, under conditions allowing the expression of the active drug and recovering and purifying the produced drug from the culture.

90. A cell comprising a CAR according to any one of claims 23 and 30-33.

91. A cell comprising a ProCAR according to any one of claims 23-33 and 37-38.

92. The cell of claim 90 or 91, wherein the cell is a T cell or a natural killer (NK) cell.

93. A method comprising transfecting a cell according to any one of claims 90-92, with a vector or an RNA comprising a nucleotide sequence encoding the CAR or the ProCAR.

94. The conditionally active chimeric antigen receptor of claim 37 or 38, wherein the conditionally active chimeric antigen receptor has a greater therapeutic index compared to a chimeric antigen receptor (CAR) that does not comprise the (a) binding moiety but is otherwise identical to the conditionally active chimeric antigen receptor.

95. The conditionally active chimeric antigen receptor of claim 94, wherein the conditionally active chimeric antigen receptor has a therapeutic index that is at least about 5-fold to about 100-fold greater than that of a chimeric antigen receptor (CAR) that does not comprise the (a) binding moiety but is otherwise identical to the conditionally active chimeric antigen receptor.

96. The conditionally active ITGB6 binding protein of any one of claims 39-44, wherein the protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

97. The conditionally active ITGB6 binding protein of any one of claims 39-44, wherein the protein comprises a sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

98. The conditionally active ITGB6 binding protein of any one of claims 39-44, wherein the protein comprises a sequence that is at least about 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

99. The conditionally active ITGB6 binding protein of any one of claims 39-44 and 96- 98, wherein the conditionally active ITGB6 binding protein has a greater therapeutic index compared to an ITGB6 binding protein that does not comprise the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active ITGB6 binding protein.

100. The conditionally active ITGB6 binding protein of claim 99, wherein the conditionally active ITGB6 binding protein has a therapeutic index that is at least about 5-fold toabout 100-fold greater than that of an ITGB6 binding protein that does not comprise the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active ITGB6 binding protein.

101. A pharmaceutical composition comprising: (i)(a) a conditionally active chimeric antigen receptor according to claim 94 or 95; or (i)(b) a conditionally active ITGB6 binding protein according to claim 99 or 100, and (ii) a pharmaceutically acceptable carrier.

102. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering the conditionally active chimeric antigen receptor according to claim 94 or 95, or the pharmaceutical composition according to claim 101, to the subject.

103. A method for treating or ameliorating a proliferative disease, or a tumorous disease, comprising administering the conditionally active ITGB6 binding protein according to claim 99 or 100, or the pharmaceutical composition according to claim 101, to the subject.

104. The method of claim 102 or 103, wherein the subject is human.

105. The method of any one of claims 102-104, wherein the tumorous disease comprises at least one of: a colorectal cancer, a prostate cancer, a neuroendocrine cancer, a thyroid cancer, a non-small cell lung cancer, a small cell lung cancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, a biliary track cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, or a combination thereof.

106. A method of increasing a therapeutic index of an ITGB6 binding domain, the method comprising conjugating the ITGB6 binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop,- wherein the non-CDR loop comprises a binding site specific for the ITGB6 binding domain,- wherein the ITGB6 binding domain is masked from binding its target by the binding moiety,- wherein the ITGB6 binding domain is able to bind its target upon cleavage of the cleavable linker.

107. The method of claim 106, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from thegroup consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296.

108. The method of claim 106 or 107, wherein the ITGB6 binding domain conjugated to the binding moiety is part of a conditionally active multispecific protein, wherein the multispecific protein further comprises a CD3 binding domain.

109. The method of any one of claims 106-108, wherein the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 728.

110. The method of claim 108 or 109, wherein the CD3 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 655.

111. The method of any one of claims 106-110, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854.

112. The method of claim any one of claims 106-111, wherein the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74.

113. The method of any one of claims 106-112, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

114. The method of any one of claims 106-112, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

115. The method of any one of claims 106-112, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

116. The method of claim 106, wherein the ITGB6 binding domain conjugated to the binding moiety is part of a conditionally active chimeric antigen receptor, wherein the conditionally active chimeric antigen receptor further comprises at least one of: a transmembrane domain, an intracellular signaling domain, and a costimulatory domain.

117. The method of claim 116, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296.

118. The method of claim 116 or 117, wherein the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 728.

119. The method of any one of claims 116-118, wherein the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74.

120. The method of any one of claims 116-118, wherein the ITGB6 binding domain comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74.

121. A method of increasing a therapeutic index of an ITGB6 binding protein comprising a first target antigen binding domain and a second target antigen binding domain, wherein at least one of the first and the second target antigen binding domain comprises an ITGB6 binding domain, the method comprising conjugating the first or the second target antigen binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop,- wherein the non-CDR loop comprises a binding site specific for the first or the second target antigen binding domain,- wherein at least one of the first or the second target antigen binding domain is masked from binding its target by the binding moiety, and- wherein the first or the second target antigen binding domain that is masked, is able to bind its target upon cleavage of the cleavable linker.

122. The method of claim 121, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ IDNOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296.

123. The method of claim 121 or 122, wherein the non-CDR loop comprises a binding site specific for the ITGB6 binding domain.

124. The method of claim 121 or 122, wherein at least one of the first or the second target antigen binding domain comprises a CD3 binding domain.

125. The method of claim 124, wherein the non-CDR loop comprises a binding site specific for the CD3 binding domain.

126. The method of claim 125, wherein the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 655.

127. The method of any one of claims 121-126, wherein the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 728.

128. The method of claim any one of claims 121-127, wherein the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74.

129. The method of any one of claims 121-128, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854.

130. The method of any one of claims 121-129, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

131. The method of any one of claims 121-129, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

132. The method of any one of claims 121-129, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

133. A method of increasing a therapeutic index of an ITGB6 binding protein comprising an ITGB6 binding domain and a CD3 binding domain, the method comprising conjugating CD3binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein the non-CDR loop comprises a binding site specific for CD3 binding domain.

134. The method of claim 133, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 75-148; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296.

135. The method of claim 133 or 134, wherein the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 655.

136. The method of any one of claims 133-135, wherein the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 728.

137. The method of claim any one of claims 133-136, wherein the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74.

138. The method of any one of claims 133-137, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854.

139. The method of any one of claims 133-138, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

140. The method of any one of claims 133-138, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

141. The method of any one of claims 133-138, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

142. An ITGB6 targeting conditionally active multispecific protein, comprising: an ITGB6 binding domain, a CD3 binding domain, an albumin binding domain, wherein the albumin binding domain comprises a non-CDR loops that comprises a binding site specific for the CD3 binding domain and a cleavable linker, wherein the ITGB6 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 75-148, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 39-76; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 149-222 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 149-222; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 223-296 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 223-296.

143. The ITGB6 targeting conditionally active multispecific protein of claim 142, wherein the albumin binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 728.

144. The ITGB6 targeting conditionally active multispecific protein of claim 142 or 143, wherein the ITGB6 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-74.

145. The ITGB6 targeting conditionally active multispecific protein of any one of claims 142-144, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 661, 664, 666, 668, 670, 673, 675, 676, 678, 680, 682, 707, 713-716, 731-772, and 846-854.

146. The ITGB6 targeting conditionally active multispecific protein of any one of claims 142-145, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

147. The ITGB6 targeting conditionally active multispecific protein of any one of claims 142-145, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

148. The ITGB6 targeting conditionally active multispecific protein of any one of claims 142-145, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 353-357.

149. A pharmaceutical composition comprising an ITGB6 targeting conditionally active multispecific protein of any one of claims 142-148.

150. The pharmaceutical composition of claim 149, further comprising a pharmaceutically acceptable carrier.

151. A process for the production of ITGB6 targeting conditionally active multispecific protein of any one of claims 142-148, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the ITGB6 targeting conditionally active multispecific protein of any one of claims 142-148, under conditions allowing the expression of the ITGB6 targeting conditionally active multispecific protein and recovering and purifying the produced protein from the culture.

152. A method for the treatment or amelioration of a proliferative disease, or a tumorous disease, compnsing the administration of an of ITGB6 targeting conditionally active multispecific protein of any one of claims 142-148, or a pharmaceutical composition according to claim 149 or 150, to a subject in need thereof.

153. The method of claim 152, wherein the tumorous disease comprises a solid tumor disease.

154. The method of claim 153, wherein the solid tumor disease is metastatic.

155. The method of any one of claims 152-154, wherein the tumorous disease comprises at least one of: a colorectal cancer, a prostate cancer, a neuroendocrine cancer, a thyroid cancer, a non-small cell lung cancer, a small cell lung cancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, a biliary track cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, or any combination thereof.

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