Identifying and targeting pathogenic extracellular matrix for the diagnosis and treatment of cancer and other diseases

Abstract

Provided herein is an agent such as antibody or chimeric antigen receptor that targets homotrimeric type I collagen.Provided is a method for treating cancer and fibroid, comprising administering an effective amount of homotrimeric type I collagen neutralizer to patients who need it.This method can further comprise administering an effective amount of chemotherapy or immunotherapy to patients.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 746,286, filed October 16, 2018, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background 1. Field The present invention relates generally to the field of medicine. More specifically, the present invention relates to a method for detecting cancer based on the presence of homotrimeric type I collagen and treating cancer by disrupting homotrimeric type I collagen and its induced signal transduction.

[0003] 2. Description of Related Art Desmoplasia, a dense stroma composed of various cell populations such as myofibroblasts, and deposition of extracellular matrix (ECM) such as type I collagen (Col1), is a defining feature of pancreatic ductal carcinoma (PDAC). However, the specific role of ECM and tumor stroma in supporting or suppressing tumorigenesis remains controversial (Mueller and Fusenig, 2004; Neesse et al., 2015). Findings support both tumor-supportive and tumor-suppressive contributions of PDAC stroma. Previous findings demonstrated that desmoplastic stroma in PDAC (such as activated pancreatic stellate cells [PSCs] / myofibroblasts and ECM) forms a pro-tumorigenic microenvironment, contributing to reduced drug delivery and therapeutic resistance. Targeting PDAC stroma has been demonstrated to reduce PDAC metaplasia and improve drug delivery by inhibition of the Sonic Hedgehog (SHH) pathway (Olive et al., 2009) or by removal of interstitial hyaluronan (Provenzano et al., 2012). However, clinical trials targeting PDAC stroma have failed to yield promising therapeutic results as expected. Furthermore, recent studies have discussed the heterogeneity of stromal fibroblasts and their multiple roles (Ohlund et al., 2014; Kalluri, 2016; Ohlund et al., 2017). Previous studies have shown that depletion of proliferating α-smooth muscle actin (αSMA)-expressing activated PSCs / myofibroblasts induces PDAC hypoxia and invasiveness despite reduced fibrosis and collagen deposition in the PDAC stroma (Ozdemir et al., 2014). Genetic ablation or smoothened inhibition of SHH also leads to more aggressive and less differentiated PDAC. These arguments are consistent with previous studies suggesting a suppressive function of the tumor stroma (Rhim et al., 2014). It has also been reported that the response of PDAC to anti-stromal therapy can vary significantly due to different PDAC genotypes and signaling, which primarily determine stromal remodeling (Laklai et al., 2016).Taken together, these various or even contradictory findings point to a complex biology and multiple roles of the PDAC stroma that go beyond previous knowledge, which will undoubtedly require further systematic investigation using new experimental systems.

[0004] Classical KPC (LSL-Kras G12D / + ;Trp53 R172H / + or Trp53 loxP / loxP Current genetically engineered PDAC mouse models (GEMMs), such as the Pdx1-Cre (Pdx1-Cre) model, provide a valuable platform that mimics the clinical situation of human PDAC and have made significant contributions to the study of PDAC and its treatment (Hingorani et al., 2005). The traditional KPC model is widely used in combination with genetic ablation (using the same pancreatic-specific Cre, such as Pdx1-Cre or P48-Cre) of floxed (loxP-flanked) genes in cancer cells or with whole-body knockout (KO) of genes. However, due to the universal Cre-loxP recombination mechanism, it is still not possible to achieve cell-type-specific genetic manipulation in stromal cell subpopulations (such as myofibroblasts or immune cells) in these GEMMs. It is also not possible to establish KPC models involving whole-body KO of genes with KO lethality, for example, Col1a1, which encodes the type I collagen α1 chain (Lohler et al., 1984). It is therefore surprising, but reasonable, that there are no PDAC GEMMs to date that would allow functional KO of Col1 in stromal cell sources to demonstrate the origin and contribution of Col1, especially considering that Col1 is such an essential component and the most abundant protein in PDAC desmoplasia and microenvironment.

[0005] Type I collagen (Col1), usually composed of α1 and α2 chains, is one of the most predominantly deposited interstitial ECM components in the PDAC microenvironment. Many studies have shown that activated PSCs / myofibroblasts are the main cellular source of Col1 and other ECM materials (Haber et al., 1999; Armstrong et al., 2004; Bachem et al., 2005; Fujita et al., 2009; Apte et al., 2012). Nevertheless, Col1 has also been shown to be produced by various types of cancer cells and promote tumor progression. Indeed, cancer cell-derived Col1 consists of a unique and MMP-resistant homotrimeric (α1)3 chain, in contrast to the (α1 / α2 / α1) heterotrimeric chains produced by fibroblasts or other normal cells (Sengupta et al., 2003; Han et al., 2008; Egeblad et al., 2010; Han et al., 2010; Makareeva et al., 2010). These findings suggested distinct structural and functional roles of cancer-derived Col1 and myofibroblast-derived Col1 in cancer. Many studies have been conducted to address the active role of PDAC stroma. However, the role of ECM components, such as Col1, with respect to specific cell origins has not been systematically verified or compared in clinically relevant transgenic PDAC models. To further understand the impact of the stroma on PDAC development, it is important to analyze the exact function of Col1 specifically derived from different cell origins, such as cancer cells and fibroblast subpopulations. Summary of the Invention

[0006] overview In one embodiment, the antibody or antibody fragment is provided herein that binds to α1 homotrimeric type I collagen.In some aspects, the antibody or antibody fragment has an affinity for α1 homotrimeric type I collagen that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the affinity for α1 / α2 / α1 heterotrimeric type I collagen.In some aspects, the antibody or antibody fragment does not detectably bind to α1 / α2 / α1 heterotrimeric type I collagen.The antibody or antibody fragment may recognize a conformation or a specific discontinuous epitope that exists in homotrimer but does not exist in heterotrimer.

[0007] In some aspects, the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. In some aspects, the antibody is a chimeric antibody or a bispecific antibody. In certain aspects, the chimeric antibody is a humanized antibody. In certain aspects, the bispecific antibody binds to both α1 homotrimeric type I collagen and CD3. In some aspects, the antibody or antibody fragment is conjugated to a cytotoxic agent. In some aspects, the antibody or antibody fragment is conjugated to a diagnostic agent.

[0008] In one embodiment, provided herein are hybridomas or engineered cells encoding the antibodies or antibody fragments of the invention embodiments. In some embodiments, provided are pharmaceutical formulations comprising one or more of the antibodies or antibody fragments of the invention embodiments.

[0009] In one embodiment, the present invention provides a method for treating a patient in need thereof, comprising administering an effective amount of α1 homotrimeric type I collagen specific antibody or antibody fragment.In some aspects, the α1 homotrimeric type I collagen specific antibody or antibody fragment is any of the antibody or antibody fragments of the present invention.

[0010] In some aspects, the patient has cancer, fibroid disease, keloid, organ fibrosis, Crohn's disease, stenosis, colitis, psoriasis, or connective tissue disorder.In some aspects, the connective tissue disorder is collagen-related connective tissue disorder.In some aspects, the collagen-related connective tissue disorder is collagen type 1-related connective tissue disorder.

[0011] In some aspects, the patient has cancer. In some aspects, the cancer patient is determined to express elevated levels of α1 homotrimeric type I collagen compared to control patients. In some aspects, the cancer is pancreatic cancer. In some aspects, the method is further defined as a method for inhibiting pancreatic cancer metastasis. In some aspects, the method is further defined as a method for inhibiting pancreatic cancer growth. 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.

[0012] In one embodiment, 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, said CAR polypeptide binding to α1 homotrimeric type I collagen. In some aspects, the antigen binding domain comprises an HCDR sequence derived from a first antibody that binds to α1 homotrimeric type I collagen, and an LCDR sequence derived from a second antibody that binds to α1 homotrimeric type I collagen. In some aspects, the antigen binding domain comprises an HCDR sequence and an LCDR sequence derived from an antibody that binds to α1 homotrimeric type I collagen. In some aspects, the antigen binding domain has an affinity for α1 homotrimeric type I collagen that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than its affinity for α1 / α2 / α1 heterotrimeric type I collagen. In some aspects, the antigen binding domain does not detectably bind to α1 / α2 / α1 heterotrimeric type I collagen.

[0013] 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.

[0014] In one embodiment, provided herein is a nucleic acid molecule encoding a CAR polypeptide of any of the embodiments of the invention. In some aspects, the sequence encoding the CAR polypeptide is operably linked to an expression control sequence.

[0015] In one embodiment, provided herein is an isolated immune effector cell comprising the CAR polypeptide or nucleic acid of the present invention.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.In one embodiment, provided herein is a pharmaceutical composition comprising a population of the cell of the present invention in a pharmaceutically acceptable carrier.

[0016] In one embodiment, provided herein is a method of treating a subject, comprising administering an antitumor effective amount of a chimeric antigen receptor (CAR) T cell expressing a CAR polypeptide according to any one of the embodiments of the present invention. In some aspects, the CAR T cell is an allogeneic cell. In some aspects, the CAR T cell is an autologous cell. In some aspects, the CAR T cell is HLA-matched to the subject. In some aspects, the subject has cancer, such as pancreatic cancer. In some aspects, the method further comprises administering a demethylation drug prior to administration of the CAR T cell to serve as a primer for immunotherapy. The demethylation drug can reverse Col1A2 hypermethylation. The demethylation drug can be 5-azacytidine or 5-aza-2'-deoxycytidine. In some aspects, the method further comprises administering a drug that disrupts the methylation of the promoter of the Col1A2 gene.

[0017] In one embodiment, provided herein is a method of treating a subject, comprising administering an antitumor effective amount of chimeric antigen receptor (CAR) NK cells expressing a CAR polypeptide according to any one of the embodiments of the present invention. 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 HLA-matched to the subject. In some aspects, the subject has cancer, such as pancreatic cancer. In some aspects, the method further comprises administering a demethylation drug prior to administration of the CAR NK cells to serve as a primer for immunotherapy. The demethylation drug can reverse Col1A2 hypermethylation. The demethylation drug can be 5-azacytidine or 5-aza-2'-deoxycytidine. In some aspects, the method further comprises administering a drug that disrupts the methylation of the promoter of the Col1A2 gene.

[0018] In one embodiment, provided herein is a method for diagnosing a patient as having a disease, comprising contacting a cancer tissue obtained from a subject with the antibody of any one of the embodiments of the present invention, and detecting the binding of the antibody to the tissue, wherein the patient is diagnosed as having cancer or fibroid disease if the antibody binds to the tissue.In some aspects, the disease is cancer, fibroid disease, keloid, organ fibrosis, Crohn's disease, stenosis, colitis, psoriasis, or connective tissue disorder.In some aspects, the connective tissue disorder is collagen-related connective tissue disorder.In some aspects, the collagen-related connective tissue disorder is collagen type 1-related connective tissue disorder.

[0019] In one embodiment, the present application provides a method for classifying patients with pancreatic ductal adenocarcinoma, comprising determining the ratio of type I collagen / CK19 in cancer tissue obtained from subject, wherein the ratio is lower than that in reference normal tissue indicates that the patient has more advanced disease state.In some aspects, the reference normal tissue is obtained from patient.

[0020] In one embodiment, provided herein is a method for treating a subject with a disease, comprising administering an antitumor effective amount of a composition that inhibits the enzyme that crosslinks α1 type I collagen homotrimer.In one embodiment, provided herein is a method for treating a subject with a disease, comprising administering an antitumor effective amount of a composition that inhibits the chaperone that promotes the formation of α1 type I collagen homotrimer.In one embodiment, provided herein is a method for treating a subject with a disease, comprising administering an antitumor effective amount of a composition that inhibits the oncogenic signaling through DDR1 receptor.In some aspects, the subject is determined to express elevated levels of α1 homotrimeric type I collagen compared to control subjects.

[0021] In some aspects, the disease is cancer, fibroid disease, keloid, organ fibrosis, Crohn's disease, stenosis, colitis, psoriasis, or connective tissue disorder.In some aspects, the connective tissue disorder is collagen-related connective tissue disorder.In some aspects, the collagen-related connective tissue disorder is collagen type 1-related connective tissue disorder.

[0022] In some aspects, the disease is cancer. In certain aspects, the cancer is pancreatic cancer. In some aspects, the method is further defined as a method for inhibiting pancreatic cancer metastasis. In some aspects, the method is further defined as a method for inhibiting the growth of pancreatic cancer. In some aspects, the method further comprises administering at least a second anti-cancer therapy. In certain aspects, the second anti-cancer therapy is chemotherapy, immunotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or cytokine therapy.

[0023] As used herein, "essentially free" is used herein to mean that, in terms of the named component, none of the named components are intentionally formulated in the composition and / or are present merely as contaminants or in trace amounts.Thus, the total amount of the named components resulting from unintentional contaminants of the composition is significantly less than 0.05%, preferably less than 0.01%.Most preferred are compositions in which the amount of the named components cannot be detected using standard analytical methods.

[0024] As used herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0025] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or to mutually exclusive alternatives, but this disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0026] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being utilized to determine the value, the variability that exists among test subjects, or a value that is within 10% of the stated value.

[0027] [The present invention 1001] A composition comprising an antibody or antibody fragment that binds to α1 homotrimeric type I collagen. [The present invention 1002] 1001. The antibody or antibody fragment of the present invention, wherein the antibody has an affinity for α1 homotrimeric type I collagen that is at least 2-fold higher than its affinity for α1 / α2 / α1 heterotrimeric type I collagen. [The present invention 1003] 1001. The antibody or antibody fragment of the present invention, wherein the antibody has an affinity for α1 homotrimeric type I collagen that is at least 5-fold higher than its affinity for α1 / α2 / α1 heterotrimeric type I collagen. [The present invention 1004] 1001. The antibody or antibody fragment of the present invention, wherein the antibody does not detectably bind to α1 / α2 / α1 heterotrimeric type I collagen. [The present invention 1005] The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab') 2 The antibody or antibody fragment of the present invention, which is an Fv fragment or an Fv fragment. [The present invention 1006] The antibody or antibody fragment of the present invention, wherein the antibody is a chimeric antibody or a bispecific antibody. [The present invention 1007] The antibody or antibody fragment of the present invention, wherein the chimeric antibody is a humanized antibody. [The present invention 1008] The antibody or antibody fragment of the present invention 1006, wherein the bispecific antibody binds to both α1 homotrimeric type I collagen and CD3. [The present invention 1009] The antibody or antibody fragment of any of claims 1001 to 1008, which is conjugated to a cytotoxic agent. [The present invention 1010] The antibody or antibody fragment of any of claims 1001 to 1008, which is conjugated to a diagnostic agent. [The present invention 1011] A hybridoma or engineered cell encoding the antibody or antibody fragment of any of claims 1001 to 1010. [The present invention 1012] A pharmaceutical formulation comprising one or more antibodies or antibody fragments of any of claims 1001-1010. [The present invention 1013] A method of treating a patient in need thereof comprising administering an effective amount of an α1 homotrimeric type I collagen specific antibody or antibody fragment. [The present invention 1014] The method of claim 1013, wherein the patient has cancer, a fibroid disease, a keloid, organ fibrosis, Crohn's disease, strictures, colitis, psoriasis, or a connective tissue disorder. [The present invention 1015] The method of claim 1014, wherein the connective tissue disorder is a collagen-associated connective tissue disorder. [The present invention 1016] The method of claim 1015, wherein the collagen associated connective tissue disorder is a type 1 collagen associated connective tissue disorder. [The present invention 1017] The method of claim 10, wherein the patient has cancer. [The present invention 1018] The method of the present invention 1013, wherein the α1 homotrimeric type I collagen specific antibody or antibody fragment is any one of the antibodies or antibody fragments of the present invention 1001 to 1010. [The present invention 1019] The method of claim 1017, wherein the cancer patient is determined to express elevated levels of α1 homotrimeric type I collagen compared to a control patient. [The present invention 1020] The method of the present invention, wherein the cancer is pancreatic cancer. [The present invention 1021] The method of claim 1020, further defined as a method of inhibiting pancreatic cancer metastasis. [The present invention 1022] The method of claim 1020, further defined as a method of inhibiting the growth of pancreatic cancer. [The present invention 1023] The method of claim 1017, further comprising administering at least a second anti-cancer therapy. [The present invention 1024] The method of claim 1023, wherein the second anti-cancer therapy is chemotherapy, immunotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or cytokine therapy. [The present invention 1025] 1. 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 α1 homotrimeric type I collagen. [The present invention 1026] A polypeptide of the present invention 1025, wherein the antigen-binding domain comprises an HCDR sequence derived from a first antibody that binds to α1 homotrimeric type I collagen, and an LCDR sequence derived from a second antibody that binds to α1 homotrimeric type I collagen. [The present invention 1027] A polypeptide of the invention 1025, wherein the antigen-binding domain comprises HCDR and LCDR sequences derived from an antibody that binds to α1 homotrimeric type I collagen. [The present invention 1028] A polypeptide of the invention 1025, wherein the antigen-binding domain has an affinity for α1 homotrimeric type I collagen that is at least two-fold higher than its affinity for α1 / α2 / α1 heterotrimeric type I collagen. [The present invention 1029] A polypeptide of the invention 1025, wherein the antigen-binding domain has an affinity for α1 homotrimeric type I collagen that is at least 5-fold higher than its affinity for α1 / α2 / α1 heterotrimeric type I collagen. [The present invention 1030] A polypeptide of the invention 1025, wherein the antigen-binding domain does not detectably bind to α1 / α2 / α1 heterotrimeric type I collagen. [The present invention 1031] The polypeptide of the invention 1025, wherein the hinge domain is a CD8a hinge domain or an IgG4 hinge domain. [The present invention 1032] The polypeptide of the invention 1025, wherein the transmembrane domain is a CD8a transmembrane domain or a CD28 transmembrane domain. [The present invention 1033] The polypeptide of the present invention, wherein the intracellular signaling domain comprises a CD3z intracellular signaling domain. [The present invention 1034] A nucleic acid molecule encoding any one of the CAR polypeptides of the present invention 1025 to 1033. [The present invention 1035] The nucleic acid molecule of the present invention, wherein the sequence encoding a CAR polypeptide is operably linked to an expression control sequence. [The present invention 1036] An isolated immune effector cell comprising a CAR polypeptide of any one of claims 1025 to 1033 or a nucleic acid of claim 1035. [The present invention 1037] The cell of claim 1036, wherein the nucleic acid is integrated into the genome of the cell. [The present invention 1038] The cell of the present invention 1036, which is a T cell. [The present invention 1039] The cell of the present invention 1036, which is a NK cell. [The present invention 1040] The cell of the present invention 1036, which is a human cell. [The present invention 1041] A pharmaceutical composition comprising a population of cells of the present invention 1036 in a pharma- ceutically acceptable carrier. [The present invention 1042] A method for treating a subject, comprising administering an antitumor effective amount of chimeric antigen receptor (CAR) T cells expressing any one of the CAR polypeptides of the present invention 1025 to 1033. [The present invention 1043] The method of claim 1042, wherein the CAR T cells are allogeneic cells. [The present invention 1044] The method of claim 1042, wherein the CAR T cells are autologous cells. [The present invention 1045] The method of claim 1042, wherein the CAR T cells are HLA-matched to the subject. [The present invention 1046] The method of claim 1042, wherein the subject has cancer. [The present invention 1047] The method of claim 1046, wherein the cancer is pancreatic cancer. [The present invention 1048] Any of the methods of claims 1042 to 1047, further comprising administering a demethylating agent prior to administration of the CAR T cells. [The present invention 1049] The method of claim 1048, wherein the demethylating agent reverses Col1A2 hypermethylation. [The present invention 1050] The method of claim 1048, wherein the demethylating agent is 5-azacytidine or 5-aza-2'-deoxycytidine. [The present invention 1051] A method for treating a subject, comprising administering an antitumor effective amount of chimeric antigen receptor (CAR) NK cells expressing any one of the CAR polypeptides of the present invention 1025 to 1033. [The present invention 1052] The method of claim 1051, wherein the CAR NK cells are allogeneic cells. [The present invention 1053] The method of claim 1051, wherein the CAR NK cells are autologous cells. [The present invention 1054] The method of claim 1051, wherein the CAR NK cells are HLA-matched to the subject. [The present invention 1055] The method of claim 1051, wherein the subject has cancer. [The present invention 1056] The method of claim 1055, wherein the cancer is pancreatic cancer. [The present invention 1057] Any of the methods of claims 1051 to 1056, further comprising administering a demethylating agent prior to administration of the CAR NK cells. [The present invention 1058] The method of claim 1057, wherein the demethylating agent reverses Col1A2 hypermethylation. [The present invention 1059] The method of claim 1057, wherein the demethylating agent is 5-azacytidine or 5-aza-2'-deoxycytidine. [The present invention 1060] contacting a cancer tissue obtained from a subject with any one of the antibodies of the present invention 1001 to 1010, and detecting binding of the antibody to the tissue. wherein if the antibody binds to the tissue, the patient is diagnosed as having cancer or a fibroid disease. [The present invention 1061] The method of claim 1060, wherein the disease is cancer, a fibroid disease, a keloid, an organ fibrosis, Crohn's disease, a stricture, colitis, psoriasis, or a connective tissue disorder. [The present invention 1062] The method of claim 1061, wherein the connective tissue disorder is a collagen-associated connective tissue disorder. [The present invention 1063] The method of claim 1062, wherein the collagen associated connective tissue disorder is a type 1 collagen associated connective tissue disorder. [The present invention 1064] 1. A method for classifying a patient having pancreatic ductal adenocarcinoma, comprising determining a type I collagen / CK19 ratio in cancer tissue obtained from a subject, wherein a ratio lower than the ratio in a reference normal tissue indicates that the patient has a more advanced disease state. [The present invention 1065] The method of claim 1064, wherein the reference normal tissue is obtained from the patient. [The present invention 1066] A method for treating a subject having a disease, comprising administering an antitumor effective amount of a composition that inhibits an enzyme that crosslinks α1 type I collagen homotrimer. [The present invention 1067] A method of treating a subject having a disease, comprising administering an antitumor effective amount of a composition that inhibits a chaperone that promotes the formation of α1 type I collagen homotrimer. [The present invention 1068] A method of treating a subject having a disease, comprising administering an antitumor effective amount of a composition that inhibits pro-oncogenic signaling via the DDR1 receptor. [The present invention 1069] The method according to any one of claims 1066 to 1068, wherein the disease is cancer, a fibroid disease, a keloid, an organ fibrosis, Crohn's disease, stenosis, colitis, psoriasis, or a connective tissue disorder. [The present invention 1070] The method of claim 1069, wherein the connective tissue disorder is a collagen-associated connective tissue disorder. [The present invention 1071] The method of claim 1070, wherein the collagen associated connective tissue disorder is a type 1 collagen associated connective tissue disorder. [The present invention 1072] The method of claim 1070, wherein the disease is cancer. [The present invention 1073] The method of any of claims 1068 to 1072, wherein the subject is determined to express elevated levels of α1 homotrimeric type I collagen compared to a control subject. [The present invention 1074] The method of claim 1072, wherein the cancer is pancreatic cancer. [The present invention 1075] The method of any one of claims 10 to 15, further defined as a method of inhibiting pancreatic cancer metastasis. [The present invention 1076] The method of claim 1074, further defined as a method of inhibiting the growth of pancreatic cancer. [The present invention 1077] The method of claim 1072, further comprising administering at least a second anti-cancer therapy. [The present invention 1078] The method of claim 1077, wherein the second anti-cancer therapy is chemotherapy, immunotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or cytokine therapy. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of example only, since various modifications and changes within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0028] The drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1A] A genetic strategy to delete type I collagen α1 (Col1a1) specifically in αSMA-expressing cell populations in the context of pancreatic cancer using KPPF;αSMA-Cre;Col1a1loxP / loxP (referred to as KPPF;Col1smaKO) mice. Control mice were littermates with the KPPF (KPPF;Cre-negative;Col1a1loxP / loxP) genotype. [Figure 1B] Serial sections of pancreatic tumors from KPPF or KPPF;Col1smaKO mice stained with hematoxylin and eosin (H&E), picrosirius red, MTS, Col1 (immunohistochemistry), and αSMA (immunohistochemistry). [Figure 2A]Representative atomic force microscopy (AFM) images of frozen sections of KPPF and KPPF;Col1smaKO tumors. Quantification of elastic modulus was based on AFM assays of three mice per group. [Figure 2B] Survival curves of KPPF and KPPF;Col1smaKO mice. [Figure 2C] Percentage of mice exhibiting abdominal distension and ascites in the indicated groups. [Figure 2D] Serial sections of pancreata from KPPF or KPPF;Col1smaKO mice at the PanIN or PDAC stage stained with H&E, CK19 (immunohistochemistry), and Col1 (immunohistochemistry). Quantification of Col1 / CK19 ratio was calculated based on the positive area percentage of CK19 and Col1. [Figure 2E] GSEA-Hallmark enrichment analysis of the most significantly upregulated cell signaling pathways, shown as normalized enrichment scores (NES), based on GSEA-Hallmark enrichment analysis of whole-transcriptome RNA sequencing (RNA-Seq) data from KPPF tumors (n = 3 mice per group) or KPPF;Col1smaKO tumors (n = 4 mice per group). [Figure 2F] Q-PCR analysis of Col1α1 in MF. [Figure 3A] Genetic strategy to induce oncogenic KrasG12D using the Pdx1-Flp-FRT recombination system in KF (FSF-KrasG12D / +;Pdx1-Flp) mice. Type I collagen alpha 1 (Col1a1) was specifically deleted in αSMA-expressing cell populations using KF;αSMA-Cre;Col1a1loxP / loxP (referred to as KF;Col1smaKO) mice (see FIG. 10A) or in Pdx1-expressing cancer cell lines using KF;Pdx1-Cre;Col1a1loxP / loxP (referred to as KF;Col1pdxKO) mice. [Figure 3B]Serial sections of pancreata from KF or KF;Col1pdxKO (age-matched 6-month-old) mice stained with H&E and Col1 (immunohistochemistry). [Figure 3C] Percentage of ADM and PanIN lesions in pancreata from age-matched 6-month-old mice with KF (left column), KF;Col1smaKO (right column) or KF;Col1pdxKO (middle column) genotypes. [Figure 3D] Col1 immunohistochemical staining in ADM lesions of KF (left column) and KF;Col1pdxKO (right column) mice. [Figure 3E] Figure 3E and F: Sox9 positivity (%) in ADM and PanIN lesions of KF (left column) and KF;Col1pdxKO (right column) mice (Figure 3E). Representative images of Sox9 immunohistochemical staining are shown in (Figure 3F). [Figure 3F] See legend to Figure 3E. [Figure 4A] A genetic strategy to delete type I collagen alpha 1 (Col1a1) in cancer cell lines in the context of pancreatic cancer using LSL-KrasG12D;Trp53loxP / loxP;Pdx1-Cre;Col1a1loxP / loxP (KPPC;referred to as Col1pdxKO) mice. LSL-KrasG12D;Trp53loxP / loxP;Pdx1-Cre (KPPC) mice served as control animals. [Figure 4B] Survival of KPPC (lower line at day 53) and KPPC;Col1pdxKO (upper line at day 53) mice. [Figure 4C] Percentage of PanIN lesion areas in the same 28-day-old KPPC (left column) and KPPC;Col1pdxKO (right column) mice. [Figure 4D] Serial sections of pancreatic tumor sections from the same 53-day-old KPPC (left column) and KPPC;Col1pdxKO (right column) mice stained with hematoxylin and eosin (H&E), Col1 (immunohistochemistry), and picrosirius red. [Figure 4E]Histological evaluation of tumors in the same 53-day-old KPPC and KPPC;Col1pdxKO mice. [Figure 4F] Pancreatic tumor burden (tumor weight / body weight) in the same 53-day-old KPPC (left column) and KPPC;Col1pdxKO (right column) mice. [Figure 5A] Figure 5A-D: Whole-transcriptome RNA sequencing (RNA-Seq) analysis was performed on KPPC tumors (n = 4) and KPPC;Col1pdxKO tumors (n = 5). GSEA plots were shown with normalized enrichment scores (NES) of upregulated gene clusters based on GSEA-Hallmark enrichment analysis of KPPC;Col1pdxKO tumors (Figure 5A) and KPPC tumors (Figure 5B) as summarized in (Figure 5C). The top upregulated genes were listed in (Figure 5D). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] Top upregulated gene networks identified based on transcripts enriched in KPPC;Col1pdxKO and KPPC tumors. [Figure 5F] Figure 5F-I: Whole-transcriptome RNA sequencing (RNA-Seq) analysis was performed on KPPC and KPPC;Col1pdxKO cell lines. GSEA plots were shown with normalized enrichment scores (NES) of upregulated gene clusters based on GSEA-Hallmark analysis of KPPC;Col1pdxKO cells (Figure 5F) and KPPC cells (Figure 5G) as summarized in (Figure 5H). The top upregulated genes were listed in (Figure 5I). [Figure 5G] See legend to Figure 5F. [Figure 5H] See legend to Figure 5F. [Figure 5I] See legend to Figure 5F. [Figure 6A] KPPC and KPPC;primary mouse cancer cell lines established from Col1pdxKO tumors. [Figure 6B] Cell proliferation of KPPC (upper line) and KPPC;Col1pdxKO (lower line) cell lines over time. Cell viability of KPPC and KPPC;Col1pdxKO cell lines in the presence of various concentrations of gemcitabine. [Figure 6C] Figure 6C and D: 3D tumor spheroids established from KPPC and KPPC;Col1pdxKO cell lines. The average diameter of spheroids from KPPC (left column) and KPPC;Col1pdxKO (right column) cell lines was quantified in (Figure 6D). [Figure 6D] See legend to Figure 6C. [Figure 6E] Gene expression profiles of different collagen types in KPPC (left column of each pair) and KPPC;Col1pdxKO (right column of each pair) cell lines examined by qRT-PCR. [Figure 6F] Methylated DNA immunoprecipitation (MeDIP) assay of Col1a1 and Col1a2 genes in primary mouse cancer cell lines established from pancreatic tumors of transgenic mouse models including KF, KPF, KPPF, KPPC, KTC, and PKT lines compared to 3T3 mouse fibroblasts. Relative expression levels of Col1a1 and Col1a2 in KPPC primary mouse cancer cell line compared to primary mouse fibroblasts sorted from KPPC tumors. Characterization of Col1α1 and Col1α2 chains of Col1 homotrimers and heterotrimers purified from cell culture medium of KPPC cancer cells, KPPC;Col1pdxKO cells, and 3T3 fibroblasts, respectively. The susceptibility of Col1 homotrimers and heterotrimers to MMP degradation was examined. [Figure 6G] Genome-wide DNA methylation analysis of human pancreatic cancer cell lines and normal human pancreatic epithelial cell lines (HPNE). DNA methylation was demonstrated in the promoter regions of the COL1A1 and COL1A2 genes. [Figure 6H] qRT-PCR examination of COL1A1 (left column of each pair) and COL1A2 (right column of each pair) genes in human pancreatic cancer cell lines compared with BJ fibroblasts. [Figure 7A] A genetic strategy to induce oncogenic KrasG12D and homozygous p53 loss using the Pdx1-Flp-FRT recombination system in KPPF (FSF-KrasG12D / +;Trp53frt / frt;Pdx1-Flp) mice. [Figure 7B] Representative pancreatic sections of normal, PanIN, and PDAC stages from KPPF mice stained by hematoxylin and eosin (H&E) and collagen type I (Col1) immunohistochemistry. [Figure 7C] A genetic strategy to induce oncogenic KrasG12D and homozygous p53 loss using the Pdx1-Cre-loxP recombination system in KPPC (LSL-KrasG12D;Trp53loxP / loxP;Pdx1-Cre) mice. [Figure 7D] Representative pancreatic sections of normal, PanIN, and PDAC stages of KPPC mice stained by hematoxylin and eosin (H&E) and collagen type I (Col1) immunohistochemistry. [Figure 7E] Figure 7E-F: Genetic strategy to induce EGFP expression in Pdx1-Flp lineage and tdTomato expression in αSMA-Cre lineage using Rosa26-CAG-loxP-frt-Stop-frt-FireflyLuc-EGFP-loxP-RenillaLuc-tdTomato (R26Dual) tracer in KPPF;αSMA-Cre;R26Dual mice. [Figure 7F] See legend to Figure 7E. [Figure 7G] Representative images of primary PDAC tumors from KPPF;αSMA-Cre;R26Dual mice examined for endogenous EGFP (in cancer cells) and tdTomato (in αSMA-expressing myofibroblasts) signals. [Figure 7H]Electrophoresis of PCR products of DNA from primary cell cultures of selected cancer cells and myofibroblasts from KPPF or KPPF;Col1smaKO mice. Detection of PCR products confirmed the specific deletion of Col1a1 by genetic recombination, specifically in myofibroblasts from KPPF;Col1smaKO mice, as shown by the expected lanes. [Figure 7I] Systemic loss of Col1a1 using CMV-Cre results in embryonic lethality. [Figure 7J] Pancreatic H&E and quantification of ADM and PanIN in the indicated groups (KF; Col1pdxKO is the left column; KF; Creneg; Col1F / F is the middle column; KF; Col1smaKO is the right column). [Figure 8A] Figures 8A and B: Serial sections of pancreata from KPPF mice during disease progression from ADM / early PanIN to PanIN (Figure 8A) or from PanIN to PDAC (Figure 8B) stained by H&E, CK19, Col1, and αSMA immunohistochemistry. [Figure 8B] See legend to Figure 8A. [Figure 8C] Quantification of CK19, Col1, and αSMA positive area rates, or Col1 / CK19 ratios, at each stage of disease progression. [Figure 9-1] Overall survival (OS) and progression-free survival (PFS) of pancreatic adenocarcinoma patients from the TCGA dataset were correlated with the ratio of COL1A1 and CK19 expression levels (RNA-Seq V2 RSEM). Patients were stratified into two groups based on the median COL1A1 / CK19 ratio (or, in control panels, by the COL1A1 / GAPDH or COL1A1 / ACTB ratios). [Figure 9-2] This is a continuation of Figure 9-1. [Figure 10A]A genetic strategy to delete type I collagen α1 (Col1a1) specifically in αSMA-expressing cell populations in the context of pancreatic cancer using KF;αSMA-Cre;Col1a1loxP / loxP (referred to as KF;Col1smaKO) mice. Littermates with the KF (KF;Cre-negative;Col1a1loxP / loxP) genotype served as control mice. [Figure 10B] Serial sections of pancreata from KF or KF;Col1smaKO (age-matched 6-month-old) mice stained with H&E, MTS, Col1 (immunohistochemistry), and αSMA (immunohistochemistry). [Figure 11A] A genetic strategy to delete type I collagen alpha 1 (Col1a1) in cancer cell lines in the context of pancreatic cancer using LSL-KrasG12D;Pdx1-Cre;Col1a1loxP / loxP (KC;referred to as Col1pdxKO) mice. LSL-KrasG12D;Pdx1-Cre (KC) mice were used as control animals. [Figure 11B] Serial sections of pancreatic tumor sections from KC or KC;Col1pdxKO mice stained with hematoxylin and eosin (H&E), picrosirius red, MTS, Col1 (immunohistochemistry), or αSMA (immunohistochemistry). [Figure 12A] Survival of KPPC (lower line at day 60), KPPC;Col1pdxKO / + (heterozygous Col1a1 deletion) (middle line at day 60), and KPPC;Col1pdxKO (upper line at day 60) mice. [Figure 12B] Serial sections of pancreatic tumor sections from endpoint stage KPPC and KPPC;Col1pdxKO mice stained with hematoxylin and eosin (H&E), Col1 (immunohistochemistry), and picrosirius red. [Figure 12C] MeDIP assay of COL1A1 and COL1A2 genes in various human pancreatic cancer cell lines compared with BJ fibroblasts. [Figure 12D]Cell viability assay of KPPC and KPPC;Col1pdxKO cells treated with the indicated concentrations of Col1 solution (heterotrimer from rat tail) for 48 h. [Figure 13] (Right panel) Relative expression levels of Col1a1 and Col1a2 in KPPC cancer cells, KPPC;Col1pdxKO cancer cells, and 3T3 mouse fibroblasts treated with the demethylating agent 5-azacytidine (5-AZA). (Left panel) Characterization of the Col1α1 and Col1α2 chains of purified Col1 homotrimers (from the Panc1 human PDAC cell line) and heterotrimers (from the BJ fibroblast cell line) by Western blot. [Figure 14A] A genetic strategy to specifically delete type I collagen alpha 1 (Col1a1) in the context of pancreatic cancer using KPPF;Fsp1-Cre;Col1a1loxP / loxP (referred to as KPPF;Col1fspKO) mice. Littermates with the KPPF (KPPF;Cre-negative;Col1a1loxP / loxP) genotype served as control mice. [Figure 14B] Survival of KPPF and KPPF;Col1fspKO mice. [Figure 14C] Relative expression levels of Col1a1 in Fsp1 antibody-sorted fibroblasts from KPPF and KPPF;Col1fspKO tumors. These fibroblasts were also examined by recombination PCR detection to confirm specific deletion of Col1a1 by genetic recombination, specifically in myofibroblasts from KPPF;Col1fspKO mice, as shown by the expected lanes. [Figure 14D] Serial sections of pancreatic tumor sections from KPPC and KPPC;Col1fspKO mice stained with hematoxylin and eosin (H&E), Col1 (immunohistochemistry), and picrosirius red. [Figure 15A] Genetic strategy to induce EGFP expression in the Pdx1-Flp line and tdTomato expression in the Fsp1-Cre line using the R26Dual tracer in KPPF;Fsp1-Cre;R26Dual mice. [Figure 15B] Representative images of Fsp1-induced endogenous tdTomato and αSMA immunofluorescence staining among fibroblasts in primary tumors from KPPF;Fsp1-Cre;R26Dual mice. [Figure 15C] Representative images of Fsp1 and αSMA immunofluorescence staining of primary tumors from KPPF; Cre-negative; R26Dual mice, which have EGFP expression but no tdTomato expression in cancer cells of the Pdx-Flp lineage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description Tumors contain both cancer cells and components of the tumor microenvironment (TME), such as fibroblasts and type I collagen. It remains unclear whether the tumor microenvironment functions as a promoter or suppressor of tumor growth. It is possible that some aspects of the TME may function as positive regulators of tumor progression, while others may function as negative regulators of tumor growth. Type I collagen (collagen I), produced by myofibroblasts, is a heterotrimer with two collagen Iα1 chains (α1(I) collagen) and one collagen Iα2 chain (α2(I) collagen), which is cancer / tumor suppressive through binding to potential receptors on cancer cells as well as other stromal cells (likely discogen domain receptor II-DDR2) and immune cells. In contrast, cancer cells produce collagen I homotrimers with three α1(I) collagen chains, which are cancer / tumor-promoting and bind to specific receptors in cancer cells, such as discogen domain receptor 1 (DDR1), inducing pro-survival, anti-apoptotic, pro-proliferative, and pro-oncogenic signals. Homotrimers (made by cancer cells) are resistant to metalloproteinases and other proteinases when compared to heterotrimers made by myofibroblasts in the tumor microenvironment. Homotrimers exhibit a different structure with different epitope exposure compared to heterotrimers, and antibodies raised against homotrimers have tumor-inhibitory properties by interfering with signaling through pro-oncogenic receptors on cancer cells, among other mechanisms. DDR1 blockade leading to specific inhibition of homotrimers to DDR1 leads to suppression of cancer progression and induces anti-survival, anti-apoptotic, anti-proliferative, and anti-oncogenic signals.

[0030] Fibrosis and prominent deposition of extracellular matrix (ECM) such as type I collagen (Col1) are defining features of pancreatic ductal adenocarcinoma (PDAC). However, the specific role of Col1, one of the most abundant proteins in PDAC, remains controversial. Herein, we used a next-generation dual recombinase system (DRS) to identify and characterize the Kras1 gene in mice. G12DWe achieved genetic ablation of Col1α1 specifically in myofibroblasts or cancer cells in the context of driving spontaneous PDAC. Interestingly, Col1 deletion in myofibroblasts expressing α-smooth muscle actin (αSMA) resulted in accelerated PDAC progression and animal death, whereas Col1α1 deletion in cancer cells of the Pdx1 lineage led to attenuation of PDAC development and prolonged survival. Cancer-derived Col1 was a unique homotrimer (α1)3 in contrast to the Col1 heterotrimer (α1 / α2 / α1) produced by fibroblasts. These different structures of Col1 (homotrimer vs. heterotrimer) led to distinct behaviors of cancer cells.

[0031] Classical KPC (LSL-Kras G12D / + ;Trp53 R172H / + or Trp53 loxP / loxP Current genetically engineered PDAC mouse models (GEMMs), such as the Pdx1-Cre (Pdx1-Cre) model, provide a valuable platform that mimics the clinical situation of human PDAC and have made significant contributions to the study of PDAC and its treatment (Hingorani et al., 2005). Conventional KPC models are widely used in combination with genetic ablation (using the same pancreatic-specific Cre, such as Pdx1-Cre or P48-Cre) of floxed (loxP-site-flanked) genes in cancer cells or with whole-body knockout (KO) of genes. However, due to the universal Cre-loxP recombination mechanism, it is still not possible to achieve cell-type-specific genetic manipulation in stromal cell subpopulations (such as myofibroblasts or immune cells) in these GEMMs. It is also not possible to establish KPC models involving whole-body KO of genes with KO lethality, for example, Col1a1, which encodes the type I collagen α1 chain (Lohler et al., 1984). Thus, there is a lack of PDAC GEMMs that would allow functional KO of Col1 in stromal cell sources to test the origin and contribution of Col1, especially considering that Col1 is such an essential component and the most abundant protein in PDAC desmoplasia and microenvironment.

[0032] To overcome such limitations in PDAC GEMMs, a next-generation dual recombinase system integrating both the Cre-loxP and Flp-FRT systems has recently been developed (Schonhuber et al., 2014). For the first time, this DRS allows deletion of Col1 in PDAC to functionally elucidate the specific role of Col1 produced by specific cell populations, e.g., myofibroblasts or cancer cells, in the context of oncogenic Kras-driven PDAC.

[0033] Col1α1 was chosen as the target for genetic ablation of Col1 because Col1α2 alone is incapable of generating any form of Col1 fiber (regardless of homotrimer or heterotrimer), and Col1α1 is essential for all Col1 fibers. DRS specifically targets αSMA lineage-activated PSCs (FSF-Kras G12D / + ;Pdx1-Flp;αSMA-Cre;Col1a1 loxP / loxP ) or Pdx1 lineage cancer cells (FSF-Kras G12D / + ;Pdx1-Flp;Pdx1-Cre;Col1a1 loxP / loxP ) to achieve genetic ablation of Col1. In parallel, Col1 was used to ablate a more acute DRS model (FSF-Kras) carrying homozygous p53 loss in αSMA lineage-activated PSCs. G12D / + ;Trp53 frt / frt ;Pdx1-Flp;αSMA-Cre;Col1a1 loxP / loxP ) and the conventional Cre-loxP-based KPC model (LSL-Kras ) with homozygous p53 loss in Pdx1 lineage cancer cells. G12D / + ;Trp53 loxP / loxP ;Pdx1-Cre;Col1a1 loxP / loxP) were used to deplete Col1. Direct comparison of the phenotypes of these PDAC GEMMs confirmed distinct contributions of Col1 in cell source and PDAC microenvironment. PanIN / PDAC development was accelerated by genetic ablation of Col1 in αSMA-lineage activated PSCs but delayed by Col1 ablation in Pdx1-lineage cancer cells. These results highlight the tumor-suppressive function of activated PSC-derived Col1 and the tumor-protective function of cancer cell-derived Col1.

[0034] Using pancreatic cancer as an example, it has been shown that pathogenic collagens are produced by cancer cells but not myofibroblasts. The collagen I produced by cancer cells is a variant called α1 homotrimer, whereas the collagen I produced by myofibroblasts is non-pathogenic and helps suppress PDAC, being an α1 / α2 / α1 heterotrimer. The homotrimer is resistant to proteases and enzymes and remains around the cancer cells, aiding their proliferation and invasion. The homotrimer binds to receptors, such as DDR1, to induce pro-survival and pro-oncogenic signals. Inhibition of DDR1 and its homotrimer formation or its ability to induce pro-oncogenic signals by small molecules or antibodies leads to the control of PDAC and suppression of tumor growth. Disruption of chaperones in cancer cells that disrupt homotrimer formation also controls PDAC. Inhibition of collagen I cross-linking enzymes specific for homotrimers can be used to control tumor growth. The generation of α1(I) collagen homotrimer-specific CAR-T constructs in autologous T cells or autologous or allogeneic NK cells as an immunotherapeutic approach will lead to eradication of early and late stage pancreatic tumors. The generation of bispecific antibodies targeting α1(I) collagen homotrimer through one arm and CD3 through the other arm will lead to immune targeting by T cells and killing of cancer cells.

[0035] I. Antibodies and Their Production An "isolated antibody" is one that has been separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would 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 the antibody as determined by the Lowry method, most preferably greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a spin cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue or silver staining. Isolated antibodies include antibodies in situ in recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0036] 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 therefore contain ten antigen-binding sites, whereas secretory IgA antibodies can polymerize to form polyvalent assemblies containing two to five basic four-chain units and a J chain. For 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 intrachain disulfide bridges. Each H chain contains a variable domain (V) at the N-terminus. H ), followed by three constant domains (C H ), μ and isotype have four C H Each L chain has a variable domain (V L ), followed by a constant domain (C L) V L is V H It is aligned with C L is the first constant domain of the heavy chain (C H1 ) and V. Certain amino acid residues are believed to form an interface between the light- and heavy-chain variable domains. H and V L The pairing of these together forms a single antigen-binding site. For the structure and properties of different antibody classes, see, e.g., 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.

[0037] The L chain from any vertebrate species comprises its constant domain (C L Depending on the amino acid sequence of the heavy chain constant domain (C), they can be assigned to one of two clearly distinct types, called κ and λ. H Depending on the amino acid sequence of the C-terminal tail, immunoglobulins can be assigned to various classes or isotypes. There are five immunoglobulin classes, IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are H They are further divided into subclasses based on relatively minor differences in sequence and function, with humans expressing the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0038] The term "variable" refers to the extensive sequence differences between antibodies in certain segments of the V domain. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the 110 amino acid span of the variable region. Instead, the V region consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter highly variable regions called "hypervariable regions" each 9-12 amino acids in length. Naturally occurring heavy and light chain variable regions each contain four FRs that are largely in a β-sheet configuration, connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as the participation of antibodies in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).

[0039] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are involved in antigen binding. A hypervariable region generally consists of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., the V L Approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in Hat about residue numbers 31-35 (H1), 50-65 (H2), and 95-102 (H3); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and / or residues from the "hypervariable loops" (e.g., residues around V when numbered according to the Chothia numbering system). L Residue numbers 24–34 (L1), 50–56 (L2), and 89–97 (L3), as well as V H residues numbered 26-32 (H1), 52-56 (H2) and 95-101 (H3); Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and / or residues from the "hypervariable loops" / CDRs (e.g., V when numbered according to the IMGT numbering system). L Residue numbers 27–38 (L1), 56–65 (L2), and 105–120 (L3), as well as V H Optionally, the antibody comprises the following residues when numbered according to AHo; Honneger, A. and Plunkthun, AJ Mol. Biol. 309:657-670 (2001): 27-38 (H1), 56-65 (H2) and 105-120 (H3); Lefranc, MP et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:219-221 (2000)). L 28, 36 (L1), 63, 74–75 (L2), and 123 (L3) in the middle, and V sub It may have symmetric insertions in H at one or more of positions 28, 36 (H1), 63, 74-75 (H2) and 123 (H3).

[0040] "Germline nucleic acid residue" refers to a nucleic acid residue that occurs naturally in a germline gene that encodes a constant or variable region. A "germline gene" is DNA found in a germ cell (i.e., a cell destined to become an egg or a sperm). A "germline mutation" refers to an inherited change in a particular DNA that occurs in a germ cell or a zygote at the single-cell stage, and when passed on to offspring, such a mutation is incorporated into all cells of the body. A germline mutation is in contrast to a somatic mutation, which is acquired by a single body cell. In some instances, a nucleotide in a germline DNA sequence that encodes a variable region is mutated (i.e., a somatic mutation) and replaced with a different nucleotide.

[0041] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible minor natural mutations. Monoclonal antibodies are directed against a single antigenic site and are highly specific. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or can be produced using recombinant DNA methods in bacteria, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567) after single-cell sorting of antigen-specific B cells, antigen-specific plasmablasts in response to infection or immunization, or capture of linked heavy and light chains from single cells in bulk-sorted antigen-specific collections. "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0042] A. General method It will be understood that monoclonal antibodies that bind to homotrimeric collagen type I have several uses. These include the detection and diagnosis of cancer, and the creation of diagnostic kits for use in the treatment of cancer. In these situations, such antibodies can be associated with diagnostic or therapeutic agents, used as capture agents or competitors in competitive assays, or used individually without additional agents attached. Antibodies can 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).

[0043] Methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step in both of these methods is the immunization of a suitable host, or the identification of a subject that is immune through previous natural infection or vaccination with an approved or experimental vaccine. As is well known in the art, a given composition for immunization may vary in its immunogenicity. Therefore, it is often necessary to boost the host immune system, as can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for coupling polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine. As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of immune response, known as adjuvants. Exemplary and preferred adjuvants in animals include complete Freund's adjuvant (a non-specific stimulator of immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant and aluminum hydroxide adjuvant, and in humans include alum, CpG, MFP59 and combinations of immune stimulating molecules ("Adjuvant Systems" such as AS01 or AS03). Additional experimental inoculation forms for inducing cancer-specific B cells are possible, including nanoparticle vaccines, or genetically encoded antigens delivered as DNA or RNA genes in physical delivery systems (e.g., on lipid nanoparticles or gold biolistic beads), delivered by needles, gene guns, transdermal electroporation devices.The antigen gene can also be carried so as to be encoded by a replication-competent or defective viral vector, such as an adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or a virus-like particle.

[0044] The amount of immunogen composition used in the production of polyclonal antibodies varies depending on the nature of the immunogen and the animal used for immunization. Various routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animal at various times after immunization. A second booster injection can also be given. The process of boosting and titering is repeated until a suitable titer is reached. Once a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and / or the animal can be used to generate MAbs.

[0045] After immunization, somatic cells capable of producing antibodies, specifically B lymphocytes (B cells), are selected for use in MAb generation protocols. These cells can be obtained from biopsied spleens, lymph nodes, tonsils or adenoids, bone marrow aspirates or biopsies, tissue biopsies from mucosal organs such as lungs or gastrointestinal tract, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal or immunized human are then fused with immortal myeloma cells, generally immortal myeloma cells of the same species as the immunized animal, or immortal myeloma cells of human cells or human / mouse chimeric cells. Myeloma cell lines suitable for use in fusion procedures to produce hybridomas preferably do not produce antibodies, have high fusion efficiency, and enzyme deficiencies that make them unable to grow in certain selective media that support the growth of only the desired fused cells (hybridomas). As known to those skilled in the art, any one of several myeloma cells can be used. HMMA2.5 cells or MFP-2 cells are particularly useful examples of such cells.

[0046] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually involve mixing somatic cells and myeloma cells in a 2:1 ratio, but the ratio can vary from about 20:1 to about 1:1, respectively, in the presence of agents (chemical or electrical) that promote cell membrane fusion. In some cases, transformation of human B cells with Epstein-Barr virus (EBV) as an initial step increases the size of the B cells and enhances fusion with myeloma cells of relatively large size. Transformation efficiency by EBV is enhanced by using CpG and Chk2 inhibitor drugs in the transformation medium. Alternatively, human B cells can be activated by co-culturing with a transfected cell line expressing CD40 ligand (CD154) in a medium containing additional soluble factors, such as IL-21 and human B cell activating factor (BAFF), a type II member of the TNF superfamily. Fusion methods using Sendai virus and fusion methods using polyethylene glycol (PEG), such as 37% (v / v) PEG, have been described. The use of electrically induced fusion methods is also suitable and has processes for better efficiency. The fusion procedure usually produces approximately 1 × 10 -6 From 1×10 -8Although viable hybrids are produced at a low frequency of 100-fold, with optimized procedures, fusion efficiencies approaching 200-fold can be achieved. However, the relatively low efficiency of fusion is not a problem, since viable fused hybrids are differentiated from the parent injected cells (especially the injected myeloma cells, which usually continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally a medium containing an agent that blocks the de novo synthesis of nucleotides in tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block the de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. When aminopterin or methotrexate are used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). When azaserine is used, the medium is supplemented with hypoxanthine. Ouabain is added when the B cell source is an EBV-transformed human B cell line to eliminate EBV-transformed lines that are not fused to a myeloma.

[0047] The preferred selection medium is HAT or HAT with ouabain. In HAT medium, only cells capable of activating the nucleotide salvage pathway can survive. Myeloma cells are defective in key enzymes of the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and cannot survive. B cells can activate this pathway, but have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selection medium are hybrids formed from myeloma cells and B cells. If the source of B cells used for fusion is a line of EBV-transformed B cells, as herein, ouabain can also be used for drug selection of the hybrids, since EBV-transformed B cells are sensitive to drug killing. In contrast, the myeloma partner used is selected for resistance to ouabain.

[0048] Culturing results in a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is performed by culturing the cells by single clone dilution in microtiter plates, followed by testing (after about 2-3 weeks) of individual clonal supernatants for the desired reactivity. The assay should be sensitive, simple, and rapid, e.g., radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunobinding assay, etc. The selected hybridomas are then serially diluted or single cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines. The clones can then be propagated indefinitely to provide mAbs. These cell lines can be utilized for MAb production in two basic ways. Hybridoma samples can be injected (often intraperitoneally) into animals (e.g., mice). Optionally, prior to injection, the animals can be primed with a hydrocarbon, particularly an oil, e.g., pristane (tetramethylpentadecane). When human hybridomas are used in this way, they are best injected into immunodeficient mice, e.g., SCID mice, to prevent tumor rejection. The injected animals develop tumors that secrete the specific monoclonal antibodies produced by the fused cell hybrids. The animal's body fluids, such as serum or ascites fluid, can then be tapped to obtain high concentrations of MAbs. Individual cell lines can also be cultured in vitro, where MAbs are naturally secreted into the culture medium and are readily available in high concentrations from the culture medium. 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 highly pure human monoclonal immunoglobulins.

[0049] MAbs produced by either means can be further purified, if desired, using filtration, centrifugation and various chromatographic methods, such as 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 by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0050] It is also contemplated that molecular cloning approaches can be used to generate monoclonal antibodies. Single B cells labeled with the antigen of interest can be physically selected using paramagnetic bead selection or flow cytometric sorting, then RNA can be isolated from the single cells, and antibody genes can be amplified by RT-PCR. Alternatively, antigen-specific bulk-sorted cell populations can be separated into microvesicles, and matched heavy and light chain variable genes can be retrieved from single cells using physical linking of heavy and light chain amplicons, or common barcoding of heavy and light chain genes from vesicles. Matched heavy and light chain genes from single cells can also be obtained from a population of antigen-specific B cells by treating cells with cell-permeable nanoparticles carrying RT-PCR primers and with barcodes to label transcripts with one barcode per cell. Antibody variable genes can also be isolated by RNA extraction of hybridoma lines, and antibody genes can be obtained by RT-PCR and cloned into immunoglobulin expression vectors. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from cell lines and phagemids expressing appropriate antibodies are selected by panning with viral antigens. The advantage of this approach over traditional hybridoma techniques is that it is approximately 10 4The advantages of this approach are that twice as many antibodies can be produced and screened in one go, and that combinations of heavy and light chains can generate new specificities, further increasing the chances of finding the right antibody.

[0051] Other U.S. patents, each of which is incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using combinatorial approaches; U.S. Pat. No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Pat. No. 4,867,973, which describes antibody-therapeutic agent conjugates.

[0052] B. Antibodies of the Present Disclosure An antibody according to the present disclosure can be defined first and foremost by its binding specificity. A person skilled in the art can determine whether a given antibody is within the scope of the claims by evaluating the binding specificity / affinity of the given antibody using techniques well known to those skilled in the art. For example, the epitope to which a given antibody binds can consist of a single continuous sequence of three 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 an antigen molecule (e.g., a linear epitope in a domain). Alternatively, an epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) located within an antigen molecule (e.g., a conformational epitope).

[0053] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" in 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, NY). Cross-blocking can be measured with various binding assays, such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide truncation analysis, high-resolution electron microscopy with single particle reconstruction, cryoEM, or tomography, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be utilized (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, hydrogen / deuterium exchange involves deuterium labeling of a protein of interest followed by binding of an antibody to the deuterium-labeled protein. The protein / antibody complex then moves into water, and exchangeable protons in amino acids protected by the antibody complex undergo back exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface will retain deuterium and therefore may exhibit a relatively high mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, which reveals deuterium-labeled residues that correspond to the 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.

[0054] The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from both adjacent or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from adjacent amino acids are usually retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are usually lost on treatment with denaturing solvents. Epitopes typically contain at least 3, more commonly at least 5 or 8-10 amino acids in a unique spatial conformation. Preferred epitopes herein are conformational epitopes present in homotrimeric type I collagen but not in heterotrimeric type I collagen.

[0055] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) against the same antigen according to the similarity of the binding profile of each antibody to a chemically or enzymatically modified antigen surface (see US 2004 / 0101920, specifically incorporated herein by reference in its entirety). Each classification may reflect a unique epitope that is distinct or partially overlapping with the epitopes represented by another classification. This technique allows for rapid filtering of genetically identical antibodies, so that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. MAP can be used to classify the antibodies of the present disclosure into groups of antibodies that bind to different epitopes.

[0056] The present disclosure includes antibodies that can bind to the same epitope or a portion of an epitope. Similarly, the present disclosure also includes antibodies that compete with any of the specific exemplary antibodies described herein for binding to a target or a fragment thereof. By using routine methods known in the art, it is easy to determine whether an antibody binds to the same epitope or competes for binding with a reference antibody. For example, to determine whether a test antibody binds to the same epitope as a reference, the reference antibody is allowed to bind to the target under saturating conditions. The ability of the test antibody to bind to the target molecule is then evaluated. If the test antibody can bind to the target molecule after 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 that the reference antibody binds to.

[0057] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably by 75%, 90% or even 99%, as measured in competitive binding assays (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody 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 reduce or eliminate the binding of the other.

[0058] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the lack of binding observed with the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. Structural studies using EM or crystallography can also demonstrate whether two antibodies competing for binding recognize the same epitope.

[0059] In another aspect, an antibody may be defined by its variable sequences, including additional "framework" regions. Furthermore, the antibody sequences may vary from these sequences, optionally using methods discussed in more detail below. For example, the nucleic acid sequences may differ from those described above in that (a) the variable regions are separated from the light and heavy chain constant domains; (b) the nucleic acids may differ from those described above without affecting the residues encoded thereby; (c) the nucleic acids may differ from those described above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology; (d) the nucleic acids may differ from those described above by their ability to hybridize under high stringency conditions, exemplified by low salt and / or high temperature conditions, such as those provided by about 0.02 M to about 0.15 M NaCl at a temperature of about 50° C. to about 70° C.; and (e) the amino acids may differ from those described above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, It may vary from those set forth above by 96%, 97%, 98% or 99% homology, or (f) it may vary from those set forth above by allowing for conservative substitutions (discussed below).

[0060] When comparing polynucleotide and polypeptide sequences, as described below, if the nucleotide or amino acid sequences in the two sequences are the same when aligned to obtain maximum agreement, the two sequences are said to be "identical".Comparison between two sequences is typically performed by comparing sequences over a comparison window to identify and compare local regions of sequence similarity.As used herein, "comparison window" refers to a segment of at least about 20 consecutive positions, usually 30 to about 75, 40 to about 50, where a sequence can be compared with a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned.

[0061] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using default parameters.This program embodies several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins--Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC 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, DG and Sharp, PM (1989) CABIOS 5:151-153; Myers, EW and Muller W. (1988) CABIOS 4:11-17; Robinson, ED (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, WJ and Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0062] Alternatively, optimal alignment of sequences for comparison may be performed 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 in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by visual inspection.

[0063] One specific example of algorithm suitable for determining percent sequence identity and percent 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 parameters described herein, to determine percent sequence identity of the polynucleotides and polypeptides of the present disclosure. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The reconstitution of the nature of antibody sequences and the variable length of each gene necessitates multiple rounds of BLAST searches of a single antibody sequence. Also, manual assembly of various 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, details rearrangement junctions, and delineates IgV domain framework regions and complementarity determining regions. IgBLAST can analyze nucleotide or protein sequences, can process sequences in batches, and allows for simultaneous searching of germline gene databases and other sequence databases to minimize the chance of missing the most likely matching germline V gene.

[0064] In one example, the cumulative score can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of word hits in each direction is stopped when the cumulative alignment score falls off its maximum achieved value by an amount X; when the accumulation of one or more negatively scoring residue alignments causes the cumulative score to become zero or below; or when either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) 50, expectation (E) 10, M=5, N=-4, and a comparison of both strands.

[0065] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score falls off by an amount X from its maximum achieved value; when the accumulation of one or more negatively scoring residue alignments causes the cumulative score to become zero or below; or when either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0066] In one approach, a "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, usually 5-15%, or 10-12% additions or deletions (i.e., gaps), compared to the reference sequence for optimal alignment of the two sequences (which does not contain additions or deletions). The percentage is calculated by determining the number of positions where an identical nucleic acid base or amino acid residue occurs in both sequences to calculate the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the size of the window), and multiplying the result by 100 to calculate the percentage of sequence identity.

[0067] Yet another way to define an antibody is as a "derivative" of any of the antibodies and antigen-binding fragments thereof described below. The term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen but contains one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications compared to the "parent" (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" encompasses variants with altered CH1, hinge, CH2, CH3 or CH4 regions to form, for example, antibodies with variant Fc regions that exhibit enhanced or impaired effector or binding properties. The term "derivative" further encompasses non-amino acid modifications, e.g., amino acids that are glycosylated (e.g., having altered content of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytically cleaved, can be linked to cellular ligands or other proteins, etc. In some embodiments, altered carbohydrate modifications modulate one or more of the following: antibody solubilization, promoting intracellular trafficking and antibody secretion, promoting antibody assembly, conformational integrity, and antibody-mediated effector function. In certain embodiments, altered carbohydrate modifications enhance antibody-mediated effector function compared to an antibody lacking the carbohydrate modification.Carbohydrate modifications that result in altered antibody-mediated effector function are well known in the art (see, e.g., Shields, RL 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 modifying carbohydrate content are known to those of skill in the art and are described, for example, in Wallick, SC 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, MH 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, EG et al. (1995) "The Effect Of Aglycosylation On The Immunogenicity Of A Humanized Therapeutic CD3 Monoclonal Antibody", Transplantation Immunol. 60(8):847-53; Elliott, S. et al. (2003) "Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering", Nature Biotechnol. 21:414-21; Shields, RL 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).

[0068] Derivative antibodies or antibody fragments can be created by manipulation of the sequence or glycosylation state to confer desired activity levels of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) function as measured by bead-based or cell-based assays or in vivo studies in animal models.

[0069] Derivative antibodies or antibody fragments 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 tunicamycin, etc. In one embodiment, an antibody derivative possesses similar or identical function as the parent antibody. In another embodiment, an antibody derivative exhibits altered activity compared to the parent antibody. For example, a derivative antibody (or fragment thereof) may bind to its epitope more strongly or be more resistant to proteolysis than the parent antibody.

[0070] C. Manipulation of antibody sequences In various embodiments, one may choose to manipulate the sequence of the identified antibody for various reasons, such as improving expression, improving cross-reactivity, or reducing off-target binding. Modified antibodies can be produced by any technique known to those skilled in the art, including expression by standard molecular biology techniques, or chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document. The following is a brief discussion of relevant targeting techniques for antibody engineering.

[0071] Hybridomas are cultured, then the cells can be lysed and total RNA can be extracted. A cDNA copy of the RNA can be made using random hexamers with RT, then PCR can be performed using a multiplex mixture of PCR primers predicted to amplify all human variable gene sequences. PCR products can be cloned into pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Binding and neutralization assays can be performed with antibodies collected from hybridoma supernatants and purified by FPLC using protein G columns.

[0072] Recombinant full-length IgG antibodies can be made by subcloning the heavy and light chain Fv DNA from a cloning vector into an IgG plasmid vector, transfected into 293 (e.g., Freestyle) or CHO cells, and the antibody can be recovered and purified from 293 or CHO cell supernatant. Other suitable host cell systems include bacteria such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without human-like glycan engineering), algae, or in the context of various non-human transgenics, e.g., mouse, rat, goat, or cow.

[0073] Expression of nucleic acids encoding antibodies is also contemplated, both for the purpose of subsequent antibody purification and for the purpose of host processing. The antibody coding sequence can be RNA, such as natural RNA or modified RNA. Modified RNA contemplates certain chemical modifications that confer improved stability and reduced immunogenicity to mRNA, thereby facilitating expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1mΨ) is superior in terms of translational capacity to several other nucleoside modifications and combinations thereof. In addition to turning off immune / eIF2α phosphorylation-dependent translation inhibition, the incorporated N1mΨ nucleotide dramatically alters the kinetics of the translation process by increasing ribosome pausing and mRNA density. The increased ribosome load of modified mRNAs makes them more permissive for initiation, by favoring either ribosome recycling or de novo ribosome recruitment on the same mRNA. Such modifications could be used to enhance antibody expression in vivo after RNA inoculation. RNA, whether natural or modified, can be delivered as naked RNA or in a delivery vehicle, such as a lipid nanoparticle.

[0074] Alternatively, DNA encoding an antibody can be used for the same purpose. The DNA is contained in an expression cassette that contains a promoter that is active in the host cell in which it is designed. The expression cassette is conveniently contained in a replicable vector, such as a conventional plasmid or a minivector. Vectors include and are contemplated as viral vectors, such as poxvirus, adenovirus, herpesvirus, adeno-associated virus and lentivirus. Also contemplated are replicons encoding antibody genes, such as alphavirus replicons based on VEE virus or Sindbis virus. Delivery of such vectors can be performed by needle injection through intramuscular, subcutaneous or intradermal routes, or by transdermal electroporation if in vivo expression is desired.

[0075] Rapid availability of antibodies produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to shorten process development programs. Lonza has developed a general method using pooled transfectants grown in CDACF medium for rapid production of small amounts (up to 50 g) of antibodies in CHO cells. Although slightly slower than true transient systems, advantages include higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of a GS-CHO pool expressing a model antibody in a single-use bioreactor: Single-use back bioreactor culture (5 L working volume) operated in fed-batch mode achieved a harvested antibody concentration of 2 g / L within 9 weeks of transfection.

[0076] Antibody molecules include fragments, e.g., fragments generated by proteolytic cleavage of mAbs (e.g., F(ab'), F(ab')2), or single chain immunoglobulins, e.g., single chain immunoglobulins that can be produced by recombinant means. F(ab') antibody derivatives are monovalent, whereas F(ab')2 antibody derivatives are divalent. In one embodiment, such fragments can be combined with each other or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Importantly, such chimeric molecules can contain substituents capable of binding different epitopes of the same molecule.

[0077] In related embodiments, the antibody is a derivative of the disclosed antibody, for example, an antibody that contains the same CDR sequence as that of the disclosed antibody (e.g., a chimeric or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into the antibody molecule. When making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological functions to a protein is generally understood in the art. It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0078] It is also understood in the art that similar amino acids can be effectively substituted based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, amino acid residues are assigned the following hydrophilicity values: 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).

[0079] It is understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity to produce a biologically or immunologically altered protein. In such changes, amino acid substitutions whose hydrophilicity values ​​are within ±2 are preferred, those whose hydrophilicity values ​​are within ±1 are particularly preferred, and those whose hydrophilicity values ​​are within ±0.5 are even more particularly preferred.

[0080] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account various of the foregoing characteristics are well known to those of skill in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0081] The present disclosure also contemplates isotype modifications. By modifying the Fc region to have different isotypes, different functionalities can be achieved. For example, changing to IgG1 can increase antibody-dependent cellular cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.

[0082] Alternatively or additionally, it may be useful to combine the amino acid modification with one or more additional amino acid modifications that alter the complement-dependent cytotoxicity (CDC) function and / or C1q binding of the Fc region of the IL-23p19 binding molecule. Particularly interesting binding polypeptides may be those that bind C1q and exhibit complement-dependent cytotoxicity. Polypeptides that have existing C1q binding activity and may optionally further have the ability to mediate CDC may be modified to enhance one or both of these activities. Amino acid modifications that alter C1q and / or modify its complement-dependent cytotoxicity function are described, for example, in WO / 0042072, which is incorporated herein by reference.

[0083] For example, the Fc region of an antibody can be designed with altered effector function by modifying C1q binding and / or FcγR binding, thereby altering CDC activity and / or ADCC activity. "Effector function" is responsible for activating or decreasing 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.).

[0084] For example, a variant Fc region of an antibody can be created that has improved C1q binding and improved FcγRIII binding (e.g., has both improved ADCC activity and improved CDC activity). Alternatively, where it is desired to reduce or eliminate effector function, a variant Fc region can be engineered with reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities is increased, and optionally the other activity may also be reduced (e.g., to create an Fc region variant with improved ADCC activity but reduced CDC activity, and vice versa).

[0085] FcRn binding Fc mutations can also be introduced and engineered to change its interaction with the neonatal Fc receptor (FcRn) and improve its pharmacokinetic properties. A collection of human Fc variants with improved binding to FcRn has been described. High-resolution mapping of the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to FcγR (J. Biol. Chem. 276:6591-6604). Several methods are known that can result in increased half-life, including amino acid modifications that can be made through techniques including alanine scanning mutagenesis, random mutagenesis, and screening to evaluate binding to neonatal Fc receptor (FcRn) and / or in vivo behavior. Computational strategies can also be used prior to mutagenesis to select one of the amino acid mutations to mutate.

[0086] Therefore, the present disclosure provides variants of antigen binding proteins that are optimized for binding to FcRn. In certain embodiments, the variants of antigen binding proteins comprise at least one amino acid modification in the Fc region of the antigen binding protein, wherein the modification is at least one amino acid modification in the Fc region of the antigen binding protein, the modification being at least one amino acid modification in the Fc region of the parent polypeptide ... 02, 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 and 447, wherein the numbering of the amino acids in the Fc region is that of the EU index of Kabat.

[0087] Furthermore, various publications describe methods for obtaining physiologically active molecules with altered half-lives by introducing FcRn-binding polypeptides into the molecules or by fusing them to antibodies or FcRn-binding domains of antibodies that have preserved FcRn-binding affinity but greatly reduced affinity for other Fc receptors.

[0088] Derivatized antibodies can be used to modify the half-life (e.g., serum half-life) of the parent antibody in a mammal, particularly a human. Such alterations can result in a half-life of more than 15 days, preferably more than 20 days, more than 25 days, more than 30 days, more than 35 days, more than 40 days, more than 45 days, more than 2 months, more than 3 months, more than 4 months, or more than 5 months. Increasing the half-life of the antibody or fragment thereof of the present disclosure in a mammal, preferably a human, results in a higher serum titer of the antibody or antibody fragment in the mammal, thus reducing the frequency of administration of the antibody or antibody fragment and / or reducing the concentration of the antibody or antibody fragment administered. Antibodies or fragments thereof with increased in vivo half-life can be produced by techniques known to those skilled in the art. For example, antibodies or fragments thereof with increased in vivo half-life can be produced by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor.

[0089] Beltramello et al. (2010) previously reported the modification of a neutralizing mAb by creating a substitution of the leucine residues at positions 1.3 and 1.2 (according to the IMGT specific numbering of the C domain) of the CH2 domain with alanine residues, which tend to enhance dengue virus infection. This modification, also known as the "LALA" mutation, abolishes antibody binding to FcγRI, FcγRII and FcγRIIIa. The variant and unmodified recombinant mAb 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 completely lacked the enhancing activity. Therefore, LALA mutations of this nature are contemplated in the context of the antibodies of the present disclosure.

[0090] Glycosylation changes A particular aspect of the present disclosure is an isolated monoclonal antibody or antigen-binding fragment thereof, which contains a substantially homogenous glycan that does not have sialic acid, galactose or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, either of which may be attached to a heavy or light chain constant region, respectively. The substantially homogenous glycan may be covalently attached to the heavy chain constant region.

[0091] Another embodiment of the present disclosure includes mAbs with novel Fc glycosylation patterns. The isolated monoclonal antibodies, or antigen-binding fragments thereof, are present in a substantially homogenous composition represented by GNGN or G1 / G2 glycotypes. Fc glycosylation plays an important role in the antiviral and anticancer properties of therapeutic mAbs. This disclosure is consistent with recent studies showing increased anti-lentivirus cell-mediated virus inhibition of fucose-free anti-HIV mAbs in vitro. This embodiment of the disclosure, using homogenous glycans lacking core fucose, showed increased protection against certain viruses by more than 2-fold. Removal of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells, but appears to have an adverse effect on the ADCC activity of polymorphonuclear cells (PMNs).

[0092] An isolated monoclonal antibody, or antigen-binding fragment thereof, comprising a substantially homogenous composition represented by GNGN or G1 / G2 glycotypes exhibits increased binding affinity to FcγRI and FcγRIII compared to the same antibody that does not have a substantially homogenous GNGN glycotype and has a glycotype that includes G0, G1F, G2F, GNF, GNGNF or GNGNFX. In one embodiment of the disclosure, the antibody has an FcγRI to FcγRIII ratio of 1×10 to 1×10. -8 M or less and FcγRIII to 1 × 10 -7 It dissociates at a Kd of M or less.

[0093] Glycosylation of the Fc region is usually either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment 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 may also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequences are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Thus, the presence of either of these peptide sequences in a polypeptide creates a potential glycosylation site.

[0094] The glycosylation pattern can be modified, for example, by deleting one or more glycosylation sites found in the polypeptide and / or adding one or more glycosylation sites not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody is conveniently accomplished by modifying the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). An exemplary glycosylation variant has an amino acid substitution of residue Asn 297 of the heavy chain. Modification can also be made by adding or substituting one or more serine or threonine residues to the original polypeptide sequence (for O-linked glycosylation sites). Additionally, one of the glycosylation sites can be removed by changing Asn 297 to Ala.

[0095] In certain embodiments, the antibody is expressed in cells expressing β(1,4)-N-acetylglucosaminyltransferase III (GnT III), so that GnT III adds GlcNAc to IL-23p19 antibody.Methods for producing antibodies in such a manner are provided in WO / 9954342, WO / 03011878, patent publication US 2003 / 0003097A1, and Umana et al., Nature Biotechnology, 17:176-180, February 1999.Genome editing technology such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) can be used to modify cell lines to enhance or reduce or eliminate certain post-translational modifications, such as glycosylation.For example, CRISPR technology can be used to eliminate genes encoding glycosylation enzymes in 293 cells or CHO cells used to express recombinant monoclonal antibodies.

[0096] Eliminating monoclonal antibody protein sequence liability Antibody variable gene sequences derived from human B cells can be engineered to enhance their manufacturability and safety. Potential protein sequence liabilities can be identified by searching for sequence motifs related to sites including: 1) unpaired Cys residue, 2) N-linked glycosylation, 3) Asn deamidation, 4) Asp isomerization, 5) SYE disconnection, 6) Met oxidation, 7) Trp oxidation, 8) N-terminal glutamic acid, 9) integrin binding, 10) CD11c / CD18 binding, or 11) Fragmentation Such motifs can be eliminated by modifying the synthetic gene of the cDNA encoding the recombinant antibody.

[0097] Protein engineering efforts in the field of therapeutic antibody development have demonstrated that certain 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 solubility-altering mutations in the literature suggests that some hydrophilic residues, such as aspartic acid, glutamic acid, and serine, contribute more favorably to protein solubility than other hydrophilic residues, such as asparagine, glutamine, threonine, lysine, and arginine.

[0098] stability Antibodies can be engineered to enhance biophysical properties. High temperatures can be used to unfold the antibody and determine its relative stability using the apparent average melting temperature. Differential scanning calorimetry (DSC) measures the heat capacity C p DSC measures the heat required to warm a molecule per degree as a function of temperature. Using DSC, the thermal stability of antibodies can be investigated. DSC data for mAbs resolves the unfolding of individual domains within the mAb structure (Fab, C). H 2, and C HThis is of particular interest as it can generate up to three peaks in the thermogram (from the unfolding of three domains). Usually, the unfolding of the Fab domain produces the most intense peak. The DSC profiles and relative stability of the Fc portion show characteristic differences for human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun. 355, 751-757, 2007). Circular dichroism (CD) performed on a CD spectrometer can also be used to determine the apparent average melting temperature. Far-UV CD spectra are measured for antibodies in the range 200-260 nm with 0.5 nm increments. The final spectrum can be determined as the average of 20 accumulations. After background subtraction, the value of residue ellipticity can be calculated. Thermal unfolding of antibodies (0.1 mg / mL) can be monitored at 235 nm from 25-95 °C and a heating rate of 1 °C / min. Dynamic light scattering (DLS) can be used to assess the tendency for aggregation. DLS is used to characterize the size of various particles, including proteins. If the system is not size-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 structures, and the particle concentration. Because DLS essentially measures the variation in scattered light intensity by the particles, the diffusion coefficient of the particles can be determined. The DLS software of commercially available DLA instruments displays particle populations of various diameters. Stability studies can be conveniently performed using DLS. DLS measurements of samples can indicate whether particles aggregate over time or with temperature changes by determining whether the hydrodynamic radius of the particles increases. If the particles aggregate, a larger population of particles with a larger radius can be examined. Stability with temperature can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include proven methodologies for characterizing antibody stability.The iCE approach can be used to resolve antibody protein charge variants due to deamidation, C-terminal lysine, sialylation, oxidation, glycosylation, and any other alteration to the protein that may result in a change in the pI of the protein. Each of the expressed antibody proteins can be assessed by high-throughput, free-solution isoelectric focusing (IEF) in a capillary column (cIEF) using a Protein Simple Maurice instrument. UV absorbance detection across the column can be performed every 30 seconds for real-time monitoring of molecules focused on their isoelectric point (pI). This approach combines the high resolution of traditional gel IEF with the quantification and automation advantages found in column-based separations, eliminating the need for a recruitment step. The technique results in a reproducible quantitative analysis of the identity, purity, and heterogeneity profile of the expressed antibodies. The results identify the charge heterogeneity and molecular sizing of the antibodies in both absorbance and native fluorescence detection modes, and with a detection sensitivity down to 0.7 μg / mL.

[0099] solubility The intrinsic solubility score of an 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) in the HCDR3 of each antibody fragment, such as scFv, can be evaluated via an online program to calculate the solubility score. Experimental techniques can also be used to determine solubility. Various techniques exist, including the addition of lyophilized protein to a solution until the solution is saturated and the solubility limit is reached, or concentration by ultrafiltration in a microconcentrator with an appropriate molecular weight cutoff. The simplest method is the induction of an amorphous precipitate, which allows protein solubility to be measured using a method involving protein precipitation with ammonium sulfate (Trevino et al., J Mol Biol, 366: 449-460, 2007). Ammonium sulfate precipitation provides rapid and accurate information on relative solubility values. Ammonium sulfate precipitation produces a precipitation solution with distinct aqueous and solid phases and requires a relatively small amount of protein. Solubility measurements, performed using the induction of an amorphous precipitate with ammonium sulfate, can also be easily performed at various pH values. Protein solubility is highly dependent on pH, and pH is considered the most important extrinsic factor affecting solubility.

[0100] Self-reactivity It is generally believed that autoreactive clones should be eliminated by negative selection during ontogeny; however, it has become clear that many natural human antibodies with autoreactivity persist in the mature repertoire of adults. HCDR3 loops in antibodies during early B cell development are often rich in positive charges and are known to display autoreactive patterns (Wardemann et al., Science 301, 1374-1377, 2003). A given antibody can be tested for autoreactivity by assessing the level of binding to human-derived cells in microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometry cell surface staining (using suspension Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity can also be examined using assessment of binding to tissues in tissue arrays.

[0101] Preferred Residues ("Human Similarity") B cell repertoire deep sequencing of human B cells from blood donors has been performed on a large scale in many recent studies. Sequence information for a significant portion of the human antibody repertoire facilitates the statistical evaluation of common antibody sequence features in healthy humans. Knowledge of the antibody sequence features in the Human Recombinant Antibody Variable Gene Reference Database allows the estimation of the position-specific degree of "Human Likeness" (HL) of antibody sequences. HL has been shown to be useful for the development of antibodies in clinical use, such as therapeutic antibodies or antibodies as vaccines. The goal is to increase the human likeness of antibodies to reduce potential side effects and anti-antibody immune responses that may lead to a significant decrease in the efficacy of antibody drugs or induce serious health effects. The antibody characteristics of the combined antibody repertoire of three healthy human blood donors, totaling about 400 million sequences, can be evaluated to create a new "relative human likeness" (rHL) score that focuses on the hypervariable regions of the antibody. The rHL score allows for an easy distinction between human (positive score) and non-human sequences (negative score). Antibodies can be engineered to eliminate residues that are uncommon in the human repertoire.

[0102] D. Single chain antibody Single chain variable fragments (scFv) are the fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, even though the constant regions have been removed and a linker peptide has been introduced. Even with this modification, the specificity usually remains unchanged. These molecules have been created throughout history to facilitate phage display, which is extremely convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be created directly from subcloned heavy and light chains from hybridomas or B cells. Single chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore lack the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L, because protein L interacts with the variable region of the kappa light chain.

[0103] Flexible linkers are generally composed of amino acid residues that promote helices and those that promote turns, e.g., alanine, serine, and glycine; however, other residues may also function. Tang et al. (1996) used phage display as a means to rapidly select tailored linkers for single-chain antibodies (scFvs) from a protein linker library. A random linker library was constructed in which genes for heavy and light chain variable domains were linked by segments encoding 18-amino acid polypeptides of differing composition. The scFv repertoire (approximately 5×10 6The 1,054 variants (10 distinct members) were displayed on filamentous phage and subjected to affinity selection with haptens. The population of selected variants showed a significant increase in binding activity, while retaining considerable sequence diversity. Subsequent individual screening of 1,054 variants yielded catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed the V H It was revealed that there is a conserved proline in the linker two residues after the C-terminus, as well as numerous arginines and prolines at other positions.

[0104] The recombinant antibody of the present disclosure may also be associated with a sequence or moiety that allows receptor dimerization or multimerization. Such sequences include sequences derived from IgA that allow multimerization with J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, these chains may be modified with agents such as biotin / avidin that allow the combination of two antibodies.

[0105] In another embodiment, single chain antibodies can be produced by linking the light and heavy chains of the receptor using a non-peptide linker or chemical unit. Typically, the light and heavy chains are produced in separate cells, purified, and then linked together in a suitable manner (i.e., the N-terminus of the heavy chain is attached to the C-terminus of the light chain via a suitable chemical bridge).

[0106] Cross-linking reagent is used to form molecular bridges that connect the functional groups of two different molecules, such as stabilizer and coagulant.However, it is contemplated that it can create heteromeric complexes that are composed of dimers or multimers of the same analog or different analogs.To link two different compounds stepwise, heterobifunctional cross-linking agents can be used, which eliminates unnecessary homopolymer formation.

[0107] Exemplary heterobifunctional crosslinkers contain two reactive groups, one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.). The crosslinker can react with lysine residues of one protein (e.g., a selected antibody or fragment) via the primary amine reactive group, and the crosslinker already attached to the first protein reacts with cysteine ​​residues (free sulfhydryl groups) of another protein (e.g., a selected agent) via the thiol reactive group.

[0108] Preferably, a crosslinker with reasonable stability in blood is utilized.Many types of disulfide bond-containing linkers are known that can be successfully used to link targeting agents and therapeutic / prophylactic agents.Containing sterically hindered disulfide bond linkers can be found to provide high stability in vivo, thus preventing the release of targeting peptide before reaching the site of action.Therefore, these linkers are one of the groups that link agents.

[0109] Another cross-linking reagent is SMPT, a bifunctional cross-linker that contains a disulfide bond that is "sterically hindered" by adjacent benzene rings and methyl groups. The steric hindrance of the disulfide bond is believed to serve the purpose of protecting the bond from attack by thiolate anions, such as glutathione, that may be present in tissues and blood, thereby helping to prevent the conjugate from dissociating before the attached drug is delivered to the target site.

[0110] The SMPT cross-linking reagent, like many other known cross-linking reagents, offers 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 potential type of cross-linker includes heterobifunctional photoreactive phenyl azides containing a cleavable disulfide bond, such as sulfosuccinimidyl-2-(p-azidosalicyamino)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups, and the phenyl azide (upon photolysis) reacts nonselectively with any amino acid residue.

[0111] In addition to hindered crosslinkers, unhindered linkers can also be utilized according to the present specification.Other useful crosslinkers that are not considered to contain or create protected disulfides include SATA, SPDP, and 2-iminothiolane.The use of such crosslinkers is well understood in the art.Another embodiment involves the use of flexible linkers.

[0112] US Patent No. 4,680,338 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and / or proteins, particularly useful for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, and the like. US Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates that include labile bonds that are cleavable under a variety of mild conditions. This linker is particularly useful because an agent of interest can be directly attached to the linker, and upon cleavage, the active agent is released. Specific applications include the addition of free amino groups or free sulfhydryl groups to proteins, e.g., antibodies, or drugs.

[0113] U.S. Patent No. 5,856,456 provides peptide linkers for use in connecting polypeptide components to create fusion proteins, e.g., single-chain antibodies. The linkers are up to about 50 amino acids in length, contain a charged amino acid (preferably arginine or lysine) followed by at least one occurrence of proline, and are characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separation methods.

[0114] E. Multispecific antibodies 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 may 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 be combined with an arm that binds to a triggering molecule on white blood cells, such as a T cell receptor molecule (e.g., CD3), or an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and Fc gamma RIII (CD16), to focus and localize the cell's defense mechanisms to the infected cell. Bispecific antibodies may also be used to localize cytotoxic agents to the infected cell. These antibodies possess an antigen-binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or a 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 bispecific anti-ErbB2 / anti-Fc gamma RIII antibodies, and U.S. Pat. No. 5,837,234 discloses bispecific anti-ErbB2 / anti-Fc gamma RI antibodies. Bispecific anti-ErbB2 / Fc α antibodies are shown in WO 98 / 02463. U.S. Pat. No. 5,821,337 teaches bispecific anti-ErbB2 / anti-CD3 antibodies.

[0115] Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually performed by affinity chromatography steps, is quite laborious and the product yield is low. Similar procedures are disclosed in WO 93 / 08829, and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0116] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is to a sequence comprising at least one of the hinge, C, or C-terminal fragments. H2 , and C H3 The fusions are with an Ig heavy-chain constant domain containing at least part of the light-chain binding region. The first heavy-chain constant region (C) containing the site necessary for light-chain binding, present in at least one of the fusions. H1 ). DNA encoding the immunoglobulin heavy chain fusions 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 unequal ratios of the three polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody. However, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector where expression of at least two polypeptide chains in equal ratios results in high yields or where the ratio does not significantly affect the yield of the desired chain combination.

[0117] In a particular embodiment of this approach, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only half of the bispecific molecule provides an easy separation method. This approach is disclosed in WO 94 / 04690. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0118] According to another approach, described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. A preferred interface is H3 The method comprises at least a portion of the domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with small ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

[0119] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several cross-linking techniques.

[0120] Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoic acid (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0121] Techniques exist that facilitate the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment was secreted separately from E. coli and subjected to direct chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was capable of binding to cells overexpressing the ErbB2 receptor and normal human T cells, as well as inducing the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0122] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been 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 from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provides an alternative mechanism for making bispecific antibody fragments. This fragment contains V domains separated by a linker that is too short to allow pairing between the two domains on the same chain. L V connected to H Therefore, the V of one fragment H and V L The domain is a complementary V L and V HThe Fv domains are forced to pair, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0123] In certain embodiments, bispecific or multispecific antibodies can 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 Examples section of each of which is incorporated herein by reference). In general, this technology utilizes the specific and high affinity binding interactions that occur between the dimerization and docking domain (DDD) sequence of the regulatory (R) subunit of cAMP-dependent protein kinase (PKA) and the anchor domain (AD) sequence 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 attached to any protein, peptide or other molecule, and because the DDD sequence spontaneously dimerizes and binds to the AD sequence, this technique allows the formation of a complex between any molecule of choice that can be attached to the DDD or AD sequence.

[0124] Antibodies with three or more valencies 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) faster than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibodies of the present disclosure can be multivalent antibodies (e.g., tetravalent antibodies) with three or more antigen binding sites, which can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. The multivalent antibody can include a dimerization domain and three or more antigen binding sites. A preferred dimerization domain includes (or consists of) an Fc region or a hinge region. In this scenario, the antibody includes an Fc region and three or more antigen binding sites amino-terminal to the Fc region. A preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), where the polypeptide chain comprises two or more variable regions. For example, the polypeptide chain may comprise VD1-(X1) 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 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain may comprise VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable region polypeptides. The multivalent antibody herein may comprise, for example, about two to about eight light chain variable region polypeptides. The light chain variable region polypeptides contemplated herein comprise a light chain variable region, and optionally a C L It may further include a domain.

[0125] Charge modifications are particularly useful in the context of multispecific antibodies, where amino acid substitutions in Fab molecules result in a reduction of mispairing of light chains with mismatched heavy chains (Bence-Jones type by-products) that can occur in the production of Fab-based bi / multispecific antigen-binding molecules with VH / VL exchange in one (or more, in the case of molecules comprising three or more antigen-binding Fab molecules) of the binding arms (see also PCT Publication No. WO 2015 / 150447, especially the Examples therein, which are incorporated herein by reference in their entirety).

[0126] F. Chimeric Antigen Receptor Chimeric antigen receptor molecules are recombinant fusion proteins that are distinguished by their ability to both bind antigen and transmit activation signals via immune receptor activation motifs (ITAMs) present in their cytoplasmic tails. (For example, receptor constructs that utilize antigen-binding moieties generated from single-chain antibodies (scFvs) offer the added advantage of being "universal" in that they bind native antigens on the surface of target cells in an HLA-independent manner.

[0127] Chimeric antigen receptor can be produced by any means known in the art, but preferably by recombinant DNA technology.The nucleic acid sequence encoding several regions of chimeric antigen receptor can be prepared and assembled into complete coding sequence by standard techniques of molecular cloning (genomic library screening, PCR, primer-assisted ligation, yeast and bacteria-derived scFv library, site-directed mutagenesis, etc.).The obtained coding region can be inserted into an expression vector and used to transform suitable expression host allogeneic or autologous immune effector cells, such as T cells or NK cells.

[0128] The embodiments of the CAR described herein include nucleic acids encoding an antigen-specific chimeric antigen receptor (CAR) polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the CAR can recognize an epitope comprised of a shared space between one or more antigens. In some embodiments, the chimeric antigen receptor comprises an extracellular domain comprising a) an intracellular signaling domain, b) a transmembrane domain, and c) an antigen binding domain. Optionally, the CAR may comprise a hinge domain disposed between the transmembrane domain and the antigen binding domain. In certain aspects, the CAR of the embodiment further comprises a signal peptide that directs expression of the CAR to the cell surface. For example, in some aspects, the CAR can comprise a signal peptide from GM-CSF.

[0129] In certain embodiments, the CAR can also be co-expressed with membrane-bound cytokines to improve persistence in the presence of low amounts of tumor-associated antigens. For example, the CAR can be co-expressed with membrane-bound IL-15.

[0130] Depending on the arrangement of the domains of the CAR and the particular 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, engineered cells selected for increased SRC, and selected cells tested for activity to identify CAR constructs predicted to have the greatest therapeutic effect.

[0131] 1. Antigen-binding domain In certain embodiments, the antigen-binding domain can comprise a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to the receptor. A "complementarity determining region (CDR)" is a short amino acid sequence found in the variable domain of an antigen receptor (e.g., immunoglobulin and T cell receptor) protein that complements an antigen and thus provides the receptor with its specificity for that particular antigen. Each polypeptide chain of an antigen receptor contains three CDRs (CDR1, CDR2, and CDR3). Since antigen receptors are usually composed of two polypeptide chains, there are six CDRs per antigen receptor that can contact the antigen--each heavy and light chain contains three CDRs. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable domains. Of these, CDR3 shows the greatest variability since it is encoded by recombination of the VJ (VDJ in the case of heavy chains and TCRαβ chains) regions.

[0132] It is contemplated that the CAR nucleic acid, particularly the scFv sequence, is a human gene to enhance 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 particular mouse, or human or humanized monoclonal antibody. The fragments can also be any number of different antigen-binding domains of an antigen-specific antibody. In more specific embodiments, the fragments are antigen-specific scFvs encoded by sequences 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 the embodiments are also provided in International (PCT) Patent Publication No. WO / 2015 / 123642, which is incorporated herein by reference.

[0133] As mentioned above, a prototypical CAR encodes an scFv that includes VH and VL domains from one monoclonal antibody (mAb) coupled to a transmembrane domain and one or more cytoplasmic signaling domains (e.g., costimulatory and signaling domains). 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 sequence of a first antibody that binds to the antigen; and (2) the LCDR1-3 sequence 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 selective binding to a particular conformation of the antigen (e.g., a conformation that is selectively associated with cancer cells compared to normal tissues).

[0134] Alternatively, it has been shown that CARs can be engineered with VH and VL chains from different mAbs to generate a panel of CAR+ T cells. The antigen-binding domain of a CAR can comprise any combination of the LCDR1-3 sequences of a first antibody and the HCDR1-3 sequences of a second antibody.

[0135] 2. Hinge domain In certain aspects, the CAR polypeptide of the embodiment can include a hinge domain disposed between the antigen binding domain and the transmembrane domain. In some cases, the hinge domain can 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 genetically modified T cell. In some aspects, the hinge domain includes a sequence that binds to an Fc receptor, such as FcγR2a or FcγR1a. For example, the hinge sequence can include an Fc domain from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE) that binds to an Fc receptor. In certain aspects, the hinge domain (and / or the CAR) does not include wild-type human IgG4 CH2 and CH3 sequences.

[0136] In some cases, the CAR hinge domain can be derived from a human immunoglobulin (Ig) constant region or a portion thereof that includes an Ig hinge, or from a human CD8α transmembrane domain and a CD8a hinge region. In one aspect, the CAR hinge domain can include the hinge-CH2-CH3 region of an antibody isotype IgG4. In some aspects, point mutations can be introduced into the antibody heavy chain CH2 domain to reduce glycosylation and non-specific Fc gamma receptor binding of CAR-T cells or any other CAR modified cells.

[0137] In certain aspects, the CAR hinge domain of the embodiment comprises an Ig Fc domain comprising at least one mutation compared to the wild-type Ig Fc domain that reduces Fc receptor binding. For example, the CAR hinge domain can comprise an IgG4-Fc domain comprising at least one mutation compared to the wild-type IgG4-Fc domain that reduces Fc receptor binding. In some aspects, 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 an L235E and / or N297Q mutation compared to the wild-type IgG4-Fc sequence. In a further aspect, the CAR hinge domain can comprise an IgG4-Fc domain having an amino acid substitution at position number L235 to an amino acid that is hydrophilic or has similar properties to "E", such as R, H, K, D, E, S, T, N or Q. In certain aspects, the CAR hinge domain can comprise an IgG4-Fc domain with an amino acid substitution at position N297 to an amino acid with similar properties to "Q", such as S or T.

[0138] In certain particular aspects, 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, a CD8a hinge domain, a CD28 hinge domain, or an engineered hinge domain.

[0139] 3. Transmembrane domain 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, human CD4 transmembrane domain, human CD28 transmembrane domain, transmembrane human CD3 zeta domain, or cysteine-mutated human CD3 zeta domain, or other transmembrane domains from other human transmembrane signaling proteins, such as CD16 and CD8 and erythropoietin receptor.In some aspects, 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 provided in US Patent Application Publication No. 2014 / 0274909 (e.g., CD8 and / or CD28 transmembrane domain) or those provided in US Patent Application No. 8,906,682 (e.g., CD8 alpha transmembrane domain), both of which are incorporated herein by reference. Transmembrane regions particularly useful in the present invention may be derived from (i.e., include at least the transmembrane regions of) 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. In certain particular aspects, the transmembrane domain may 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.

[0140] 4. Intracellular signaling domains The intracellular signaling domain of the chimeric antigen receptor of the embodiment is involved in the activation of 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 function of a differentiated cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. The effector function in naive, memory, or memory T cells includes antigen-dependent proliferation. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. In some aspects, the intracellular signaling domain is derived from the intracellular signaling domain of a natural receptor. Examples of such natural receptors include the zeta chain of the T cell receptor or any of 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 portion of other members of the family of activating proteins can be used. Usually, the entire intracellular signaling domain is utilized, but in many cases, it is not necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may have utility, such a truncated portion may be used in place of the intact chain, so long as it transmits an effector function signal. Thus, the term "intracellular signaling domain" is meant to include a truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal when the CAR binds to a target. In a preferred embodiment, the human CD3 zeta intracellular domain is used as the intracellular signaling domain of the CAR of the embodiment.

[0141] In certain embodiments, the intracellular receptor signaling domain in the CAR includes those of the T cell antigen receptor complex, such as the zeta chain of CD3, either alone or in series with CD3zeta, as well as the Fcγ RIII costimulatory signaling domain, CD28, CD27, DAP10, CD137, OX40, CD2. In certain embodiments, the intracellular domain (which may be referred to as the cytoplasmic domain) includes part or all of one or more of the TCR zeta chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12, and CD40. In some embodiments, any portion of the endogenous T cell receptor complex in the intracellular domain is utilized. The so-called third generation CARs have at least two or three signaling domains fused together for additive or synergistic effects, so that one or more cytoplasmic domains can be utilized, for example, CD28 and 4-1BB can be combined in a CAR construct.

[0142] In some embodiments, the CAR comprises additional co-stimulatory domains. The 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 CD3zeta, the additional signal provided by the human co-stimulatory receptor inserted into the human CAR is important for the complete activation of T cells and can help improve in vivo persistence and therapeutic success of adoptive immunotherapy.

[0143] In certain particular aspects, the intracellular signaling domain comprises a sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a domain comprising a CD3zeta intracellular domain, a CD28 intracellular domain, a CD137 intracellular domain, or a CD28 intracellular domain fused to a 4-1BB intracellular domain.

[0144] G. ADC Antibody-drug conjugates or ADCs are a new class of highly potent biopharmaceutical drugs designed as targeted therapy to treat people with disease. ADCs are composite molecules composed of an antibody (whole mAb or an antibody fragment, e.g., single-chain variable fragment, or scFv) linked by a labile bond via a stable chemical linker to a biologically active cytotoxic / antiviral payload or drug. Antibody-drug conjugates are examples of bioconjugates and immunoconjugates.

[0145] By combining the inherent targeting capabilities of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs, antibody-drug conjugates can discriminate between healthy and diseased tissues with high sensitivity, meaning that, in contrast to traditional systemic approaches, antibody-drug conjugates target and attack diseased cells in such a way that healthy cells are less affected than cancer cells.

[0146] In antitumor therapy based on development ADC, anticancer drugs (e.g., cytotoxins or cytotoxic agents) are coupled to antibodies that specifically target certain cell markers (e.g., proteins that are ideally found only in or on infected cells). The antibodies locate these proteins in the body and attach to the surface of cancer cells. A biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, and the antibody is then absorbed or internalized together with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released, killing the cell or impairing cell replication. This targeting ideally allows the drug to have fewer side effects than other agents and a wider therapeutic window.

[0147] A stable link between the antibody and the cytotoxic agent is a key aspect of ADCs. Linkers are based on chemical motifs including disulfides, hydrazones, or peptides (cleavable), or thioethers (non-cleavable), which control the distribution and delivery of the cytotoxic agent to the target cells. Cleavable and non-cleavable types of linkers have been found to be safe in preclinical and clinical trials. Brentuximab vedotin contains an enzyme-sensitive cleavable linker that delivers the synthetic anti-cancer drug, the potent and highly toxic microtubule inhibitor monomethyl auristatin E or MMAE, to human specific CD30-positive malignant cells. Due to this high toxicity, MMAE, which inhibits cell division by blocking tubulin polymerization, cannot be used as a single agent chemotherapy agent. However, the combination of MMAE linked to an anti-CD30 monoclonal antibody (cAC10, a cell membrane protein of the tumor necrosis factor or TNF receptor) was found to be stable in extracellular fluids, cleavable by cathepsins, and safe for therapy. Another approved ADC, trastuzumab emtansine, is a combination of the microtubule inhibitor mertansine (DM-1), a derivative of maytansine, and the antibody trastuzumab (Herceptin® / Genentech / Roche), attached by a stable, non-cleavable linker.

[0148] The availability of better and more stable linkers has changed the function of chemical bonds. Cleavable or non-cleavable linkers confer certain properties to cytotoxic (anti-cancer) drugs. For example, non-cleavable linkers keep the drug intracellular. As a result, the whole antibody, linker and cytotoxic agent enter the target cancer cell, where the antibody is degraded to the amino acid level. The resulting conjugate of amino acids, linker and cytotoxic agent is then the active drug. In contrast, cleavable linkers are catalyzed by enzymes present in the host cell, releasing the cytotoxic agent inside the host cell.

[0149] Another type of cleavable linker currently being developed adds an extra molecule between the cytotoxic drug and the cleavage site. Using this linker technology, researchers can create ADCs with greater flexibility without worrying about altering the cleavage kinetics. Researchers are also developing new peptide cleavage methods based on Edman degradation, a method for sequencing the amino acids of a peptide. Future directions in ADC development also include the development of site-directed conjugation (TDC) to further improve stability and therapeutic index, as well as alpha-radioimmunoconjugates and antibody-conjugated nanoparticles.

[0150] H. BiTES Bi-specific T-cell engagers (BiTEs) are a type of artificial bispecific monoclonal antibody being investigated for use as anti-cancer drugs. They target the host's immune system, specifically the cytotoxic activity of T cells, against infected cells. BiTE is a registered trademark of Micromet AG.

[0151] BiTEs are fusion proteins consisting of two single-chain variable fragments (scFv) of different antibodies or amino acid sequences from four different genes on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, while the other binds to infected cells via a specific molecule.

[0152] Like other bispecific antibodies, but unlike conventional monoclonal antibodies, BiTEs form a link between T cells and target cells, which allows T cells to exert cytotoxic activity on infected cells by producing proteins like perforin and granzymes, independent of the presence of MHC I or costimulatory molecules. These proteins enter infected cells and initiate cellular apoptosis, mimicking the physiological processes observed during T cell attack.

[0153] I. Intrabody In certain embodiments, the antibody is a recombinant antibody suitable for intracellular action, such antibodies are known as "intrabodies". These antibodies can interfere with target function by various mechanisms, such as modifying intracellular protein trafficking, interfering with enzyme function, and blocking protein-protein or protein-DNA interactions. Their structure is in many ways similar to or mimics the structure of the single-chain and single-domain antibodies described above. Indeed, being a single transcript / single chain is a key feature that allows intracellular expression in target cells, and also makes protein trafficking through cell membranes more feasible. However, other features are also necessary.

[0154] Two major issues that affect the realization of intrabody therapy are delivery, including cell / tissue targeting, and stability. With regard to delivery, various approaches have been utilized, such as tissue-specific delivery, the use of cell type-specific promoters, viral-based delivery, and the use of cell-permeable / membrane-translocating peptides. One means of delivery includes the use of lipid-based nanoparticles, or exosomes, as taught in US Patent Application Publication No. 2018 / 0177727, the entirety of which is incorporated herein by reference. With regard to stability, approaches are generally taken toward either brute-force screening, including methods that involve phage display and may include sequence maturation or consensus sequence development, or more direct modifications, such as the insertion of stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide substitutions / modifications.

[0155] An additional feature that intrabodies may require is a signal for intracellular targeting. Vectors that can target intrabodies (or other proteins) to subcellular regions such as the cytoplasm, nucleus, mitochondria, and ER have been designed and are commercially available (Invitrogen Corp.).

[0156] Intrabody has additional uses that cannot be achieved by other types of antibodies due to its ability to enter cells.In the case of the antibody of the present invention, its ability to interact with DDR1 cytoplasmic domain in living cells can prevent DDR1-related functions, such as signaling function (binding to other molecules) or oligomerization.In particular, it is contemplated that such antibody can be used to inhibit type I collagen homotrimerization, for example, by disrupting related chaperone or cross-linking enzyme function.

[0157] J. Purification The antibody of the present disclosure may be purified. The term "purified" as used herein is intended to refer to a composition that can be isolated from other components, where the protein is purified to any degree compared to the state in which it is naturally available. Thus, a purified protein also refers to a protein that is free of the environment in which it may naturally occur. When the term "substantially purified" is used, this indication refers to a composition in which the protein or peptide forms the major component of the composition, for example, making up about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0158] Protein purification methods are well known to those skilled in the art. These techniques involve, at one level, crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. After separating the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic methods to partially or completely purify (or to homogeneity). Analytical methods that are particularly suitable for the preparation of pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or heat denaturation followed by precipitation by centrifugation; gel filtration, reverse phase, hydroxylapatite, and affinity chromatography; and combinations of such techniques with other techniques.

[0159] In purifying the antibody 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 it is believed that the method will still result in a suitable method for preparing substantially purified protein or peptide.

[0160] Typically, intact antibodies are fractionated using agents that bind to the Fc portion of the antibody (i.e., Protein A). Alternatively, antigens may be used to simultaneously purify and select suitable antibodies. Such methods often utilize a selection agent bound to a support such as a column, filter, or beads. The antibody is bound to the support, contaminants are removed (e.g., washed away), and the antibody is released by application of conditions (salt, heat, etc.).

[0161] Various methods for quantifying the degree of purification of a protein or peptide will be known to those of skill in the art in view of the present disclosure. These methods include, for example, determining the specific activity of an active fraction or assessing the amount of polypeptides in a 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, thus calculating the degree of purity. The actual units used to express the amount of activity will, of course, depend on the particular assay technique chosen to track the purification, whether the expressed protein or peptide shows detectable activity or not.

[0162] It is known that migration of polypeptides can vary, sometimes significantly, under different conditions of SDS / PAGE, and therefore it is understood that the apparent molecular weight of purified or partially purified expression products can vary under different electrophoretic conditions.

[0163] K. Antibody conjugates The antibody of the present disclosure may be linked to at least one agent to form an antibody conjugate. It is conventional to link or covalently bind or conjugate at least one desired molecule or moiety to enhance the efficacy of an antibody molecule as a diagnostic or therapeutic agent. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules include molecules with a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules attached to an antibody include toxins, antitumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelators, 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 attached to an antibody include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or ligands, e.g., biotin.

[0164] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostic agents are generally divided into two classes: antibody diagnostic agents for use in in vitro diagnostics, such as various immunoassays, and antibody diagnostic agents for use in in vivo diagnostic protocols commonly known as "antibody-directed imaging". Many suitable imaging agents are known in the art, as are methods for attaching imaging agents to antibodies (see, for example, U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioisotopes, fluorescent dyes, substances detectable by NMR, and X-ray imaging agents.

[0165] In the case of paramagnetic ions, by way of example, mention may be made of 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), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II) and, in particular, bismuth (III).

[0166] 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 In some embodiments, 125 I is often preferred, but technetium 99m and / or indium 111 is also often preferred due to its low energy and suitability for long-distance detection. Radioactively labeled monoclonal antibodies of the present disclosure can be made by methods well known in the art. For example, monoclonal antibodies can be iodinated by contacting with sodium iodide and / or potassium iodide and 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 iodinated with technetium by a ligand exchange process, for example, by reducing pertechnate with a stannous solution, chelating the reduced technetium with a Sephadex column, and applying the antibody to the column. 99m Alternatively, direct labeling techniques may be used, for example, by incubating the antibody with pertechnate, a reducing agent, such as SNCl2, a buffer solution, such as sodium-potassium phthalate solution, and the antibody. Intermediate functional groups often used to bind radioisotopes that exist as metal ions to antibodies are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0167] Among the fluorescent labels contemplated for use as conjugates are 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.

[0168] Another type of antibody conjugate contemplated in the present disclosure is an antibody conjugate intended primarily for in vitro use. In this case, the antibody is linked to a secondary binding ligand and / or an enzyme (enzyme tag) that produces a colored product when contacted with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and 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.

[0169] Yet another known method of site-specifically attaching molecules to antibodies involves reacting the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous as it may cause loss of antigen binding by the antibody conjugate.

[0170] Molecules containing azide groups may also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light. In particular, 2-azido and 8-azido analogs of purine nucleotides have been used as site-specific photoprobes to identify nucleotide-binding proteins in crude cell extracts. 2- and 8-azido nucleotides have also been used to map nucleotide-binding domains in purified proteins and may be used as antibody binders.

[0171] Several methods are known in the art for attaching or binding an antibody to its conjugate moiety. Some attachment methods involve, for example, the use of organic chelating agents attached to the antibody, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or metal chelate complexes with tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is accomplished using a monoclonal antibody, and a detectable imaging moiety is attached to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0172] In other embodiments, it is contemplated that immunoglobulins are derivatized by selectively introducing sulfhydryl groups into the Fc region of immunoglobulins using reaction conditions that do not alter the antibody binding site. Antibody conjugates produced according to this method are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which the reporter or effector molecule is bound to carbohydrate residues in the Fc region, has also been disclosed in the literature. This approach has been reported to produce antibodies with diagnostic and therapeutic potential that are currently undergoing clinical evaluation.

[0173] II. Treatment Method Certain aspects of the present invention can be used to prevent or treat diseases or disorders associated with the presence of homotrimeric type I collagen, such as pancreatic ductal adenocarcinoma. The function of homotrimeric type I collagen can be reduced by any suitable drug. Preferably, such a substance will be an anti-homotrimeric type I collagen antibody.

[0174] "Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject, or the administration of a medical treatment or modality to a subject, for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment can include the administration of a pharmacologic effective amount of an antibody that inhibits homotrimeric type I collagen, alone or in combination with the administration of chemotherapy, immunotherapy or radiation therapy, the administration of surgery, or any combination thereof.

[0175] The term "subject" as used herein refers to any individual or patient on whom the subject method is carried out.Generally, subject is human, but as understood by those skilled in the art, subject can also be animal.Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans and gorillas), are included in the definition of subject.

[0176] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the health of a subject with respect to the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease, such as cancer or fibroid disease. For example, treating cancer can involve, for example, reducing the size of a tumor, reducing the invasiveness of a tumor, reducing the rate of growth of a cancer, or preventing metastasis. Treating cancer can also refer to extending the survival of a subject with cancer.

[0177] The term "cancer" as used herein 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, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testicles, tongue, or uterus.

[0178] The cancer may specifically be of the following histological types, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Nonencapsulating sclerosing carcinoma; Adrenal cortical carcinoma; Endometrioid carcinoma; Adnexal carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease, breast; Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Theca cell tumor, malignant; Granulosa cell tumor, malignant; Androblastoma, malignant; Sertoli cell tumor; Leydig cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma, malignant; Extra-mammary paraganglioma, malignant; Pheochromocytoma; Hemangiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant;Mullerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Embryonic carcinoma; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Hemangiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Anaplastic neuroectodermal; Cerebellar sarcoma; Ganglioblastoma; Neuroblastoma; Retinoblastoma; Olfactory nerve tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Lateral granuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, diffuse large cell; Malignant lymphoma, follicular; Mycosis fungoides; Other non-Hodgkin's lymphomas specified; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemia; Lymphocytic leukemia; The leukemias that may be treated include, but are not limited to, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myelosarcoma, and hairy cell leukemia. However, it is recognized that the present invention may also be used to treat non-cancerous diseases (e.g., fungal infections, bacterial infections, viral infections, neurodegenerative diseases, and / or genetic disorders);

[0179] B. Formulation and Administration The present disclosure provides pharmaceutical compositions comprising an antibody that selectively binds to homotrimeric type I collagen. Such compositions comprise a prophylactically or therapeutically effective amount of the antibody or fragment thereof and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly for use in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic substance is administered. 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, and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.

[0180] The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, and sustained release formulations. Oral formulations may contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences." Such compositions contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier to provide the form for administration suitable for the patient. The formulation should be suited to the mode of administration, which may be oral, intravenous, intraarterial, buccal, intranasal, aerosol, bronchial inhalation, rectal, vaginal, topical, or delivered by mechanical ventilation.

[0181] Passive transfer of antibodies generally involves the use of intravenous or intramuscular injections. The antibody may be in the form of a monoclonal antibody (MAb). Such immunity generally lasts only a short time and there are potential risks of hypersensitivity reactions and serum sickness, especially from gamma globulins of non-human origin. The antibody is formulated in a carrier suitable for injection, i.e., sterile and injectable.

[0182] In general, the components of the composition of the present disclosure are supplied in unit dosage form, for example as lyophilized dry powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active agent, either individually or mixed together.When the composition is administered by injection, it can be dispensed into an injection 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 before administration.

[0183] The compositions of the present disclosure may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0184] C. Kits and Diagnostics In various aspects of the embodiments, kits containing the therapeutic agent and / or other therapeutic and delivery agents are envisioned. The embodiments of the present invention contemplate kits for preparing and / or administering the therapeutic methods of the embodiments. The kits may include one or more sealed vials containing any of the pharmaceutical compositions of the embodiments of the present invention. The kits may include, for example, at least one homotrimeric type I collagen antibody, and reagents for preparing, formulating, and / or administering the components of the embodiments, or for carrying out one or more steps of the methods of the present invention. In some embodiments, the kits may also include suitable containers, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes, which are containers that do not react with the components of the kit. The containers may be made of sterilizable materials, such as plastic or glass.

[0185] The kit may further include instructions outlining the procedural steps of the methods described herein, following substantially the same procedures as described herein or known to those of skill in the art. The instruction information may be in a computer readable medium containing machine readable instructions that, when executed using a computer, result in the display of a real or virtual procedure for delivering a pharma- ceutically effective amount of a therapeutic agent.

[0186] D. ADCC Antibody-dependent cellular cytotoxicity (ADCC) is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an antibody or a fragment thereof comprising an Fc region specifically binds, typically via a protein portion that is N-terminal to the Fc region. By "antibody with increased / reduced antibody-dependent cellular cytotoxicity (ADCC)" is meant an antibody with increased / reduced ADCC as determined by any suitable method known to one of skill in the art.

[0187] As used herein, the term "increase / reduction of ADCC" is defined as either an increase / reduction in the number of target cells lysed in a given time at a given concentration of antibody in the medium surrounding the target cells by the mechanism of ADCC defined above, and / or a decrease / increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time by the mechanism of ADCC. The increase / reduction of ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cell, but not engineered, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art). For example, the increase in ADCC mediated by an antibody produced by a host cell engineered to have a modified glycosylation pattern (e.g., to express glycosyltransferase, GnTIII, or other glycosyltransferase) by the methods described herein is relative to the ADCC mediated by the same antibody produced by a non-engineered host cell of the same type.

[0188] E. CDC Complement-dependent cytotoxicity (CDC) is a function of the complement system. It is a process in the immune system that kills pathogens 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, which causes lethal colloid osmotic swelling, or CDC. This is one of the mechanisms by which antibodies or antibody fragments exert their cytotoxic effects.

[0189] F. Combination Therapy In certain embodiments, the compositions and methods of the present invention comprise antibodies or antibody fragments against homotrimeric type I collagen that inhibit its activity in combination with a second or additional therapy, such as chemotherapy or immunotherapy.Such therapy can be applied in the treatment of any disease associated with the elevation of homotrimeric type I collagen.For example, the disease can be cancer or fibroid disease.

[0190] The methods and compositions, including combination therapy, enhance the therapeutic or prophylactic effect and / or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. The therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve a desired effect, such as killing of cancer cells and / or inhibiting cell hyperproliferation. The process can include contacting cells with both the antibody or antibody fragment and the second therapy. The tissue, tumor, or cells can be contacted with one or more compositions or pharmacological formulations that include one or more of the agents (i.e., antibody or antibody fragment or anti-cancer agent), or the tissue, tumor, and / or cells can be contacted with two or more different compositions or formulations, such that 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 also contemplated that such combination therapy can be used with chemotherapy, radiation therapy, surgery, or immunotherapy.

[0191] The terms "contacted" and "exposed," as applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or placed directly adjacent to a target cell. To achieve cell killing, for example, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0192] The therapeutic antibody may be administered prior to, during, after, or in various combinations with respect to the anti-cancer treatment. Administration may be spaced apart from simultaneously, to within minutes, days, or weeks. In embodiments where the antibody or antibody fragment is provided to the patient separately from the anti-cancer drug, it will generally be ensured that there is no time lapse between each delivery so that the two compounds can still exert a beneficially combined effect on the patient. In such instances, it is contemplated that the antibody therapy and the anti-cancer therapy may be provided to the patient within about 12-24 or 72 hours of each other, and more specifically, within about 6-12 hours of each other. In some circumstances, it may be desirable to extend the treatment period significantly, with days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapsing between each administration.

[0193] In certain embodiments, the course of treatment will last from 1 to 90 days or more (such ranges include intervening days). It is contemplated that one agent can be administered on any of days 1 to 90 (such ranges include intervening days) or any combination thereof, and another agent can be administered on any of days 1 to 90 (such ranges include intervening days) or any combination thereof. Within a single day (24 hours), the patient may receive one or more doses of the agent. It is further contemplated that after the course of treatment, there will be a period during which no anti-cancer treatment is administered. This period may last from 1 to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or more (such ranges include intervening days), depending on the condition of the patients, such as their prognosis, strength, health, etc. It is expected that the treatment cycles will be repeated as necessary.

[0194] Various combinations may be utilized. In the example below, the antibody therapy is "A" and the anti-cancer therapy is "B". TIFF0007599716000001.tif17128

[0195] Administration of any of the compounds or therapies of the present invention to a patient will follow standard protocols for administration of such compounds, taking into account the toxicity, if any, of the drug. Thus, in some embodiments, there is a step of monitoring for toxicity due to the combination therapy.

[0196] 1. Chemotherapy A wide variety of chemotherapeutic agents can be used according to the embodiments of the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to compounds or compositions administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within cells, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0197] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and biceresin synthetic analogs); Cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictyin; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega I1); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino -doxorubicin, including 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, or zorubicin; Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; Bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide;Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, verracurin A, roridin 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 inhibitors RFS 2000; Difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharma- ceutically acceptable salts, acids, or derivatives of any of the foregoing;

[0198] 2. Radiation therapy Other agents that cause DNA damage and are widely used include what are commonly known as gamma radiation, X-rays, and / or specific delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also considered, such as microwaves, proton beam irradiation (U.S. Patents Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents most likely affect a wide range of DNA damage, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dose ranges for X-rays range from daily doses of 50-200 roentgens over an extended period (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0199] 3. Immunotherapy Those skilled in the art will understand that immunotherapy can be used in combination with or with the methods of this embodiment. In the context of cancer treatment, immunotherapeutics generally rely 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 to some marker on the surface of tumor cells. The antibody alone can act as an effector of therapy or can recruit other cells to actually affect cell killing. The antibody can also be conjugated with a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply act as a targeting agent. Alternatively, the effector can be a lymphocyte with a surface molecule that interacts either directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0200] In one aspect of immunotherapy, tumor cells must have some markers that are amenable to targeting, i.e., not present on the majority of other cells. Many tumor markers exist, and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0201] Examples of immunotherapies currently under investigation or in use are immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169); cytokine therapy, such as interferon alpha, beta, and gamma, IL-1, GM-CSF, and TNF; gene therapy, such as TNF, IL-1, IL-2, and p53 (U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be utilized in conjunction with the antibody therapies described herein.

[0202] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints can either increase a signal (e.g., a costimulatory molecule) or decrease a signal. Inhibitory immune checkpoints that can be targeted by immune checkpoint inhibition include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte antigen 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 particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0203] The immune checkpoint inhibitor may be a drug, e.g., a small molecule, a recombinant ligand or receptor, or may be, in particular, an antibody, e.g., a human antibody (e.g., International Patent Publication WO2015016718, incorporated herein by reference). Known immune checkpoint protein inhibitors or their analogs may be used, in particular chimeric, humanized, or human antibodies. As known by those skilled in the art, alternative and / or equivalent names may be used for certain antibodies mentioned in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by alternative and / or equivalent names, MK-3475 and pembrolizumab.

[0204] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its ligand binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its ligand binding partner. In certain aspects, the PDL2 binding partner is PD-1. The antagonist may 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.

[0205] 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 an extracellular portion or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, 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.

[0206] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte 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 acts 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 CD28, a T cell costimulatory protein, and both molecules bind to CD80, also known as B7-1, and CD86, also known as B7-2, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 increases the expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0207] 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.

[0208] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the invention can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; formerly also known as 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 disclosure of each of the aforementioned publications is 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 Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0209] An exemplary anti-CTLA-4 antibody is 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 heavy and light chain CDRs or VRs 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 binding to the same epitope on CTLA-4 as the aforementioned antibodies and / or binds to the same epitope on CTLA-4 as the aforementioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to an antibody described above (e.g., at least about 90%, at least about 95%, or at least about 99% variable region identity to ipilimumab).

[0210] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796, and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference, and immunoadhesins, such as those described in U.S. Pat. No. 8,329,867, which is incorporated herein by reference.

[0211] In some embodiments, immunotherapy can be adoptive immunotherapy, involving the transfer of autoantigen-specific T cells generated ex vivo. T cells used for adoptive immunotherapy can be generated by expanding antigen-specific T cells or by redirecting T cells by genetic engineering (Park, Rosenberg et al. 2011). Isolation and transfer of tumor-specific T cells has been shown to be successful in melanoma treatment. Novel specificities in T cells have been successfully generated by gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety linked to one or more signaling domains in the form of a fusion molecule. In general, the binding moiety of CARs consists of the light chain fragment of a monoclonal antibody and the antigen-binding domain of a single chain antibody (scFv), in which the variable fragment is connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains of first-generation CARs are derived from the cytoplasmic region of CD3ζ or the Fc receptor γ chain. CARs have been used successfully to redirect T cells to antigens expressed on the surface of tumor cells from a variety of neoplasms, including lymphomas and solid tumors.

[0212] In one embodiment, the present application provides a combination therapy for treating cancer, comprising adoptive T cell therapy and checkpoint inhibitors.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.

[0213] 4. Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventive, diagnostic, or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies, such as treatments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs surgery).

[0214] Removal of part or all of the cancer cells, tissues, or tumors may result in the formation of a cavity in the body. Treatment may be performed by perfusion, direct injection, or local application of additional anticancer therapy to the area. Such treatment may 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 may also be at various dosages.

[0215] 5. Other agents It is contemplated that other agents may be used in combination with certain aspects of the embodiments of the present invention to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions increases the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. It is contemplated that cell adhesion inhibitors improve the efficacy of the embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the embodiments of the present invention to improve the efficacy of the treatment.

[0216] III. Immunodetection Methods In yet another embodiment, the present disclosure relates to immunodetection methods for binding, quantifying, or otherwise generally detecting homotrimeric type I collagen.Other immunodetection methods include specific assays for determining the presence of homotrimeric type I collagen in a subject.A wide variety of assay formats are contemplated, but specifically, those that are used to detect homotrimeric type I collagen in tissue samples obtained from a subject, such as biopsies.These assays can be packaged in the form of a kit with appropriate reagents and instructions to enable use.

[0217] Some immunodetection methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluorescent immunoassay, chemiluminescence assay, bioluminescence assay, and Western blot, to name a few. The steps of various useful immunodetection methods are described in the scientific literature. In general, the immunobinding method includes obtaining a sample suspected of containing homotrimeric type I collagen, and contacting the sample with a first antibody according to the present disclosure, optionally under conditions effective to allow the formation of an immune complex.

[0218] The immunodetection method also includes a method for detecting and quantifying the amount of homotrimeric type I collagen or related components in a sample, and a method for detecting and quantifying any immune complexes formed during the binding process.Here, a sample suspected of containing homotrimeric type I collagen is obtained, the sample is contacted with an antibody that binds to homotrimeric type I collagen, and then the amount of immune complexes formed under certain conditions is detected and quantified.With regard to antigen detection, the biological sample analyzed can be any sample suspected of containing homotrimeric type I collagen, such as tissue section or specimen, homogenized tissue extract, or biological fluid.

[0219] Contacting a selected biological sample with an antibody under conditions effective to form immune complexes (primary immune complexes) and for a period of time sufficient to form immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibody to form immune complexes, i.e., bind to homotrimeric collagen I. After this time, the sample-antibody composition, e.g., tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove non-specifically bound antibody species, thereby allowing only those antibodies to bind specifically in the primary immune complexes to be detected.

[0220] In general, the detection of immune complex formation is well known in the art and can be achieved by applying a large number of approaches.These methods are generally based on the detection of labels or markers, such as radioactive tags, fluorescent tags, biological tags, and enzyme tags.Patents relating to the use of such labels include 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.Of course, as known in the art, further advantages may be found by using secondary binding ligands, such as second antibodies and / or biotin / avidin ligand binding sequences.

[0221] The antibody itself used in detection may be linked to a detectable label. This label can then be easily detected, thereby allowing the amount of primary immune complexes in the composition to be determined. Alternatively, the first antibody bound in the primary immune complex may be detected by a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand itself is often an antibody, and therefore this antibody may be called a "secondary" antibody. The primary immune complex is contacted with a labeled secondary binding ligand or antibody under conditions effective to form secondary immune complexes and for a period of time sufficient to form secondary immune complexes. The secondary immune complex is then generally washed to remove non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complex is then detected.

[0222] Further methods include detecting primary immune complexes by a two-step approach. A second binding ligand, for example an antibody having binding affinity to the antibody, is used to form secondary immune complexes as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody having binding affinity to the second antibody, also under conditions effective to form immune complexes (tertiary immune complexes) and for a period of time sufficient to form immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, thereby detecting the tertiary immune complexes thus formed. If desired, the system may amplify the signal.

[0223] In one immunodetection method, two different antibodies are used. A first biotinylated antibody is used to detect the target antigen, and then a second antibody is used to detect the biotin attached to the complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the first stage antibody. If the target antigen is present, a portion of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, each stage adding additional biotin sites to the antibody / antigen complex. The amplification stages are repeated until a suitable level of amplification is reached. At this time, the sample is incubated in a solution containing a second stage antibody against biotin. This second stage antibody is labeled, for example with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogenic substrate. With appropriate amplification, macroscopically visible conjugates can result.

[0224] Another known immunodetection method utilizes immuno-PCR (polymerase chain reaction). PCR is similar to the Cantor method up to the incubation with biotinylated DNA, but instead of multiple incubations of streptavidin with biotinylated DNA, the DNA / biotin / streptavidin / antibody complex is washed away using a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to perform a PCR reaction with appropriate primers and appropriate controls. At least in theory, the enormous amplification power and specificity of PCR can be exploited to detect a single antigen molecule.

[0225] A. ELISA Immunoassay, in its simplest and most direct sense, is binding assay.Some preferred immunoassays are various types of enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) known in the art.Immunohistochemical detection using tissue sections is also particularly useful.However, it is easily understood that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analysis, etc. may also be used.

[0226] In one exemplary ELISA, the antibody of the present disclosure is immobilized on a well on a selected surface, e.g., a polystyrene microtiter plate, that exhibits protein affinity. A test composition suspected of containing homotrimeric type I collagen is then added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be accomplished by adding another anti-homotrimeric type I collagen antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be accomplished by adding a second anti-homotrimeric type I collagen antibody, followed by a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label.

[0227] In another exemplary ELISA, a sample suspected of containing homotrimeric type I collagen is immobilized on a well surface and then contacted with the anti-homotrimeric type I collagen antibody of the present disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-homotrimeric type I collagen antibody is detected. If the first anti-homotrimeric type I collagen antibody is linked to a detectable label, the immune complex may be detected directly. Again, the immune complex may be detected using a second antibody that has binding affinity for the first anti-homotrimeric type I collagen antibody, and the second antibody is linked to a detectable label.

[0228] Regardless of the format used, ELISAs have certain features in common, such as coating, incubation and binding, washing to remove non-specifically bound species, and detection of bound immune complexes, which are described below.

[0229] When coating a plate with an antigen or antibody, the wells of the plate are generally incubated with a solution of the antigen or antibody overnight or for a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. The remaining available well surfaces are then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include bovine serum albumin (BSA), casein, or milk powder solution. The coating blocks nonspecific adsorption sites on the immobilizing surface, thus reducing the background caused by nonspecific binding of the antiserum to the surface.

[0230] In ELISA, it is common to use secondary or tertiary detection means rather than a direct procedure. Thus, after the protein or antibody is bound to the well, coated with a non-reactive material to reduce background, and washed to remove unbound material, the immobilization surface is contacted with the biological sample to be tested under conditions effective to form immune complexes (antigen / antibody). Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody and a labeled tertiary antibody or third binding ligand.

[0231] By "under conditions effective to form immune complexes (antigen / antibody)" it is meant that the conditions preferably include diluting the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween. These added agents also tend to help reduce nonspecific background.

[0232] "Appropriate" conditions also mean that incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically for about 1-2-4 hours, preferably at temperatures of about 25°C to 27°C, or may be overnight at about 4°C.

[0233] After all incubation steps in an ELISA, the contacted surface is washed to remove uncomplexed material. A preferred washing procedure involves washing with a solution such as PBS / Tween, or borate buffer. After formation of specific immune complexes between the test sample and the originally bound material, followed by washing, further traces of immune complexes may be determined.

[0234] In order to provide detection means, the second antibody or the third antibody has a label attached thereto that allows detection.Preferably, this is an enzyme that develops color when incubated with a suitable color-developing substrate.Thus, it is desirable to contact or incubate the first immune complex and the second immune complex with an antibody that is conjugated with urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase, for a period and under conditions that favor the occurrence of further immune complex formation (e.g., incubation at room temperature for 2 hours in a PBS-containing solution such as PBS-Tween).

[0235] After incubation with the labeled antibody and washing to remove unbound material, the amount of label is quantified, for example, by incubating with a chromogenic substrate, such as urea, or bromocresol purple, or 2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H2O2 in the case of peroxidase as the enzyme label. Quantitation is then accomplished by measuring the extent of color development, for example, using a visible spectrum spectrophotometer.

[0236] In another embodiment, the present disclosure contemplates the use of a competitive format.This is particularly useful in detecting Norovirus antibodies in samples.In competitive-based assays, unknown amounts of analyte or antibody are determined by their ability to displace known amounts of labeled antibody or analyte.Therefore, quantifiable signal loss is a representation of the amount of unknown antibody or analyte in the sample.

[0237] B. Western Blot Western blot (or protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. Western blot uses gel electrophoresis to separate native or denatured proteins by polypeptide length (denaturing conditions) or by the 3D structure of the protein (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF) where they are probed (detected) with an antibody specific for the target protein.

[0238] Samples may be taken from whole tissues or from cell cultures. In most cases, solid tissues are first mechanically disintegrated using a blender (for large sample volumes), a homogenizer (for small volumes), or by sonication. Cells may also be pried open by one of the mechanical methods mentioned above. Miscellaneous detergents, salts, and buffers may be used to promote cell lysis and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the sample from being digested by its own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0239] Proteins in a sample are separated using gel electrophoresis. Proteins may be separated by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The type of separation depends on the sample processing and the gel. This is a very useful technique to ascertain proteins. It is also possible to use two-dimensional (2D) gels that spread the proteins from a single sample in two dimensions. Proteins are separated according to their isoelectric point (pH at which they have a neutral net charge) in the first dimension and according to molecular weight in the second dimension.

[0240] To make the proteins available for antibody detection, the proteins are transferred from the inside of the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). A membrane is placed on top of the gel, and a stack of filter papers is placed on top of the membrane. The whole stack is then placed in a buffer solution, which rises up the paper by capillary action, thereby carrying the proteins with it. Another method for transferring proteins is called electroblotting, which uses an electric current to pull the proteins from the gel to a PVDF or nitrocellulose membrane. The proteins move from the inside of the gel to the top of the membrane while maintaining the structure they had inside the gel. As a result of this blotting process, the proteins are exposed to a thin surface layer for detection (see below). Both types of membranes are selected for their nonspecific protein binding properties (i.e., they bind all proteins equally). Protein binding is based on hydrophobic interactions and charged interactions between the membrane and the proteins. Nitrocellulose membranes are cheaper than PVDF, but are much more fragile and less resistant to repeated interrogations. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once transferred, the proteins are detected using a labeled primary antibody or an unlabeled primary antibody followed by indirect detection using either labeled Protein A or binding of a secondary labeled antibody to the Fc region of the primary antibody.

[0241] C. Lateral flow assay Lateral flow assays, also known as lateral flow immunochromatographic assays, are simple devices aimed at detecting the presence (or absence) of a target analyte in a sample (matrix) without the need for specialized and expensive equipment, but there are many laboratory-based applications supported by a reading device. Typically, these tests are used as low-resource medical diagnostics, either for at-home testing, point-of-care immediate testing, or laboratory use. A well-known and widespread application is the home pregnancy test.

[0242] The technology is based on a series of capillary beds, such as pieces of porous paper or sintered polymers. Each of these elements has the ability to spontaneously transport fluids (e.g. urine). The first element (sample pad) acts as a sponge and holds excess sample liquid. When immersed, the fluid moves to the second element (conjugate pad), which is stocked by the manufacturer with bioactive particles (see below), so-called conjugates, in a dried form in a salt-sugar matrix that contains everything to ensure an optimized chemical reaction between the target molecule (e.g. antigen) and its chemical partner (e.g. antibody) immobilized on the surface of the particle. The sample fluid dissolves the salt-sugar matrix, but also the particles, and in one combined transport action, the sample and the conjugates mix as they flow through the porous structure. In this way, the analyte binds to the particles as it moves further through the third capillary bed. This material has one or more areas (often called stripes) on which the manufacturer immobilizes a third molecule. By the time the sample and conjugate mixture reaches these strips, the analyte is bound to the particles and a third "capture" molecule binds to the complex. After a while, as more fluid passes through the stripes, particles accumulate and the stripe area changes color. There are usually at least two stripes: one (control) that captures any particles and thereby indicates that the reaction conditions and technique worked properly, and another that contains a specific capture molecule and only captures particles on which the analyte molecules are immobilized. After passing through these reaction zones, the fluid enters the wick, a final porous material that simply functions as a waste container. Lateral flow tests can function as either competitive or sandwich assays. Lateral flow assays are disclosed in U.S. Pat. No. 6,485,982.

[0243] D. Immunohistochemistry The antibodies of the present disclosure may also be used with fresh frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). Methods for preparing tissue blocks from these particulate preparations have been used successfully in previous IHC studies of various prognostic factors and are well known to those of skill in the art.

[0244] Briefly, frozen sections may be prepared by rehydrating 50 ng of "pulverized" frozen tissue in phosphate buffered saline (PBS) at room temperature in a small plastic capsule; pelleting the particles by centrifugation; resuspending in viscous embedding medium (OCT); inverting the capsule and / or repelleting by centrifugation; flash freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen tissue cylinder; fixing the tissue cylinder to a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, the entire frozen tissue sample may be used to cut the serial sections.

[0245] Permanent sections may be prepared by a similar method involving rehydrating 50 mg of sample in a plastic microcentrifuge tube, pelleting, resuspending in 10% formalin for 4 hours fixation, washing / pelleting, resuspending in warm 2.5% agar, pelleting, chilling in ice water to set the agar, removing the tissue / agar block from the tube, infiltrating and / or embedding the block in paraffin, and / or cutting up to 50 serial permanent sections. Again, the entire tissue sample may be substituted.

[0246] E. Immunodetection Kits In yet another embodiment, the present disclosure relates to an immunodetection kit for use in the above immunodetection method.Antibody can be included in the kit, since it can be used to detect homotrimeric type I collagen.Accordingly, the immunodetection kit includes, in suitable container means, a first antibody that binds to homotrimeric type I collagen, and optionally an immunodetection reagent.

[0247] In certain embodiments, the homotrimeric collagen type I antibody may be pre-immobilized on a solid support, for example, a column matrix and / or a well of a microtiter plate.The immunodetection reagent of the kit may take any one of a variety of formats, including a detectable label that is bound or linked to a given antibody.Detectable labels that are bound or attached to secondary binding ligands are also contemplated.An exemplary secondary ligand is a secondary antibody that has binding affinity for the first antibody.

[0248] Further suitable immunodetection reagents for use in the kits of the present invention include two-component reagents comprising a second antibody having binding affinity for the first antibody and a third antibody having binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, several exemplary labels are known in the art, and all such labels may be utilized in connection with the present disclosure.

[0249] The kit may further include an appropriately dispensed composition of homotrimeric collagen type I, labeled or unlabeled, which may also be used to generate a standard curve for the detection assay. The kit may include the antibody-label conjugate in fully conjugated form, in the form of an intermediate, or as a separate moiety that is conjugated by the user of the kit. The components of the kit may be packaged in a dissolved state in an aqueous medium or in a lyophilized form.

[0250] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody may be placed, and preferably suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials may be held. EXAMPLES

[0251] IV. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples demonstrate techniques that the inventors have discovered work well in the practice of the invention, and thus are believed to constitute preferred modes for its practice. However, in light of this disclosure, it should be understood by those skilled in the art that many changes can be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the invention.

[0252] material and method mouse FSF-Kras G12D / + (Schonhuber et al., 2014), Pdx1-Flp (Schonhuber et al., 2014), Trp53 frt / + (Lee et al., 2012), LSL-Kras G12D / + (Hingorani et al., 2005), Trp53 loxP / + (Chen et al., 2005), Pdx1-Cre (Hingorani et al., 2005), αSMA-Cre (LeBleu et al., 2013), and Fsp1-Cre (Xue et al., 2003; Bhowmick et al., 2004) mouse lines have been established to date. loxP / loxPThe mouse strain (containing loxP-flanked exons 2–5) was Col1a1, purchased from the European Mouse Mutant Cell Repository (EuMMCR). tm1a(EUCOMM)Wtsi The line was established from Rosa26-CAG-loxP-frt-Stop-frt-FireflyLuc-EGFP-loxP-RenillaLuc-tdTomato (R26 Dual The mouse strain, R26, Dual Dual fluorescent reporter alleles are included, allowing EGFP expression under the control of the Pdx1-Flp transgene, or tdTomato expression under the control of the αSMA-Cre and Fsp1-Cre transgenes. G12D / + ;Pdx1-Flp (called KF) or FSF-Kras G12D / + ;Trp53 frt / frt Characterization of the genotype and disease phenotype of KF and KPPF mice was performed as previously described (Schonhuber et al., 2014). KF and KPPF mice were transfected with αSMA-Cre, Pdx1-Cre, Fsp1-Cre, Col1a1 loxP / loxP , or R26 Dual The mouse strain was crossed with KF;αSMA-Cre;Col1a1 loxP / loxP (KF;Col1 smaKO (also called KF; Pdx1-Cre; Col1a1) loxP / loxP (KF;Col1 pdxKO (also called KPPF;αSMA-Cre;Col1a1) loxP / loxP (KPPF;Col1 smaKO ), and KPPF;Fsp1-Cre;Col1a1 loxP / loxP (KPPF;Col1 fspKO These mice allow deletion of Col1a1 in PDAC-associated fibroblast subpopulations expressing αSMA or Fsp1. G12D ;Pdx1-Cre (known as KC) or LSL-Kras G12D ;Trp53 loxP / loxPPdx1-Cre (KPPC) mice were transfected with Col1a1 loxP / loxP The mouse strain was crossed with KC;Col1a1 loxP / loxP (KC;Col1 pdxKO (also called KPPC; Col1a1) loxP / loxP (KPPC;Col1 pdxKO This study resulted in the generation of mice (referred to as Col1a1-positive mice) that allow for the deletion of Col1a1 in PDAC cells. The aforementioned experimental mice with the desired genotype were monitored and analyzed without randomization or blinding. Both female and male mice with the desired genotype for PDAC were used for the experiments. All mice were housed under standard housing conditions in the MD Anderson Cancer Center (MDACC) animal facility, and all animal procedures were reviewed and approved by the MDACC Institutional Animal Care and Use Committee.

[0253] Example 1. The Dual Recombinase System (DRS) mouse model induces spontaneous pancreatic cancer and allows genetic modulation in various target cell populations. The novel dual recombinase system (DRS) mouse model of pancreatic cancer utilizes the flippase-FRT (Flp-FRT) system to mediate oncogenic Kras expression and p53 loss in the Pdx1 lineage (FSF-Kras G12D / + ;Trp53 frt / frt ;Pdx1-Flp) and is induced in the widely used KPC (LSL-Kras G12D / + ;Trp53 R172H / + or Trp53 loxP / loxP This Flp-FRT-based DRS (FSF-Kras ;Pdx1-Cre) mouse model replaced the conventional Cre-loxP system. G12D / + ;Trp53 frt / frt The KPPF mouse model is a conven- tional Cre-loxP-based (LSL-Kras ;Pdx1-Flp, collectively referred to as “KPPF”) mouse model, as also substantiated by the original DRS model study (Schonhuber et al., 2014). G12D / + ;Trp53 loxP / loxPBoth DRS mice develop pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC) in a manner nearly identical to the Flp-FRT-based (DRS-Pdx1-Cre, broadly referred to as "KPPC") mouse model (Figure 7A-D). As expected, both pancreatic cancer mouse model systems showed prominent collagen type I (Col1) deposition during disease development. Importantly, this new DRS model system allows for the addition of another genetic engineering system with a Cre transgene and a floxed (flanked by loxP sites) allele, independent of the spontaneous PDAC induced by the Flp-FRT-based system.

[0254] To test the functionality of this DRS mouse model carrying both the Cre-loxP and Flp-FRT systems, we introduced a novel lineage-tracing dual reporter (Rosa26-CAG-loxP-frt-Stop-frt-FireflyLuc-EGFP-loxP-RenillaLuc-tdTomato, hereafter R26). Dual (hereinafter referred to as KPPF;αSMA-Cre;R26 Dual In PDAC tissues from this mouse line, cancer cells in the Pdx1 lineage expressed EGFP, and PSCs activated in the αSMA lineage expressed tdTomato (Fig. 7F and G), confirming genetic recombination by Pdx-Flp and αSMA-Cre, respectively.

[0255] Example 2. Type I collagen (Col1) deposition changes along the developmental stages of PanIN / PDAC Using IHC staining on serial sections, the expression of Col1, compared to the expression levels of CK19 and αSMA (markers of cancer cells and activated PSCs, respectively), was examined during the development of PanIN / PDAC (Figure 8A-B). The expression of the aforementioned proteins revealed dynamic changes (Figure 8C). Normal pancreatic tissue revealed minimal / negligible presence of CK19, αSMA, or Col1. Upon the appearance of ADM (or early PanIN) lesions, αSMA quickly rose to its highest levels due to the activation of PSCs in response to abnormalities in the pancreatic epithelium. These activated PSCs started to generate interstitial Col1, resulting in peak levels of Col1 fibers in the next stage of PanIN. As the disease continued to develop from PanIN to PDAC, the cancer cell population became larger than the stromal component, coinciding with a decrease in the presence of αSMA or Col1 positive areas.

[0256] Notably, CK19 levels were constantly increased throughout PanIN / PDAC development. αSMA reached highest levels at the acinar-ductal metaplasia (ADM) or early PanIN stage, indicating immediate recruitment and / or activation of αSMA-positive PSCs in response to the very early stages of disease progression. In contrast, Col1 levels reached highest levels during the PanIN stage and then decreased during development to the PDAC stage. PDAC tissues revealed the presence of predominant cancer cells (CK19-positive areas) along with a dilution / decrease in the presence of Col1- and αSMA-positive activated PSCs.

[0257] Notably, despite the nonlinear dynamics of Col1 levels, the Col1 / CK19 ratio continually decreased as the disease progressed. These results suggest that a decrease in the Col1 / CK19 ratio may indicate a decreased host restraint on PDAC development and a more advanced disease state.

[0258] Example 3. Col1 deletion in activated PSCs expressing αSMA accelerates PDAC development and shortens animal survival The above findings were consistent with previous studies that implicated activated PSC populations (rather than pancreatic cancer cells) as the major producers of Col1 in PDAC stroma. Therefore, genetic ablation of Col1 in activated PSC populations was explored using the new DRS mouse model. As shown in Figure 1A, Col1 was expressed in the KPPF;Col1 smaKO (FSF-Kras G12D / + ;Trp53 frt / frt ;Pdx1-Flp;αSMA-Cre;Col1a1 loxP / loxP ) was specifically deleted in αSMA-expressing activated PSCs of PDAC in mice. Deletion of Col1 in αSMA-expressing activated PSCs resulted in reduced levels of fibrous Col1, desmoplasia, and stiffness in PDAC tissues, as shown by IHC staining in serial sections (Figure 1B).

[0259] Col1 deletion in activated PSCs expressing αSMA in the context of PDAC significantly shortened animal survival (Figure 2B) and led to a higher incidence of ascites at the end-point stage (Figure 2C). smaKO Reduced Col1 levels in tumors were observed at both PanIN and PDAC stages (Figure 2F), which was greater in KPPF;Col1 tumors than in KPPF tumors. smaKO This was accompanied by a significantly decreased Col1 / CK19 ratio in the tumors (Figure 2D). These findings further support the notion that a lower Col1 / CK19 ratio correlates with reduced host restraint on PDAC development and a more advanced disease state. We next examined the Col1 / CK19 ratio by comparing the mRNA levels of Col1a1 and CK19 (RNA Seq V2 RSEM) in human PDAC samples from the TCGA database. A lower Col1 / CK19 ratio correlated with significantly worse overall survival (OS) and progression-free survival (PFS), consistent with findings in transgenic mouse models (Figure 9).

[0260] Carrying oncogenic Kras mutations but not p53 loss, KF;Col1 smaKO (FSF-KrasG12D / + ;Pdx1-Flp;αSMA-Cre;Col1a1 loxP / loxP ) mice were generated to observe the effects on the early stages of PDAC development (ADM and / or PanIN) (Figure 10A). Age-matched (6-month-old) animals were examined for the development of ADM and PanIN lesions. As shown in Figure 10B, KF;Col1 mice were generated to observe the effects on the early stages of PDAC development (ADM and / or PanIN) (Figure 10A). Age-matched (6-month-old) animals were examined for the development of ADM and PanIN lesions. smaKO Mice exhibited significantly larger areas of ADM and PanIN lesions than KF littermate control mice. Collectively, these findings are consistent with previous findings demonstrating a tumor-suppressive function of myofibroblast subpopulations in the PDAC microenvironment.

[0261] Example 4. Deletion of Col1 in pancreatic cancer cells delays the development of ADM and PanINs While some studies have proposed cancer-associated fibroblasts as the primary producers of Col1, others have highlighted the potentially unique composition and function of cancer cell-derived Col1 (Sengupta et al., 2003; Han et al., 2008; Egeblad et al., 2010; Han et al., 2010; Makareeva et al., 2010). To achieve genetic ablation of Col1 in cancer cells, we developed another DRS mouse model, KF;Col1 pdxKO (FSF-Kras G12D / + ;Pdx1-Flp;Pdx1-Cre;Col1a1 loxP / loxP ) was established. KF;Col1 pdxKO The lines had the same KF background but incorporated the Pdx1-Cre transgene (Figure 3A) to replace the previous KF;Col1 smaKO The αSMA-Cre transgene was replaced in the line.

[0262] Notably, KF;Col1 pdxKO The mouse model is KF;Col1 smaKO The same control mice (KF; Cre-negative; Col1a1 loxP / loxP), and at the same age of 6 months, the three lineages (KF control, KF with Col1 deletion in αSMA-expressing myofibroblasts as shown in FIG. 10A; Col1 smaKO group, and KF;Col1 with Col1 deletion in Pdx1 lineage cancer cells pdxKO Interestingly, the Pdx1 lineage cancer cells with Col1 deletion were significantly more aggressive than the KF;Col1 pdxKO Mice were: KF;Col1 smaKO In contrast to the accelerated disease progression in KF;Col1 mice, we revealed a significant delay in ADM and PanIN development compared to KF control mice (Figures 3B-C and 7J). pdxKO Although pancreatic tissues from KF mice revealed significantly better histology and fewer ADM / PanIN areas than KF control mice, Col1 deposition levels within any given field at the same PanIN stage (Figure 3B, 20x magnification panel) did not differ between these two mouse groups. These results suggest that cancer-derived Col1 may have important cancer-supportive functions, although its presence may often be obscured by abundant Col1 produced by myofibroblasts at the PanIN stage.

[0263] Nevertheless, KF;Col1 at early stages of disease progression (ADM) was observed when PSCs were just activated and did not deposit large amounts of Col1. pdxKO A decrease in Col1 levels was observed in KF;Col1 mice (Fig. 3D). pdxKO ADM lesions in mice not only showed reduced Col1 deposition but also significantly decreased levels of Sox9, an essential marker for pancreatic organogenesis and pancreatic cancer development (Seymour et al., 2007; Kopp et al., 2012) (Figure 3E-F). These findings suggest that Col1 deposition by cancer-initiating cells supports the early development of pancreatic cancer.

[0264] KF;Col1 pdxKOIn addition to the mouse model, another mouse model was generated in parallel to achieve genetic deletion of Col1 in Pdx1 lineage cancer cells. Here, KC (LSL-Kras G12D / + ;Pdx1-Cre) control mice using a conventional Cre-loxP-based system, compared with KC;Col1 pdxKO (LSL-Kras G12D / + ;Pdx1-Cre;Col1a1 loxP / loxP ) mouse line was established (Figure 11A). Consistent results were obtained using the KC;Col1 pdxKO Mice were obtained from KC mice and showed significantly delayed development of ADM and PanINs compared with KC control mice at the same age of 6 months (Figure 11B).

[0265] Example 5. Deletion of Col1 in pancreatic cancer cells delays PDAC development and animal survival Next, KPPC;Col1 pdxKO (LSL-Kras G12D / + ;Trp53 loxP / loxP ;Pdx1-Cre;Col1a1 loxP / loxP We generated a KPPC;Col1 mouse model, which carried both oncogenic Kras mutations and homozygous loss of p53 induced by the conventional Cre-loxP system (Figure 4A). These mice, which have a KPPC genetic background, developed acute PDAC within 45 days, leading to animal death at approximately 55 days of age. Consistent with previous findings (Figures 3A-F and 11A-B), KPPC;Col1 pdxKO Deletion of Col1 in cancer cell lines in mice significantly prolonged animal survival and delayed the development of PDAC when compared with KPPC control mice (Figure 4B). loxP Further KPPC with deletion;Col1 pdxKO / + Strain (LSL-Kras G12D / + ;Trp53 loxP / loxP ;Pdx1-Cre;Col1a1 loxP / + ) were also generated and showed animal survival similar to that of the KPPC control line (Figure 12A).

[0266] The early stages of disease development were observed in the same 28-day-old KPPC;Col1pdxKO The study was carried out in KPPC;Col1 mice and KPPC control mice. pdxKO The pancreata of KPPC;Col1 mice revealed significantly fewer ADM and PanIN lesions than KPPC control mice (Figure 4C). pdxKO (LSL-Kras G12D / + ;Trp53 loxP / loxP ;Pdx1-Cre;Col1a1 loxP / loxP ) revealed significantly better histology (Fig. 4D and E) and reduced pancreatic tumor burden (Fig. 4F) compared with age-matched KPPC control mice.

[0267] RNA sequencing analysis was performed on KPPC;Col1 at the same age of 53 days. pdxKO Gene set enrichment analysis (GSEA) was performed on total RNA from tumor tissues of KPPC;Col1 mice (n = 5) and KPPC control mice (n = 4). pdxKO We revealed significantly upregulated transcriptional signatures in distinctive pathways related to interferon response, inflammatory response, mesenchymal signature, IL6 / IL2 pathway, and Kras downregulation signaling in tumors (Figure 5C and D). These results demonstrate elevated immune response, immune infiltration, and stromal response upon Col1 deletion in cancer cells, which further contribute to suppression of PDAC progression. This is surprising given that inflammation has been shown to directly contribute to PDAC development, but these results reveal upregulation of inflammatory pathways with delayed PDAC development with better histology upon Col1 deletion in cancer cells. In contrast, GSEA also revealed significantly upregulated transcriptional signatures in distinctive pathways related to TGF-β signaling and mitotic spindle regulation in KPPC tumors, consistent with more advanced PDAC stage in these tumors.

[0268] RNA sequencing analysis revealed that KPPC and KPPC;Col1 pdxKOThis was also performed on total RNA from primary cancer cell lines. We observed significant changes in gene expression profiles upon Col1a1 deletion in cancer cells (Fig. 5H and I).

[0269] Example 6. PDAC cancer cells exhibited significant phenotypic changes upon Col1 deletion The primary pancreatic cancer cell lines were also KPPC;Col1, respectively. pdxKO As shown in Figure 6A, KPPC;Col1 mice and KPPC control mice were established from tumor tissues. pdxKO The primary cancer cell lines revealed reduced cell adhesion and different cell morphology (spindle-shaped cells) when compared to the KPPC cancer cell line (cobblestone-shaped cells growing in colonies).

[0270] KPPC;Col1 in 2D cell culture system pdxKO The proliferation of the primary cancer cell lines was significantly slower than that of the KPPC cancer cell lines (MTT; Figure 6B). pdxKO Primary cancer cell lines also demonstrated impaired ability to form tumor spheres in 3D Matrigel (Figure 6C and D).

[0271] Interestingly, primary PDAC cells derived from KPPC mice revealed detectable expression levels of Col1a1, but not Col1a2, consistent with the notion that cancer cells in some cancer types express Col1 homotrimer(α1)3 due to DNA hypermethylation of the Col1a2 gene and loss of Col1a2 expression (Figure 6E). pdxKO Primary PDAC cells derived from mice showed efficient knockdown of Col1a1 but significantly increased expression levels of Col4a1, Col5a2, and Col9a1, possibly due to a complementation mechanism ( Fig. 6E ).

[0272] To examine the DNA methylation level of the Col1a2 gene, we performed methylated DNA immunoprecipitation (MeDIP) assays in multiple PDAC cell lines established from tumors of various PDAC transgenic mouse models, including KF, KPF, KPPF, KPC, and KPPC lines (Figure 6F). MeDIP assays revealed DNA hypermethylation of the Col1a2 gene, but not the Col1a1 gene, in these mouse primary PDAC cells (Figure 6F), a consistent finding in human cancer cell lines (Figure 12C). In contrast, fibroblasts isolated from KPPC mouse tumors revealed very low levels of Col1a2 DNA methylation (Figure 6F) and expressed high levels of both Col1a1 and Col1a2 at similar levels (Figure 13). Furthermore, treatment with the demethylating agent 5-azacytidine partially restored the expression level of Col1a2 in cancer cells, but not in fibroblasts (Figure 13). These results confirmed the suppression of Col1a2 expression in cancer cells by DNA hypermethylation.

[0273] Next, KPPC and KPPC;Col1 pdxKO We investigated cell proliferation in cancer cell lines when treated with various concentrations of Col1. Interestingly, Col1 treatment slightly inhibited cell proliferation in the KPPC cancer cell line, whereas KPPC;Col1 pdxKO The Col1a1 homotrimer significantly inhibited the proliferation of cancer cell lines (Figure 12D). This is intriguing, given that Col1 isolated from rat tail tendons is a heterotrimer, in contrast to homotrimeric Col1 derived from cancer cells. These findings are consistent with the results that heterotrimeric Col1 derived from myofibroblasts suppresses pancreatic tumor growth, especially when cancer cells are deleted for their own Col1a1 homotrimer. These results indicate distinct functions of Col1 homotrimers derived from cancer cells and Col1 heterotrimers derived from normal tissues.

[0274] Interestingly, KPPC;Col1 pdxKOCancer cells revealed an unexpected increase in DDR1, one of the receptors for Col1, in epithelial and cancer cells. Next, we examined the effect of a DDR1 inhibitor (3-(2-(pyrazolo(1,5-a)pyrimidin-6-yl)-ethynyl)benzamide compound (7rh)) on KPPC and KPPC;Col1 in the presence of Col1 supplement (Col1 heterotrimer solution from rat tail). pdxKO Interestingly, both KPPC and Col1 cancer cell lines were tested. pdxKO Cancer cells responded to 7rh differently from KPPC control cells, and showed a significant increase in cell proliferation at lower doses of 7rh. This result suggests that low concentrations of 7rh could inhibit the proliferation of KPPC;Col1 by the Col1 heterotrimer solution provided. pdxKO It is suggested that 7rh can reverse the proliferation inhibition on cancer cells, whereas higher concentrations of 7rh can ultimately block this signaling pathway and significantly inhibit cell proliferation.

[0275] Example 7. Deletion of Col1 in Fsp1-expressing fibroblast subpopulations did not affect PDAC progression Given our previous findings showing that Col1 deletion in activated PSCs expressing αSMA results in accelerated PanIN development, we next asked whether Col1 deletion in another fibroblast subpopulation within PDAC could also lead to a similar phenotype. fspKO (FSF-Kras G12D / + ;Trp53 frt / frt ;Pdx1-Flp;Fsp1-Cre;Col1a1 loxP / loxP ) mice (Figure 14A) were generated with a fibroblast-specific Fsp1-Cre transgene. Interestingly, KPPF;Col1 mice (Figure 14B) were generated with a fibroblast-specific Fsp1-Cre transgene, allowing Col1 deletion in Fsp1-expressing fibroblasts. fspKO Mice demonstrated no difference in animal survival and PDAC progression when compared to KPPC littermate control mice (Figure 14B). fspKOThe system efficiently deleted Col1 in Fsp1-expressing fibroblasts, as confirmed in isolated primary Fsp1-expressing fibroblasts (Figure ​(Figure14C). 14C). However, the overall levels of Col1 in PDAC tissues were significantly lower in KPPF;Col1 mice compared to KPPF control mice. fspKO The Fsp1-expressing fibroblast subpopulation was not significantly decreased in PDAC mice (Figure S14D), suggesting that the Fsp1-expressing fibroblast subpopulation may not be the major contributor to Col1 in the PDAC stroma. These results support the heterogeneity of fibroblast subpopulations in the PDAC microenvironment and their various contributions to collagen deposition.

[0276] To further investigate the heterogeneity of fibroblast subpopulations, we cloned KPPF;Fsp1-Cre;R26, a mouse model in which Pdx1 cancer cells express EGFP and Fsp1 fibroblasts express tdTomato. Dual We generated Fsp1-Cre mice (Figure 15A). The specificity and efficacy of the Fsp1-Cre transgene in this mouse model was confirmed by the colocalization between Fsp1 antibody staining and Fsp1-Cre-induced tdTomato signal (Figure 15B). Interestingly, Fsp1-expressing fibroblasts revealed a stromal localization pattern in the PDAC stroma, which was significantly different from the peritumoral localization of αSMA-expressing activated PSCs (Figure 15B). Such minimal colocalization between Fsp1 and αSMA fibroblast subpopulations was also confirmed by immunofluorescence staining with αSMA and Fsp1 antibodies (Figure 15C).

[0277] All of the methods disclosed and claimed herein can be made and carried out without undue experimentation in light of the present invention. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and to the steps or sequence of 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 agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0278] References The following references, to the extent that they provide exemplary procedural details or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007599716000002.tif183150TIFF0007599716000003.tif223150TIFF0007599716000004.tif217150TIFF0007599716000005.tif17150

Claims

1. A pharmaceutical composition for treating pancreatic cancer in a patient in need thereof, comprising an antibody that binds to α1 homotrimeric type I collagen or an antibody fragment that binds to α1 homotrimeric type I collagen.

2. The pharmaceutical composition of claim 1, wherein the cancer patient has been determined to express elevated levels of α1 homotrimeric type I collagen compared to a control patient.

3. 13. The pharmaceutical composition of claim 1, for use in a method for inhibiting pancreatic cancer metastasis.

4. 13. The pharmaceutical composition of claim 1, for use in a method for inhibiting the growth of pancreatic cancer.

5. 13. The pharmaceutical composition of claim 1, used in combination with at least a second anti-cancer therapy.

6. 6. The pharmaceutical composition of claim 5, wherein the second anti-cancer therapy is chemotherapy, immunotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or cytokine therapy.

7. the antibody has an affinity for α1 homotrimeric type I collagen that is at least 2-fold greater, or at least 5-fold greater, than it has affinity for α1 / α2 / α1 heterotrimeric type I collagen, or The antibody does not detectably bind to α1 / α2 / α1 heterotrimeric type I collagen 2. The pharmaceutical composition of claim 1.

8. 1. A composition for use in a method of treating pancreatic cancer in a patient, comprising chimeric antigen receptor (CAR) T cells or CAR NK cells, wherein the CAR T cells or CAR NK cells 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, and wherein the CAR polypeptide binds to alpha 1 homotrimeric type I collagen.

9. The composition of claim 8, wherein the CAR T cells or CAR NK cells are allogeneic cells.

10. The composition of claim 8, wherein the CAR T cells or CAR NK cells are autologous cells.

11. The composition of claim 8, wherein the CAR T cells or CAR NK cells are HLA-matched to the subject.

12. The composition of any one of claims 8-11, wherein the method further comprises administering a demethylating agent prior to administration of the CAR T cells.

13. The composition of claim 12, wherein the demethylating agent reverses Col1A2 hypermethylation.

14. 13. The composition of claim 12, wherein the demethylating agent is 5-azacytidine or 5-aza-2'-deoxycytidine.

15. 1. A method for classifying a patient having pancreatic ductal adenocarcinoma, the method comprising the step of determining a type I collagen / CK19 ratio in cancer tissue obtained from the subject, wherein a ratio lower than the ratio in a reference normal tissue indicates that the patient has a more advanced disease state.

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