Targeting of α3β1 integrin for treatment of cancer and other diseases

Abstract

To provide targeting of α3β1 integrin for treatment of cancer and other diseases.SOLUTION: Provided herein, in some aspects, are agents, such as antibodies, chimeric antigen receptors, or RNA interference molecules that target the interaction between α3β1 integrin and α1 homotrimeric type I collagen. One aspect is directed to a method of treating cancer and fibroids comprising administering to a patient in need thereof an effective amount of an agent that disrupts the interaction between α3β1 integrin and α1 homotrimeric type I collagen. The method can further include administering an effective amount of chemotherapy or immunotherapy to said patient.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 864,611, filed on Jun. 21, 2019, the entire content of which is hereby expressly incorporated by reference in its entirety. Background 1. Field

[0002] Aspects of the present invention generally relate to the field of medicine. Certain aspects relate to methods of treating cancer by interfering with the interaction of homotrimeric type I collagen and α3β1 integrin.

Background Art

[0003] 2. Background Type I collagen (col1), a fibrous collagen, is the most abundant protein in the human body and is most abundant in bone, tendon, and skin. The basic functional unit of col1 is a heterotrimer composed of two a1 chains and one a2 chain, which together form a triple helix structure. Each a-chain polypeptide is synthesized in the cytosol and binds to the other two a-chains to produce triple helix type I procollagen with N-terminal and C-terminal propeptides. Subsequently, the procollagen molecule is secreted into the extracellular space, where the N-terminal and C-terminal propeptides are cleaved by procollagenases to produce the basic functional unit of Col1. Col1 triple helix rod-like molecules interact with each other to form fibrils, which are further cross-linked to form large bundles of fibers.

[0004] During embryogenesis, many organs are thought to express Col1 to promote cell migration, differentiation, and structural compartmentalization, but Col1 is almost absent in adult tissue parenchyma and organs (Hay, 1981). Global deletion of the Col1a1 gene (resulting in complete absence of type I collagen) causes embryonic lethality (Lohler et al., 1984). In pathogenic conditions such as organ fibrosis and cancer, Col1 accumulates robustly in the affected tissues (Apte et al., 2012; Armstrong et al., 2004; Bachem et al., 2005; Fujita et al., 2009; Haber et al., 1999). Col1 associated with tumor tissue is known to create a biophysically "stiff" environment around cancer cells, promote cell migration through "tracks" of conductive fibers, promote abnormal cell interactions, and induce cancer cell proliferation and survival (Apte et al., 2012; Armstrong et al., 2004; Bachem et al., 2005; Egeblad et al., 2010; Fujita et al., 2009; Haber et al., 1999; Levental et al., 2009). In this regard, Col1 is a major component of the tumor stroma / microenvironment associated with pancreatic cancer (Mollenhauer et al., 1987). aSMA associated with PDAC +Myofibroblasts (MFs) are presumed to contribute significantly to the production of Col1 and have been proposed to impede drug delivery to cancer cells (Apte et al., 2012; Armstrong et al., 2004; Bachem et al., 2005; Egeblad et al., 2010; Fujita et al., 2009; Haber et al., 1999; Levental et al., 2009; Provenzano et al., 2012). Recent studies have suggested that stromal fibroblasts in PDAC exhibit context-dependent functions and may influence tumor promotion and suppression (Biffi et al., 2019; Kalluri, 2016; Laklai et al., 2016; Lee et al., 2014; Mueller and Fusenig, 2004; Neesse et al., 2015; Ohlund et al., 2014; Ohlund et al., 2017; Olive et al., 2009; Ozdemir et al., 2014; Provenzano et al., 2012; Rhim et al., 2014; Sugimoto et al., 2006). In this regard, the exact function of type I collagen produced by activated stellate cells / myofibroblasts in the initiation and progression of PDAC remains unclear.

Summary of the Invention

Means for Solving the Problems

[0005] Abstract In some embodiments, provided herein is a composition comprising an antibody or antibody fragment that binds to α3β1 integrin. In some aspects, the antibody or antibody fragment binds to α3β1 integrin in epithelial cells. In some aspects, the antibody or antibody fragment binds to α3β1 integrin in fibroblasts. In some aspects, the antibody or antibody fragment interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. In some aspects, the antibody or antibody fragment inhibits prosurvival signaling by α3β1 integrin.

[0006] In some embodiments, the antibody is a chimeric antibody or a bispecific antibody. In some embodiments, the antibody is a chimeric antibody, and the chimeric antibody is a humanized antibody. In some embodiments, the bispecific antibody binds to both α3β1 integrin and CD3. In some embodiments, the antibody or antibody fragment is conjugated to a cytotoxic agent. In some embodiments, the antibody or antibody fragment is conjugated to a diagnostic agent.

[0007] In some embodiments, provided herein is a hybridoma or engineered cell encoding an antibody or antibody fragment of any one of these embodiments. In some embodiments, provided herein is a pharmaceutical formulation comprising an antibody or antibody fragment of any one of these embodiments.

[0008] In some embodiments, provided herein is a method of treating a patient in need of treatment, the method comprising administering an effective amount of an α3β1 integrin-specific antibody or antibody fragment. In some embodiments, the antibody or antibody fragment binds to α3β1 integrin on epithelial cells. In some embodiments, the antibody or antibody fragment binds to α3β1 integrin on fibroblasts. In some embodiments, the antibody or antibody fragment interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. In some embodiments, the antibody or antibody fragment inhibits survival-promoting signaling by α3β1 integrin. In some embodiments, the α3β1 integrin-specific antibody or antibody fragment is an antibody or antibody fragment of any one of these embodiments.

[0009] In some embodiments, the patient has cancer, fibroma, tissue injury, keloid, organ fibrosis, Crohn's disease, stricture, colitis, psoriasis, or connective tissue disorder. In some embodiments, the patient is in need of tissue injury repair or tissue regeneration. In some embodiments, the connective tissue disorder is a connective tissue disorder comprising collagen. In some embodiments, the connective tissue disorder comprising collagen is a connective tissue disorder comprising type I collagen.

[0010] In certain embodiments, the patient has cancer. In some embodiments, the cancer patient was determined to express high levels of α1 homotrimeric type I collagen compared to control patients. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the method is further defined as a method of inhibiting metastasis of pancreatic cancer. In some embodiments, the method is further defined as a method of inhibiting the growth of pancreatic cancer.

[0011] In some embodiments, the method further comprises administering at least a second anti-cancer therapy. In some embodiments, the second anti-cancer therapy is chemotherapy, immunotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. In some embodiments, the second anti-cancer therapy is immunotherapy. In some embodiments, the immunotherapy is checkpoint blockade therapy. In some embodiments, the checkpoint blockade therapy comprises administering an anti-PD-1 antibody or antibody fragment. In some embodiments, the method further comprises administering an integrin signaling inhibitor. In some embodiments, the integrin signaling inhibitor inhibits FAK and / or PYK2. In some embodiments, the integrin signaling inhibitor is VS-4718 (PND-1086).

[0012] In some embodiments, provided herein is a method of treating a patient in need of treatment, the method comprising administering an effective amount of an agent that inhibits pro-survival signaling through α3β1 integrin. In some embodiments, the agent is an antibody or antibody fragment that interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. In some embodiments, the α3β1 integrin-specific antibody or antibody fragment is any one of the antibodies or antibody fragments of any one of these embodiments. In some embodiments, the agent is an antisense oligonucleotide that inhibits the expression of α3β1 integrin.

[0013] In some embodiments, the patient has cancer, fibroid tumors, tissue injury, keloids, organ fibrosis, Crohn's disease, strictures, colitis, psoriasis, or connective tissue disorders. In some embodiments, the patient requires tissue injury repair or tissue regeneration. In some embodiments, the connective tissue disorder is a connective tissue disorder involving collagen. In some embodiments, the connective tissue disorder involving collagen is a connective tissue disorder involving type I collagen.

[0014] In some embodiments, the patient has cancer. In some embodiments, the cancer patient was determined to express a high level of α1 homotrimeric type I collagen compared to control patients. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the method is further defined as a method for inhibiting the metastasis of pancreatic cancer. In some embodiments, the method is further defined as a method for inhibiting the growth of pancreatic cancer.

[0015] In some embodiments, the method further comprises administering at least a second anti-cancer therapy. In some embodiments, the second anti-cancer therapy is chemotherapy, immunotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or cytokine therapy. In some embodiments, the method further comprises administering an integrin signaling inhibitor. In some embodiments, the integrin signaling inhibitor inhibits FAK and / or PYK2. In some embodiments, the integrin signaling inhibitor is VS-4718 (PND-1086).

[0016] In some embodiments, provided herein is a method of treating a subject, comprising administering an agent that inhibits the expression of α3β1 integrin in an antitumor effective amount. In some aspects, the agent is an siRNA that targets the mRNA of α3β1 integrin. In some aspects, the agent is formulated in lipid nanoparticles. In some aspects, the lipid nanoparticles are exosomes. In some aspects, the subject has cancer. In some aspects, the cancer is pancreatic cancer. In some aspects, the method further comprises administering an integrin signaling inhibitor. In some aspects, the integrin signaling inhibitor inhibits FAK and / or PYK2. In some aspects, the integrin signaling inhibitor is VS-4718 (PND-1086).

[0017] In some embodiments, provided herein is a chimeric antigen receptor (CAR) polypeptide comprising, from N-terminus to C-terminus, an antigen-binding domain; a hinge domain; a transmembrane domain and an intracellular signaling domain, wherein the CAR polypeptide binds to α3β1 integrin. In some aspects, the antigen-binding domain comprises the HCDR sequences of a primary antibody that binds to α3β1 integrin and the LCDR sequences of a secondary antibody that binds to α3β1 integrin. In some aspects, the antigen-binding domain comprises the HCDR sequences and LCDR sequences of an antibody that binds to α3β1 integrin. In some aspects, the CAR interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. In some aspects, the hinge domain is a CD8a hinge domain or an IgG4 hinge domain. In some aspects, the transmembrane domain is a CD8a transmembrane domain or a CD28 transmembrane domain. In some aspects, the intracellular signaling domain comprises a CD3z intracellular signaling domain.

[0018] In some embodiments, provided herein is a nucleic acid molecule encoding any one of the CAR polypeptides of this embodiment. In some aspects, the sequence encoding the CAR polypeptide is operably linked to an expression control sequence.

[0019] In some embodiments, provided herein is an isolated immune effector cell comprising any one CAR polypeptide of this embodiment, or any one nucleic acid of this embodiment. In some aspects, the nucleic acid is integrated into the genome of the cell. In some aspects, the cell is a T cell. In some aspects, the cell is a NK cell. In some aspects, the cell is a human cell.

[0020] In some embodiments, provided herein is a pharmaceutical composition comprising a population of cells according to any one of this embodiment in a pharmaceutically acceptable carrier.

[0021] In some embodiments, provided herein is a method of treating a subject comprising administering an anti-tumor effective amount of chimeric antigen receptor (CAR) T cells expressing any one CAR polypeptide of this embodiment. In some aspects, the CAR T cells are allogeneic cells. In some aspects, the CAR T cells are autologous cells. In some aspects, the CAR T cells are HLA compatible with the subject. In some aspects, the subject has cancer. In some aspects, the cancer is pancreatic cancer. In some aspects, the method further comprises administering an integrin signaling inhibitor. In some aspects, the integrin signaling inhibitor inhibits FAK and / or PYK2. In some aspects, the integrin signaling inhibitor is VS-4718 (PND-1086).

[0022] In some embodiments, provided herein is a method of treating a subject, comprising administering an anti-tumor effective amount of chimeric antigen receptor (CAR) NK cells that express a CAR polypeptide according to any one of these embodiments. In some aspects, the CAR NK cells are allogeneic cells. In some aspects, the CAR NK cells are autologous cells. In some aspects, the CAR NK cells are compatible with the subject's HLA. In some aspects, the subject has cancer. In some aspects, the cancer is pancreatic cancer. In some aspects, the method further comprises administering an integrin signaling inhibitor. In some aspects, the integrin signaling inhibitor inhibits FAK and / or PYK2. In some aspects, the integrin signaling inhibitor is VS-4718 (PND-1086). In some aspects, the method further comprises administering at least a second anti-cancer therapy. In some aspects, the second anti-cancer therapy is chemotherapy, immunotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. In some aspects, the method further comprises administering an integrin signaling inhibitor. In some aspects, the integrin signaling inhibitor inhibits FAK and / or PYK2. In some aspects, the integrin signaling inhibitor is VS-4718 (PND-1086).

[0023] In some embodiments, provided herein is a method of treating a subject having cancer, the method comprising: (a) administering to the subject an effective amount of an α3β1 integrin-specific antibody or antibody fragment; (b) administering to the subject an anti-tumor effective amount of chimeric antigen receptor (CAR) T cells that express a CAR polypeptide comprising, from N-terminus to C-terminus, an antigen-binding domain; a hinge domain; a transmembrane domain; and an intracellular signaling domain, wherein the CAR polypeptide binds to α3β1 integrin; or (c) administering to the subject an anti-tumor effective amount of an agent that inhibits the expression of α3β1 integrin; wherein the cancer cells of the subject are determined to have increased expression of α3 integrin as compared to healthy cells or control cells. In some embodiments, the method comprises administering to the subject an effective amount of the α3β1 integrin-specific antibody or antibody fragment of (a), wherein the antibody or antibody fragment interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. In some embodiments, the method comprises administering to the subject an anti-tumor effective amount of the chimeric antigen receptor (CAR) T cells of (b). In some embodiments, the method comprises administering to the subject an anti-tumor effective amount of the agent of (c).

[0024] As used herein, "essentially free of" with respect to a specified component means that the specified component is not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from unintentional contamination of the composition is far below 0.05%, preferably below 0.01%. Most preferably, the composition is one in which the specified component cannot be detected by standard analytical methods.

[0025] As used herein, "a" or "an" can mean one or more. In one or more claims herein, when used in conjunction with the word "comprising", the words "a" or "an" can mean one or more.

[0026] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer only to alternatives or the alternatives are not mutually exclusive, but the present disclosure supports definitions that refer only to alternatives and "and / or". As used herein, "another" can mean at least a second or more.

[0027] Throughout this application, the term "about" is used to indicate that a value includes variations due to the inherent error of the device, the method used to determine the value, and variations that exist between the subjects of study.

[0028] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, so it should be understood that the detailed description and specific examples, while showing embodiments of the present invention, are described for illustrative purposes only. In embodiments of the present invention, for example, the following items are provided. (Item 1) A composition comprising an antibody or antibody fragment that binds to α3β1 integrin. (Item 2) The composition according to item 1, wherein the antibody or antibody fragment interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. (Item 3) The composition according to item 1 or 2, wherein the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, F(ab’) 2 fragment, or an Fv fragment. (Item 4) The composition according to item 1 or 2, wherein the antibody is a chimeric antibody or a bispecific antibody. (Item 5) The composition according to item 4, wherein the antibody is a chimeric antibody and the chimeric antibody is a humanized antibody. (Item 6) The composition according to item 4, wherein the bispecific antibody binds to both α3β1 integrin and CD3. (Item 7) The composition according to any one of items 1 to 6, wherein the antibody or antibody fragment is bound to a cytotoxic agent. (Item 8) The composition according to any one of items 1 to 6, wherein the antibody or antibody fragment is bound to a diagnostic agent. (Item 9) A hybridoma or engineered cell encoding an antibody or antibody fragment of the composition according to any one of items 1 to 8. (Item 10) A pharmaceutical preparation comprising the composition according to any one of items 1 to 8. (Item 11) A method for treating a patient in need of treatment, the method comprising administering an effective amount of an α3β1 integrin-specific antibody or antibody fragment. (Item 12) The method according to item 11, wherein the antibody or antibody fragment interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. (Item 13) The method according to item 11, wherein the antibody or antibody fragment inhibits survival-promoting signal transduction by α3β1 integrin. (Item 14) The method according to item 11, wherein the patient has cancer, fibroma, tissue injury, keloid, organ fibrosis, Crohn's disease, stricture, colitis, psoriasis, or connective tissue disorder. (Item 15) The method according to item 14, wherein the connective tissue disorder comprises collagen. (Item 16) The method according to item 15, wherein the connective tissue disorder comprising collagen is a connective tissue disorder comprising type I collagen. (Item 17) The method according to item 15, wherein the patient has cancer. (Item 18) The method according to item 11, wherein the α3β1 integrin-specific antibody or antibody fragment is the antibody or antibody fragment of the composition according to any one of items 1 to 8. (Item 19) The method according to item 17, wherein the cancer patient is determined to express a high level of α1 homotrimeric type I collagen as compared with a control patient. (Item 20) The method according to item 17, wherein the cancer is pancreatic cancer. (Item 21) The method according to item 20, further defined as a method for inhibiting the metastasis of pancreatic cancer. (Item 22) The method according to item 20, further defined as a method for inhibiting the growth of pancreatic cancer. (Item 23) The method according to item 17, further comprising administering at least a second anti-cancer therapy. (Item 24) The method according to item 23, wherein the second anti-cancer therapy is chemotherapy, immunotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. (Item 25) The method according to item 24, wherein the second anti-cancer therapy is immunotherapy. (Item 26) The method according to item 25, wherein the immunotherapy is checkpoint blockade therapy. (Item 27) The method according to item 26, wherein the checkpoint blockade therapy comprises administering an anti-PD-1 antibody or antibody fragment. (Item 28) The method according to any one of items 17 to 27, further comprising administering an integrin signaling inhibitor. (Item 29) The method according to item 28, wherein the integrin signaling inhibitor inhibits FAK and / or PYK2. (Item 30) The method according to item 28, wherein the integrin signaling inhibitor is VS-4718 (PND-1086). (Item 31) A method of treating a patient in need of treatment, the method comprising administering an effective amount of an agent that inhibits survival-promoting signaling through α3β1 integrin. (Item 32) The method according to item 31, wherein the agent is an antibody or antibody fragment that interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. (Item 33) The method according to item 31, wherein the agent is an antisense oligonucleotide that inhibits the expression of α3β1 integrin. (Item 34) The method according to item 31, wherein the patient has cancer, fibroid, tissue injury, keloid, organ fibrosis, Crohn's disease, stricture, colitis, psoriasis, or connective tissue disorder. (Item 35) The method according to item 34, wherein the connective tissue disorder is a connective tissue disorder containing collagen. (Item 36) The method according to item 35, wherein the connective tissue disorder containing collagen is a connective tissue disorder containing type I collagen. (Item 37) The method according to item 35, wherein the patient has cancer. (Item 38) The method according to item 31, wherein the α3β1 integrin-specific antibody or antibody fragment is the antibody or antibody fragment according to any one of items 1 to 8. (Item 39) The method according to item 37, wherein the cancer patient is determined to express a high level of α1 homotrimeric type I collagen compared to a control patient. (Item 40) The method according to item 37, wherein the cancer is pancreatic cancer. (Item 41) The method according to item 40, further defined as a method of inhibiting the metastasis of pancreatic cancer. (Item 42) The method according to item 40, further defined as a method of inhibiting the growth of pancreatic cancer. (Item 43) The method according to item 37, further comprising administering at least a second anti-cancer therapy. (Item 44) The method according to item 43, wherein the second anti-cancer therapy is chemotherapy, immunotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. (Item 45) The method according to any one of items 37 to 44, further comprising administering an integrin signaling inhibitor. (Item 46) The method according to item 45, wherein the integrin signaling inhibitor inhibits FAK and / or PYK2. (Item 47) The method according to item 45, wherein the integrin signaling inhibitor is VS-4718 (PND-1086). (Item 48) A chimeric antigen receptor (CAR) polypeptide, comprising, from the N-terminus to the C-terminus, an antigen-binding domain; a hinge domain; a transmembrane domain and an intracellular signaling domain, wherein the CAR polypeptide binds to α3β1 integrin. (Item 49) The polypeptide according to item 48, wherein the antigen-binding domain comprises the HCDR sequence of a first antibody that binds to α3β1 integrin and the LCDR sequence of a second antibody that binds to α3β1 integrin. (Item 50) The polypeptide according to item 48, wherein the antigen-binding domain comprises the HCDR sequence and the LCDR sequence of an antibody that binds to α3β1 integrin. (Item 51) The polypeptide according to any one of items 48 to 50, wherein the CAR interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. (Item 52) The polypeptide according to any one of items 48 to 51, wherein the hinge domain is a CD8a hinge domain or an IgG4 hinge domain. (Item 53) The polypeptide according to any one of items 48 to 52, wherein the transmembrane domain is a CD8a transmembrane domain or a CD28 transmembrane domain. (Item 54) The polypeptide according to any one of items 48 to 53, wherein the intracellular signaling domain comprises a CD3z intracellular signaling domain. (Item 55) A nucleic acid molecule encoding a CAR polypeptide according to any one of items 48 to 54. (Item 56) The nucleic acid molecule according to item 55, wherein the sequence encoding the CAR polypeptide is operably linked to an expression control sequence. (Item 57) An isolated immune effector cell comprising a CAR polypeptide according to any one of items 48 to 54, or a nucleic acid according to item 55 or 56. (Item 58) The cell according to item 57, wherein the nucleic acid is integrated into the genome of the cell. (Item 59) The cell according to item 57 or 58, wherein the cell is a T cell. (Item 60) The cell according to item 57 or 58, wherein the cell is an NK cell. (Item 61) The cell according to any one of items 57 to 60, wherein the cell is a human cell. (Item 62) A pharmaceutical composition comprising a cell according to any one of items 58 to 61 and a pharmaceutically acceptable carrier, the cell being in a population of cells. (Item 63) A method of treating a subject, comprising administering an anti-tumor effective amount of chimeric antigen receptor (CAR) T cells expressing a CAR polypeptide according to any one of items 48 to 54. (Item 64) The method according to item 63, wherein the CAR T cells are allogeneic cells. (Item 65) The method according to item 63, wherein the CAR T cells are autologous cells. (Item 66) The method according to item 63, wherein the CAR T cells are HLA-compatible with the subject. (Item 67) The method according to any one of items 63 to 66, wherein the subject has cancer. (Item 68) The method according to item 67, wherein the cancer is pancreatic cancer. (Item 69) The method according to any one of items 63 to 68, further comprising administering an integrin signaling inhibitor. (Item 70) The method according to item 69, wherein the integrin signaling inhibitor inhibits FAK and / or PYK2. (Item 71) The method according to item 69, wherein the integrin signaling inhibitor is VS-4718 (PND-1086). (Item 72) The method according to any one of items 63 to 71, further comprising administering at least a second anti-cancer therapy. (Item 73) The method according to item 72, wherein the second anti-cancer therapy is chemotherapy, immunotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. (Item 74) The method according to item 73, wherein the second anti-cancer therapy is immunotherapy. (Item 75) The method according to item 74, wherein the immunotherapy is checkpoint blockade therapy. (Item 76) The method according to item 75, wherein the checkpoint blockade therapy comprises administering an anti-PD-1 antibody or antibody fragment. (Item 77) A method of treating a subject, comprising administering an antitumor-effective amount of an agent that inhibits the expression of α3β1 integrin. (Item 78) The method according to item 77, wherein the agent is an siRNA targeting α3β1 integrin mRNA. (Item 79) The method according to item 77, wherein the agent is an shRNA targeting α3β1 integrin mRNA. as described. (Item 80) The method according to item 77 or 78, wherein the agent is formulated in lipid nanoparticles. (Item 81) The method according to item 77, wherein the lipid nanoparticles are exosomes. (Item 82) The method according to item 81, wherein the exosomes are exosomes derived from mesenchymal stem cells. (Item 83) The method according to any one of items 77 to 81, wherein the subject has cancer. (Item 84) The method according to item 83, wherein the cancer is pancreatic cancer. (Item 85) The method according to any one of items 77 to 84, further comprising administering an integrin signaling inhibitor. (Item 86) The method according to item 85, wherein the integrin signaling inhibitor inhibits FAK and / or PYK2. (Item 87) The method according to item 85, wherein the integrin signaling inhibitor is VS-4718 (PND-1086). (Item 88) The method according to any one of items 77 to 87, further comprising administering at least a second anti-cancer therapy. (Item 89) The method according to item 88, wherein the second anti-cancer therapy is chemotherapy, immunotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. (Item 90) The method according to item 89, wherein the second anti-cancer therapy is immunotherapy. (Item 91) The method according to item 90, wherein the immunotherapy is checkpoint blockade therapy. (Item 92) The method according to item 91, wherein the checkpoint blockade therapy comprises administering an anti-PD-1 antibody or antibody fragment. (Item 93) A method of treating a subject with cancer, the method comprising: (a) administering to the subject an effective amount of an α3β1 integrin-specific antibody or antibody fragment; (b) administering to the subject an anti-tumor effective amount of chimeric antigen receptor (CAR) T cells expressing a CAR polypeptide comprising, from N-terminus to C-terminus, an antigen-binding domain; a hinge domain; a transmembrane domain and an intracellular signaling domain, wherein the CAR polypeptide binds to α3β1 integrin; or (c) administering to the subject an anti-tumor effective amount of an agent that inhibits the expression of α3β1 integrin; comprising, The method, wherein the cancer cells of the subject are determined to have increased expression of α3 integrin compared to healthy cells or control cells. (Item 94) The method according to item 93, wherein the method comprises administering to the subject the effective amount of the α3β1 integrin-specific antibody or antibody fragment of (a), and the antibody or antibody fragment interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. (Item 95) The method according to item 94, wherein the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, a Fab fragment, an F(ab’) 2 fragment, or an Fv fragment. (Item 96) The method according to item 94 or 95, wherein the antibody is a chimeric antibody or a bispecific antibody. (Item 97) The method according to item 96, wherein the antibody is a chimeric antibody and the chimeric antibody is a humanized antibody. (Item 98) The method according to item 96, wherein the antibody is a bispecific antibody and the bispecific antibody binds to both α3β1 integrin and CD3. (Item 99) The method according to any one of Items 94 to 98, wherein the antibody or antibody fragment is bound to a cytotoxic agent. (Item 100) The method according to any one of Items 94 to 98, wherein the antibody or antibody fragment is bound to a diagnostic agent. (Item 101) The method according to any one of Items 93 to 100, wherein the method comprises administering to the subject the chimeric antigen receptor (CAR) T cells of (b) in the anti-tumor effective amount. (Item 102) The method according to Item 101, wherein the antigen-binding domain comprises the HCDR sequence of a first antibody that binds to α3β1 integrin and the LCDR sequence of a second antibody that binds to α3β1 integrin. (Item 103) The method according to Item 101, wherein the antigen-binding domain comprises the HCDR sequence and the LCDR sequence of an antibody that binds to α3β1 integrin. (Item 104) The method according to any one of Items 101 to 103, wherein the CAR interferes with the interaction between α3β1 integrin and α1 homotrimeric type I collagen. (Item 105) The method according to any one of Items 101 to 104, wherein the hinge domain is a CD8a hinge domain or an IgG4 hinge domain. (Item 106) The method according to any one of Items 101 to 105, wherein the transmembrane domain is a CD8a transmembrane domain or a CD28 transmembrane domain. (Item 107) The method according to any one of Items 101 to 106, wherein the intracellular signaling domain comprises a CD3z intracellular signaling domain. (Item 108) The method according to any one of Items 93 to 107, wherein the method comprises administering to the subject the agent of (c) in the anti-tumor effective amount. (Item 109) The method according to item 108, wherein the agent is an siRNA targeting α3β1 integrin mRNA. (Item 110) The method according to item 108, wherein the agent is an shRNA targeting α3β1 integrin mRNA. (Item 111) The method according to any one of items 108 to 110, wherein the agent is formulated in lipid nanoparticles. (Item 112) The method according to item 111, wherein the lipid nanoparticles are exosomes. (Item 113) The method according to item 112, wherein the exosomes are exosomes derived from mesenchymal stem cells. (Item 114) The method according to any one of items 93 to 113, wherein the subject has cancer. (Item 115) The method according to item 114, wherein the cancer is pancreatic cancer. (Item 116) The method according to any one of items 93 to 115, further comprising administering an integrin signaling inhibitor. (Item 117) The method according to item 116, wherein the integrin signaling inhibitor inhibits FAK and / or PYK2. (Item 118) The method according to item 117, wherein the integrin signaling inhibitor is VS-4718 (PND-1086). (Item 119) The method according to any one of items 93 to 118, further comprising administering at least a second anti-cancer therapy. (Item 120) The method according to item 119, wherein the second anti-cancer therapy is chemotherapy, immunotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy. (Item 121) The method according to item 120, wherein the second anti-cancer therapy is immunotherapy. (Item 122) The method according to item 121, wherein the immunotherapy is checkpoint blockade therapy. (Item 123) The method according to item 122, wherein the checkpoint blockade therapy comprises administering an anti-PD-1 antibody or antibody fragment.

[0029] The following drawings form a part of this specification and are included to further demonstrate specific embodiments of the present invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0039] Detailed Description Aspects of the present disclosure relate to the fact that cancer cells specifically produce a variant of type I collagen. Without wishing to be bound by theory, it is understood that healthy muscle fibroblasts produce an α1 / α2 / α1 heterotrimer that binds to the DDR receptor and suppresses tumor growth. On the other hand, as disclosed herein, cancer cells produce an α1 / α1 / α1 homotrimer that binds to α3β1 integrin, thereby inducing pro-oncogenic signals.

[0040] The Col1 homotrimer potently induces phosphorylation of DDR1 and activates FAK, AKT, and ERK1 / 2 when compared to the heterotrimer. In an initial assessment, this is in contrast to what has been previously published (Armstrong et al., 2004; Bachem et al., 2005; Fujita et al., 2009). However, in all such studies, the contribution of Col1 produced by cancer cells cannot be ignored. Inhibition of DDR1 leads to continuous activation of FAK, AKT, and ERK1 / 2. This suggests that the Col1 homotrimer can activate other receptors in parallel or as a compensatory mechanism. Single-cell RNA sequencing analysis suggested that pancreatic cancer cells can express Col1-binding integrins such as α1β1, α2β1, and α3β1. Previous studies have shown that integrins α1β1, α2β1, and α3β bind to Col1 (Ruoslahti, 1991; Takada et al., 2007). The present disclosure shows that the Col1 homotrimer can interact with integrin α3β1 and continuously induce survival-promoting signals. Furthermore, inhibition of DDR1 increases the expression of integrin α3β1. Also disclosed is the role of the Col1 homotrimer in suppressing T cell infiltration in cancer, which can be reversed by decreasing or eliminating Col1 homotrimer expression in cancer cells. Collectively, these studies suggest that the Col1 homotrimer interacts with integrin α3β1 in early pancreatic cancer-initiating cells to induce proliferation and survival and suppress infiltration of immune cells. Inhibition of FAK at the early stage of pancreatic cancer in KPPC mice leads to significant disease control, verifying the importance of this signaling axis in the initiation and progression of PDAC. In summary, these studies have identified a new oncogenic variant of Col1, a new bimodal contribution of Col1 in the progression of PDAC, and are suggested to influence the development of new therapeutic strategies.

[0041] Thus, it is contemplated that, for example, the use of antibodies, small molecules, siRNA, antisense oligos, CAR-T cells, CAR-NK cells, bispecific antibodies to interfere with the binding of homotrimers and α3β1 integrin can be used to treat cancer or fibrosis. The inventors have identified several means for treating cancer by exploiting this difference. This includes (1) the use of antibodies that specifically bind to α3β1 integrin; (2) the use of agents that inhibit signal transduction by α3β1 integrin; (3) siRNA or antisense oligos that inhibit the expression of α3β1 integrin; (4) the use of CAR-T cells that target α3β1 integrin; (5) the use of CAR NK cells that target α3β1 integrin; and (6) the use of bispecific antibodies that target both α3β1 integrin and CD3 to direct T cells to cancer cells. Further contemplated is the use of such methods in combination with immunotherapies such as checkpoint blockade therapies (e.g., anti-PD-1 therapy) to interfere with the binding of Col1 homotrimers and α3β1 integrin. I. Antibodies and Their Production

[0042] An "isolated antibody" is an antibody that has been separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified (1) to greater than 95% by weight of antibody, particularly greater than 99% by weight, as measured by the Lowry method; (2) to a degree sufficient to obtain at least a 15-residue N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibodies include intrabodies in recombinant cells because at least one component of the antibody's natural environment is not present. However, typically, isolated antibodies are prepared by at least one purification step.

[0043] The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, and thus contain ten antigen-binding sites, while secretory IgA antibodies can polymerize to form multivalent aggregates containing two to five basic four-chain units together with the J chain. In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H-chain isotype. Each H and L chain also has regularly spaced intra-chain disulfide bridges. Each H chain has a variable region (V H ) at the N-terminus, followed by three constant domains (C H ) in each of the α and γ chains and four C H domains in the μ and isotypes. Each L chain has a variable region (V L ) at the N-terminus and a constant domain (C L ) at its other end. V L aligns with V H , and C L aligns with the first constant domain (C H1 ) of the heavy chain. Certain amino acid residues are thought to form the interface between the variable regions of the light and heavy chains. The pairing of V H and V L forms a single antigen-binding site. For information on the structures and properties of the various classes, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.

[0044] The L chain of any vertebrate species has a constant domain (C LBased on the amino acid sequence of (), it can be assigned to one of two distinct types called kappa and lambda. The constant domain of its heavy chain (C H ) Depending on the amino acid sequence of, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having a heavy chain called α, δ, ε, γ, and μ, respectively. The gamma and alpha classes are further classified into subclasses based on relatively small differences in sequence and function, and humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. H Based on relatively small differences in sequence and function, it is further classified into subclasses, and humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0045] The term "variable" refers to the fact that the sequences of certain segments of the V domain vary greatly among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acid length of the variable region. Instead, the V region is composed of relatively invariant stretches called framework regions (FRs) of 15 - 30 amino acids, separated by extremely variable, short regions called "hypervariable regions" that are 9 - 12 amino acids long each. The variable regions of native heavy and light chains each contain four FRs, which mainly adopt a β - sheet conformation and are connected by three hypervariable regions. These hypervariable regions form loops that connect the β - sheet structures and, in some cases, form part of the β - sheet structure. The hypervariable regions of each chain are held in proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen - binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest, 5th (See Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).

[0046] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally include amino acid residues from "complementary determining regions" or "CDRs" (e.g., when numbered according to the Kabat numbering system, residues around positions L about 24 - 34 (L1), 50 - 56 (L2), and 89 - 97 (L3) of V H and residues around positions about 31 - 35 (H1), 50 - 65 (H2), and 95 - 102 (H3) of V L ; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991)); and / or amino acid residues from "hypervariable loops" (e.g., when numbered according to the Chothia numbering system, residues 24 - 34 (L1), 50 - 56 (L2), and 89 - 97 (L3) of V H and residues 26 - 32 (H1), 52 - 56 (H2), and 95 - 101 (H3) of V L ; Chothia and Lesk, J.Mol.Biol.196:901 - 917(1987)); and / or amino acid residues from "hypervariable loops" / CDRs (e.g., when numbered according to the IMGT numbering system, 27 - 38 (L1), 56 - 65 (L2), and 105 - 120 (L3) of V H27 - 38 (H1), 56 - 65 (H2) and 105 - 120 (H3); Lefranc, M.P. et al., Nucl. Acids Res. 27:209 - 212 (1999), Ruiz, M. et al., Nucl. Acids Res. 28:219 - 221 (2000)). Optionally, when numbered according to AHo, the antibody has one or more of the following points, V L 28, 36 (L1), 63, 74 - 75 (L2) and 123 (L3), and V sub has insertions symmetric to 28, 36 (H1), 63, 74 - 75 (H2) and 123 (H3) of H; Honneger, A. and Plunkthun, A. J. Mol. Biol. 309:657 - 670 (2001)).

[0047] "Germline nucleic acid residue" means a nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region. A "germline gene" is DNA found in germ cells (i.e., cells destined to become eggs or sperm). "Germline mutation" refers to a genetic change in specific DNA that occurs in germ cells or zygotes at the single - cell stage, and such mutations are transmitted to offspring and incorporated into all cells of the body. Germline mutations are contrasted with somatic mutations that are acquired in one somatic cell. In some cases, the nucleotides of a germline DNA sequence encoding a variable region mutate (i.e., somatic mutation) and are replaced by different nucleotides.

[0048] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of antibodies that are substantially homogeneous, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are made against a single antigenic site. Further, in contrast to polyclonal antibody preparations, which contain different antibodies against different determinants (epitopes), each monoclonal antibody is made against a single determinant on the antigen. Monoclonal antibodies are advantageous in that, in addition to their specificity, they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring the production of antibodies by a particular method. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or by using recombinant DNA methods after single cell sorting of antigen-specific B cells, i.e., antigen-specific plasmablasts, in bacteria, eukaryotic animals or plant cells in response to infection or immunization (see, e.g., U.S. Patent No. 4,816,567), or by using capture of linked heavy and light chains from single cells in a collection bulk selected antigen-specifically. Also, "monoclonal antibodies" may be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991). B. General Methods

[0049] It is understood that monoclonal antibodies that bind to α3β1 integrin have several uses. These include the detection and diagnosis of cancer, and the manufacture of diagnostic kits for use in the treatment of cancer. In these situations, such antibodies can be conjugated to diagnostic or therapeutic agents, used as capture agents or competitors in competitive assays, or used alone without the conjugation of additional agents. The antibodies may be mutated or modified, as further discussed below. Methods for preparing and characterizing antibodies are well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent No. 4,196,265).

[0050] Methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as methods for preparing polyclonal antibodies. The first step in both of these methods is the identification of a suitable host that has been immunized, either through natural infection in the past or vaccination with an approved or experimental vaccine, or through vaccination with an appropriate composition. As is well known in the art, a given composition for immunization may have different immunogenicities. Thus, it is often necessary to enhance the host's immune system so that it can be achieved by binding the immunogen of a peptide or polypeptide to a carrier. Examples of carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), and other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for binding a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine. Also, as is well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by the use of non-specific stimulators of the immune response known as adjuvants. Examples of adjuvants in animals include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant and aluminum hydroxide adjuvant. In humans, examples include combinations of alum, CpG, MFP59 and immunostimulatory molecules (the "adjuvant system", such as AS01 or AS03). Additional experimental forms of inoculation are possible to induce cancer-specific B cells, including nanoparticle vaccines, or genetic encoded antigens delivered as DNA or RNA genes in physical delivery systems (such as lipid nanoparticles or gold microparticle bombardment beads), and genetic encoded antigens delivered by needles, gene guns, or transcutaneous electroporation devices.The antigen gene can also be carried as encoded by a replicating or defective viral vector such as an adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or a virus-like particle.

[0051] The amount of the immunogenic composition used in the production of polyclonal antibodies varies depending on the nature of the immunogen and the animal used for immunization. The immunogen can be administered using various routes (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animals at various time points after immunization. A second booster injection may also be given. The process of boosting and titer measurement is repeated until a suitable titer is achieved. Once the desired level of immunogenicity is obtained, the immunized animals can be bled, the serum isolated, stored, and / or used to produce Mabs using the animals.

[0052] Following immunization, somatic cells that have the potential to produce antibodies, particularly B lymphocytes (B cells), are selected for use in the MAb production protocol. These cells can be obtained from biopsied spleen, lymph nodes, tonsils or adenoids, bone marrow aspirates or biopsies, tissue biopsies from mucosal organs such as the lung or gastrointestinal tract, or circulating blood. Next, the antibody-producing B lymphocytes of the immunized animal or human are fused with immortal myeloma cells, generally cells of the same species as the immunized animal or human or human / mouse chimeric cells. A myeloma cell line suitable for use in the hybridoma production fusion method is preferably one that is non-antibody-producing, has a high fusion efficiency, and cannot grow in a specific selection medium due to an enzyme deficiency, supporting the growth of only the desired fused cells (hybridomas). As is known to those skilled in the art, any one of a number of myeloma cells can be used. HMMA2.5 cells or MFP-2 cells are particularly useful examples of such cells.

[0053] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells generally involve mixing somatic cells and myeloma cells at a ratio of 2:1, although this ratio can vary from about 20:1 to about 1:1, respectively, in the presence of one or more agents (chemical or electrical) that promote cell membrane fusion. In some cases, as a first step, human B cells are transformed with Epstein-Barr virus (EBV), which increases the size of the B cells and promotes fusion with relatively large-sized myeloma cells. The transformation efficiency by EBV is improved by using CpG and Chk2 inhibitors in the transformation medium. Alternatively, human B cells can be activated by co-culturing them with a transfected cell line expressing CD40 ligand (CD154) in a medium containing additional soluble factors such as IL-21 and B cell-activating factor of the TNF superfamily (BAFF), which are type II members of the TNF superfamily. A fusion method using Sendai virus has been described, and a fusion method using polyethylene glycol (PEG), such as 37% (v / v) PEG, has been described. The use of an electrically induced fusion method is also appropriate, and there are processes for better efficiency. The fusion procedure usually involves about 1×10 -6 to 1×10 -8generate viable hybrids at a low frequency, but using an optimized procedure, a fusion efficiency approaching 1 / 200 can be achieved. However, since viable fusion hybrids differentiate from the parental injected cells (especially the injected myeloma cells that usually divide indefinitely) by culturing in a selective medium, the relatively low fusion efficiency is not a problem. The selective medium generally contains in the tissue culture medium an agent that blocks de novo nucleotide synthesis. Examples of the agent are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as nucleotide sources (HAT medium). When azaserine is used, the medium is supplemented with hypoxanthine. When the B cell source is an EBV-transformed human B cell line, ouabain is added to remove the EBV-transformed strain that has not fused to the myeloma.

[0054] Examples of the selective medium are HAT or HAT containing ouabain. Only cells that can manipulate the nucleotide salvage pathway can survive in HAT medium. Myeloma cells are defective in an important enzyme of the salvage pathway, such as hypoxanthine phosphoribosyl transferase (HPRT), and cannot survive. B cells can manipulate this pathway, but their culture lifespan is limited and they usually die within about two weeks. Therefore, the only cells that can survive in the selective medium are hybrids formed from myeloma cells and B cells. When the source of B cells used for fusion is a strain of B cells transformed with EBV as in this case, since the B cells transformed with EBV are susceptible to killing by the drug, ouabain can also be used for drug selection of the hybrids, but the myeloma partner used is selected to be ouabain-resistant.

[0055] A population of hybridomas is obtained by culturing, from which specific hybridomas are selected. Usually, the selection of hybridomas is carried out by culturing cells by single clone dilution in microtiter plates and then testing the individual clone supernatants (after about 2 - 3 weeks) for the desired reactivity. The assay should be highly sensitive, simple and rapid, and examples include radioimmunoassay, enzyme immunoassay, cytotoxicity test, plaque assay, dot immunobinding assay, etc. Next, the selected hybridomas are made into single cells by serial dilution or fluorescence-activated cell sorting and cloned into individual antibody-producing cell lines. Then, the clones can be propagated indefinitely to obtain mAbs. These cell lines can be used to produce MAb by two basic methods. A sample of hybridomas can be injected into an animal (e.g., a mouse), often intraperitoneally. If necessary, the animal is stimulated with an oil such as a hydrocarbon, especially pristane (tetramethylpentadecane), before injection. When using human hybridomas in this method, it is optimal to inject them into immunodeficient mice such as SCID mice to prevent tumor rejection reactions. The injected animal develops a tumor that secretes a specific monoclonal antibody produced by the fused cell hybrid. Next, high concentrations of MAb can be obtained by removing the body fluids of the animal such as serum and ascites. Individual cell lines can also be cultured in vitro, and MAb is naturally secreted into the culture medium, from which MAb can be easily obtained at high concentrations. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. The cell lines can be adapted to grow in serum-free medium to optimize the ability to recover high-purity human monoclonal immunoglobulins.

[0056] MAb produced by any means may be further purified, if necessary, using various chromatography methods such as filtration, centrifugation, and FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibodies by methods including digestion with enzymes such as pepsin or papain and / or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0057] It is also contemplated to use a molecular cloning approach to produce monoclonal antibodies. A single B cell labeled with the antigen of interest can be physically sorted using paramagnetic bead selection or fluorescence-activated cell sorting, and then RNA can be isolated from the single cell and the antibody gene amplified by RT-PCR. Alternatively, an antigen-specific, bulk-sorted cell population can be separated into microvesicles, and paired heavy and light chain variable genes can be recovered from single cells using physical linkage of heavy and light chain amplicons or common barcoding of the heavy and light chain genes of the vesicles. Paired heavy and light chain genes obtained from single cells can also be obtained from an antigen-specific B cell population by treating the cells with cell-permeable nanoparticles having RT-PCR primers and a barcode for marking the transcript with one barcode per cell. Also, antibody variable genes can be separated by RNA extraction from hybridoma strains, the antibody genes obtained by RT-PCR, and cloned into immunoglobulin expression vectors. Alternatively, a combinatorial immunoglobulin phagemid library is prepared from RNA isolated from a cell line and phagemids expressing appropriate antibodies are selected by panning with a viral antigen. Compared to the conventional hybridoma method, the advantage of this approach is that about 10 4It is possible to produce and screen multiple times more antibodies, and new specificities are generated by the combination of the H chain and the L chain, further increasing the probability of finding the appropriate antibody.

[0058] Other U.S. patents that teach the production of antibodies useful in the present disclosure and are hereby incorporated by reference herein include U.S. Patent No. 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent No. 4,816,567, which describes recombinant immunoglobulins; and U.S. Patent No. 4,867,973, which describes antibody-therapeutic agent conjugates. C. Antibodies of the Present Disclosure

[0059] Antibodies according to the present disclosure can first be defined by their binding specificities. One of ordinary skill in the art can determine whether such an antibody is within the scope of the claims by evaluating the binding specificity / affinity of a given antibody using techniques well known to those of ordinary skill in the art. For example, the epitope to which a given antibody binds may consist of a single continuous 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) amino acids located within the antigen molecule (e.g., a linear epitope within a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the antigen molecule (e.g., a conformational epitope).

[0060] Using a variety of techniques known to those of ordinary skill in the art, it can be determined whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include routine cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Cross-blocking can be measured in a variety of binding assays such as ELISA, biolayer interferometry, surface plasmon resonance. Other methods include alanine scanning mutagenesis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, single particle reconstruction, cryo-EM, or high resolution electron microscopy techniques using tomography, crystallographic studies, and NMR analysis. Further, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids within a polypeptide that interact with an antibody is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves labeling the protein of interest with deuterium and then binding an antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and exchangeable protons within amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface may retain deuterium and thus may exhibit a relatively high mass compared to amino acids not included in the interface. After the antibody dissociates, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing deuterium-labeled residues corresponding to specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0061] The term "epitope" refers to the site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from both adjacent and non - adjacent amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from adjacent amino acids are generally retained even when exposed to denaturing solvents, while epitopes formed by tertiary folding are generally lost when treated with denaturing solvents. Epitopes generally contain at least 3, more commonly at least 5 or 8 - 10 amino acids in a characteristic spatial conformation. Here, in some embodiments, the epitope is a linear or conformational epitope present in α3β1 integrin.

[0062] Modification - Assisted Profiling (MAP), also known as Antigen - Structure - based Antibody Profiling (ASAP), is a method of classifying a large number of monoclonal antibodies (mAbs) made against the same antigen according to the similarity of the binding profiles of each antibody to a chemically or enzymatically modified antigen surface (see US2004 / 0101920, the entire text of which is specifically incorporated herein by reference). Each category may reflect unique epitopes that are clearly different from or partially overlap with epitopes represented by another category. This technique enables the rapid filtering of genetically identical antibodies and allows for the characterization of genetically different antibodies. When applied to hybridoma screening, MAP may facilitate the identification of rare hybridoma clones that produce mAbs with desired properties. Using MAP, the antibodies of the present disclosure can be classified into groups of antibodies that bind to different epitopes.

[0063] The present disclosure includes antibodies that can bind to the same epitope or a portion of an epitope. Similarly, the present disclosure includes antibodies that compete with any of the specific exemplary antibodies described herein for binding to a target or a fragment thereof. Whether an antibody binds to the same epitope as a reference antibody or competes with the reference antibody for binding can be readily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is bound to the target under saturating conditions. Next, the ability of the test antibody to bind to the target molecule is evaluated. If the test antibody can bind to the target molecule following saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the target molecule after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody.

[0064] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, preferably 75%, 90%, or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if substantially all amino acid mutations of the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

[0065] Next, additional routine experiments (e.g., peptide mutagenesis and binding assays) can be performed to confirm whether the lack of binding of the test antibody was actually due to binding to the same epitope as the reference antibody or whether steric hindrance (or another phenomenon) was the cause of the lack of binding observed. This type of experiment can be carried out using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art. Structural studies using EM or crystallography can also show whether two antibodies that compete for binding recognize the same epitope.

[0066] In another aspect, antibodies may be defined by their variable sequences that include additional "framework" regions. Further, the antibody sequences may differ from these sequences and, if necessary, use the methods discussed in more detail below. For example, the nucleic acid sequences may (a) have variable regions that may be separated from the constant domains of the light and heavy chains, (b) the nucleic acids may differ from the nucleic acids shown above without affecting the residues encoded thereby, (c) the nucleic acids may differ from the nucleic acids shown above with a given percentage of homology, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, (d) the nucleic acids may differ from the nucleic acids shown above by their ability to hybridize under high stringency conditions exemplified by low salt and / or high temperature conditions such as provided by about 0.02 M to about 0.15 M NaCl at a temperature of about 50°C to about 70°C, (e) the amino acids may differ from the nucleic acids shown above with a given percentage of homology, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or (f) the amino acids may differ from the nucleic acids shown above in that they allow for conservative substitutions (described below).

[0067] When comparing polynucleotide sequences with polypeptide sequences, as described hereinafter, two sequences are said to be "identical" if the nucleotide or amino acid sequences of the two sequences are the same when aligned to maximize matches. The comparison between two sequences is generally performed by comparing the sequences in a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20, usually from 30 to about 75, 40 to about 50 adjacent positions that can be compared to a reference sequence of the same number of adjacent positions after the two sequences are optimally aligned.

[0068] The optimal array alignment for comparison can be performed using the Megalign program of the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wisconsin) with default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins--Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogeny pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D.G. and Sharp, P.M. (1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E.D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W.J. and Lipman, D.J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0069] Alternatively, the optimal alignment for comparison may be carried out by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA of the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

[0070] One specific example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine the percent sequence identity of the polynucleotides and polypeptides of the present disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Due to the rearranged nature of antibody sequences and the variable length of each gene, multiple rounds of BLAST searches are required for one antibody sequence. Also, manually assembling different genes is difficult and error-prone. The sequence analysis tool IgBLAST (world wide web at ncbi.nlm.nih.gov / igblast / ) identifies matches to germline V, D, and J genes and shows the depiction of Ig V domain framework regions and complementarity-determining regions of the rearranged junctions. IgBLAST can analyze nucleotide or protein sequences, process sequences in batches, and search germline gene databases and other sequence databases simultaneously, thus minimizing the likelihood of missing the most likely germline V gene.

[0071] In one exemplary example, for nucleotide sequences, the cumulative score can be calculated using parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). The extension of the search term hit in each direction is stopped when: the cumulative alignment score has decreased by an amount X from its maximum achieved value; the cumulative score has become zero or less due to the accumulation of residue alignments with a negative score of 1 or more; or when the end of either sequence has been reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915). The alignment uses (B) 50, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands.

[0072] For amino acid sequences, the cumulative score can be calculated using a scoring matrix. The extension of the search term hit in each direction is stopped when: the cumulative alignment score has decreased by an amount X from its maximum achieved value; the cumulative score has become zero or less due to the accumulation of residue alignments with a negative score of 1 or more; or when the end of either sequence has been reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment.

[0073] In one approach, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window is compared to a reference sequence (excluding additions or deletions) for optimal alignment of the two sequences and can include 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent additions or deletions (i.e., gaps). This percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue is present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0074] Yet another way to define an antibody is to define it as a "derivative" of either of the antibodies and their antigen-binding fragments described below. A "derivative" is an antibody or its antigen-binding fragment that binds immunospecifically to an antigen and that contains one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications as compared to the "parent" (or wild-type) molecule. Such amino acid substitutions or additions may involve the introduction of naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" encompasses, for example, variants having modified CH1, hinge, CH2, CH3 or CH4 regions that form, for example, antibodies having variant Fc regions that exhibit enhanced or diminished effector or binding properties. The term "derivative" further encompasses amino acids that have been subjected to non-amino acid modifications, such as glycosylation (e.g., changes in the content of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolylneuraminic acid, etc.), acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to a cell ligand or other protein, etc. In some embodiments, the modified carbohydrate modifications modulate one or more of antibody solubilization, promotion of intracellular transport and secretion of the antibody, promotion of antibody aggregation, conformational integrity, and antibody-mediated effector functions. In certain embodiments, the modified carbohydrate modifications modulate antibody-mediated effector functions relative to an antibody lacking hydrocarbon modifications.Carbohydrate modifications that lead to changes in effector functions mediated by the altered antibody are well known in the art (e.g., Shields, R.L. et al., (2002) ''Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity,'', J.Biol.Chem. 277(30):26733-26740; Davies J. et al., (2001) ''Expression Of GnTIII In A Recombinant Anti-CD20 CHO Production Cell Line:Expression Of Antibodies With Altered。 Glycoforms Leads To An Increase In ADCC Through Higher Affinity For FC Gamma RIII,'', Biotechnology&Bioengineering 74(4):288-294). Methods for varying the carbohydrate content are known to those skilled in the art, e.g., Wallick, S.C. et al., (1988) ''Glycosylation Of A VH Residue Of A Monoclonal Antibody Against Alpha(1----6)Dextran Increases Its Affinity For Antigen,'', J.Exp.Med. 168(3):1099-1109; Tao, M.H. et al., (1989) ''Studies Of "Aglycosylated Chimeric Mouse-Human IgG. Role Of Carbohydrate In The Structure And Effector Functions Mediated By The Human IgG Constant Region,’’ J. Immunol. 143(8):2595-2601; Routledge, E.G. et al., (1995) ‘‘The Effect Of Aglycosylation On The Immunogenicity Of A Humanized Therapeutic CD3 Monoclonal Antibody,’’ Transplantation 60(8):847-53; Elliott, S. et al., (2003) ‘‘Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering,’’ Nature Biotechnol. 21:414-21; Shields, R.L. et al., (2002) ‘‘Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity,’’ J. Biol. Chem. 277(30):26733-26740). See also

[0075] Derivative antibodies or antibody fragments can be made using engineered sequences or glycosylation states to confer a favorable level of activity on antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) functions, as measured in bead-based or cell-based assays or in in vivo studies in animal models.

[0076] The derivative antibody or antibody fragment may be modified by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of geldanamycin, etc. In one embodiment, the antibody derivative will have the same or identical function as the parent antibody. In another embodiment, the antibody derivative will exhibit altered activity compared to the parent antibody. For example, the derivative antibody (or fragment thereof) may bind more strongly to its epitope than the parent antibody or may be more resistant to proteolysis. D. Manipulation of Antibody Sequences

[0077] In various embodiments, one may choose to manipulate the sequence of the identified antibody for a variety of reasons, such as improved expression, improved cross-reactivity, or reduced off-target binding. The modified antibody may be made by any technique known to those skilled in the art, including expression by standard molecular biology techniques or chemical synthesis of the polypeptide. Methods for recombinant expression are addressed elsewhere in this document. The following is a general description of target techniques related to antibody engineering.

[0078] Hybridomas can be cultured and then the cells lysed and total RNA extracted. Random hexamers can be used in RT to generate cDNA copies of the RNA and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. The PCR products can be cloned into a pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Binding and neutralization assays can be performed using antibodies collected from the hybridoma supernatant and purified by FPLC using a Protein G column.

[0079] The recombinant full-length IgG antibody can be prepared by subcloning the heavy and light chain Fv DNAs from a cloning vector into an IgG plasmid vector, transfecting 293 (e.g., Freestyle) cells or CHO cells, and collecting and purifying the antibody from the 293 or CHO cell supernatant. Other suitable host cell lines include bacteria such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco with or without engineering for human-like glycans), algae, or those in various non-human transgenic contexts such as mice, rats, goats, or cows.

[0080] For both the purposes of subsequent antibody purification and host treatment, it is also contemplated to express the nucleic acid encoding the antibody. The antibody-encoding sequence can be RNA such as native RNA or modified RNA. Modified RNA contemplates specific chemical modifications that bring about improved stability and reduced immunogenicity to mRNA, thereby promoting the expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1mΨ) is superior to several other nucleoside modifications and their combinations in terms of translational ability. In addition to turning off the immune / eIF2α phosphorylation-dependent translational inhibition, the incorporated N1mΨ nucleotides dramatically alter the kinetics of the translation process by increasing ribosome pausing and density on the mRNA. The increased ribosome loading of the modified mRNA enhances the permissiveness to the initiation of mRNA by supporting either the recycling of ribosomes on the same mRNA or the recruitment of new ribosomes. Such modifications can be used to enhance in vivo antibody expression after RNA inoculation. The RNA, whether natural or modified, can be delivered as naked RNA or in a delivery vehicle such as a lipid nanoparticle.

[0081] Alternatively, DNA encoding an antibody may be used for the same purpose. This DNA is included in an expression cassette containing a promoter active in the designed host cell. The expression cassette is preferably included in a replicable vector such as a conventional plasmid or minivector. The vector includes viral vectors, such as poxviruses, adenoviruses, herpesviruses, adeno-associated viruses, and lentiviruses, being contemplated. Also contemplated are replicons encoding antibody genes, such as alphavirus replicons based on VEE virus or Sindbis virus. Delivery of such vectors can be carried out by a needle through intramuscular, subcutaneous, or intradermal routes or, if in vivo expression is desired, by subcutaneous electroporation.

[0082] The ability to rapidly utilize antibodies produced in the same host cells and cell culture processes as the final cGMP manufacturing process may shorten the duration of the process development program. Lonza has developed a common method using pooled transfectants grown in CDACF medium for the rapid production of small amounts (up to 50 g) of antibody in CHO cells. Although slightly slower than true transient systems, it has advantages such as high product concentration and the ability to use the same host and process as the manufacturing cell line. Example of growth and productivity of a GS-CHO pool expressing a model antibody in a disposable bioreactor: In a disposable bag bioreactor culture (working volume 5 L) operating in fed-batch mode, a recovered antibody concentration of 2 g / L was achieved within 9 weeks from transfection.

[0083] Antibody molecules include, for example, fragments (F(ab’), F(ab’) 2 etc.) generated by proteolytic cleavage of mAbs, or, for example, single-chain immunoglobulins that can be generated via recombinant means. F(ab’) antibody derivatives are monovalent, but F(ab’) 2The antibody derivative is bivalent. In one embodiment, such fragments can form "chimeric" binding molecules with each other or in combination with other antibody fragments or receptor ligands. Importantly, such chimeric molecules may contain substituents that can bind to different epitopes of the same molecule.

[0084] In related embodiments, the antibody is a derivative of the disclosed antibody, e.g., an antibody that contains the same CDR sequences as the disclosed antibody (e.g., a chimeric or CDR-grafted antibody). Alternatively, modifications such as introducing conservative changes into the antibody molecule may be made. When making such changes, the hydropathy index of the amino acids may be considered. The importance of the hydropathy index of amino acids in conferring interactive biological functions to proteins is generally understood in the art. The relative hydropathicity of amino acids contributes to the secondary structure of the resulting protein, which in turn has been recognized to define the interaction of the protein with other molecules, e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0085] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference, states that the maximum local average hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of that protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0 ± 1), glutamic acid (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, non-ionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4); sulfur-containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0086] It is understood that amino acids can be substituted with another amino acid having similar hydrophilicity to produce a biologically or immunologically modified protein. In such changes, substitutions of amino acids with hydrophilicity values within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0087] As outlined above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various properties described above are well known to those skilled in the art and include arginine and lysine, glutamic acid and aspartic acid, serine and threonine, glutamine and asparagine, and valine, leucine, and isoleucine.

[0088] The present disclosure also contemplates isotype modification. By modifying the Fc region to have different isotypes, different functions can be achieved. For example, IgG 1 when changed to can enhance antibody-dependent cell cytotoxicity, when switched to class A can improve tissue distribution, and when switched to class M can improve avidity.

[0089] Alternatively, or in addition thereto, it may be useful to combine amino acid modifications with one or more additional amino acid modifications that alter C1q binding and / or complement-dependent cytotoxicity (CDC) function of the Fc region of the IL-23p19 binding molecule. Particularly notable binding polypeptides may be those that bind to C1q and exhibit complement-dependent cytotoxicity. Polypeptides having existing C1q binding activity and, optionally, further having the ability to mediate CDC may be modified such that one or both of these activities are enhanced. Amino acid modifications that alter C1q and / or modify its complement-dependent cytotoxicity function are described, for example, in International Publication No. WO 0042072, which is incorporated herein by reference.

[0090] For example, by modifying C1q binding and / or FcγR binding, thereby altering CDC activity and / or ADCC activity, an Fc region of an antibody with altered effector function can be designed. "Effector function" is responsible for activation or decrease of biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require combining the Fc region with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays (e.g., Fc binding assay, ADCC assay, CDC assay, etc.).

[0091] For example, a mutant Fc region of an antibody having improved C1q binding and improved FcγRIII binding (e.g., having both improved ADCC activity and improved CDC activity) can be produced. Alternatively, when it is desired to reduce or eliminate effector function, a mutant Fc region with reduced CDC activity and / or ADCC activity can be designed. In other embodiments, only one of these activities may be increased and, if necessary, the other activity may be decreased (e.g., to produce an Fc region variant where ADCC activity is improved but CDC activity is decreased, or vice versa).

[0092] FcRn-binding Fc mutations can also be introduced and engineered to alter the interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc mutants with improved binding to FcRn has been described. High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR (High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn, and design of IgG1 variants with improved binding to the FcγR) (J. Biol. Chem. 276:6591-6604). Many methods are known that can result in an increase in half-life, including amino acid modifications that can be generated by techniques such as alanine scanning mutagenesis, random mutagenesis, and screening to evaluate binding to the neonatal Fc receptor (FcRn) and / or behavior in vivo. A computational strategy followed by mutagenesis may be used to select one of the amino acid mutations to mutate.

[0093] Accordingly, the present disclosure provides variants of antigen-binding proteins with optimized binding to FcRn. In certain embodiments, said variants of the antigen-binding protein comprise at least one amino acid modification in the Fc region of said antigen-binding protein, said modification being at positions 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 selected from the group consisting of the Fc regions of 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447. Here, the numbering of the amino acids in the Fc region is that of the Kabat EU index. In a further aspect of the present disclosure, the modification is M252Y / S254T / T256E.

[0094] Furthermore, various publications describe methods for obtaining bioactive molecules with altered half-lives by introducing FcRn-binding polypeptides into molecules, or by fusing molecules with antibodies that retain FcRn-binding affinity but have significantly reduced affinity for other Fc receptors, or by fusing the FcRn-binding domain of an antibody with a molecule.

[0095] Derivatized antibodies can be used to alter the half-life (e.g., serum half-life) of the parent antibody in mammals, particularly humans. Such changes can result in a half-life exceeding 15 days, preferably exceeding 20 days, exceeding 25 days, exceeding 30 days, exceeding 35 days, exceeding 40 days, exceeding 45 days, exceeding 2 months, exceeding 3 months, exceeding 4 months, or exceeding 5 months. By increasing the half-life of the antibodies or fragments thereof of the present disclosure in mammals, preferably humans, the serum titer of said antibodies or antibody fragments in the mammal is increased, and thus the frequency of administration of said antibodies or antibody fragments is decreased and / or the concentration of said antibodies or antibody fragments administered is reduced. Antibodies or fragments thereof with an increased in vivo half-life can be produced by techniques known to those skilled in the art. For example, by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor, antibodies or fragments thereof with an increased in vivo half-life can be produced.

[0096] Beltramello et al. (2010) previously reported a modification by creating a version in which the leucine residues at positions 1.3 and 1.2 of the CH 2 domain (according to the IMGT unique numbering for the C domain) of a neutralizing mAb were substituted with alanine residues. This modification, also known as the "LALA" mutation, abrogates antibody binding to FcγRI, FcγRII, and FcγRIIIa. Mutant and unmodified recombinant mAbs were compared for their ability to neutralize and enhance infection by four dengue virus serotypes. The LALA variant retained the same neutralizing activity as the unmodified mAb but had no enhancing activity. Thus, the LALA mutation with this property is contemplated in the context of the antibodies currently disclosed.

[0097] Changes in glycosylation. Certain embodiments of the present disclosure are isolated monoclonal antibodies or antigen-binding fragments thereof that comprise a substantially homogeneous glycan that does not contain sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy-chain variable region and a light-chain variable region, both of which may be linked to a heavy-chain or light-chain constant region, respectively. The aforementioned substantially homogeneous glycan may be covalently bound to the heavy-chain constant region.

[0098] Another embodiment of the present disclosure comprises an mAb having a novel Fc glycosylation pattern. The isolated monoclonal antibody, or antigen-binding fragment thereof, is present in a substantially homogeneous composition represented by the GNGN or G1 / G2 glycoform. Fc glycosylation plays an important role in the antiviral and anti-cancer properties of therapeutic mAbs. The present disclosure is consistent with recent studies showing an increase in the anti-lentiviral cell-mediated viral inhibition of fucose-free anti-HIV mAbs in vitro. This embodiment of the present disclosure using homogeneous glycans lacking core fucose showed an increase in the protection rate of more than twofold against certain viruses. Removal of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells, but appears to have the opposite effect on the ADCC activity of polymorphonuclear cells (PMNs).

[0099] An isolated monoclonal antibody, or antigen-binding fragment thereof, comprising a substantially homogeneous composition represented by the GNGN or G1 / G2 glycoform shows an increased binding affinity for FcγRI and FcγRIII compared to the same antibody comprising substantially homogeneous GNGN glycoform and containing G0, G1F, G2F, GNF, GNGNF, or GNGNFX glycoforms. In one embodiment of the present disclosure, the antibody dissociates from FcγRI with a Kd of 1×10 -8 M or less and from FcγRIII with a Kd of 1×10 -7 M or less.

[0100] Glycosylation in the Fc region is usually either N-linked or O-linked. N-linked refers to the linkage of the carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the linkage of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. The recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequence are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Thus, if any of these peptide sequences are present in a polypeptide, a glycosylation site may be created.

[0101] The glycosylation pattern can be altered, for example, by removing one or more glycosylation sites present in the polypeptide and / or by adding one or more glycosylation sites not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody can be readily achieved by altering the amino acid sequence to include one or more of the above tripeptide sequences (in the case of N-linked glycosylation sites). Exemplary glycosylation variants have an amino acid substitution at residue Asn297 of the heavy chain. This change can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original polypeptide (in the case of O-linked glycosylation sites). Additionally, changing Asn297 to Ala can remove one of the glycosylation sites.

[0102] In certain embodiments, the antibody is expressed in cells that express β(1,4)-N-acetylglucosaminyltransferase III (GnT III), such that GnT III adds GlcNAc to the IL-23p19 antibody. Methods for making antibodies in such a manner are described in WO 99 / 54342, WO 03 / 011878, US 2003 / 0003097 (A1), and Umana et al., Nature Biotechnology, 17:176-180, February 1999. Cell lines can be modified using genome editing techniques such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to enhance or decrease or remove specific post-translational modifications such as glycosylation. For example, in 293 cells or CHO cells used for the expression of recombinant monoclonal antibodies, CRISPR technology can be used to remove genes encoding glycosylation enzymes.

[0103] Removal of liability of monoclonal antibody protein sequences. Antibody variable gene sequences obtained from human B cells can be engineered to enhance their manufacturability and safety. The liability of potential protein sequences can be identified by searching for sequence motifs related to sites including the following. 1) Unpaired Cys residues, 2) N-linked glycosylation, 3) Deamidation of Asn, 4) Isomerization of ASP, 5) Truncation of SYE, 6) Oxidation of Met, 7) Oxidation of Trp, 8) N-terminal glutamic acid, 9) Integrin binding, 10) CD11c / CD18 binding, or 11) Fragmentation Such motifs can be removed by altering the synthetic gene of the cDNA encoding the recombinant antibody.

[0104] Protein engineering efforts in the field of therapeutic antibody development have clearly shown that specific sequences or residues are associated with differences in solubility (Fernandez-Escamilla et al., Nature Biotech., 22(10), 1302-1306, 2004; Chennamsetty et al., PNAS, 106(29), 11937-11942, 2009; Voynov et al., Biocon.Chem., 21(2), 385-392, 2010). Evidence of mutations that alter solubility in the literature indicates that some hydrophilic residues such as aspartic acid, glutamic acid, and serine contribute significantly more favorably to protein solubility than other hydrophilic residues such as asparagine, glutamine, threonine, lysine, and arginine.

[0105] Stable antibodies can be designed to enhance biophysical properties. Antibodies can be unfolded at high temperature and the relative stability can be determined using the average apparent melting temperature. Differential scanning calorimetry (DSC) measures the heat capacity C p (the heat required to raise the temperature of the molecule by one degree) as a function of temperature. DSC can be used to examine the thermal stability of antibodies. DSC data for mAbs can sometimes resolve the unfolding of individual domains within the mAb structure and generate up to three peaks (from the unfolding of the Fab, C H 2, and C H 3 domains) in the thermogram, which is particularly interesting. Generally, the unfolding of the Fab domain generates the strongest peak. The DSC profile and the relative stability of the Fc portion are for human IgG 1 , IgG 2 , IgG 3 , and IgG 4Characteristic differences of subclasses are shown (Garber and Demarest, Biochem. Biophys. Res. Commun. 355, 751 - 757, 2007). The apparent melting temperature on average can also be determined using circular dichroism (CD) performed on a CD spectrometer. The far - UV CD spectrum will be measured for antibodies in the range of 200 - 260 nm at 0.5 nm intervals. The final spectrum can be determined as the average of 20 accumulations. The value of the residue ellipticity can be calculated after subtracting the background. Thermal unfolding of the antibody (0.1 mg / mL) can be monitored at 235 nm from 25 to 95 °C and at a heating rate of 1 °C / min. Dynamic light scattering (DLS) can be used to evaluate the tendency of aggregation. DLS is used to characterize the sizes of various particles including proteins. When the size of the system is not dispersed, the average effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of the surface structure, and the particle concentration. Since DLS basically measures the fluctuations in the scattered light intensity by particles, the diffusion coefficient of the particles can be determined. The DLS software installed in commercially available DLA devices displays populations of particles of different diameters. The stability test can be easily performed using DLS. In the DLS measurement of the sample, by judging whether the hydrodynamic radius of the particles increases, it can be shown whether the particles aggregate over time or with temperature changes. When the particles aggregate, a larger population of particles with a larger radius can be seen. Temperature - dependent stability can be analyzed by controlling the temperature in insights. The capillary electrophoresis (CE) technique includes a proven methodology for determining the characteristics of antibody stability. Using the iCE approach, antibody protein charge variants can be resolved by deamidation, C - terminal lysine, sialylation, oxidation, glycosylation, and other changes to the protein that may lead to changes in the pI of the protein.Each expressed antibody protein can be evaluated by high-throughput free solution isoelectric focusing (IEF) in a capillary column (cIEF) using a Protein Simple Maurice instrument. To monitor molecules migrating to their isoelectric point (pI) in real time, UV absorption detection across the entire column can be performed every 30 seconds. This approach combines the high resolution of conventional gel IEF with the quantification and automation advantages seen in column-based separations, eliminating the need for manual steps. This technique enables the reproducible quantitative analysis of the identity, purity, and heterogeneity profiles of the expressed antibodies. These results identify the charge heterogeneity and molecular sizing of the antibodies with a detection sensitivity down to 0.7 μg / mL in both absorbance and native fluorescence detection modes.

[0106] The intrinsic solubility score of a solubility antibody sequence can be determined. The intrinsic solubility score can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The amino acid sequence of residues 95-102 (Kabat numbering) of the HCDR3 of each antibody fragment such as scFv can be evaluated via an online program to calculate the solubility score. Solubility can also be determined using laboratory techniques. There are various techniques, including adding lyophilized protein to a solution until the solution is saturated and reaches the solubility limit, or concentrating by ultrafiltration with a suitable molecular weight cut-off in a microconcentrator. The simplest method is the induction of amorphous precipitation, using a method involving protein precipitation with ammonium sulfate to measure the solubility of the protein (Trevino et al., J Mol Biol, 366:449-460, 2007). Ammonium sulfate precipitation provides rapid and accurate information on the specific solubility value. Ammonium sulfate precipitation produces a precipitation solution with a distinct aqueous and solid phase and requires a relatively small amount of protein. Solubility measurements using the induction of amorphous precipitation by ammonium sulfate can also be easily performed at different pH values. Protein solubility is highly dependent on pH, which is considered the most important extrinsic factor affecting solubility.

[0107] Generally speaking of autoreactivity, autoreactive clones are thought to be eliminated during ontogeny by negative selection. However, it has become clear that many naturally occurring human antibodies with autoreactivity persist in the mature repertoire of adults. It has been pointed out that the HCDR3 loops of antibodies during early B cell development are often rich in positive charges and show autoreactive patterns (Wardemann et al., Science 301, 1374 - 1377, 2003). The autoreactivity of a given antibody can be tested by evaluating the level of binding to human-derived cells by microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometry cell surface staining (using Jurkat T cells and 293S human embryonic kidney cell suspensions). Autoreactivity can also be investigated using the evaluation of binding to tissues within tissue arrays.

[0108] Preferred residues ("human-likeness"). Deep sequencing of the B cell repertoire of human B cells from blood donors has recently been carried out on a large scale in many studies. Sequence information on most of the human antibody repertoire facilitates the statistical evaluation of the characteristics of antibody sequences common to healthy humans. By knowing the characteristics of the antibody sequences in the human recombinant antibody variable gene reference database, the position-specific "human-likeness" (HL) degree of antibody sequences can be estimated. HL has been shown to be useful for the development of antibodies used clinically, such as therapeutic antibodies or antibodies as vaccines. The goal is to increase the human-likeness of antibodies and reduce the harmful effects and anti-antibody immune responses that can lead to a significant decrease in the efficacy of antibody drugs or induce serious health effects. It is possible to evaluate the antibody characteristics of the combined antibody repertoire of approximately 400 million sequences from three healthy human blood donors and create a new "relative human-likeness" (rHL) score focusing on the hypervariable regions of antibodies. The rHL score makes it possible to easily distinguish human sequences (positive scores) from non-human sequences (negative scores). Antibodies can be designed to remove residues that are not common in the human repertoire. E. Single-chain antibody

[0109] A single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked by a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin despite removal of the constant regions and introduction of a linker peptide. This modification usually does not alter specificity. These molecules were historically created to facilitate phage display, which is very convenient for expressing antigen-binding domains as a single peptide. Alternatively, scFvs can also be made directly from subcloned heavy and light chains derived from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region found in complete antibody molecules and thus lack the common binding sites (e.g., protein A / G, etc.) used to purify antibodies. Since protein L interacts with the variable region of the κ light chain, these fragments can often be purified / immobilized using protein L.

[0110] Flexible linkers generally consist of amino acid residues that promote helices and turns, such as alanine, serine, and glycine. However, other residues may also function. Tang et al. (1996) used phage display as a means to rapidly select linkers made for single-chain antibodies (scFvs) from a protein linker library. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by segments encoding 18-amino acid polypeptides of variable composition. The scFv repertoire (about 5×10 6 different members) was displayed on filamentous phage and affinity selection with a hapten was performed. The selected population of mutants showed a significant increase in binding activity but retained considerable sequence diversity. Subsequently, when 1054 individual mutants were screened, a catalytically active scFv that was efficiently produced in soluble form was obtained. Sequence analysis revealed that a conserved proline in the linker two residues after the V H C terminus, and arginine and proline abundant at other positions, were the only common features of the selected tethers.

[0111] The recombinant antibodies of the present disclosure may also include sequences or moieties that allow for receptor dimerization or multimerization. Such sequences include those derived from IgA, which allows for multimer formation together with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin / avidin that allow for the combination of two antibodies.

[0112] In another embodiment, single-chain antibodies can be made by binding the receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains are produced and purified in separate cells and then joined together in an appropriate manner (i.e., the N-terminus of the heavy chain is bound to the C-terminus of the light chain via an appropriate chemical crosslink).

[0113] Crosslinking reagents are used to form a molecular crosslink that connects the functional groups of two different molecules, such as stabilizers and coagulants. However, it is contemplated that dimers or other multimers of the same analog, or heteromeric complexes consisting of different analogs, can be made. To sequentially bind two different compounds, a heterobifunctional crosslinking agent can be used to eliminate the formation of unwanted homopolymers.

[0114] Exemplary heterobifunctional crosslinking agents contain two reactive groups, one of which reacts with a primary amine group (e.g., N-hydroxysuccinimide) and the other of which reacts with a thiol group (e.g., pyridyldisulfide, maleimide, halogen, etc.). Through the primary amine-reactive group, the crosslinking agent may react with a lysine residue of one protein (e.g., a selected antibody or fragment), and through the thiol-reactive group, the crosslinking agent already bound to the first protein reacts with a cysteine residue (free sulfhydryl group) of the other protein (e.g., a selection agent).

[0115] It is preferred that a cross-linking agent having appropriate stability in the blood be used. Many types of disulfide bond-containing linkers are known that can be successfully used to bind targeting agents and therapeutic / preventive agents. Linkers containing sterically hindered disulfide bonds can provide higher stability in vivo and may be able to prevent the release of the target peptide before reaching the site of action. Thus, these linkers are one group of linking agents.

[0116] Another cross-linking reagent is SMPT. This is a bifunctional cross-linking agent containing a disulfide bond with "steric hindrance" due to adjacent benzene rings and methyl groups. The steric hindrance of the disulfide bond serves to protect the bond from attack by thiolate anions such as glutathione that may be present in tissues and blood, thereby helping to prevent the dissociation of the complex before delivering the attached drug to the target site.

[0117] The SMPT cross-linking reagent, like many other known cross-linking reagents, helps in the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the ε-amino group of lysine). Another possible type of cross-linking agent includes heterobifunctional photoreactive phenyl azides containing cleavable disulfide bonds such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxysuccinimidyl group reacts with primary amino groups, and the phenyl azide (upon photolysis) reacts non-selectively with any amino acid residue.

[0118] In addition to hindered cross-linking agents, unhindered linkers can also be employed according to the present specification. Other useful cross-linking agents that do not involve or generate protected disulfides include SATA, SPDP, and 2-iminothiolane. The use of such cross-linking agents is well understood in the art. Another embodiment involves the use of flexible linkers.

[0119] U.S. Patent No. 4,680,338 describes a bifunctional linker useful for generating complexes of ligands with amine-containing polymers and / or proteins, particularly useful for forming antibody complexes with chelating agents, drugs, enzymes, detectable labels, etc. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable complexes containing labile linkages that are cleavable under various mild conditions. This linker is particularly useful in that the agent of interest may be directly attached to the linker and the active agent is released upon cleavage. Certain applications involve adding free amino or free sulfhydryl groups to proteins such as antibodies or drugs.

[0120] U.S. Patent No. 5,856,456 provides a peptide linker for use in connecting polypeptide components to make fusion proteins, such as single-chain antibodies. The linker is up to about 50 amino acids in length and includes at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by an occurrence of proline, and is characterized by higher stability and less aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in various immuno-diagnostic and separation techniques. F. Multispecific Antibodies

[0121] In certain embodiments, the antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen-binding site with a binding site for a second antigen. Alternatively, the antigen-specific arm may bind an arm to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD3), or an Fc receptor of IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγIII (CD16), thereby concentrating and localizing the cellular defense mechanism to infected cells. Bispecific antibodies can be used to localize cytotoxic agents to infected cells. These antibodies have an antigen-binding arm and an arm that binds to a cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate, or radioisotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab’).sub.2 bispecific antibodies). WO 96 / 16673 describes a bispecific anti-ErbB2 / anti-FcγRIII antibody, and U.S. Patent No. 5,837,234 discloses a bispecific anti-ErbB2 / anti-FcγRI antibody. A bispecific anti-ErbB2 / Fcα antibody is shown in WO 98 / 02463. U.S. Patent No. 5,821,337 teaches a bispecific anti-ErbB2 / anti-CD3 antibody.

[0122] Methods for making bispecific antibodies are known in the art. The production of conventional full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Since the heavy and light chains of immunoglobulins are randomly sorted, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually done in an affinity chromatography step, but it is quite cumbersome and the yield of the product is low. Similar procedures are disclosed in WO 93 / 08829, and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0123] According to another approach, antibody variable regions (antibody-antigen binding sites) having the desired binding specificities are fused to immunoglobulin constant domain sequences. Preferably, the fusion is to an Ig heavy chain constant domain comprising at least a portion of the hinge, C H2 , and C H3 regions. A first heavy chain constant region (C H1 ) containing the site necessary for light chain binding is preferably present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, optionally, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host cell. This provides greater flexibility in adjusting the mutual ratios of the three polypeptide fragments in embodiments where the unequal ratios of the three polypeptide chains used in the construction provide an optimal yield of the desired bispecific antibody. However, if expressing at least two polypeptide chains in equal ratios results in a high yield, or if the ratio does not significantly affect the yield of the desired chain combination, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0124] In certain embodiments of this approach, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain having a first binding specificity for one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) for the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of immunoglobulin light chains only in half of the bispecific molecule provides an easy separation method. This approach is disclosed in WO 94 / 04690. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0125] According to another approach described in U.S. Patent No. 5,731,168, the interface between antibody molecule pairs can be designed to maximize the proportion of heterodimers recovered from recombinant cell culture. Preferred interfaces include at least a portion of the C H3 domain. In this method, one or more small amino acid side chains from the interface of the primary antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with small amino acid side chains (e.g., alanine or threonine), compensatory "holes" of the same or similar size to one or more of the large side chains are created at the interface of the secondary antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

[0126] Bispecific antibodies include cross-linked antibodies or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can bind to avidin and the other can bind to biotin. Such antibodies have been proposed, for example, for the targeting of immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (International Publication No. 91 / 00360, International Publication No. 92 / 200373, and European Patent No. 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 along with many cross-linking techniques.

[0127] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229:81 (1985) describe a procedure for proteolytically cleaving intact antibodies to produce F(ab’) 2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab’ fragments are then converted to thionitrobenzoic acid (TNB) derivatives. One of the Fab’-TNB derivatives is then reconverted to Fab’-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab’-TNB derivative to form a bispecific antibody. The bispecific antibody produced can be used as an agent for selectively immobilizing enzymes.

[0128] There are techniques that facilitate the direct recovery of Fab’-SH fragments from Escherichia coli and their chemical linkage to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe a humanized bispecific antibody F(ab’) 2It describes the production of molecules. Each Fab' fragment was secreted separately from E. coli and underwent direct chemical coupling in vitro to form a bispecific antibody. The bispecific antibody thus formed could bind to cells overexpressing the ErbB2 receptor and normal human T cells, and could induce the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0129] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell cultures are also described (Merchant et al., Nat. Biotechnol. 16, 677-681 (1998)). For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547-1553, 1992). The leucine zipper peptides of Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced in the hinge region to form monomers, which were then re-oxidized to form antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provided an alternative mechanism for producing bispecific antibody fragments. These fragments have V H and V L connected by a linker, but this linker is too short to allow pairing between two domains on the same chain. Thus, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for producing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0130] In certain embodiments, bispecific or multispecific antibodies may be formed as DOCK-AND-LOCK™ (DNL™) complexes (see, e.g., U.S. Patent Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the respective disclosures of which are incorporated herein by reference). Generally, this technique exploits the specific and high-affinity binding interaction that occurs between the dimerization and docking domain (DDD) sequence of the regulatory (R) subunit of cAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie et al., FEBS Letters. 2005; 579:3264; Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The DDD and AD peptides can be conjugated to any protein, peptide, or other molecule. Since the DDD sequence spontaneously dimerizes and binds to the AD sequence, this technique enables the formation of complexes between selected molecules that can bind to the DDD or AD sequence.

[0131] Antibodies having a valence greater than 2 are contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147:60, 1991; Xu et al., Science, 358(6359):85-90, 2017). Multivalent antibodies can be internalized (and / or catabolized) more rapidly than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibodies of the present disclosure can be multivalent antibodies having 3 or more antigen-binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of a nucleic acid encoding the polypeptide chains of the antibody. Multivalent antibodies can comprise a dimerization domain and 3 or more antigen-binding sites. Preferred dimerization domains comprise (or consist of) the Fc region or the hinge region. In this scenario, the antibody comprises the Fc region and 3 or more antigen-binding sites at the amino terminus of the Fc region. Preferred multivalent antibodies herein comprise (or consist of) from 3 to about 8, preferably 4 antigen-binding sites. Multivalent antibodies comprise at least one polypeptide chain (preferably 2 polypeptide chains), and one or more polypeptide chains comprise 2 or more variable regions. For example, one or more polypeptide chains can comprise VD1-(X1).sub.n-VD2-(X2) n -Fc, where VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of the Fc region, X1 and X2 are amino acids or polypeptides, and n is 0 or 1. For example, one or more polypeptide chains can comprise VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibodies herein preferably further comprise at least 2 (preferably 4) light chain variable region polypeptides. The multivalent antibodies herein can comprise, for example, from about 2 to about 8 light chain variable region polypeptides. Light chain variable region polypeptides contemplated herein comprise a light chain variable region and, optionally, a C L domain further.

[0132] Charge modification is particularly useful in the context of multispecific antibodies, and amino acid substitutions in Fab molecules can reduce the mispairing of light and heavy chains that do not match (Bence Jones-type by-products) that can occur in the production of Fab-based bispecific / multispecific antigen-binding molecules that perform VH / VL exchange in one of their binding arms (one or more in the case of a molecule containing three or more antigen-binding Fab molecules) (see also PCT Publication No. WO2015 / 150447, the entire text of which is incorporated herein by reference, especially the examples therein). G. Chimeric Antigen Receptors

[0133] Chimeric antigen receptor molecules are recombinant fusion proteins, distinguished by their ability to both bind antigen and transmit activation signals via immunoreceptor activation motifs (ITAMs) present at the cytoplasmic terminus. Receptor constructs that utilize antigen-binding moieties (e.g., generated from single-chain antibodies (scFvs)) offer the additional advantage of being "universal" in that they bind to native antigens on target cell surfaces in an HLA-independent manner.

[0134] Chimeric antigen receptors can be made by any means known in the art, but are preferably made using recombinant DNA techniques. Nucleic acid sequences encoding some regions of the chimeric antigen receptor can be prepared by standard techniques of molecular cloning (genomic library screening, PCR, primer-assisted ligation, scFv libraries from yeast and bacteria, site-directed mutagenesis, etc.) and assembled into the complete coding sequence. The resulting coding region can be inserted into an expression vector and used to transform allogeneic or autologous effector cells of a suitable expression host such as T cells and NK cells.

[0135] The embodiments of the CAR described herein include nucleic acids encoding antigen - specific chimeric antigen receptor (CAR) polypeptides that include an intracellular signaling domain, a transmembrane domain, and an extracellular domain that includes one or more signaling motifs. In certain embodiments, the CAR can recognize an epitope consisting of a shared space between one or more antigens. In some embodiments, the chimeric antigen receptor includes a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain that includes an antigen - binding domain. Optionally, the CAR can include a hinge domain disposed between the transmembrane domain and the antigen - binding domain. In certain aspects, the CAR of the embodiments further includes a signal peptide that directs the expression of the CAR to the cell surface. For example, in some aspects, the CAR can include a signal peptide from GM - CSF.

[0136] In certain embodiments, the CAR can be co - expressed with a membrane - bound cytokine to improve persistence when the amount of tumor - associated antigen is low. For example, the CAR can be co - expressed with membrane - bound IL - 15.

[0137] Depending on the arrangement of the domains of the CAR and the specific sequences used in the domains, immune effector cells expressing the CAR may have different levels of activity against target cells. In some aspects, different CAR sequences can be introduced into immune effector cells to generate engineered cells, and those engineered cells can be selected for an increase in SRC, and the selected cells can be tested for activity to identify a CAR construct predicted to have maximum therapeutic efficacy. 1. Antigen - binding domain

[0138] In certain embodiments, the antigen-binding domain can include the complementarity-determining regions of a monoclonal antibody, the variable regions of a monoclonal antibody, and / or antigen-binding fragments thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor. A "complementarity-determining region (CDR)" is a short amino acid sequence found in the variable domain of an antigen receptor (e.g., an immunoglobulin and a T cell receptor) protein that complements an antigen and thereby confers specificity for that particular antigen on the receptor. Each polypeptide chain of an antigen receptor contains three CDRs (CDR1, CDR2, and CDR3). Since an antigen receptor generally consists of two polypeptide chains, there are six CDRs that can potentially contact an antigen per antigen receptor (three CDRs in each of the heavy and light chains). Because most sequence variations associated with immunoglobulins and T cell receptors are found in the CDRs, these regions are sometimes called hypervariable domains. Among these, CDR3 exhibits the greatest variability because it is encoded by the recombination of the VJ (VDJ in the case of heavy chains and TCRαβ chains) regions.

[0139] CAR nucleic acids, particularly scFv sequences, are contemplated to be human genes for enhancing cellular immunotherapy in human patients. In certain embodiments, full-length CAR cDNA or coding regions are provided. The antigen-binding region or domain can include fragments of the VH and VL chains of a single-chain variable fragment (scFv) derived from a specific mouse, or human or humanized monoclonal antibody. Also, the fragment can be any number of different antigen-binding domains of an antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells. In certain aspects, the VH and VL domains of the CAR are separated by a linker sequence such as a Whitlow linker. CAR constructs that can be modified or used according to embodiments are also provided in International (PCT) Patent Publication No. WO / 2015 / 123642, which is incorporated herein by reference.

[0140] As described above, the prototype CAR encodes a scFv comprising a VH domain and a VL domain derived from one monoclonal antibody (mAb) that is linked to a transmembrane domain and one or more cytoplasmic signaling domains (e.g., a co-stimulatory domain and a signaling domain). Thus, the CAR may include the LCDR1-3 and HCDR1-3 sequences of an antibody that binds to an antigen of interest, such as a tumor-associated antigen. However, in a further aspect, two or more antibodies that bind to an antigen of interest are identified and a CAR is constructed that includes: (1) the HCDR1-3 sequences of a first antibody that binds to the antigen; and (2) the LCDR1-3 sequences of a second antibody that binds to the antigen. Such a CAR that includes HCDR and LCDR sequences from two different antigen-binding antibodies may have the advantage of preferentially binding to a specific conformation of the antigen (e.g., a conformation that is preferentially associated with cancer cells relative to normal tissue).

[0141] Alternatively, it is also shown that CARs can be designed using VH and VL chains derived from different mAbs to generate a panel of CAR+ T cells. The antigen-binding domain of the CAR can include any combination of the LCDR1-3 sequences of a first antibody and the HCDR1-3 sequences of a second antibody. 2. Hinge Domain

[0142] In certain embodiments, the CAR polypeptide of the embodiments can include a hinge domain disposed between the antigen-binding domain and the transmembrane domain. Optionally, the hinge domain may be included in the CAR polypeptide to provide an appropriate distance between the antigen-binding domain and the cell surface or to reduce steric hindrance that may adversely affect the antigen-binding or effector function of the CAR gene-modified T cells. In some embodiments, the hinge domain includes a sequence that binds to an Fc receptor such as FcγR2a or FcγR1a. For example, the hinge sequence may include an Fc domain derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE) that binds to an Fc receptor. In certain embodiments, the hinge domain (and / or CAR) does not include wild-type human IgG4 CH2 and CH3 sequences.

[0143] Optionally, the CAR hinge domain may be derived from a human immunoglobulin (Ig) constant region or a portion thereof that includes the Ig hinge, or from the human CD8α transmembrane domain and the CD8a hinge region. In one embodiment, the CAR hinge domain is the hinge CH 4 of the antibody isotype IgG 2 -CH 3 region. In some embodiments, point mutations can be introduced into the antibody heavy chain CH 2 domain to reduce glycosylation and non-specific Fc gamma receptor binding of the CAR-T cells or any other CAR-modified cells.

[0144] In certain embodiments, the CAR hinge domain comprises an Ig Fc domain that contains at least one mutation compared to the wild-type Ig Fc domain that reduces Fc receptor binding. For example, the CAR hinge domain comprises an IgG4-Fc domain that contains at least one mutation compared to the wild-type IgG4-Fc domain that reduces Fc receptor binding. In some embodiments, the CAR hinge domain comprises an IgG4-Fc domain having a mutation (such as an amino acid deletion or substitution) at a position corresponding to L235 and / or N297 compared to the wild-type IgG4-Fc sequence. For example, the CAR hinge domain can comprise an IgG4-Fc domain having L235E and / or N297Q mutations compared to the wild-type IgG4-Fc sequence. In further embodiments, the CAR hinge domain can comprise an IgG4-Fc domain having an amino acid substitution at position L235 to an amino acid that is hydrophilic (such as R, H, K, D, E, S, T, N, or Q, etc.) or an amino acid having properties similar to "E" (such as D, etc.). In certain embodiments, the CAR hinge domain can comprise an IgG4-Fc domain having an amino acid substitution at position N297 to an amino acid having properties similar to "Q" (such as S or T, etc.).

[0145] In certain embodiments, the hinge domain comprises a sequence that is about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an IgG4 hinge domain, CD8a hinge domain, CD28 hinge domain, or engineered hinge domain. 3. Transmembrane Domain

[0146] The antigen-specific extracellular domain and the intracellular signaling domain can be linked by a transmembrane domain. Polypeptide sequences that can be used as part of the transmembrane domain include, but are not limited to, the human CD4 transmembrane domain, the human CD28 transmembrane domain, the transmembrane human CD3ζ domain, or the cysteine mutant human CD3ζ domain, or other transmembrane domains from other human transmembrane signaling proteins such as CD16 and CD8 and the erythropoietin receptor. In some embodiments, for example, the transmembrane domain comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of those described in U.S. Patent Application Publication No. 2014 / 0274909 (such as the CD8 and / or CD28 transmembrane domains) or U.S. Patent No. 8,906,682 (such as the CD8α transmembrane domain), both of which are incorporated herein by reference. The transmembrane regions particularly used in the present invention may be derived from the α, β or ζ chains of the T cell receptor, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., include at least their transmembrane regions). In certain embodiments, the transmembrane domain can be 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CD8a transmembrane domain or the CD28 transmembrane domain. 4. Intracellular signaling domain

[0147] The intracellular signaling domain of the chimeric antigen receptor of the present embodiment is responsible for activating at least one of the normal effector functions of immune cells engineered to express the chimeric antigen receptor. The term "effector function" refers to the specialized functions of differentiated cells. The effector functions of T cells can be, for example, cytolytic activity or helper activity including cytokine secretion. The effector functions of naive, memory, or memory-like T cells include antigen-dependent proliferation. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector function signals and instructs the cell to perform specialized functions. In some embodiments, the intracellular signaling domain is derived from the intracellular signaling domain of a native receptor. Examples of such native receptors include either the ζ chain of the T cell receptor or its homologs (e.g., η, δ, γ, or ε), the MB1 chain, B29, Fc RIII, Fc RI, and combinations of signaling molecules such as CD3ζ and CD28, CD27, 4-1BB, DAP-10, OX40, and combinations thereof, as well as other similar molecules and fragments. The intracellular signaling portions of other members of the family of activating proteins can be used. Usually, the entire intracellular signaling domain is used, but in many cases, it may not be necessary to use the entire intracellular polypeptide. To the extent that truncated portions of the intracellular signaling domain can be used, such truncated portions may be used in place of the intact chain as long as they still transmit effector function signals. Thus, the term "intracellular signaling domain" means including truncated portions of the intracellular signaling domain sufficient to transmit effector function signals when the CAR binds to the target. In a preferred embodiment, the human CD3ζ intracellular domain is used as the intracellular signaling domain of the CAR of these embodiments.

[0148] In certain embodiments, the intracellular receptor signaling domain of the CAR includes, for example, the signaling domain of the T cell antigen receptor complex such as the ζ chain of CD3, and also the FcγRIII co-stimulatory signaling domain, CD28, CD27, DAP10, CD137, OX40, CD2, either alone or in series with CD3ζ. In certain embodiments, the intracellular domain (sometimes referred to as the cytoplasmic domain) includes one or more of TCRζ chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12, and a portion or all of CD40. In some embodiments, any part of the endogenous T cell receptor complex of the intracellular domain is used. So-called third-generation CARs can use one or more cytoplasmic domains because at least two or three signaling domains are fused together for additive or synergistic effects. For example, CD28 and 4-1BB can be combined in a CAR construct.

[0149] In some embodiments, the CAR includes additional other co-stimulatory domains. Other co-stimulatory domains can include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, and 4-1BB (CD137). In addition to the primary signal initiated by CD3ζ, the additional signals provided by the human co-stimulatory receptors inserted into the human CAR are important for the complete activation of T cells, and can help improve persistence in vivo and the therapeutic success of adoptive immunotherapy.

[0150] In certain aspects, the intracellular signaling domain includes a domain that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a domain comprising the CD3ζ intracellular domain, the CD28 intracellular domain, the CD137 intracellular domain, or the CD28 intracellular domain fused to the 4-1BB intracellular domain. H.ADC

[0151] An antibody-drug conjugate or ADC is a new class of very potent biopharmaceuticals designed as a targeted therapy for treating people with diseases. An ADC is a complex molecule composed of an antibody (a full mAb, or an antibody fragment such as a single-chain variable fragment, or scFv), which is linked via a stable chemical linker with a labile bond to a biologically active cytotoxic / antiviral payload, i.e., a drug. Antibody-drug conjugates are examples of bioconjugates and immunoconjugates.

[0152] By combining the unique targeting ability of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, antibody-drug conjugates can highly sensitively distinguish between healthy tissue and diseased tissue. This means that, in contrast to conventional systemic approaches, antibody-drug conjugates target and attack diseased cells, so healthy cells are not as severely affected.

[0153] In the development of ADC-based anti-tumor therapies, an anti-cancer agent (e.g., a cell toxin or cytotoxin) is conjugated to an antibody that specifically targets a particular cell marker (e.g., ideally, a protein found only in or on infected cells). The antibody tracks these proteins in the body and attaches to the surface of cancer cells. Due to the biochemical reaction between the antibody and the target protein (antigen), tumor cells induce a signal and absorb or internalize the antibody along with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released, killing the cells or impairing cell replication. This targeting ideally results in this drug having fewer side effects and a broader therapeutic window than other agents.

[0154] A stable bond between an antibody and a cytotoxic agent is an important aspect of an ADC. Linkers are based on chemical motifs such as disulfide, hydrazone or peptide (cleavable), or thioether (non-cleavable), and control the distribution and delivery of the cytotoxic agent to target cells. Both cleavable and non-cleavable types of linkers have proven to be safe in preclinical and clinical trials. Brentuximab vedotin contains an enzyme-sensitive and cleavable linker that delivers monomethyl auristatin E, a potent and highly toxic microtubule inhibitor, or MMAE, a synthetic antitumor agent, to human-specific CD30-positive malignant cells. Since MMAE, which inhibits cell division by inhibiting tubulin polymerization, is highly toxic, it cannot be used as a single-agent chemotherapy drug. However, the combination of MMAE conjugated to an anti-CD30 monoclonal antibody (cAC10, a cell membrane protein of tumor necrosis factor or TNF receptor) has been shown to be stable in extracellular fluid, cleavable by cathepsin, and safe for treatment. Trastuzumab emtansine is another approved ADC, a combination of the antibody trastuzumab (Herceptin® / Genentech / Roche) conjugated with mertansine (DM-1), a microtubule formation inhibitor and a derivative of maytansine, via a stable non-cleavable linker.

[0155] The availability of better and more stable linkers has changed the function of the chemical bond. Depending on the type of cleavable or non-cleavable linker, specific properties are imparted to the cytotoxic (anticancer) drug. For example, non-cleavable linkers retain the drug inside the cell. As a result, all the antibody, linker, and cytotoxic agent enter the target cancer cell, where the antibody is degraded down to the amino acid level. The resulting complex (amino acid, linker, and cytotoxic agent) then becomes the active drug here. In contrast, cleavable linkers are catalyzed by enzymes within the host cell to release the cytotoxic agent.

[0156] Another type of cleavable linker currently under development adds an extra molecule between the cytotoxic drug and the cleavage site. This linker technology allows researchers to create more flexible ADCs without worrying about variations in cleavage rates. Researchers are also developing a new method of peptide cleavage based on Edman degradation, a method for sequencing amino acids in peptides. Future directions in ADC development also include site-specific conjugation (TDC) to further improve stability and therapeutic index, and the development of alpha-emitting immunoconjugates and antibody-conjugated nanoparticles. I. BiTE

[0157] Bispecific T-cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies being investigated for use as anti-cancer agents. BiTEs direct the cytotoxic activity of the host immune system, more specifically T cells, against infected cells. BiTE is a registered trademark of Micromet AG.

[0158] A BiTE is a fusion protein consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of approximately 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor and the other binds to infected cells via a specific molecule.

[0159] Like other bispecific antibodies and unlike normal monoclonal antibodies, BiTEs form a link between T cells and target cells. This allows T cells to exert cytotoxic activity against infected cells by producing proteins such as perforin and granzyme, independent of the presence of MHC I or costimulatory molecules. These proteins enter the infected cells and initiate apoptosis. This action mimics the physiological process observed while T cells attack infected cells. J. Intrabodies

[0160] In certain embodiments, the antibody is a recombinant antibody suitable for intracellular action, and such antibodies are known as "intrabodies." These antibodies may inhibit target functions by a variety of mechanisms, such as altering intracellular protein transport, inhibiting enzyme function, or blocking protein-protein or protein-DNA interactions. In many respects, these structures mimic or are equivalent to the structures of the single-chain and single-domain antibodies described above. Indeed, the single transcript / single-chain is an important feature that enables intracellular expression in target cells and makes proteins that cross the cell membrane more feasible. However, additional features are required.

[0161] Two major problems that affect the implementation of intrabody therapy are delivery, including cell / tissue targeting, and stability. With regard to delivery, a variety of approaches have been employed, including tissue-directed delivery, the use of cell-type specific promoters, virus-based delivery, and the use of cell-penetrating / membrane translocation peptides. One means of delivery involves the use of lipid-based nanoparticles or exosomes, as taught in U.S. Patent Application Publication No. 2018 / 0177727, the entire disclosure of which is incorporated herein by reference. With regard to stability, the approaches generally involve screening by brute force, which may involve phage display and methods that may include sequence maturation or the development of consensus sequences, or additional directed modifications such as the insertion of stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide substitution / modification.

[0162] An additional feature that intrabodies may require is a signal for intracellular targeting. Vectors that can target intrabodies (or other proteins) to intracellular regions such as the cytoplasm, nucleus, mitochondria, and ER have been designed and are commercially available (Invitrogen). K. Purification

[0163] The antibodies of the present disclosure may be purified. As used herein, the term "purified" is intended to refer to a composition separable from other components, where the protein is purified to some extent relative to its naturally occurring state. Thus, a purified protein also refers to a protein separated from its naturally occurring environment. When the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, e.g., a composition that comprises about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0164] Protein purification techniques are well known to those of skill in the art. These techniques involve, at some level, a crude fractionation of the polypeptide and non-polypeptide fractions of the cellular environment. After separating the polypeptide from other proteins, the polypeptide of interest can be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, size exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or heat denaturation followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such techniques with other techniques.

[0165] When purifying the antibodies of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column that binds to the tagged portion of the polypeptide. As is generally known in the art, the order in which the various purification steps are performed may be changed or certain steps may be omitted, and still a suitable method for the preparation of a substantially purified protein or peptide is obtained.

[0166] Generally, intact antibodies are fractionated using an agent that binds to the Fc portion of the antibody (i.e., Protein A). Alternatively, an antigen may be used to simultaneously purify and select the appropriate antibody. In such methods, a selector bound to a support such as a column, filter, beads, etc. is often utilized. The antibody is bound to the support, contaminants are removed (e.g., washed), and the antibody is released by applying conditions (such as salt, heat, etc.).

[0167] Various methods for quantifying the degree of purification of a protein or peptide will be known to those skilled in the art in light of the present disclosure. These include, for example, determining the specific activity of the active fraction or evaluating the amount of polypeptide within the fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction and compare it to the specific activity of the initial extract to calculate the degree of purity. The actual units used to represent the amount of activity will, of course, depend on the specific assay technique chosen to track the purification and whether the expressed protein or peptide exhibits a detectable activity.

[0168] It is known that the migration of peptides can vary significantly in some cases depending on the different conditions of SDS / PAGE. Thus, it will be understood that if the electrophoresis conditions are different, the apparent molecular weight of a purified or partially purified expression product can change. L. Antibody complex

[0169] The antibodies of the present disclosure can be linked to at least one agent to form an antibody conjugate. To enhance the effectiveness of antibody molecules as diagnostic or therapeutic agents, conventional methods involve linking, covalently binding, or complexing at least one desired molecule or moiety. Such a molecule or moiety can be, but is not limited to, at least one effector or reporter molecule. Effector molecules include molecules having a desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules conjugated to an antibody include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, anti-viral agents, chelating agents, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to an antibody include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or ligands such as biotin.

[0170] Antibody conjugates are generally preferably used as diagnostic agents. Antibody diagnostic agents are generally classified into two classes, those for use in in vitro diagnostics, such as various immunoassays, and those for use in in vivo diagnostic protocols generally referred to as "antibody-directed imaging." Many suitable contrast agents are known in the art, as are methods for conjugating them to antibodies (see, for example, U.S. Patent Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moiety used can be a paramagnetic ion, a radioisotope, a fluorescent dye, an NMR-detectable substance, and an X-ray contrast agent.

[0171] In the case of paramagnetic ions, examples thereof include ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III), and gadolinium is particularly preferred. Ions useful in other situations such as X-ray imaging include, but are not limited to, lanthanum (III), gold (III), lead (II), and particularly bismuth (III).

[0172] In the case of radioisotopes for therapeutic and / or diagnostic use, astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 cobalt, copper 67 , 152 Eu, gallium 67 , 3 hydrogen, iodine 123 , iodine 125 , iodine 131 , indium 111 , 59 iron, 32 phosphorus, rhenium 186 , rhenium 188 , 75 selenium, 35 sulfur, technetium 99m and / or yttrium 90 can be mentioned. 125 I is often preferred for use in certain embodiments, technetium 99m and / or indium 111Also, it is often preferred due to its compatibility with low energy and long-distance detection. The radiolabeled monoclonal antibodies of the present disclosure may be manufactured according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contacting sodium iodide and / or potassium iodide with a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent such as lactoperoxidase. Monoclonal antibodies according to the present disclosure can be labeled with technetium in a ligand exchange process, for example, by reducing pertechnate with a tin solution, chelating the reduced technetium on a Sephadex column, and applying the antibody to this column. 99m Alternatively, for example, direct labeling techniques can be used by incubating pertechnate, a reducing agent such as SNCl 2 , a buffer such as a sodium-potassium phthalate solution, and the antibody. Intermediate functional groups often used to bind radioisotopes present as metal ions to antibodies are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0173] Fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, rhodamine green, rhodamine red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

[0174] Additional types of antibodies contemplated by the present disclosure are primarily intended for in vitro use, where the antibody is linked to an enzyme (enzyme tag) that contacts a secondary binding ligand and / or a chromogenic substrate to produce a colored product. Examples of suitable enzymes include urease, alkaline phosphatase, horseradish peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin, avidin, streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0175] Yet another method of site-specific binding of a molecule to an antibody involves the reaction of the antibody with a hapten-based affinity label. Basically, a hapten-based affinity label reacts with the amino acids of the antigen-binding site, thereby disrupting this site and blocking a specific antigen reaction. However, this may not be advantageous as it results in the loss of antigen binding by the antibody complex.

[0176] Molecules containing azide groups can also be used to form covalent bonds to proteins through reactive nitrene intermediates generated by low-intensity ultraviolet light. In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-specific photo-probes to identify nucleotide-binding proteins in crude cell extracts. 2- and 8-azido nucleotides have also been used to map the nucleotide-binding domains of purified proteins and can be used as antibody binders.

[0177] Several methods for conjugating or binding an antibody to its conjugate moiety are known in the art. Some conjugation methods involve, for example, the use of metal chelate complexes using organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenediaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 conjugated to an antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Complexes with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, and the detectable imaging moiety is conjugated to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0178] In other embodiments, derivatization of immunoglobulins is contemplated by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody binding site. Antibody conjugates prepared according to this methodology have been disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific binding of effector or reporter molecules, where the reporter or effector molecule is bound to a carbohydrate residue of the Fc region, has also been disclosed in the literature. This approach has been reported to generate diagnostically and therapeutically promising antibodies currently in clinical evaluation. II. Treatment Methods

[0179] Certain aspects of the present embodiment can be used to prevent or treat diseases or disorders associated with the presence of homotrimeric type I collagen, such as pancreatic ductal adenocarcinoma (PDAC). The function of homotrimeric type I collagen can potentially be reduced by a suitable agent that interferes with the interaction between homotrimeric type I collagen and α3β1 integrin. For example, such substances may include anti-α3β1 integrin antibodies, α3 integrin siRNA, Col1 siRNA, and the like.

[0180] "Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject, or the performance of a procedure or modality on a subject, for the purpose of obtaining a therapeutic benefit in the treatment of a disease or a health-related condition. For example, treatment may include the administration of a pharmaceutically effective amount of an antibody that targets α3β1 integrin alone or in combination with the administration of chemotherapy, immunotherapy, or radiation therapy, the performance of surgery, or any combination thereof.

[0181] As used herein, the term "subject" refers to any individual or patient on whom the method of interest is performed. Generally, the subject is a human, but as will be understood by those skilled in the art, the subject may be an animal. Thus, other animals including mammals, such as rodents (including mice, rats, hamsters, guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas), etc. are included in the definition of the subject.

[0182] As used throughout this application, the term "therapeutic benefit" or "therapeutically effective" refers to anything that promotes or enhances the well-being of a subject with respect to the medical treatment of a condition. This includes, but is not limited to, a decrease in the frequency or severity of the signs or symptoms of a disease such as cancer or a fibrotic condition. For example, the treatment of cancer may include, for example, a decrease in tumor size, a decrease in tumor invasiveness, a decrease in the cancer growth rate, or the prevention of metastasis. The treatment of cancer may also refer to prolonging the survival of a subject having cancer.

[0183] As used herein, the term "cancer" can be used to describe solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, cancer can occur in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, rectum, anus, gingiva, head, kidney, liver, lung, oropharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.

[0184] Cancer can specifically be of the following histological types, but is not limited thereto: neoplasm, malignant; carcinoma; undifferentiated carcinoma; carcinoma of giant and spindle cells; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; trichoblastoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; coexistence of hepatocellular carcinoma and cholangiocarcinoma; cord adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyp; adenocarcinoma, familial adenomatous polyposis of the colon; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; anaplastic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; carcinoma of skin appendages; apocrine adenocarcinoma; sebaceous gland carcinoma; adenocarcinoma of the ear canal; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; serous papillary cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma of breast; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant androblastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant lipoid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomus angiosarcoma; malignant melanoma; amelanotic melanoma; superficially spreading melanoma; malignant melanoma of large pigmented nevus; epitheloid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; müllerian duct mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; undifferentiated embryonal cell tumor; embryonal carcinoma; malignant teratoma; malignant ovarian teratoma; choriocarcinoma; malignant mesonephroma; angiosarcoma; angioendothelioma, malignant; Kaposi sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; malignant odontogenic tumor; ameloblastic odontogenic sarcoma; malignant ameloblastic epithelioma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma, ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioblastoma;Primitive neuroectodermal; cerebellar sarcoma; gangliocytoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's sarcoidosis; small lymphocyte malignant lymphoma; diffuse large cell type malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasmacytic leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblast leukemia; myelosarcoma; and hairy cell leukemia. Nevertheless, it is also recognized that the present invention can also be used to treat non-cancerous diseases (e.g., fungal infections, bacterial infections, viral infections, neurodegenerative diseases, and / or genetic disorders).; B. Formulations and Administration

[0185] The present disclosure provides a pharmaceutical composition comprising an antibody that selectively binds to α3β1 integrin. Such a composition comprises a prophylactically or therapeutically effective amount of the antibody or a fragment thereof, and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory authority or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more specifically in humans. The term "carrier" refers to a diluent, excipient, or vehicle administered together with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Physiological saline and dextrose and glycerol aqueous solutions can also be used particularly as liquid carriers for injection solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.

[0186] The composition may optionally contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceuticals are described in "Remington’s Pharmaceutical Sciences". Such compositions will contain an amount of carrier suitable to provide a form for the appropriate administration to a patient of a prophylactically or therapeutically effective amount of an antibody or fragment thereof, preferably in purified form. The formulation must be suitable for the mode of administration, such as oral, intravenous, intraarterial, buccal, intranasal, spray, bronchial inhalation, rectal, vaginal, topical, or delivery by artificial respiration.

[0187] Passive transfer of antibodies generally involves the use of intravenous or intramuscular injection. The form of the antibody can be a monoclonal antibody (MAb). Such immunity generally lasts only a short time and there is also the potential risk of hypersensitivity reactions and serum sickness, especially with non-human derived gamma globulin. Antibodies are formulated in a carrier suitable for injection, i.e., a sterile injectable carrier.

[0188] Generally, the components of the compositions of the present disclosure are supplied separately or mixed together and supplied as a dry lyophilized powder or anhydrous concentrate in a unit dosage form, e.g., in a sealed container such as an ampoule or sachet indicating the amount of the active agent. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0189] The compositions of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions derived from, for example, hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. C. Kits and Diagnostic Agents

[0190] In various aspects of the embodiments, kits containing a therapeutic agent and / or other therapeutic agents and delivery agents are envisioned. This embodiment contemplates a kit for preparing and / or administering the treatment of the embodiment. The kit may include one or more sealed vials containing any of the pharmaceutical compositions of the present embodiment. The kit may include, for example, at least one α3β1 integrin antibody and reagents for preparing, formulating and / or administering the components of the present embodiment, or reagents for performing one or more steps of the method of the present invention. In some embodiments, the kit may include a suitable container that does not react with the components of the kit, such as an Eppendorf tube, assay plate, syringe, bottle, or tube, etc. The container may be made of a sterilizable material such as plastic or glass.

[0191] The kit can further include instructions that outline the procedural steps of the methods described herein, and will follow substantially the same procedures as described herein, or procedures known to those skilled in the art. The instructional information may be in a computer-readable medium containing machine-readable instructions that, when executed using a computer, display the actual or virtual procedures for delivering a pharmaceutically effective amount of the therapeutic agent. D. ADCC

[0192] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. The target cells are generally cells to which an antibody or a fragment thereof containing an Fc region specifically binds via a protein moiety that is the N-terminus of the Fc region. The term "antibody with increased / decreased antibody-dependent cell-mediated cytotoxicity (ADCC)" means an antibody having an increase / decrease in ADCC determined by a suitable method known to those skilled in the art.

[0193] As used in the specification, the term "increased / decreased ADCC" is defined as an increase / decrease in the number of target cells lysed within a predetermined time by the ADCC mechanism as defined above at a predetermined antibody concentration in the culture medium surrounding the target cells, and / or as a decrease / increase in the antibody concentration in the culture medium surrounding the target cells necessary to effect the lysis of a predetermined number of target cells within a predetermined time by the ADCC mechanism. The increase / decrease in ADCC is compared to the ADCC mediated by the same antibody produced by the same type of host cells using the same standard manufacturing, purification, formulation, and storage methods (which are known to those skilled in the art) but not manipulated. For example, the increase in ADCC mediated by an antibody produced by host cells engineered to have an altered glycosylation pattern (e.g., to express a glycosyltransferase, GnTIII, or other glycosyltransferases) by the methods described herein is compared to the ADCC mediated by the same antibody produced by the same type of unengineered host cells. E. Complement-dependent cytotoxicity (CDC)

[0194] Complement-dependent cytotoxicity (CDC) is a function of the complement system. It is an immune system process by which pathogens are killed by damaging their membranes without the involvement of antibodies or cells of the immune system. There are three main processes. All three insert one or more membrane attack complexes (MACs) into the pathogen, causing lethal colloidal osmotic swelling, i.e., CDC. This is one of the mechanisms by which antibodies or antibody fragments exert a cytotoxic effect. F. Combination therapy

[0195] In certain embodiments, the compositions and methods of this embodiment include an antibody or antibody fragment against α3β1 integrin for inhibiting the activity of α3β1 integrin, in combination with a second or additional treatment such as chemotherapy or immunotherapy (e.g., checkpoint blockade therapy). Such treatments can be applied to the treatment of any disease associated with elevated homotrimeric type I collagen. For example, the disease can be cancer or a fibroid.

[0196] Methods and compositions comprising combination therapies enhance the therapeutic or protective effect and / or increase the therapeutic effect of another anti-cancer or anti-proliferative treatment. Therapeutic and prophylactic methods and compositions can be provided in a combined dosage effective to achieve a desired effect such as killing cancer cells and / or suppressing cell overgrowth. This process may involve contacting the cells with both the antibody or antibody fragment and the second treatment. Tissues, tumors, or cells can be contacted with one or more compositions or pharmaceutical formulations containing one or more agents (i.e., an antibody or antibody fragment or an anti-cancer agent), or alternatively, can be contacted by contacting the tissue, tumor, and / or cells with two or more separate compositions or formulations. Here, one composition provides 1) an antibody or antibody fragment, 2) an anti-cancer agent, or 3) both an antibody or antibody fragment and an anti-cancer agent. It is contemplated that such combination therapies can be used in combination with chemotherapy, radiation therapy, surgical treatment, or immunotherapy.

[0197] When applied to cells, the terms "contact" and "exposure" are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are directly juxtaposed to the target cell. For example, to achieve cell killing, both agents are delivered to the cells in a combined dosage effective to kill the cells or prevent cell division.

[0198] Therapeutic antibodies may be administered before, during, after, or in various combinations compared to anti-cancer treatment. Administration can be carried out at intervals ranging from simultaneous to a few minutes to several days to several weeks. In embodiments where the antibody or antibody fragment is provided to the patient separately from the anti-cancer agent, generally, sufficient time elapses between each delivery such that the two compounds can still exert an advantageous combined effect on the patient before the expiration. In such cases, it is contemplated that antibody therapy and anti-cancer therapy can be provided to the patient within about 12 - 24 or 72 hours of each other, more specifically within about 6 - 12 hours of each other. In some situations, when several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapse between each administration, it may be desirable to significantly extend the treatment period.

[0199] In certain embodiments, the treatment process will continue for 1 - 90 days or more (such ranges include intervening days). It is contemplated that one agent is administered on any day from day 1 to day 90 (such ranges include intervening days) or any combination thereof, and another agent is administered on any day from day 1 to day 90 (such ranges include intervening days) or any combination thereof. Within one day (24 hours), a patient can receive one or more administrations of one or more agents. Further, it is contemplated that there will be a period after a series of treatments during which anti-cancer treatment is not administered. This period may continue for 1 - 7 days, and / or 1 - 5 weeks, and / or 1 - 12 months or more (such ranges include intervening days), depending on the patient's symptoms, such as prognosis, physical strength, health status, etc. Treatment cycles are expected to be repeated as necessary.

[0200] Various combinations can be used. In the following examples, antibody therapy is "A" and anti-cancer therapy is "B". A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0201] Administration of the compounds or therapies of the present embodiment to a patient will follow the general protocol for administration of such compounds, taking into account the toxicity of the agent, if present. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from combination therapy. 2. Chemotherapy

[0202] A wide variety of chemotherapeutic agents can be used in accordance with the present embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered during the treatment of cancer. These agents or drugs are classified by their mode of activity within the cell, e.g., whether they affect the cell cycle and at which stage. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intervene between DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0203] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bradykin and bradykidinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammaI and calicheamicin omegaI1); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein engyin antibiotics chromophore, actinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carvicin, calminomycin, cardinophyllin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, for example mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin; antimetabolites, for example methotrexate and 5-fluorouracil (5-FU); folic acid analogs, for example denopterin, pteropterin, and trimethoprim; purine analogs, for example fludarabine, 6-mercaptopurine, thiampurine, and thioguanine; pyrimidine analogs, for example ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, and floxuridine; androgens, for example calusterone, drostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenals, for example mitotane and trilostane; folic acid supplements, for example folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;Maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; lysocine; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, lolitrem A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitor, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are included.; 3. Radiation therapy

[0204] Other factors that cause DNA damage and are widely used include gamma rays, X-rays, and / or those generally known as the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation are also contemplated. All of these factors are most likely to affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range of X-rays is in the range from a daily dose of 50 to 200 roentgens for a long period (3 - 4 weeks) to a single dose of 2000 - 6000 roentgens. The dose range of radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the uptake by the newly formed cells. 4. Immunotherapy

[0205] One of ordinary skill in the art will understand that immunotherapy may be used in combination with, or in conjunction with, the methods of the embodiments. In the context of treating cancer, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody may act as a therapeutic effector alone or may recruit other cells to actually affect cell death. The antibody may also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and may simply act as a targeting agent. Alternatively, the effector may be a lymphocyte bearing surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0206] In one aspect of immunotherapy, tumor cells must have several markers that are suitable for targeting, i.e., that are not present in most other cells. There are many tumor markers, and any of these could be suitable for targeting in the context of the present embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine an anti-cancer effect with an immune-stimulating effect. There are also immune-stimulating molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, etc., chemokines such as MIP-1, MCP-1, IL-8, etc., and growth factors such as FLT3 ligand.

[0207] Immunotherapies currently under investigation or in use include immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169); cytokine therapy such as interferons α, β, and γ, IL-1, GM-CSF, and TNF; gene therapy such as TNF, IL-1, IL-2, and p53 (U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185 (U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies can be used in conjunction with the antibody therapy described herein.

[0208] In some embodiments, the immunotherapy may include an immune checkpoint inhibitor (i.e., it may be checkpoint blockade therapy). Immune checkpoints either raise or lower signals (e.g., co-stimulatory molecules). Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In certain embodiments, the immune checkpoint inhibitor targets the PD-1 axis and / or CTLA-4. In some embodiments, the immunotherapy of the present disclosure includes anti-PD-1 checkpoint blockade therapy (e.g., an anti-PD-1 antibody).

[0209] The immune checkpoint inhibitor can be a drug such as a small molecule, a recombinant of a ligand or a receptor, or an antibody such as a human antibody in particular (e.g., International Patent Publication WO2015016718, which is incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs may be used, particularly chimeric, humanized, or human forms of antibodies. As known to those skilled in the art, alternative and / or equivalent names may be used for certain antibodies referred to in the present disclosure. Such alternative names and / or equivalent names are interchangeable in the context of the present disclosure. For example, lambrolizumab is also known as an alternative name and equivalent name for MK-3475 and pembrolizumab.

[0210] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific embodiment, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific embodiment, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific embodiment, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art as described in U.S. Patent Application Publication Nos. 20140294898, 2014022021, and 20110008369, all of which are incorporated herein by reference.

[0211] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0212] Another immune checkpoint that can be targeted by the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and functions as an “off” switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell co-stimulatory protein CD28, and both molecules bind to CD80 and CD86 on antigen-presenting cells, called B7-1 and B7-2, respectively. CTLA4 transmits an inhibitory signal to T cells, while CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. Activation of T cells by the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0213] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0214] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the present invention can be generated using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, International Publication No. 01 / 14424, International Publication No. 98 / 42752; International Publication No. 00 / 37504 (CP675,206, also known as tremelimumab; former name ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al., (2004) J Clin Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al., (1998) Cancer Res 58:5301-5304, can be used in the methods disclosed herein. The teachings of each of the foregoing publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Applications No. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0215] Exemplary anti-CTLA-4 antibodies include ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the CDRs or VRs of the heavy and light chains of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for and / or binds to the same epitope on CTLA-4 as the above-described antibody. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to the above-described antibody (e.g., at least about 90%, 95%, or 99% variable region identity to ipilimumab).

[0216] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors as described in U.S. Patent Nos. 5,844,905, 5,885,796 and International Patent Applications WO1995001994 and WO1998042752, which are all incorporated herein by reference, and immunoadhesins as described in U.S. Patent No. 8,329,867, which is incorporated herein by reference.

[0217] In some embodiments, the immunotherapy can be adoptive immunotherapy involving the transfer of autologous antigen-specific T cells generated ex vivo. The T cells used in adoptive immunotherapy can be generated either by the expansion of antigen-specific T cells or by genetic engineering of T cell redirection (Park, Rosenberg et al., 2011). The isolation and transfer of tumor-specific T cells have been shown to be successful in the treatment of melanoma. Novel specificities of T cells have been successfully generated by the gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al., 2010). A CAR is a synthetic receptor composed of a targeting moiety associated with one or more signaling domains within a single fusion molecule. Generally, the binding portion of a CAR is composed of the antigen-binding domain of a single-chain antibody (scFv), including the variable fragment of the light chain of a monoclonal antibody linked by a flexible linker. Binding portions based on receptor or ligand domains have also been successfully used. The signaling domain of the first-generation CAR is derived from the cytoplasmic region of CD3ζ or the Fc receptor γ chain. CARs have been successful in redirecting T cells against antigens expressed on the surface of tumor cells of various malignancies, including lymphoma and solid tumors.

[0218] In one embodiment, the present application provides a combination therapy for the treatment of cancer, the combination therapy comprising adoptive T cell therapy and a checkpoint inhibitor. In one aspect, the adoptive T cell therapy comprises autologous and / or allogeneic T cells. In another aspect, the autologous and / or allogeneic T cells are targeted against tumor antigens. 5. Surgery

[0219] Approximately 60% of cancer patients will undergo some type of surgery, including prophylactic surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection to physically remove, excise, and / or destroy all or part of the cancerous tissue, and can be combined with other therapies such as the treatment of this embodiment, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical procedures include laser surgery, cryosurgery, electrocautery, and microsurgical control surgery (Mohs surgery).

[0220] Removing part or all of the cancerous cells, tissue, or tumor may create a cavity in the body. The treatment may be achieved by perfusion, direct injection, or local application of the area by additional anti-cancer therapies. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can also be of various dosages as well. 6. Other drugs

[0221] It is contemplated that other agents may be used in combination with particular aspects of the present embodiment to improve the therapeutic effect of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cell growth inhibitors and differentiating agents, inhibitors of cell adhesion, agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing substances, or other biological agents. An increase in intercellular signaling by increasing the number of gap junctions increases the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, a cell growth inhibitor or differentiating agent may be used in combination with particular aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of intercellular adhesion are contemplated to improve the effectiveness of the present embodiment. Examples of intercellular adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Furthermore, it is contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with particular aspects of the present embodiment to potentially improve treatment efficacy.

Examples

[0222] III. Examples The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the invention. However, those skilled in the art should understand that, in light of the present disclosure, many modifications may be made in the specific embodiments disclosed and still obtain similar or analogous results without departing from the spirit and scope of the invention. Example 1 - Targeting of Alpha 3 Beta 1 (α3β1) Integrin for the Treatment of Cancer Materials and Methods

[0223] Histology and immunohistochemistry. For paraffin-fixed samples, mouse tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at a thickness of 5 μm. Sections were processed for hematoxylin and eosin (H&E) staining. Masson's trichrome staining (MTS) was performed using a Gomori's trichrome staining kit (38016SS2, Leica Biosystems). Picrosirius red staining of collagen was performed using 0.1% picrosirius red (Direct Red80; Sigma) and counterstained with Weigert's hematoxylin. Images were captured with an MC120HD microscope camera equipped with a Leica DM1000LED microscope and LasV4.4 software (Leica). Formalin-fixed, paraffin-embedded sections were processed for immunohistochemical staining as previously documented (Chen et al., 2018). Sections were incubated with primary antibodies: αSMA (M0851, Dako, 1:100), CK19 (ab52625, Abcam, 1:200), collagen I (ab34710, Abcam, 1:200), integrin α3 (ab131055, Abcam, 1:300), Sox9 (ab185966, Abcam, 1:200), then biotinylated secondary antibodies, and streptavidin HRP (Biocare Medical). In all immunolabeling experiments, sections were developed with DAB and counterstained with hematoxylin.

[0224] Fresh tumor tissues of single-cell RNA sequencing (sc-RNA-seq) KPPC mice were minced finely with a sterile lancet, digested with collagenase IV (17104019, Gibco, 4 mg / mL) / dispase II (17105041, Gibco, 4 mg / mL) / DMEM at 37 °C for 0.5 h, filtered through a 70-μm cell strainer, and resuspended as a single-cell suspension in PBS / 2% FBS. This single-cell suspension was stained with Live / Dead viability dye eFluor 780 (65-0865-14, eBioscience), filtered through a 40-μm mesh, and then analyzed on an Aria in the South Campus Flow Cytometry Core Laboratory of MDACC Live cells were sorted using an II sorter (BD Biosciences). A total of 4,064 cells from two mice were analyzed for tumors in KPPF (FSF-KrasG12D / +; Trp53frt / frt; Pdx1-Flp) mice. A total of 3,989 cells from two mice were analyzed for tumors in KPPC (LSL-KrasG12D; Trp53loxP / loxP; Pdx1-Cre) mice. Sc-RNA-seq of these samples was performed using a Chromium Controller and a Single Cell 3’ Reagent Kit v2 (10X Genomics) at the sequencing and microarray facility of MDACC. Generation of single cell gel beads in emulsion (GEM), barcoding, clean-up and cDNA amplification after GEM-RT, library construction, and preparation of sequence libraries compatible with Illumina were prepared according to the manufacturer's guidelines. The concentrations of cDNA and libraries were estimated using a High-Sensitivity dsDNA Qubit kit. An HS DNA Bioanalyzer was used for quantification of cDNA. A DNA 1000 Bioanalyzer was used for quantification of libraries. “c-loupe” files were generated using the Cell Ranger software pipeline according to the manufacturer's guidelines. Unsorted tumor cells were encapsulated using a 10X Genomics Chromium Controller and Single Cell 3’ Reagent Kit v2. Following capture and lysis, cDNA was synthesized and amplified to construct an Illumina sequencing library. Libraries obtained from approximately 1,000 cells per sample were sequenced on an Illumina Nextseq 500. The run format was 26 cycles for Read 1, 8 cycles for Index 1, and 124 cycles for Read 2. The sc-RNA-seq data was processed by the sequencing and microarray facility of the MD Anderson Cancer Center. Further data analysis was performed using the R package software of the Bioconductor project.

[0225] The type I collagen (Col1) solution extracted by Western blotting and solubilized Matrigel (growth factor reduced, 354230, Corning) were prepared with 6x reducing SDS Laemmli Sample Buffer (Bio-world) and denatured at 95°C for 20 minutes. Next, the samples were electrophoresed using a Mini-PROTEAN TGX precast polyacrylamide gel (Bio-Rad) and transferred to a polyvinylidene fluoride (PVDF) membrane using a Trans-Blot Turbo transfer system (Bio-Rad). Col1 was blotted with a goat anti-col1 antibody (1310, SouthernBiotech, 1:1000) and an HRP-labeled donkey anti-goat secondary antibody. Type IV collagen (Col4) was blotted with a rabbit anti-Col4 antibody (ab52235, Abcam, 1:300) and an HRP-labeled secondary goat anti-rabbit antibody.

[0226] In vivo FAK inhibitor (FAKi) treatment. Twenty-one-day-old KPPC mice were randomly divided into a control group or a FAKi (VS-4718 or PND-1186, Selleckchem) treatment group for further treatment. Mice in the FAKi treatment group were treated by forced oral administration twice a day with 50 mg / kg of FAKi resuspended in 0.5% carboxymethylcellulose (Sigma-Aldrich) and 0.1% Tween-80 (Sigma-Aldrich) in sterile water as previously described (Jiang et al., 2016). Mice in the control group were administered the vehicle using the same dosing strategy. Pancreatic tissues were collected from the mice and examined at the same age of 28 days after 7 days of treatment. Results

[0227] Tumors contain both cancer cells and components of the tumor microenvironment (TME), such as fibroblasts and type I collagen. Whether the tumor microenvironment functions as a promoter or inhibitor of tumor growth remains unclear. Some aspects of the TME may function as positive regulators of tumor progression, while other aspects may function as negative regulators of tumor growth. Type I collagen (collagen I) produced by myofibroblasts is a heterotrimer containing two α1 chains of collagen I (α1(I) collagen) and one α2 chain of collagen I (α2(I) collagen), and suppresses cancer / tumors by binding to potential receptors (presumably discoidin domain receptor II - DDR2) of cancer cells and other stromal cells and immune cells. In contrast, cancer cells produce a collagen I homotrimer with three α1(I) collagen chains that promotes cancer / tumors and binds to specific receptors of cancer cells to induce survival - promoting signals, anti - apoptotic signals, proliferation signals, growth - promoting signals, and cancer - promoting signals. The homotrimer (produced by cancer cells) is resistant to metalloproteinases and other proteinases compared to the heterotrimer produced by myofibroblasts in the tumor microenvironment. The homotrimer exhibits a different structure exposing different epitopes compared to the heterotrimer, and antibodies generated against this homotrimer will have tumor - suppressing properties, among other mechanisms, by interfering with signaling through cancer - promoting receptors on cancer cells.

[0228] Since integrins have been widely studied as collagen receptors on cancer cells (Egeblad et al., 2010; Leitinger, 2011; Yeh et al., 2012), the direct functional involvement of integrins in the survival-promoting signaling induced by col1 homotrimers was investigated. Col1 homotrimers induced the phosphorylation of DDR1, FAK, AKT, and ERK. However, even when DDR1 was knocked down with siRNA, the activation of FAK, AKT, and ERK by col1 homotrimers was not inhibited, probably due to the compensatory upregulation of integrins (especially α3β1) after DDR1 knockdown (Figures 1A, 1B). On the other hand, when integrin β1 was suppressed with siRNA, the activation of FAK, AKT, and ERK induced by col1 homotrimers was significantly reduced, suggesting an important role of integrin β1 in mediating the survival-promoting function of col1 homotrimers in cancer cells (Figure 1C, Figure 2A).

[0229] Further examinations using siRNAs against integrin α1, α2, and α3 subunits revealed that siRNA against integrin α3 (Figures 1C, 2B), but not the α1 integrin subunit, significantly inhibited the phosphorylation of FAK, AKT, and ERK induced by col1 homotrimers, while siRNA against integrin α2 moderately inhibited the phosphorylation induced by col1 homotrimers. RNA-seq data obtained from mouse (Figure 2C) and human (Figure 2D, CCLE database) pancreatic cancer cell lines confirmed that integrin α3 is the most abundant α-integrin subunit expressed by pancreatic cancer cells. Integrins α10 and α11, also known as collagen-binding integrin subunits, were found to have minimal expression in pancreatic cancer cells together with their β1 integrin subunit partners (Figures 2C, 2D). The widespread expression pattern of integrin α3 in cancer cells is consistent with the KPPC tumor (LSL-Kras G12D ; Trp53 loxP / loxPSingle-cell RNA sequencing analysis in (Pdx1-Cre) (Figs. 2E and 3A - 3X) and further confirmed by IHC staining in mouse and human tumors (Figs. 2F and 4A - 4F).

[0230] Next, to test the role of col1 homotrimer-induced FAK phosphorylation in the etiology of early lesions associated with pancreatic cancer, its effect on col1 homotrimer-induced survival-promoting signaling was evaluated using VS-4718 (PND-1186), a dual inhibitor targeting FAK and PYK2. VS-4718 inhibited FAK and PYK2 (downstream mediators of integrin and DDR1-mediated signaling pathways) and suppressed the activation of FAK, AKT, and ERK induced by col1 homotrimer (Fig. 2G). Treatment of KPPC mice with VS-4718 also inhibited early PanIN progression, consistent with previous findings (Jiang et al., 2016) (Fig. 2H). Collectively, these results revealed that col1 homotrimer promotes PDAC cell proliferation by inducing sustained activation of FAK, AKT, and ERK via collagen-binding integrin α3β1. Example 2 - Identification of α3 integrin expression in human PDAC patients and correlation with survival outcomes Methods

[0231] All human PDAC sections were fixed on tissue microarray slides containing three representative 1-mm cores (two from tumor and one from benign pancreatic tissue) per patient. The staining intensity of integrin α3 was quantified on a scale of 0–3 (3 - very strong, 2 - strong, 1 - weak, and 0 - negative) by visual scoring of the staining. The IHC score of integrin α3 for all samples was graded by a score combining the intensity of staining and the proportion of positive tumor cells. The formula for the staining score was used: S = p1x1 + p2x2 + p3x3, where p1, p2, and p3 represent the proportion of tumor cells in each staining category of 1, 2, and 3, respectively. The mean score of integrin α3 (ITGA3) expression was 1.87 across the cohort. The expression of integrin α3 was classified as ITGA3-high (n = 68) and ITGA3-low (n = 62) using a mean composite score of 1.87 as the cutoff. Results

[0232] Human PDAC sections (n = 141) fixed on tissue microarray slides were examined for the IHC score of integrin α3 (Figure 5A), and the mean score across the cohort was 1.87 (Figure 5B). Most (97%) of the human PDAC sections revealed very high or high integrin α3 expression (Figure 5C). High integrin α3 expression levels were significantly correlated with a decrease in the overall survival (Figure 5D) and a decrease in the progression-free survival (Figure 5E) of the patients. Patients with high ITGA3 are shown by the lower lines in Figures 5D and 5E, while patients with low ITGA3 are shown by the upper lines in Figures 5D and 5E. Example 3 - In Vitro Itga3 siRNA Treatment of PDAC Cells Methods

[0233] KPPC (LSL-Kras G12D ;Trp53 loxP / loxP ;Pdx1-Cre) and KPPC;Col1 pdxKO (KPPC;Col1a1 loxP / loxP ) cells were seeded into 96-well plates (3 × 10 per well in 100 μL RPMI containing 1% FBS 3Cells were then treated with the siRNA shown below for 48 hours. The cell viability / count in each well of 6-well or 96-well plates was determined using Cell Counting Kit-8 (CCK8; Abcam ab228554), and examined at OD 450 nm using a microplate reader according to the manufacturer's instructions. Results

[0234] Suppression of α3 integrin with siRNA decreases the proliferation of KPPC cancer cells, but not KPCC;Col1 pdxKO does not decrease (Figure 6). This indicates that inhibition of the binding between Col1 homotrimer and α3 integrin in cancer cells affects their proliferation. Example 4 - In vitro Itga3 siRNA treatment. Method

[0235] Exosomes were produced from human mesenchymal stem cells. 10 9 total exosomes (measured by Nanosight™ analysis) and 1 μg of siRNA (scrambled siRNA-control or siRNA-Itga3) were mixed in 400 μL of electroporation buffer (1.15 mM potassium phosphate pH 7.2, 25 mM potassium chloride, 21% Optiprep). These exosomes were electroporated using a single 4 mm cuvette and the Gene Pulser Xcell Electroporation System (Bio-Rad, 165-2081). Mice were injected with 10 8 exosomes per injection at a volume of 100 μL. This dosage corresponds to an exosome protein loading of approximately 0.15 - 0.20 μg per injection into mice every 48 hours. Results

[0236] Treatment with exosomes containing siRNA targeting integrin α3 significantly prolonged the survival of KPPC mice compared to exosomes containing scrambled siRNA control (Figure 7). Example 5 - Deletion of type I collagen (Col1) in pancreatic cancer cells increases the accumulation of immune cells and the sensitivity to checkpoint blockade Method Mouse

[0237] FSF-Kras G12D / + (5), Pdx1-Flp(5), Trp53 frt / + (6), LSL-Kras G12D / + (7), Trp53 loxP / + (8), Pdx1-Cre(7), αSMA-Cre(9), and Fsp1-Cre(10, 11) mouse strains have been previously documented. Col1a1 loxP / loxP Mouse strain (including loxP-flanked exons 2 - 5) was obtained from the European Mouse Mutant Cell Repository (EuMMCR) Col1a1 tm1a(EUCOMM)Wtsi Embryonic stem cells were used to establish in the transgenic mouse facility of MD Anderson Cancer Center (MDACC).

[0238] Rosa26-CAG-loxP-frt-Stop-frt-FireflyLuc-EGFP-loxP-RenillaLuc-tdTomato (R26 Dual Also called) mouse strain contains a novel R26 that enables EGFP expression under the control of the Pdx1-Flp transgene, or tdTomato expression under the control of the αSMA-Cre and Fsp1-Cre transgenes Dual Double-fluorescent reporter alleles are included (12). FSF-Kras G12D / + ;Pdx1-Flp (also called KF) or FSF-Kras G12D / + ;Trp53 frt / frt; Genotyping of Pdx1-Flp (also called KPPF) mice and characterization of disease phenotypes were performed as previously described by Saur et al. (5). Osteogenesis imperfecta mouse (OIM) strains with Col1a2 mutations were purchased from The Jackson Laboratory (001815; B6C3Fe a / a-Col1a2oim / J). The inventors crossed KF mice and KPPF mice with αSMA-Cre, Pdx1-Cre, Fsp1-Cre, Col1a1 loxP / loxP , or R26 Dual mouse strains, resulting in the generation of KF;αSMA-Cre;Col1a1 loxP / loxP (also called KF;Col1 smaKO ), KF;Pdx1-Cre;Col1a1 loxP / loxP (also called KF;Col1 pdxKO ), KPPF;αSMA-Cre;Col1a1 loxP / loxP (also called KPPF;Col1 smaKO ), KPPF;Fsp1-Cre;Col1a1 loxP / loxP (also called KPPF;Col1 fspKO ) mice. These mice allow for deletion of Col1a1 in either αSMA+ myofibroblasts (MF) or Fsp1+ cell populations in the context of spontaneous PDAC. KF;Col1 pdxKO mice and KF;Col1 smaKO mice share the same control mice (KF; Cre-negative; Col1a1l oxP / loxP ), enabling direct comparison of disease progression among these three strains (KF control group, Col1 smaKO group with Col1 deletion in αSMA-expressing myofibroblasts, and KF;Col1 pdxKO group with Col1 deletion in Pdx1 lineage cancer cells). The inventors also crossed LSL-Kras G12D ;Pdx1-Cre (also called KC), LSL-Kras G12D / + ;Trp53 R172H / + ;Pdx1-Cre;Col1a1 loxP / loxP (also called KPC), or LSL-Kras G12D ;Trp53 loxP / loxP; Pdx1-Cre (also called KPPC) mice were crossed with the Col1a1 loxP / lox mouse strain, resulting in the generation of KC;Col1a1 loxP / loxP (also called KC;Col1 pdxKO ), KPC;Col1a1 loxP / loxP (also called KPC;Col1 pdxKO ), and KPPC;Col1a1 loxP / loxP (also called KPPC;Col1 pdxKO ). KF;Col1 pdxKO , KC;Col1 pdxKO , and KPPC;Col1 pdxKO mice allow for deletion of Col1a1 in PDAC cells. The aforementioned experimental mice with the desired genotypes were monitored and analyzed without randomization or blinding. For the experimental mice, both male and female mice with one or more genotypes desirable for PDAC were used. Anti-PD1 (BE0273, 29F.1A12, BioXCell) antibody was administered intraperitoneally to each mouse at a dose of 100 μg, at 3-day intervals (starting at 35 days of age) for a total of 3 injections. All mice were housed under standard breeding conditions in the MDACC animal facility, and all animal procedures were reviewed and approved by the MDACC Institutional Animal Care and Use Committee. Total mRNA sequencing

[0239] For mRNA sequencing (RNA-seq) analysis of tumor tissues, freshly dissected tumor samples were frozen in RNase-free tubes using liquid nitrogen and stored at -80°C. Samples were homogenized using a bead tube containing ceramic beads with a Fisherbrand Bead Mill 24 homogenizer (Fisher Scientific). For RNA-seq of cell lines, KPPC and / or KPPC;Col1pdxKO cancer cells were cultured in 6-well plates (Corning) with vehicle (PBS containing 0.5 M glycerol), homotrimeric Col1 (50 μg / mL), or heterotrimeric Col1 (50 μg / mL). Cells were harvested after 48 hours of culture. For both tissues and cells, total RNA was extracted using the Direct-zol RNA Kit (Zymo Research).

[0240] Quality control analysis was performed on the Bioanalyzer 2100 (Agilent) using the RNA 6000 NanoKit. Whole mRNA sequencing was performed using the Illumina TrueSeq stranded mRNAseq library and NextSeq 500 (Illumina) High-Output sequencing PE 75x75 nt by the MDACC Sequencing and ncRNA Program core facility. Raw sequencing data from the Illumina platform was converted to Fastq files and aligned to the reference genome mm10 using the Spliced Transcripts Alignment to a Reference (STAR) algorithm. Next, HTSeq-count was utilized to generate raw counts for each gene. Raw counts were then analyzed by DESeq2 for data processing, normalization, and differential expression analysis following standard procedures. Functional classification and pathway reconstruction from the RNA-seq data were performed using gene set enrichment analysis (GSEA; Broad Institute) and Ingenuity Pathway Analysis (IPA) software (Qiagen). All analyses were performed in R. Flow cytometry

[0241] For the characterization of immune infiltration, fresh tumor tissues (53-day-old KPPC and KPPC; derived from Col1pdxKO, respectively) were weighed, minced with a gentleMACS Dissociator, and digested with 2 mL of a solution containing 1 mg / mL Liberase TL (Roche) and 0.2 mg / mL DNase I in RPMI medium at 37 °C for 30 minutes. Before immunostaining, the tissue lysates were filtered through a 100-μm mesh. The subsequent single-cell suspensions were stained with Fixable Viability Dye eFluor 780 (eBioscience) and appropriate antibodies. The samples were filtered through a 40-μm mesh and examined using a BD LSR Fortessa X20. The percentage of positive cells was analyzed by FlowJo 10.1 and gated on CD45 positivity. Unstained, viability staining only, and single-stained beads (eBioscience) were used as compensation controls. Multispectral images of multiplexed stained sections

[0242] Multiplex staining procedures, spectral unmixing, and cell segmentation using Nuance and inForm imaging software have been described previously (1). Multiplexed stained slides were imaged using a Vectra Multispectral Imaging System with Vectra software, version 3.0.3 (PerkinElmer). The entire tissue section was scanned using a 4× objective lens. Using Phenochart software (PerkinElmer), up to 80 regions (at 20×) per section were selected for multispectral imaging. Each multiplex field was scanned at 10-nm emission spectra across the entire range of each emission filter cube. The filter cubes used for multispectral imaging were DAPI (440–600 nm), FITC (520 nm–680 nm), Cy3 (570–690 nm), Texas Red (580–700 nm), and Cy5 (680–720 nm).

[0243] A spectral library was created using multi - spectral images obtained from slides stained with a single marker containing the corresponding fluorophore using Nuance Image Analysis software (PerkinElmer). Since this library contained the peaks of the emission spectra of all fluorophores, the image analysis software inForm 2.2 was used to unmix (spectral decomposition) each multi - spectral image into its six individual components. To ensure consistent and reliable capture of positive signals in all controls, the detection thresholds for various markers were adjusted among different cohorts. All images of each cohort were processed using the same threshold for positive staining. Co - culture of mouse spleen lymphocytes and PDAC cancer cells

[0244] KPPC and KPPC;Col1 pdxKO Cancer cells (2×10 4 cells / well) were seeded in 96 - well plates. The spleens of 4 healthy 2.5 - month - old mice (KPPC and KPPC;Col1 pdxKO mice with the same genetic background) were minced, filtered through a 40 - μm mesh, washed with ice - cold PBS, then resuspended in 5 mL of red blood cell lysis solution (sc - 296258, Santa Cruz) for 5 minutes on ice and washed with PBS. Spleen lymphocytes were counted and seeded in round - bottom 96 - well plates (2×105 cells / well) with or without activation for 24 hours, then transferred to 96 - well plates containing KPPC or KPPC;Col1 pdxKO cancer cells (or culture medium only without cancer cells). After 24 - hour co - culture, spleen lymphocytes were collected, stained using the above - mentioned flow cytometry method, and examined for lymphocyte activation. Anti - CD3 (553057, BD Biosciences) and anti - CD28 (553294, BD Biosciences) antibodies (1 μg / mL) were used for in vitro activation of T cells. Statistics

[0245] Statistical analysis of flow cytometry and quantification of immunostaining were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) by unpaired two-sided t-test, one-way ANOVA with Tukey's multiple comparison test, or Fisher's exact probability test. χ2 analysis was performed by comparing the metastasis frequencies across multiple histological parameters of the mice.

[0246] Kaplan–Meier plots were drawn for survival analysis, and statistical differences were evaluated using the log-rank Mantel–Cox test. The data satisfied the prerequisites of each statistical test, and Welch's correction for unequal variances was applied when the variances were unequal (determined by F-test). A P-value < 0.05 was considered statistically significant. Error bars represent the standard error of the mean (SEM) when multiple fields were averaged to generate a single value for each animal, and then averaged again to represent the mean bar of the group for each graph. Results

[0247] As revealed by gene set enrichment analysis (GSEA), KPPF;Col1 smaKO RNA-seq of total RNA from tumor tissues of KPPF;Col1 mice and KPPF control mice showed that deletion of Col1 in myofibroblasts led to KPPF;Col1 smaKO It was revealed that the immune response pathways (such as lymphocyte activation / mobilization pathways) were significantly decreased in tumors. Furthermore, by IPA (Ingenuity Pathway Analysis), KPPF;Col1 smaKO Genes related to T cell responses in tumors, such as CD3g, CD3e, PdCD1, Il2rg, CD80, and CD86, were significantly decreased (Figure 8A - 8C). In contrast, KPPC;Col1 pdxKO RNA-seq of total RNA from tumor tissues of KPPC;Col1 mice and KPPC control mice showed that deletion of Col1 in cancer cells led to KPPC;Col1 pdxKO It was revealed that the immune response pathways (such as lymphocyte activation / mobilization pathways) were significantly increased in tumors. By IPA, KPPC;Col1 pdxKOGenes that were significantly increased and associated with T cell responses in tumors were identified, including Cd3d, Cd3g, Cd4, Cd8a, Ctla4, Pdcd1, and Il2ra (Figures 8D - 8F). Next, KPPC, KPPC;Col1 pdxKO , KPPF, and KPPF;Col1 smaKO Tumor sections from mice were examined by TSA multispectral imaging (1). CD3, CD4, and CD8 T cell infiltration was increased in KPPC;Col1 pdxKO tumors but decreased in KPPF;Col1 smaKO tumors compared to KPPC and KPPF control tumors, respectively (Figures 8G and 8H). Collectively, Col1 deletion in myofibroblasts of KPPF;Col1 smaKO tumors decreased both stromal Col1 levels and T cell infiltration, while Col1 deletion in cancer cells of KPPC;Col1 pdxKO increased T cell infiltration.

[0248] Flow cytometry analysis of fresh tissues showed that KPPC;Col1 pdxKO tumors increased T cell infiltration and related T cell activation markers compared to KPPC tumors (Figures 9A - 9H). Specifically, a significant increase in CD4 + / PD - 1 + (Figure 9F) and CD8 + / PD - 1 + cells (Figure 9H) was observed in KPPC;Col1 pdxKO tumors. To determine whether such an increase in PD - 1 pdxKO T cells in KPPC;Col1 + tumors was functionally important (2), KPPC mice and KPPC;Col1 pdxKO mice with advanced PDAC were treated with anti - PD - 1 antibody. Similar to previous reports (3, 4), KPPC mice were refractory to anti - PD - 1 treatment (Figure 9I). However, KPPC;Col1 pdxKO showed a positive response to anti - PD - 1 treatment and an extended overall survival (Figure 9I).

[0249] Using an in vitro co - culture system of mouse spleen lymphocytes and PDAC cancer cells (a schematic diagram thereof is shown in Fig. 9J), it was further demonstrated that KPPC cancer cells significantly suppressed the activation and proliferation of mouse spleen lymphocytes co - cultured with them. In contrast, KPPC;Col1 cancer cells lacking the Col1 homotrimer pdxKO lost such immunosuppressive effects on co - cultured mouse spleen lymphocytes (Figs. 9K - 9N).

[0250] In summary, these results reveal that the deletion of oncogenic Col1 homotrimers in cancer cells alleviates the immunosuppressive PDAC microenvironment, promotes T - cell infiltration into tumors, and enhances the efficacy of anti - PD - 1 checkpoint blockade therapy. * * *

[0251] All of the methods disclosed and claimed in this specification can be made and executed without undue experimentation in light of the present disclosure. The compositions and methods of the present invention are described with respect to specific embodiments, but it will be apparent to those skilled in the art that changes may be made in the methods described herein and in the steps or the order of the steps of the methods described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically or physiologically related agents may be used in place of the agents described herein while achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims. References The following references are specifically incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement those described herein.

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Claims

[Claim 1] The invention described in the specification.

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