Anti-variable muc1*antibody and use thereof

Abstract

To provide an antibody for diagnosis, treatment, or prevention of cancer.SOLUTION: There is provided an antibody or a fragment thereof for diagnosis, treatment, or prevention of cancer, the antibody specifically binding to a PSMGFR peptide (SEQ ID NO: 2) or a fragment of the peptide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application relates to human, humanized, and non-human anti-MUC1* antibodies, and methods of making and using them. This application also relates to the use of immune cells transfected or transduced with a cleavage enzyme for the treatment of cancer. The invention also relates to the use of immune cells transfected or transduced with a CAR and another protein for the treatment of cancer. [Background technology]

[0002] The present inventors previously discovered that a truncated form of the MUC1 (SEQ ID NO: 1) transmembrane protein is a growth factor receptor that drives the growth of more than 75% of all human cancers. * A truncated form of MUC1, termed MUC1 (pronounced muk-1-star), is a potent growth factor receptor. Cleavage and release of most of the extracellular domain of MUC1 results in the formation of the ligand dimer NME1, NME6, NME7, and NME7. AB , NME7-X1, or NME8, which are aberrantly expressed in over 75% of all cancers and may be overexpressed in an even higher percentage of metastatic cancers, making them ideal targets for anticancer drugs (Mahanta et al., (2008) A Minimal Fragment of MUC1 Mediates Growth of Cancer Cells. PLoS ONE 3(4):e2054. doi:10.1371 / journal.pone.0002054; Fessler et al., (2009) "MUC1 *"MUC1 is a determinant of trastuzumab (Herceptin) resistance in breast cancer cells" Breast Cancer Res Treat. 118(1):113-124). After MUC1 cleavage, most of its extracellular domain is shed from the cell surface. The remaining portion has a truncated extracellular domain that contains most or all of the primary growth factor receptor sequence called PSMGFR (SEQ ID NO: 2).

[0003] Antibodies are increasingly being used to treat human diseases. Antibodies generated in non-human species, such as equine antibodies, have historically been used as human therapeutics. More recently, antibodies have been engineered or selected to contain most or all human sequences to avoid the typical rejection of foreign antibodies. The process of engineering the recognition fragment of a non-human antibody into a human antibody is commonly referred to as "humanizing." The amount of non-human sequence used to replace human antibody sequences determines whether they are referred to as chimeric, humanized, or fully human.

[0004] Alternative technologies exist that allow for the generation of humanized or fully human antibodies. These strategies involve screening libraries of human antibodies or antibody fragments and identifying those that bind to the target antigen, rather than immunizing animals with an antigen. Another approach is to engineer the variable region(s) of an antibody into an antibody-like molecule. Another approach involves immunizing a humanized animal. The present invention also provides a method for the production of humanized or fully human antibodies, which the inventors have developed to date. * It is intended to encompass these approaches for use with recognition fragments of antibodies determined to bind to the extracellular domain of .

[0005] In addition to treating patients with antibodies, cancer immunotherapy has recently proven effective in treating hematologic cancers. One cancer immunotherapy, called CAR T (chimeric antigen receptor T cell) therapy, involves engineering T cells to express chimeric receptors with an extracellular domain that recognizes tumor antigens, a transmembrane domain, and a cytoplasmic tail containing T cell signaling and costimulatory components (Dai H, Wang Y, Lu X, Han W (2016) Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy. J Natl Cancer Inst. 108(7):djv439). Such receptors consist of a single-chain antibody fragment (scFv) that recognizes tumor antigens and is linked to T cell transmembrane, signaling, and costimulatory domain(s). When the receptor binds to a cancer-associated antigen, a signal is transmitted, resulting in T cell activation, proliferation, and targeted killing of cancer cells. In practice, T cells are isolated from a patient or donor, transduced with a CAR, expanded, and then injected back into the patient. If from a donor, the immune cells can be mutated or engineered so that they do not induce graft-versus-host disease in the recipient. When the CAR T cells bind to an antigen on the cancer cells, they attack the cancer cells and then expand their population of T cells.

[0006] To date, CAR T therapy has been highly successful in treating hematologic cancers, but its efficacy against solid tumors in humans has yet to be demonstrated. Because most hematologic cancers are B-cell malignancies, CAR T cells can eliminate only a patient's B cells without causing significant harm to the patient. Solid tumors have no B-cell equivalent. Most tumor-associated antigens are expressed on normal tissues and at higher levels in cancerous tissues. Therefore, the challenge is to develop antibodies that recognize epitopes on tumor-associated antigens that are somehow different in the tumor context compared to normal tissues. To further minimize the risk of off-tumor / on-target killing of normal tissues, antibodies should recognize and bind to cancerous tissue at least twice as often as normal tissues. Antibodies with less cancer selectivity may be used therapeutically if they are inducibly expressed at the tumor site.

[0007] Another cancer treatment incorporating cancer-selective antibodies is a bispecific T cell engager, also known as a BiTE. The BiTE approach attempts to eliminate the risk of off-tumor / on-target effects associated with CAR T. Unlike CAR T, BiTEs are bispecific antibodies that should not pose any greater risks than conventional antibody-based therapies. However, unlike typical anti-cancer antibodies that bind and block cancer antigens, BiTEs are designed to simultaneously bind to antigens on tumor cells and immune cells, such as T cells. In this way, BiTEs recruit T cells to tumors. BiTEs are engineered proteins that simultaneously bind to cancer-associated antigens and T cell surface proteins, such as CD3-epsilon. BiTEs are antibodies created by genetically linking the scFv of an antibody that binds to a T cell antigen, such as anti-CD3-epsilon, to the scFv of a therapeutic monoclonal antibody that binds to a cancer antigen (Patrick A. Baeuerle, and Carsten Reinhardt (2009) Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res. 69(12):4941-4944). A drawback of BiTE technology is that, unlike CAR T cells, they do not expand within the patient's body, limiting their persistence.

[0008] Yet another cancer treatment incorporating cancer-selective antibodies is antibody-drug conjugate technology, also known as ADC. In this case, a toxin, or a precursor to a toxin, is linked to the cancer-selective antibody. Unlike CART cells, which exploit the natural death of CD8+ T cells to kill cancer cells, ADCs deliver a toxic payload to tumors. The drawbacks of ADCs are the possibility that the toxic payload may be delivered to normal cells, and the fact that most ADCs must bind to cell surface molecules that are then internalized after binding, requiring approximately 10,000 surface molecules for eventual cell death. Summary of the Invention [Problem to be solved by the invention]

[0009] In one aspect, the present invention provides a non-human, human, or humanized anti-MUC1 antibody that binds to a region on the extracellular domain or cleavage product of a MUC1 isoform that lacks the tandem repeat domain. * Antibodies or antibody fragments, or antibody-like proteins. Non-human, human or humanized anti-MUC1 * The antibody or antibody fragment or antibody-like protein may specifically bind to: (I) PSMGFR region of MUC1; (ii) PSMGFR peptide; (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10) (SEQ ID NO: 3); (iv) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFSAQSGA(N-19) (SEQ ID NO: 4); (v) a peptide having the amino acid sequence NLTISDVSVSDVPFPFSAQSGA(N-23) (SEQ ID NO: 5); (vi) a peptide having the amino acid sequence ISDVSVSDVPFPFSAQSGA(N-26) (SEQ ID NO: 6); (vii) a peptide having the amino acid sequence SVSDVPFPFSAQSGA(N-30) (SEQ ID NO: 7); (viii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5) (SEQ ID NO: 8); (ix) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) (SEQ ID NO: 9); (x) A peptide having the amino acid sequence FPFSAQSGA (SEQ ID NO: 10).

[0010] The non-human, human or humanized antibody may be an IgG1, IgG2, IgG3, IgG4 or IgM. The human or humanized antibody fragment or antibody-like protein may be an scFv or scFv-Fc.

[0011] As described above, the murine, camelid, human or humanized antibody, antibody fragment or antibody-like protein may comprise heavy and light chain variable regions derived from murine monoclonal MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11 antibodies and have at least 80%, 90%, or 95% or 98% sequence identity to the murine monoclonal MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11 antibodies. The heavy chain variable regions of CDR1 and CDR2 may have at least 90%, 95%, or 98% sequence identity to the specifically designated antibody heavy chain variable region sequences set forth in the Sequence Listing section of this application, and the light chain variable regions of CDR1 and CDR2 may have at least 90%, 95%, or 98% sequence identity to the specifically designated antibody heavy chain variable region sequences set forth in the Sequence Listing section of this application. The heavy chain variable region of CDR3 may have at least 80%, 85%, or 90% sequence identity to the specifically designated antibody heavy chain variable region sequences set forth in the Sequence Listing section of this application, and the light chain variable region of CDR3 may have at least 80%, 85%, or 90% sequence identity to the specifically designated antibody heavy chain variable region sequences set forth in the Sequence Listing section of this application.

[0012] A murine, camelid, human or humanized antibody, antibody fragment or antibody-like protein according to the above may comprise complementarity determining regions (CDRs) in the heavy and light chain variable regions having at least 90%, 95%, or 98% sequence identity to the specifically designated antibody heavy chain CDR1, CDR2 or CDR3 region and light chain CDR1, CDR2 or CDR3 region sequences (as set out in the Sequence Listing section of this application).

[0013] In another aspect, the present invention provides an anti-MUC1 *The present invention relates to an extracellular domain antibody or anti-N-10 antibody, which may be any of the above antibodies consisting of a sequence represented by a humanized IgG2 heavy chain or a humanized IgG1 heavy chain paired with a humanized kappa or lambda light chain. The humanized IgG2 heavy chain may be SEQ ID NO: 53, the humanized IgG1 heavy chain may be SEQ ID NO: 57, the humanized kappa light chain may be SEQ ID NO: 108, and the humanized lambda light chain may be SEQ ID NO: 112, or a sequence having 90%, 95%, or 98% sequence identity thereto.

[0014] In another aspect, the present invention provides an anti-MUC1 antibody comprising the sequence of humanized MN-C2 represented by a humanized IgG1 or IgG2 heavy chain paired with a humanized lambda or kappa light chain. * It relates to extracellular domain antibodies or anti-N-10 antibodies.

[0015] In another aspect, the invention provides an anti-MUC1 antibody consisting of a humanized MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 sequence represented by a humanized IgG1 or IgG2 heavy chain paired with a humanized lambda or kappa light chain. * It relates to extracellular domain antibodies or anti-N-10 antibodies.

[0016] In another aspect, the present invention relates to antibodies "similar" to MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, which have the same or very similar pattern of binding to a subset of peptides derived from PSMGFR peptides, and which do not recognize linear epitopes and do not bind to MUC1. * to NME1 or NME7 ABcompetitively inhibits binding of MUC1, recognizes the MUC1 transmembrane cleavage product generated by cleavage by MMP9, or comprises CDR sequences that are at least 80% homologous to the MN-E6, MN-C2, MN-18G12, MN-20A10, MN-25E6, MN-28F9, MN-5C6F3, MN-3C2B1, and MN-1E4 CDR consensus sequences.

[0017] In another aspect, the present invention relates to an antibody that binds to the extracellular domain of MUC1 lacking the tandem repeat domain, which may be a cleavage product. In one aspect of the invention, the antibody binds to a peptide having the sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10). In one aspect of the invention, the antibody binds to a peptide having the sequence ASRYNLTISDVSVSDVPFPFSAQSGA(N-19). In one aspect of the invention, the antibody binds to a peptide having the sequence SVSDVPFPFSAQSGA(N-30). In one aspect of the invention, the antibody binds to a peptide having the sequence FPFSAQSGA(N-36). Examples of such antibodies include, but are not limited to, monoclonal antibodies MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11. The heavy and light chain complementarity determining region sequences of these antibodies are set forth in the Sequence Listing section of this application.

[0018] In one embodiment of the present invention, one or more of these antibodies are administered to a patient diagnosed with or at risk of developing cancer. The antibody may be a human or humanized antibody. The antibody may be a murine or camelid antibody. The antibody may be bivalent or monovalent. The antibody may be a fragment, including a single-chain fragment of an antibody, an scFv. The antibody or antibody fragment may be administered directly to the patient or incorporated into a bispecific antibody, a bispecific T cell inducer (BiTE), or an antibody-drug conjugate (ADC). The antibody or antibody fragment may be incorporated into a T cell receptor (TCR). The antibody or antibody fragment sequence may be incorporated into a chimeric antigen receptor, "CAR," or other similar entity, which is then introduced into immune cells ex vivo and then administered to a patient diagnosed with or at risk of developing cancer. The immune cells, which may be T cells or natural killer cells, may be derived from a donor or a patient. In one embodiment, the immune cells are derived from stem cells instructed to differentiate into that immune cell type in vitro. In one embodiment, an antibody or CAR comprising an antibody sequence can be expressed from an inducible promoter. In some cases, the antibody or CAR is expressed upon activation of T cells or other immune cells. In one example, the antibody or CAR of the present invention is expressed from an NFAT response element. In another example, CAR recognition of target tumor cells activates immune cells, resulting in the NFAT-inducible expression of cytokines such as IL-12 or IL-18, or the expression of checkpoint inhibitors such as PD1 inhibitors or PDL-1 inhibitors. In yet another embodiment, CAR recognition of target tumor cells activates immune cells, resulting in the NFAT-inducible expression of a second CAR comprising a sequence of a second antibody.

[0019] In another aspect, the present invention provides a murine, camelid, human, or humanized anti-MUC1 antibody that binds to the N-10 peptide, as described above. * The antibody or antibody fragment or antibody-like protein is an NME protein MUC1 * NME inhibits the binding of NME1, NME6, and NME7 AB , NME7-X1, NME7 or NME8.

[0020] In yet another aspect, the present invention provides a method for the production of a medicament comprising administering to a mammalian subject the invention, comprising administering to a mammalian subject the invention, a method for the production of a medicament ... * The present invention relates to single-chain variable fragments (scFv) that further comprise CDRs of an antibody that binds to the extracellular domain. The CDRs can be derived from MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11. The scFv can have SEQ ID NOs: 233, 235, and 237 (MN-E6), and SEQ ID NOs: 239, 241, and 243 (MN-C2).

[0021] In yet another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising an scFv or humanized variable region that binds to the extracellular domain of MUC1 without the tandem repeats, a linker molecule, a transmembrane domain, and a cytoplasmic domain. The single chain antibody fragment may bind to: (I) PSMGFR region of MUC1; (ii) PSMGFR peptide; (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10) (SEQ ID NO: 3); (iv) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFSAQSGA(N-19) (SEQ ID NO: 4); (v) a peptide having the amino acid sequence NLTISDVSVSDVPFPFSAQSGA(N-23) (SEQ ID NO: 5); (vi) a peptide having the amino acid sequence ISDVSVSDVPFPFSAQSGA(N-26) (SEQ ID NO: 6); (vii) a peptide having the amino acid sequence SVSDVPFPFSAQSGA(N-30) (SEQ ID NO: 7); (viii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5) (SEQ ID NO: 8); (ix) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) (SEQ ID NO: 9); (x) A peptide having the amino acid sequence FPFSAQSGA(N-36) (SEQ ID NO: 10).

[0022] In the above-mentioned CAR, any part of the variable region described above or a combination thereof can be used in the extracellular domain of the CAR.The CAR also includes a transmembrane region and a cytoplasmic tail containing a sequence motif that signals immune system activation.The extracellular domain can be composed of mouse, camelid, human, non-human, or humanized single-chain antibody fragments of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11. Additional antibodies that can be generated from single chain antibody fragments can include, but are not limited to, monoclonal antibodies similar to MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11, which have the same or very similar pattern of binding to a subset of peptides derived from the PSMGFR peptide and do not recognize linear epitopes or do not recognize NME1 or NME7. AB MUC1 * or recognize the MUC1 transmembrane cleavage product generated by cleavage by MMP9, or may comprise CDR sequences that are at least 80% homologous to the MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11 CDR consensus sequences.

[0023] In the above-described CARs, the extracellular domain may comprise a murine, camelid, human, non-human, or humanized single-chain antibody fragment of MN-E6 scFv (set forth as SEQ ID NOs: 233, 235, or 237), MN-C2 scFv (set forth as SEQ ID NOs: 239, 241, or 243), or 20A10 scFv (set forth as SEQ ID NOs: 1574-1575), 25E6 scFv (set forth as SEQ ID NOs: 1598-1599).

[0024] In any of the above CARs, the cytoplasmic tail can be composed of one or more of the signaling sequence motifs CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7. In any of the above CARs, the cytoplasmic tail can contain mutations that attenuate signaling. Such mutations include, but are not limited to, tyrosine mutated to inhibit phosphorylation and signaling (Salter et al., 2018). In any of the above CARs, the ITAM of CD3-zeta can be mutated to inhibit or attenuate signaling (Feucht et al., 2019). In any of the above CARs, the CD3 in the cytoplasmic tail can contain mutations in the ITAM, such as those called 1XX. In any of the above CARs, T cells can be engineered to overexpress c-Jun as a way to inhibit T cell exhaustion (Lynn et al., 2019).

[0025] In any of the above CARs, the sequence is selected from the group consisting of CAR MN-E6 CD28 / CD3z (SEQ ID NO: 298); CAR MN-E6 4-1BB / CD3z (SEQ ID NO: 301); CAR MN-E6 OX40 / CD3z (SEQ ID NO: 617); CAR MN-E6 CD28 / 4-1BB / CD3z (SEQ ID NO: 304); CAR MN-E6 CD28 / OX40 / CD3z (SEQ ID NO: 619); CAR MN-C2 CD3z (SEQ ID NO: 607); CAR MN-C2 CD28 / CD3z SEQ ID NO: 609); CAR MN-C2 4-1BB / CD3z (SEQ ID NO: 611 and SEQ ID NO: 719); CAR MN-C2 OX40 / CD3z (SEQ ID NO: 613); CAR MN-C2 CD28 / 4-1BB / CD3z (SEQ ID NO: 307); CAR MN-C2 CD28 / OX40 / CD3z (SEQ ID NO: 615) or CAR It may be MN-C3 4-1BB / CD3z (SEQ ID NO: 601).

[0026] In another aspect, the present invention relates to a composition comprising at least two CARs with different extracellular domain units transfected into the same cells, which may be immune cells derived from a patient in need of cancer treatment. Expression of the second CAR may be induced or driven by target recognition by the first CAR. The nucleic acid encoding the second CAR may be linked to an inducible promoter. Expression of the second CAR may be induced by an event that specifically occurs when immune cells initiate an immune response against target tumor cells. One or both antibody fragments of the CARs may bind to MUC1. * The antibody fragments of the first and second CARs can be directed against MUC1-positive tumors. The antibody fragments of the first and second CARs are directed against MUC1, which is generated when MUC1 is cleaved by two different cleavage enzymes. * Expression of the second CAR by an inducible promoter allows the antibody fragment of the first CAR to bind to MUC1 or MUC1 on the tumor. * The NFAT protein can be induced when it engages or binds to the NFAT protein. One way to do this is to induce the expression of the second CAR when, or immediately after, the NFAT protein is expressed or translocated to the nucleus. For example, a sequence derived from the NFAT promoter region is placed upstream of the gene of the second CAR. In this way, when transcription factors that bind to the promoter of the NFAT protein are present at a concentration sufficient to bind to the NFAT protein and induce its transcription, they also bind to the same promoter operated in front of the sequence for transcription of the second CAR. The NFAT protein can be NFAT1, also known as NFATc2, NFAT2, also known as NFATc or NFATc1, NFAT3, also known as NFATc4, NFAT4, also known as NFATc3, or NFAT5. In one embodiment of the present invention, the NFAT is NFATc1, NFATc3, or NFATc2. In one embodiment of the present invention, the NFAT is NFAT2, also known as NFATc1. SEQ ID NO: 646 shows the nucleic acid sequence of the transcriptional regulatory region upstream of NFAT2. The recognition unit of the second CAR can be an antibody fragment or a peptide, where the recognition unit can bind to NME7, PD-1, PDL-1, or a checkpoint inhibitor.

[0027] In at least two CARs, one CAR does not have a tumor antigen targeting recognition unit, and the other CAR has a tumor antigen targeting recognition unit. In another embodiment of the present invention, one of the extracellular domain recognition units is MUC1. * In another embodiment of the present invention, one of the extracellular domain recognition units can be an antibody fragment, and the other can be a peptide, which can lack transmembrane and signaling motifs, and the peptide can be a single-chain antibody fragment or an antibody. In another embodiment of the present invention, one of the recognition units can bind PD-1 or PDL-1. In another embodiment of the present invention, one of the extracellular domain recognition units is an anti-MUC1 antibody selected from the group consisting of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11. * The other recognition unit may be an antibody, antibody fragment, or scFv. The other recognition unit may be a CAR or an anti-NME7 antibody.

[0028] In another aspect, the invention relates to a cell comprising a CAR having an extracellular domain that binds to the extracellular domain of a MUC1 molecule lacking tandem repeats. * The present invention relates to cells comprising a CAR having an extracellular domain that binds to cells transfected or transduced with the CAR. The CAR-containing cell can be an immune system cell, preferably a T cell, a natural killer cell (NK), a dendritic cell, or a mast cell.

[0029] In another aspect, the invention relates to engineered antibody-like proteins.

[0030] In another aspect, the invention relates to a method for treating a disease in a subject, the method comprising administering an antibody of any of the above claims to a human suffering from the disease, wherein the subject aberrantly expresses MUC1. The disease can be cancer, such as breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer or esophageal cancer.

[0031] In another aspect, the present invention relates to an antibody, antibody fragment, or scFv comprising a variable domain fragment derived from an antibody that binds to the extracellular domain or cleavage product of a MUC1 isoform lacking the tandem repeat domain. In a preferred embodiment, the antibody or antibody fragment binds to the N-10 peptide. The variable domain fragment can be derived from the murine monoclonal antibody MN-E6 (SEQ ID NOs: 13 and 66) or humanized MN-E6 (SEQ ID NOs: 39 and 94), or the MN-E6 scFv (SEQ ID NOs: 233, 235, and 237). Alternatively, the variable domain fragment can be derived from the murine monoclonal antibody MN-C2 (SEQ ID NOs: 119 and 169) or humanized MN-C2 (SEQ ID NOs: 145 and 195), or the MN-C2 scFv (SEQ ID NOs: 239, 241, and 243). Alternatively, the variable domains can be derived from monoclonal antibodies MN-18G12, MN-20A10, MN-25E6, MN-28F9, MN-5C6F3, MN-3C2B1, or MN-1E4, whose heavy and light chain complementarity determining region sequences are also set forth in the sequence listing herein.

[0032] In another aspect, the present invention provides a method for the production of MUC1 or MUC1 * A method for treating a human diagnosed with, suspected of having, or at risk of developing positive cancer, the method comprising administering to the human an effective amount of an antibody, antibody fragment, or scFv as described above, wherein the species can be murine, camelid, human, or humanized.

[0033] In another aspect, the invention relates to a polypeptide comprising at least two different scFv sequences, wherein one of the scFv sequences binds to the extracellular domain or cleavage product of a MUC1 isoform that does not contain the tandem repeat domain. The peptide may bind to: (I) PSMGFR region of MUC1; (ii) PSMGFR peptide; (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10) (SEQ ID NO: 3); (iv) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFSAQSGA(N-19) (SEQ ID NO: 4); (v) a peptide having the amino acid sequence NLTISDVSVSDVPFPFSAQSGA(N-23) (SEQ ID NO: 5); (vi) a peptide having the amino acid sequence ISDVSVSDVPFPFSAQSGA(N-26) (SEQ ID NO: 6); (vii) a peptide having the amino acid sequence SVSDVPFPFSAQSGA(N-30) (SEQ ID NO: 7); (viii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5) (SEQ ID NO: 8); (ix) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) (SEQ ID NO: 9); (x) A peptide having the amino acid sequence FPFSAQSGA(N-36) (SEQ ID NO: 10).

[0034] The polypeptide may bind to a receptor on an immune cell such as a T cell, in particular CD3 on a T cell.

[0035] In another embodiment, the present invention provides a method for detecting MUC1, comprising contacting a sample of cells with the scFv-Fc described above and detecting the presence of binding of the scFv-Fc to the cells. * The present invention relates to a method for detecting the presence of a cell that abnormally expresses the gene. The cell may be a cancer cell.

[0036] In another aspect, the invention relates to a method for testing a subject's cancer for suitability for treatment with a composition comprising an antibody of the invention, wherein the antibody can be a murine, camelid, human, or humanized antibody, or a fragment thereof, or a portion of the variable region of antibody MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, the method comprising contacting a body specimen from the patient with the antibody in vitro, ex-vivo, or in vivo, and determining whether the patient has MUC1 or MUC2+ / MUC1 ... * The method includes determining whether the antibody exhibits abnormal expression of the antibody. The antibodies used in these diagnostics may be conjugated to an imaging agent.

[0037] In another aspect, the invention relates to a method of treating a subject suffering from a disease, the method comprising: inducing T cells from the subject or a donor to induce MUC1 * By exposing T cells to peptides, they undergo various rounds of maturation to MUC1 * Developing specific receptors, exposing, generating adapted T cells, expanding adapted T cells, and MUC1 * Diagnosed or suspected MUC1 positive cancer * and administering the matched T cells to a donor patient who has or is at risk of developing MUC1 positive cancer. * The peptide is selected from the following group: (I) PSMGFR region of MUC1; (ii) PSMGFR peptide; (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10); (iv) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFSAQSGA(N-19); (v) a peptide having the amino acid sequence NLTISDVSVSDVPFPFSAQSGA(N-23); (vi) a peptide having the amino acid sequence ISDVSVSDVPFPFSAQSGA(N-26); (vii) a peptide having the amino acid sequence SVSDVPFPFSAQSGA(N-30); (viii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5); (ix) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFS(N-19 / C-5); (x) A peptide having the amino acid sequence FPFSAQSGA(N-36).

[0038] In one aspect of the invention, MUC1 or MUC1 * The antibody administered to patients for the treatment or prevention of positive cancer is selected according to its ability to bind to the N-10 peptide of PSMGFR. The antibody can be administered alone as a monovalent antibody, as an scFv, or the fragment of the antibody can be incorporated into CAR, BiTE or ADC.

[0039] In one aspect of the invention, MUC1 or MUC1 * The antibodies administered to patients for the treatment or prevention of MUC1 or MUC1 positive cancers * The antibody is selected for its inability to recognize a linear epitope of the antibody. The antibody can be administered alone as a monovalent antibody, as an scFv, or a fragment of the antibody can be incorporated into a CAR, BiTE, or ADC.

[0040] In one aspect of the invention, MUC1 or MUC1 * Antibodies administered to patients for the treatment or prevention of MUC1-positive cancers are selected for their ability to recognize the MUC1 transmembrane cleavage product after cleavage by MMP9. The antibody can be administered alone as a monovalent antibody, as an scFv, or a fragment of the antibody can be incorporated into a CAR, BiTE, or ADC.

[0041] In one aspect of the invention, MUC1 or MUC1 *The antibody administered to patients for the treatment or prevention of MUC1-positive cancers is NME7, which is directed against the extracellular domain of MUC1 that does not contain the tandem repeats. AB or selected for its ability to competitively inhibit the binding of NME7-X1. The antibody may be administered alone as a monovalent antibody, as an scFv, or a fragment of the antibody can be incorporated into a CAR, BiTE, or ADC.

[0042] In another aspect, the invention relates to a method of treating cancer in a patient, the method comprising administering to the patient any of the immune cells described above in combination with a checkpoint inhibitor.

[0043] The above methods can use any of the following antibodies or variable regions thereof: MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11.

[0044] The above method can use any of the variable regions listed below: (i) an anti-MUC1 antibody consisting of the sequence of humanized MN-E6 expressed by a humanized IgG2 or IgG1 heavy chain paired with a humanized kappa or lambda light chain; * extracellular domain antibodies or anti-N-10 antibodies; (ii) the antibody of (i), wherein the humanized IgG2 heavy chain is SEQ ID NO: 53, the humanized IgG1 heavy chain is SEQ ID NO: 57, the humanized kappa light chain is SEQ ID NO: 108, and the humanized lambda light chain is SEQ ID NO: 112, or a sequence with 90%, 95%, or 98% sequence identity thereof; (iii) anti-MUC1 consisting of the sequence of humanized MN-C2 represented by humanized IgG1 and IgG2 heavy chains paired with humanized lambda and kappa light chains; * extracellular domain antibodies or anti-N-10 antibodies; (iv) The antibody of (iii), wherein the humanized IgG1 heavy chain MN-C2 (SEQ ID NO: 159) or IgG2 heavy chain (SEQ ID NO: 164) is paired with a lambda light chain (SEQ ID NO: 219) or a kappa light chain (SEQ ID NO: 213), or a sequence having 90%, 95%, or 98% sequence identity thereto.

[0045] In the above methods, in the CAR, the extracellular domain may be composed of a humanized single-chain antibody fragment of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. The extracellular domain may be composed of a humanized single-chain antibody fragment of MN-E6 scFv, MN-C2 scFv (SEQ ID NO: 239, 241, or 243), set forth as SEQ ID NO: 233, 235, or 237. In the CAR, the cytoplasmic tail may be composed of one or more signaling sequence motifs: CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7.

[0046] The above method can include at least two CARs with different extracellular domain units transfected into the same cell. One of the extracellular domain recognition units is MUC1. * The extracellular domain recognition units may bind to the extracellular domain. One of the extracellular domain recognition units may bind to PD-1. One of the extracellular domain recognition units may be an antibody fragment, and the other may be a peptide or anti-MUC1 * It may be an antibody fragment.

[0047] This method can include immune cells transfected or transduced with a plasmid encoding a CAR and a plasmid encoding a non-CAR species expressed from an inducible promoter. The non-CAR species can be expressed from an inducible promoter activated by elements of activated immune cells. The non-CAR species can be expressed from an NFAT inducible promoter. The NFAT can be NFATc1, NFATc3, or NFATc2. The cleavage enzyme can be MMP2, MMP3, MMP9, MMP13, MMP14, MMP16, ADAM10, ADAM17, or ADAM28, or a catalytically active fragment thereof. The non-CAR species can be a cytokine. The cytokine can be IL-7, IL-12, IL-15, or IL-18.

[0048] The present invention relates to an antibody or a fragment thereof for the diagnosis, treatment or prevention of cancer, wherein the antibody specifically binds to the PSMGFR peptide (SEQ ID NO: 2) or a fragment thereof of the peptide.

[0049] The antibody binds to the N-10 peptide (SEQ ID NO:3), the N-19 peptide (SEQ ID NO:4), the N-23 peptide (SEQ ID NO:5), the N-26 peptide (SEQ ID NO:6), the N-30 peptide (SEQ ID NO:7), the N-10 / C-5 peptide (SEQ ID NO:8), the N-19 / C-5 peptide (SEQ ID NO:9), or the C-5 peptide (SEQ ID NO:825).

[0050] The antibody interacts with a peptide containing the conformational epitopes SVSDV (SEQ ID NO: 1751) and FPSA (SEQ ID NO: 1747) within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), and mutation or deletion of FPFS (SEQ ID NO: 1747) disrupts binding of the antibody or fragment thereof to the N-26 peptide.

[0051] The antibody interacts with a peptide containing the conformational epitopes ASRYNLT (SEQ ID NO: 1745) and SVSDV (SEQ ID NO: 1751) and FPSA (SEQ ID NO: 1747) within the N-19 sequence ASRYNLT ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4), and mutation or deletion of ASRYNLT (SEQ ID NO: 1745) disrupts binding of the antibody or fragment thereof to the N-26 peptide.

[0052] The antibody does not bind to the C-10 peptide (SEQ ID NO: 825).

[0053] The antibody binds to the N-10 peptide (SEQ ID NO:3) but not to the C-10 peptide (SEQ ID NO:825).

[0054] The antibody is NME7 AB and MUC1 * inhibits the interaction of

[0055] The antibody is NME7 AB and PSMGFR peptide (SEQ ID NO: 2).

[0056] The antibody is NME7 AB and the N-10 peptide (SEQ ID NO: 3), N-19 peptide (SEQ ID NO: 4), N-23 peptide (SEQ ID NO: 5), N-26 peptide (SEQ ID NO: 6), N-30 peptide (SEQ ID NO: 7), N-10 / C-5 peptide (SEQ ID NO: 8), N-19 / C-5 peptide (SEQ ID NO: 9), or C-5 peptide (SEQ ID NO: 825).

[0057] The antibody recognizes the MUC1 transmembrane enzyme cleavage product.

[0058] In the above, the cleavage enzyme is MMP14 or MMP9 or a catalytically active fragment of the enzyme.

[0059] The antibody binds to PSMGFR (SEQ ID NO: 2) or a fragment thereof, and the presence of an amino acid sequence within PSMGFR (SEQ ID NO: 2) induces the antibody to bind to PSMGFR.

[0060] The amino acid sequence of the binding conformation-inducing peptide is present within the N-10 peptide (SEQ ID NO: 3).

[0061] The antibody does not bind to the linear form of the bound conformation-guided peptide sequence, the linear form of the peptide being the denatured form.

[0062] The binding conformation-inducing peptide sequence is in the N-26 peptide sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), and mutation or deletion of FPFS (SEQ ID NO: 1747) disrupts binding of the antibody or fragment thereof to the N-26 peptide.

[0063] The binding conformation-inducing peptide sequence is located within the N-19 sequence ASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4), and mutation or deletion of ASRYNLT (SEQ ID NO: 1745) disrupts binding of the antibody or fragment thereof to the N-19 peptide.

[0064] The binding-inducing peptide sequence can be located within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), where a mutation or deletion within FPFS (SEQ ID NO: 1747) disrupts binding of the antibody or fragment thereof to PSMGFR.

[0065] The antibody may have a consensus sequence.

[0066] The heavy chain CDR1 comprises a consensus sequence at least 90% identical to the following sequence: F or I at position 1, T at position 2, F at position 3, S at position 4, T, G, or R at position 5, Y at position 6, A, G, or T at position 7, M at position 8, and S at position 9.

[0067] Heavy chain CDR2 comprises a T at position 1, an I or S at position 2, an I or S at position 3, a G or R at position 5, a G or A at position 6, a T or I at position 9, a Y at position 10, a Y at position 11, a P or S at position 12, and a consensus sequence at positions 13-17 that is at least 90% identical to the sequence DSVKG:

[0068] The heavy chain CDR3 comprises a consensus sequence at least 90% identical to the sequence at position 2: G, L, or N, at position 4: G or T, at position 7: Y, at position 12: D or E, at position 14: A, and at position 15: Y.

[0069] The light chain CDR1 comprises a consensus sequence at least 90% identical to the following sequence: K or R at position 1, A or S at position 2, S at position 3, K or Q at position 4, S at position 5, L or V at position 6, L at position 7, T or S at position 10, Y at position 15, and I, L, or M at position 16.

[0070] The light chain CDR2 contains a consensus sequence at least 90% identical to the sequence: L or W or S at position 1, A or T at position 2, S at position 3, N or T at position 4, L or R at position 5, E or A at position 6, and S at position 7:

[0071] The light chain CDR3 comprises a consensus sequence at least 90% identical to the following sequence: Q at position 1, H or Q at position 2, S, Q or R at position 3, R, S or Y at position 4, E, L, or S at position 5, L or S at position 6, P or S at position 7, F or L at position 8, and T at position 9.

[0072] The antibody-binding conformation-guiding peptide is within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), and mutations or deletions within FPFS (SEQ ID NO: 1747), SVSDV (SEQ ID NO: 1751), or ASRYNLT (SEQ ID NO: 1745) disrupt binding of the antibody or fragment thereof to PSMGFR.

[0073] The antibody may have additional consensus sequences, heavy chain CDR1 comprises a consensus sequence at least 90% identical to the following sequence: F or I at position 1, T or A at position 2, F at position 3, S at position 4, T, G, or R at position 5, Y or F at position 6, A, G, or T at position 7, M at position 8, and S at position 9; heavy chain CDR2 comprises a consensus sequence at least 90% identical to the sequence of T or A at position 1, I or S at position 2, I or S at position 3, N, S, T, or G at position 4, G or R at position 5, G or A at position 6, G, T, or D at position 7, Y, K, H, or S at position 8, T or I at position 9, Y or F at position 10, Y at position 11, P or S at position 12, and D at position 13, S or T at position 14, V or L at position 15, and KG at positions 16-17; the heavy chain CDR3 comprises G, L, or N at position 2, G, T, or Y at position 3, G or T at position 4, Y at position 7, Y, A, or G at position 10, M, D, or F at position 11, D or E at position 12, and a consensus sequence at positions 14-15 that is at least 90% identical to the sequence AY; the light chain CDR1 comprises a consensus sequence at least 90% identical to the following sequence at position 1: K or R, at position 2: A or S, at position 3: S or R, at position 8: S, Y, I, or V, at position 10: T or S, at position 12: G, S, D, or Q, at position 13: V, Y, K, or N, at position 14: N, S, or T, at position 15: Y or F, and at position 16: I, L, or M; the light chain CDR2 comprises a consensus sequence at least 90% identical to the following sequence: A, T, or V at position 2, S at position 3, N, T, or K at position 4, L or R at position 5, E, A, F, or D at position 6, and S at position 7; The light chain CDR3 comprises a consensus sequence at least 90% identical to the following sequence: Q, F, or W at position 1; H or Q at position 2; R, S, T, Y, or N at position 4; E, L, S, or H at position 5; L, S, V, D, or Y at position 6; P or S at position 7; and T at position 9.

[0074] The antibody may be: MNC2 with: heavy chain CDR1 containing the consensus sequence FTFSGYAMS; heavy chain CDR2 containing the consensus sequence TISSGGTYIYYPDSVKG; heavy chain CDR3 containing the consensus sequence -LGGDNYYEYFDV--; light chain CDR1 containing the consensus sequence RASKS--VSTSGYSYMH; a light chain CDR2 containing the consensus sequence LASNLES; and Light chain CDR3 containing the consensus sequence QHSRELPFT. MNE6, which has: heavy chain CDR1 containing the consensus sequence FTFSRYGMS; heavy chain CDR2 containing the consensus sequence TISGGGTYIYYPDSVKG; heavy chain CDR3 containing the consensus sequence DNYGRNYDYGMDY--; Light chain CDR1 containing the consensus sequence ---SATSSVSYIH; a light chain CDR2 containing the consensus sequence STSNLAS; and Light chain CDR3 containing the consensus sequence QQRSSSPFT. B2 with: heavy chain CDR1 containing the consensus sequence FAFSTFAMS; heavy chain CDR2 containing the consensus sequence AISNGGGYTYYPDTLKG; heavy chain CDR3 containing the consensus sequence ----RYYDLYFDL--; light chain CDR1 containing the consensus sequence RSSQNIV-HSNGNTYLE; a light chain CDR2 containing the consensus sequence KVSNRFS; and Light chain CDR3 containing the consensus sequence FQDSHVPLT. B7 with: heavy chain CDR1 containing the consensus sequence FTFSRYGMS; heavy chain CDR2 containing the consensus sequence TISSGGTYIYYPDSVKG; heavy chain CDR3 containing the consensus sequence DNYGSSYDYAMDY--; light chain CDR1 containing the consensus sequence RSSQTIV-HSNGNTYLE; a light chain CDR2 containing the consensus sequence KVSNRFS; and Light chain CDR3 containing the consensus sequence FQDSHVPLT. B9 with: heavy chain CDR1 containing the consensus sequence FTFSRYGMS; heavy chain CDR2 containing the consensus sequence TISSGGTYIYYPDSVKG; heavy chain CDR3 containing the consensus sequence DNYGSSYDYAMDY--; Light chain CDR1 containing the consensus sequence ---SASSSVSYMH; a light chain CDR2 containing the consensus sequence TTSNLAS; and Light chain CDR3 containing the consensus sequence QQRSSYPF-. 8C7F3 with: heavy chain CDR1 containing the consensus sequence FTFSTYAMS; heavy chain CDR2 containing the consensus sequence AISNGGGYTYYPDSLKG; heavy chain CDR3 containing the consensus sequence ----RYYDHYFDY--; Light chain CDR1 containing the consensus sequence--RASESVATYGNNFMQ; a light chain CDR2 containing the consensus sequence LASTLDS; and Light chain CDR3 containing the consensus sequence QQNNEDPPT. H11 with: heavy chain CDR1 containing the consensus sequence FAFSTFAMS; heavy chain CDR2 containing the consensus sequence AISNGGGYTYYPDTLKG; heavy chain CDR3 containing the consensus sequence ----RYYDLYFDL--; light chain CDR1 containing the consensus sequence RSSQNIV-HSNGNTYLE; a light chain CDR2 containing the consensus sequence KVSNRFS; and Light chain CDR3 containing the consensus sequence FQDSHVPLT. B12 with: heavy chain CDR1 containing the consensus sequence SYGVH; heavy chain CDR2 containing the consensus sequence VIWPGGSTNYNSTLMSRM; a heavy chain CDR3 containing the consensus sequence DRTPRVGAWFAY; and light chain CDR1 containing the consensus sequence RASESVATYGNNFMQ; a light chain CDR2 containing the consensus sequence LASTLDS; and Light chain CDR3 containing the consensus sequence QQNNEDPPT. 20A10 with: heavy chain CDR1 containing the consensus sequence FTFSTYAMS; heavy chain CDR2 containing the consensus sequence -SIGRAGSTYYSDSVKG; Heavy chain CDR3 containing the consensus sequence ---GPIYNDYDEFAY; light chain CDR1 containing the consensus sequence KSSQSVLYSSNQKNYLA; a light chain CDR2 containing the consensus sequence WASTRES; and Light chain CDR3 containing the consensus sequence HQYLSSLT. 3C2B1 with: heavy chain CDR1 containing the consensus sequence ITFSTYTMS; heavy chain CDR2 containing the consensus sequence TISTGGDKTYYSDSVKG; heavy chain CDR3 containing the consensus sequence -GTTAMYYYAMDY; light chain CDR1 containing the consensus sequence RASKS---ISTSDYNYIH; a light chain CDR2 containing the consensus sequence LASNLES; and Light chain CDR3 containing the consensus sequence QHSRELPLT.

[0075] In another aspect, the present invention relates to an antibody or fragment thereof for the diagnosis, treatment or prevention of cancer requiring the presence of the antibody-binding conformation-guiding peptide ASRYNLT (SEQ ID NO: 1745) of PSMGFR (SEQ ID NO: 2). The antibody may be 25E6 having: heavy chain CDR1 containing the consensus sequence FTFSSYGMS; heavy chain CDR2 containing the consensus sequence TISNGGRHTFYPDSVKG; heavy chain CDR3 containing the consensus sequence QTGTEGWFAY; light chain CDR1 containing the consensus sequence KSSQSLLDSDGKTYLN; light chain CDR2 containing the consensus sequence LVSKLDS_; Light chain CDR3 containing the consensus sequence WQGTHFPQT.

[0076] In another aspect, the present invention relates to an antibody or fragment thereof for the diagnosis, treatment or prevention of cancer requiring the presence of the antibody-binding conformation-guiding peptide SVSDV (SEQ ID NO: 1761) of PSMGFR (SEQ ID NO: 2). The antibody may be 5C6F3 having the following: heavy chain CDR1 containing the consensus sequence FTFSTYAMS; heavy chain CDR2 containing the consensus sequence AISNGGGYTYYPDSLKG; heavy chain CDR3 containing the consensus sequence RYYDHYFDY; light chain CDR1 containing the consensus sequence RSSQTIVHSNGNTYLE; a light chain CDR2 containing the consensus sequence KVSNRFS; and Light chain CDR3 containing the consensus sequence FQDSHVPLT.

[0077] The antibodies or fragments thereof according to all of the above may be murine, camelid, human, or humanized. The antibody fragments may be scFv or scFv-Fc. The variable regions thereof may be murine, camelid, human, or humanized.

[0078] In another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising the above-mentioned antibody fragment, which may further comprise a mutation in the costimulatory domain or the CD3-zeta signaling domain. The tyrosine may be mutated in CD28 or 4-1BB. The CD3-zeta may comprise a 1XX mutation.

[0079] In another aspect, the present invention relates to an immune cell comprising the above-described CAR. The immune cell can be a T cell, an NK cell, a dendritic cell, or a mast cell.

[0080] In another aspect, the present invention relates to a cell composition expressed in a cell comprising the above-described CAR, and a second entity having a biological recognition unit with a different specificity from the CAR, which can bind PD-1, PDL-1, other checkpoint inhibitors, NME7, or cytokines such as IL-12 or IL-18, or c-Jun.

[0081] In yet another aspect, the present invention relates to an immune cell engineered to express a nucleic acid encoding the above-mentioned CAR and a nucleic acid encoding a second entity according to any of the above claims, wherein the second entity is expressed from an inducible promoter. The second entity may be expressed from an inducible promoter activated by an element of an activated immune cell. The second entity may be expressed from an NFAT inducible promoter. The NFAT may be NFATc1, NFATc3, or NFATc2. The second entity may be a cytokine such as IL-7, IL-15, or IL-18. The nucleic acid encoding the second entity may be inserted into a Foxp3 promoter or enhancer region, and the cytokine is IL-18. The cytokine may be expressed from an NFAT inducible promoter.

[0082] In another aspect, the invention relates to a BiTE construct comprising the antibody fragment described above.

[0083] In yet another aspect, the present invention relates to an antibody-drug conjugate (ADC) comprising the above-described antibody or antibody fragment.

[0084] The present invention relates to an antibody or fragment thereof that specifically binds to PSMGFR (SEQ ID NO: 2) and N-10 (SEQ ID NO: 3); Does not bind to full-length MUC1 not bound to C-10 (SEQ ID NO: 825); NME1 or NME7 AB MUC1 * competitively inhibits binding to the extracellular domain or PSMGFR peptide; MUC1 generated by cleavage with a cleavage enzyme * Do you recognize; recognize conformational epitopes rather than linear epitopes, or Cancer can be screened for by immunohistochemistry of the tissue.

[0085] Four of criteria (i)-(vi) may be met. Five of criteria (i)-(vi) may be met. Six of criteria (i)-(vi) may be met. At least criterion (vi) may be met. The cleavage enzyme may be MMP-9.

[0086] In all of the above, the cancer may be breast cancer, pancreatic cancer, ovarian cancer, lung cancer, colon cancer, stomach cancer, or esophageal cancer.

[0087] The present invention also relates to a method for the treatment of atopic dermatitis in which MUC1 is abnormally expressed and MUC1 is expressed. * The present invention also relates to methods of diagnosing, treating, or preventing cancer by administering the antibodies and fragments disclosed herein to a cancer patient in need thereof, the cancer patient having been identified as expressing a truncated MUC1 such as

[0088] These and other objects of the present invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto, and the claims appended hereto. [Brief explanation of the drawings]

[0089] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0090] The present invention will be more fully understood from the detailed description set forth herein below and the accompanying drawings, which are given by way of example only and therefore are not intended to be limiting of the invention. [Figure 1]Figure 1 shows cell growth assay graphs of MUC1*-positive cells treated with either bivalent "bv" anti-MUC1* antibodies, monovalent "mv" or Fab, NM23-H1 dimer, or NME7-AB. Bivalent anti-MUC1* antibodies stimulate cancer cell growth, while monovalent Fab inhibits growth (Figures 1A-1B). The classic bell-shaped curve indicates that ligand-induced dimerization stimulates growth. Dimeric NM23-H1, also known as NME1, stimulates growth of MUC1*-positive cancer cells, whereas siRNA to silence MUC1 expression abolishes this effect (Figure 1C). NME7-AB also stimulates growth of MUC1*-positive cells (Figure 1D). [Figure 2] Results of ELISA assays are shown. MUC1* peptide PSMGFR, PSMGFR minus 10 amino acids subtracted from the N-terminus (also known as N-10), or PSMGFR minus 10 amino acids subtracted from the C-terminus (also known as C-10), are immobilized on plates and assayed for binding to: NME7-AB (Figure 2A), MN-C2 monoclonal antibody (Figure 2B), MN-E6 monoclonal antibody (Figure 2C), or dimeric NME1 (Figure 2D). These assays demonstrate that NME1, NME7-AB, and monoclonal antibodies MN-C2 and MN-E6 all require the first 10 membrane-proximal amino acids of the MUC1* extracellular domain for binding. MUC1* peptides PSMGFR, minus 10 amino acids from the N-terminus (also known as N-10), or PSMGFR, minus 10 amino acids from the C-terminus (also known as C-10), are immobilized on plates and assayed for binding to the following: MN-C3 (Figure 2E) and MN-C8 (Figure 2F). Figure 2G shows the amino acid sequence of the PSMGFR peptides. Figure 2H shows the amino acid sequence of the N-10 peptide. Figure 2I shows the amino acid sequence of the C-10 peptide. [Figure 3]The results of a competitive ELISA assay are shown. The PSMGFR MUC1* peptide was immobilized on a plate, and the dimer NM23-H1, also known as NME1, was added alone or after the addition of the MN-E6 antibody (Figure 3A). The same experiment was performed in which NM23-H7, NME7-AB, was added alone or after the addition of MN-E6 (Figure 3B). The results show that MN-E6 competitively inhibits the binding of the MUC1* activating ligands NME1 and NME7. In a similar experiment (Figure 3C), PSMGFR or PSMGFR minus 10 amino acids from the N-terminus, also known as N-10, was immobilized on a plate. The dimer NM23-H1 was then added. The anti-MUC1* antibodies MN-E6, MN-C2, MN-C3, or MN-C8 were then tested for their ability to compete with NM23-H1. The results show that all three antibodies bind to the PSMGFR peptide, but MN-E6 and MN-C2 competitively inhibit the binding of MUC1* activating ligands. [Figure 4]Figure 4A shows FACS scans of the anti-MUC1* antibody huMN-C2scFv, which specifically binds to MUC1*-positive cancer cells and MUC1*-transfected cells but not to MUC1*- or MUC1*-negative cells. ZR-75-1, also known as 1500, MUC1*-positive breast cancer cells were stained with 1:2 or 1:10 dilutions of 1.5 μg / ml humanized MN-C2. After two washes, cells were stained with a secondary antibody, an anti-Penta-His antibody conjugated to Alexa 488 (Qiagen), at 1:200 (Figure 4A), 1:50 (Figure 4B), or 1:10 (Figure 4C), to detect the 6xHis tag on huMN-C2scFv. Figure 4A shows huMN-C2 binding to ZR-75-1 breast cancer cells when the secondary antibody was added at a 1:200 dilution. Figure 4B shows huMN-C2 binding to ZR-75-1 breast cancer cells when the secondary antibody was added at a 1:50 dilution. Figure 4C shows huMN-C2 binding to ZR-75-1 breast cancer cells when the secondary antibody was added at a 1:10 dilution. Flow cytometry analysis revealed a concentration-dependent shift in cell subsets, demonstrating specific binding that was not seen in the absence of MN-C2 scFv (Figures 4A-4C). Figure 4D shows anti-MUC1* antibody MN-E6 staining of MUC1-negative HCT-116 colon cancer cells transfected with empty vector, single-cell clone #8. Figure 4E shows anti-MUC1* antibody MN-E6 staining of HCT-116 colon cancer cells transfected with MUC1* single-cell clone #10. Figure 4F shows anti-MUC1* antibody MN-E6 staining of ZR-75-1, also known as 1500, MUC1*-positive breast cancer cells. As shown by FACS scans, both MN-C2 and MN-E6 stain only MUC1* positive cells and do not stain MUC1 or MUC1* negative cells. [Figure 5] Figure 1 shows a graph of an ELISA in which surfaces are coated with either the MUC1* PSMGFR peptide or a control peptide. Humanized MN-C2 scFv is then incubated with the surface, washed, and detected according to standard methods. The ELISA shows that huMAN-C2 scFv binds to the MUC1* peptide with an EC-50 of approximately 333 nM. [Figure 6]Figure 6 shows graphs of cancer cell growth inhibition by the MUC1* antibody variable region fragment, humanized MN-C2 scFv. hMN-C2 scFv potently inhibited the growth of ZR-75-1, also known as 1500, MUC1*-positive breast cancer cells (Figure 6A) and T47D MUC1*-positive breast cancer cells (Figure 6B), with an EC-50 value similar to that of in vitro ELISA. [Figure 7] Figure 7A shows a graph of tumor growth in immunocompromised mice implanted with human tumors and then treated with the anti-MUC1* antibody MN-E6Fab or mock treatment. Female nu / nu mice implanted with estrogen pellets for 90 days were implanted with 6 million T47D human breast cancer cells mixed 50 / 50 with Matrigel. Mice with tumors that reached a tumor volume of at least 150 mm3 and grew three consecutive times were selected for treatment. Animals were injected subcutaneously with 80 mg / kg of MN-E6Fab twice weekly, and an equal number of mice meeting the same selection criteria were injected with vehicle alone (Figure 7A). Male NOD / SCID mice were implanted with 6 million DU-145 human prostate cancer cells mixed 50 / 50 with Matrigel. Mice with tumors that reached a tumor volume of at least 150 mm3 and grew three consecutive times were selected for treatment. Animals were injected subcutaneously with 160 mg / kg of MN-E6Fab every 48 hours, and an equal number of mice meeting the same selection criteria were injected with vehicle alone (Figure 7B). Tumors were measured and recorded twice weekly independently by two investigators. Statistics were calculated blindly by independent statisticians and assigned a P value of 0.0001 for each. Anti-MUC1*Fab inhibited breast and prostate cancer growth. Treatment had no effect on body weight, bone marrow cell type, or number. [Figure 8] Graphs of ELISAs are shown in which surfaces were immobilized with either PSMGFR peptide, PSMGFR with 10 amino acids subtracted from the N-terminus, or PSMGFR with 10 amino acids subtracted from the C-terminus. huMN-E6 scFv-Fc bound to the PSMGFR peptide and the PSMGFR N-10 peptide, but not to the PSMGFRC-10 peptide. The parental MN-E6 antibody and humanized MN-E6 require the C-terminal 10 amino acids of PSMGFR for binding. [Figure 9] Figure 9 shows graphs of an ELISA in which the assay plate surface was immobilized with either the PSMGFR peptide, PSMGFR minus 10 amino acids from the N-terminus, or PSMGFR minus 10 amino acids from the C-terminus. The MN-C3 antibody variants were then assayed for binding to various MUC1* peptides. Figure 9A shows the purified murine monoclonal MN-C3 antibody, and Figure 9B shows the humanized MN-C3scFv-Fc. The ELISA demonstrates binding to the PSMGFR peptide and specific deletion peptides. [Figure 10] Figures 10A and 10B are photographs of breast cancer tissue arrays. Figure 10A was stained with VU4H5, which recognizes MUC1-FL (full-length). Figure 10B was stained with MN-C2, a mouse monoclonal antibody that recognizes cancerous MUC1*. After automated staining (Clarient Diagnostics), tissue staining was scored using the Allred scoring method, which combines intensity and distribution scores. Figures 10C and 10F are color-coded graphs showing the calculated scores for MUC1 full-length staining of each patient's tissue. Figures 10G and 10J are color-coded graphs showing the calculated scores for MUC1* staining of each patient's tissue. [Figure 11] Figures 11A and 11B are photographs of breast cancer tissue arrays. Figure 11A was stained with VU4H5, which recognizes MUC1-FL (full-length). Figure 11B was stained with MN-C2, a mouse monoclonal antibody that recognizes cancerous MUC1*. After automated staining (Clarient Diagnostics), tissue staining was scored using the Allred scoring method, which combines intensity and distribution scores. Figures 11C-11F are color-coded graphs showing the calculated scores for MUC1 full-length staining of each patient's tissue. Figures 11G-11J are color-coded graphs showing the calculated scores for MUC1* staining of each patient's tissue. [Figure 12]Figures 12A-12H show photographs of normal breast tissue and breast cancer tissue stained with 2.5 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUC1* antibody followed by a secondary streptavidin-HRP antibody. Figure 12A shows normal breast tissue. Figures 12B-12D show breast cancer tissue from patients as indicated. Figures 12E-12H show photographs of corresponding serial sections stained with the secondary antibody alone. [Figure 13] Figures 13A-13C show photographs of normal breast tissue and breast cancer tissue stained with 2.5 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUC1* antibody followed by a secondary streptavidin-HRP antibody. Figure 13A shows normal breast tissue. Figures 13B-13C show breast cancer tissue from patients as indicated. Figures 13D-13F show photographs of corresponding serial sections stained with secondary antibody only. [Figure 14] Photographs of breast cancer tissues stained with 10 μg / mL of MN-E6 anti-MUC1* antibody followed by a rabbit anti-mouse secondary HRP antibody are shown. Figures 14A-14D show breast cancer tissues from patient #300. Figures 14E-14H show breast cancer tissues from metastatic patient #291. [Figure 15] Photographs of normal and lung cancer tissues stained with 2.5 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUC1* antibody followed by a secondary streptavidin-HRP antibody are shown. Figure 15A shows normal lung tissue. Figures 15B-15C show lung cancer tissues from patients as indicated. Figures 15D-15F show corresponding serial sections stained with the secondary antibody alone. [Figure 16] Photographs of normal and lung cancer tissues stained with 2.5 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUC1* antibody followed by a secondary streptavidin-HRP antibody are shown. Figure 16A shows normal lung tissue. Figures 16B-16C show lung cancer tissues from patients as indicated. Figures 16D-16F show corresponding serial sections stained with secondary antibody alone. [Figure 17]Photographs of normal and lung cancer tissues stained with 25 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUC1* antibody followed by a secondary streptavidin-HRP antibody are shown. Figure 17A shows normal lung tissue. Figures 17B-17C show lung cancer tissues from patients as indicated. Figures 17D-17F show corresponding serial sections stained with secondary antibody alone. [Figure 18] Photographs of normal and lung cancer tissues stained with 25 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUC1* antibody followed by a secondary streptavidin-HRP antibody are shown. Figure 18A shows normal lung tissue. Figures 18B-18C show lung cancer tissues from patients as indicated. Figures 18D-18F show corresponding serial sections stained with the secondary antibody alone. [Figure 19] Figure 19 shows photographs of normal and cancerous small intestinal tissue stained with 5 μg / mL of humanized MN-E6-scFv-Fc biotinylated anti-MUC1* antibody followed by a secondary streptavidin-HRP antibody. Figure 19A shows normal small intestinal tissue. Figure 19B shows small intestinal cancer tissue from the patient indicated. Figures 19C-19D show photographs of corresponding serial sections stained with the secondary antibody alone. [Figure 20] Photographs of normal small intestinal tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody are shown in Figures 20A-20D. Photographs of corresponding serial sections stained with secondary antibody alone are shown in Figures 20E-20H. [Figure 21] Photographs of cancerous small intestine tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody are shown. Figures 21A-21D show cancerous small intestine tissue from a patient, as shown. Figures 21E-21H show corresponding serial sections stained with the secondary antibody alone. [Figure 22]Photographs of cancerous small intestine tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody are shown. Figures 22A-22D show cancerous small intestine tissue from a patient, as shown. Figures 22E-22H show corresponding serial sections stained with the secondary antibody alone. [Figure 23] Figures 23A-23H show photographs of normal colon tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody. Figures 23A-23D show normal colon. Figures 23E-23H show corresponding serial sections stained with secondary antibody alone. [Figure 24] Photographs of colon cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody are shown. Figures 24A-24D show colon tissue from a metastatic patient, as indicated. Figures 24E-24H show corresponding serial sections stained with the secondary antibody alone. [Figure 25] Photographs of colon cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody are shown. Figures 25A-25D show colon cancer tissue from a grade 2 patient, as shown. Figures 25E-25H show corresponding serial sections stained with the secondary antibody alone. [Figure 26] Photographs of colon cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody are shown. Figures 26A-26D show colon tissue from a metastatic patient, as indicated. Figures 26E-26H show corresponding serial sections stained with the secondary antibody alone. [Figure 27] Photographs of prostate cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by a secondary goat anti-human HRP antibody are shown. Figures 27A-27D show prostate cancer tissue from a patient, as indicated. Figures 27E-27H show corresponding serial sections stained with the secondary antibody alone. [Figure 28]Photographs of prostate cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody are shown. Figures 28A-28D show prostate cancer tissue from a patient, as indicated. Figures 28E-28H show corresponding serial sections stained with secondary antibody alone. [Figure 29] Photographs of prostate cancer tissue stained with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody are shown. Figures 29A-29D show prostate cancer tissue from a patient, as indicated. Figures 29E-29H show corresponding serial sections stained with secondary antibody alone. [Figure 30-1] Figures 30A-B show photographs of triple-negative breast cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of arrays scored as zero, weak, moderate, or strong is graphed as a pie chart. Figure 30A shows a pie chart of the scores for anti-MUC1* antibody staining. Figure 30B shows a photograph of an array stained with the antibody. [Figure 30-2] Figures 30C-F show photographs of triple-negative breast cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of the array scored as zero, weak, moderate, or strong is graphed as a pie chart. Figures 30C-30D show magnified photographs of two breast cancer specimens from the array. Figures 30E-30F show closer magnifications of the boxed specimen portions. [Figure 31-1]Figures 31A-B show photographs of ovarian cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of sequences scored as zero, weak, moderate, or strong is graphed as a pie chart. Figure 31A shows a pie chart of the anti-MUC1* antibody staining scores. Figure 31B shows a photograph of an array stained with the antibody. [Figure 31-2] Figures 31C-F show photographs of ovarian cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of sequences scored as zero, weak, moderate, or strong is graphed as a pie chart. Figures 31C-D show magnified photographs of two breast cancer specimens from the array. Figures 31E-F show closer magnifications of the boxed specimen portions. [Figure 32-1] Figures 32A-B show photographs of pancreatic cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of sequences scored as zero, weak, moderate, or strong is graphed as a pie chart. Figure 32A shows a pie chart of the scores for anti-MUC1* antibody staining. Figure 32B shows a photograph of an array stained with the antibody. [Figure 32-2] Figures 32C-F show photographs of pancreatic cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of sequences scored as zero, weak, moderate, or strong is graphed as a pie chart. Figures 32C-D show magnified photographs of two breast cancer specimens from the array. Figures 32E-F show closer magnifications of the boxed specimen portions. [Figure 33-1]Figures 33A-B show photographs of lung cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of sequences scored as zero, weak, moderate, or strong is graphed as a pie chart. Figure 33A shows a pie chart of the scores for anti-MUC1* antibody staining. Figure 33B shows a photograph of an array stained with the antibody. [Figure 33-2] Figures 33C-F show photographs of lung cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of arrays scored as zero, weak, moderate, or strong is graphed as a pie chart. Figures 33C-D show magnified photographs of two breast cancer specimens from the array. Figures 33E-F show closer magnifications of the boxed specimen portions. [Figure 34] Photographs of normal tissue stained with the anti-MUC1* antibody huMNC2scFv are shown. [Figure 35] Figure 35A shows FACS scans of cells that do not express either MUC1, MUC1*, or full-length MUC1; the cells were probed with either MNC2 or VU4H5. Figure 35A shows MUC1-negative HCT-116 colon cancer cells probed with antibody MNC2. Figure 35B shows HCT cells transfected with MUC1*, where the extracellular domain is the sequence of the PSMGFR peptide, probed with antibody MNC2. Figure 35C shows HCT-MUC1-18 cells (also referred to herein as HCT-MUC1-41TR), a cleavage-resistant single-cell clone of HCT cells transfected with full-length MUC1; the cells were probed with antibody MNC2. Figure 35D shows HCT-MUC1-18 cells probed with antibody VU4H5, an antibody that recognizes hundreds of tandem repeat epitopes within full-length MUC1. As can be seen in the figure, MNC2 recognizes an ectopic epitope that is inaccessible to full-length MUC1. [Figure 36]Figure 36A shows a Western blot and corresponding FAC analysis of HCT-116 cells, a MUC1-negative colon cancer cell line stably transfected with either MUC1* or full-length MUC1. The single-cell clones shown are HCT-MUC1-41TR and HCT-MUC1*. Figure 36B shows a Western blot of the parental cell lines HCT-116, HCT-MUC1-41TR, and HCT-MUC1*. The gel was probed with the rabbit polyclonal antibody SDIX, which recognizes only cleaved MUC1. A visible band at 25-35 kDa is readily visible in lane 6, where HCT-MUC1* is loaded, while there are only faint bands in lanes 4 and 5, indicating that only a small amount of MUC1 is cleaved in HCT-MUC1-41Tr cells. No cleaved MUC1 is present in the parental cell line HCT-116 loaded in lanes 2 and 3. Figure 36B is a Western blot probed with the mouse monoclonal antibody VU4H5, which recognizes the tandem repeats of full-length MUC1. As can be seen, only HCT-MUC1-41TR contains full-length MUC1. Figure 36C shows a FACS scan demonstrating that HCT-MUC1* is 95.7% positive for SDIX, which binds only to MUC1* and essentially no binding to full-length MUC1. Figure 36D shows a FACS scan demonstrating that HCT-MUC1-41TR cells are 95% positive for full-length MUC1 and only about 11% positive for the truncated form, MUC1*. [Figure 37]Figure 37 shows a Western blot and bar graph of a FACS analysis assessing the ability of MNC2 to recognize full-length MUC1 after cleavage by MMP9. Figure 37A shows a Western blot of HCT-MUC1-18 cells, a cleavage-resistant cell line, to which the cleavage enzyme MMP9 was added. Cell lysate fractions were run on a gel and probed with a polyclonal anti-PSMGFR antibody. The photograph shows that MMP9 cleaved MUC1 into a ∼25 kDa species, MUC1*, in a dose-dependent manner. Figure 37B shows a Western blot of conditioned medium from the same experiment. The photograph shows that addition of the cleavage enzyme MMP9 increased the release of the tandem repeat domain into the conditioned medium in a dose-dependent manner. Figure 37C shows a FACS analysis of the experiment. The graph shows that addition of MMP9 dose-dependently increased recognition of the cleavage products and decreased recognition of full-length MUC1, including the tandem repeat domain, by the anti-MUC1* antibody MNC2. [Figure 38] Figure 1 shows a photograph of a Western blot in which HCT-MUC1-18 cells, designated here as HCT-18, a cleavage-resistant single-cell clone of HCT cells transfected with full-length MUC1, were treated with varying amounts of catalytically active ADAM17 or MMP14. The released MUC1 tandem repeat domain of full-length MUC1 was immunoprecipitated from conditioned medium, run on a gel, and probed with VU4H5, which binds to the tandem repeat epitope. As can be seen, MMP14 also efficiently cleaves full-length MUC1, releasing the tandem repeat, including the extracellular domain, into the conditioned medium. The cleavage enzyme ADAM17 did not cleave MUC1. [Figure 39]Fluorescence-activated cell sorting (FACS) assay results for human CD34+ hematopoietic stem cells from human bone marrow stained with the anti-MUC1* monoclonal antibodies MNC3, MNC2, and MNE6 or an isotype control antibody are shown. The histogram and bar graph showing the data from the FACS assay demonstrate that MUC1*-positive cells in bone marrow are recognized by one anti-MUC1* antibody, MNC3, but not by MNE6 or MNC2. All three antibodies bind to the PSMGFR peptide. The significant difference in specificity between these antibodies suggests that MNC3 recognizes a MUC1*-like form created when MUC1 is cleaved by an enzyme distinct from MMP9. [Figure 40] Figure 40A shows details of FACS analysis of hematopoietic stem cells probed with either MNC3 or MNE6. Figure 40A shows a FACS scatterplot of whole bone marrow cells. Figure 40B shows a FACS scatterplot of CD34+ cells. Figure 40C shows a FACS histogram of CD34+ cells. Figure 40D shows a FACS scatterplot of earliest hematopoietic stem cells that are CD34+ / CD38- stained with either MNC3 or MNE6. Figure 40E shows a histogram of the experiment. Figure 40F shows a histogram overlay of MNC3 binding to CD34+ / CD38- cells versus MNE6. Figure 40G shows a bar graph of the FACS experiment. [Figure 41]Figure 41A shows detailed FACS analysis of CD34+ / CD38- / lo hematopoietic stem cells probed with polyclonal anti-PSMGFR antibody SDIX, MNE6, or MNC2. Figure 41A shows a FACS scatter plot of the CD34+ / CD38- / lo cell population. Figure 41E shows a detailed analysis table. Figure 41B shows a FACS scatter plot of the CD34+ / CD38- / lo population of cells probed with anti-PSMGFR polyclonal antibody SDIX. Figure 41F shows a detailed analysis table. Figure 41C shows a FACS scatter plot of the CD34+ / CD38- / lo population of cells probed with MNE6. Figure 41G shows a detailed analysis table. Figure 41D shows a FACS scatter plot of the CD34+ / CD38- / lo population of cells probed with MNC2. Figure 41H shows a detailed analysis table. [Figure 42] Photographs of DU145 prostate cancer cells or T47D breast cancer cells treated with Fabs of either the anti-MUC1* antibodies MNC2, MNE6, MNC3, or MNC8 are shown. The images show that the cancer-specific antibodies MNC2 and MNE6 effectively kill prostate and breast cancer cells, whereas the monoclonal antibodies MNC3 and MNC8 do not kill these cells. [Figure 43] Figure 43 shows graphs of PCR experiments comparing the expression of a wide range of cleavage enzymes expressed in various cell lines. These values ​​are normalized to the values ​​expressed in the breast cancer cell line T47D. The cell lines compared are the prostate cancer cell line DU145, HCT-MUC1-41TR (a MUC1-negative colon cancer cell line transfected with MUC1 whose extracellular domain is truncated after 41 tandem repeat units and is not cleaved to the MUC1* form), the T47D breast cancer cell line, and CD34+ bone marrow cells. Figure 44 shows graphs of PCR experiments measuring the expression levels of various cleavage enzymes in DU145 prostate cancer cells, HCT116+MUC1FL (a cell line expressing full-length MUC1, also known as HCT-MUC1-18), T47D breast cancer cells, and bone marrow CD34+ hematopoietic stem cells. Fold expression is relative to the expression of each cleavage enzyme in T47D breast cancer cells and is set as 1. [Figure 44] Figure 43 shows a graph of the PCR experiment, with the maximum value of the Y axis set to 5. [Figure 45]Photographs of CAR T co-culture assays are shown, in which the targeting antibody fragment of the CAR is huMNC2scFv. CAR44 has a CD8 transmembrane domain followed by 41BB-3 zeta, and CAR50 has a CD4 transmembrane domain followed by 41BB-3 zeta. The target cancer cells are HCT-FLR, HCT-116 cells transfected with MUC1* 45, and HCT-MUC1-41TR, a stable single-cell clone HCT-116 cell line that expresses MUC1 with a truncated extracellular domain after 41 tandem repeats and that is not itself cleaved to the MUC1* form. HCT-MUC1-41TR cancer cells were also incubated with conditioned medium from cells transfected with MMP9 or ADAM17 before co-culture with CAR T cells. Conditioned medium from cells expressing MMP9 or ADAM17 was also incubated with APMA, an activator of these cleavage enzymes. The images shown are a 4x brightfield image overlaid with the same fluorescent image showing cancer cells stained with a red CMTMR lipophilic dye. Figure 45A, Figure 45E, Figure 45I, Figure 45M show photographs of cells co-cultured with untransduced human T cells. Figure 45B, Figure 45F, Figure 45J, Figure 45N show photographs of cells co-cultured with human T cells transduced with anti-MUC1* CAR44 at an MOI of 10. Figure 45C, Figure 45G, Figure 45K, Figure 45O show photographs of cells co-cultured with human T cells transduced with anti-MUC1* CAR50 at an MOI of 10. Figure 45D, Figure 45H, Figure 45L, Figure 45P show photographs of cells co-cultured with human T cells transduced with anti-MUC1* CAR44 at an MOI of 50, which increases transduction efficiency. Figures 45B, 45C, and 45D show that both CAR44- and CAR50-transduced T cells recognized and bound to MUC1* expressed on these cancer cells, induced clustering, and killed many cancer cells. Figures 45F, 45G, and 45H show that neither CAR44- nor CAR50-transduced T cells recognized full-length MUC1 expressed on HCT-MUC1-41TR cancer cells. There was no clustering induced by the T cells, and the number of cancer cells was not reduced.Figures 45J, 45K, and 45L show that activated MMP9 cleaves full-length MUC1 into the MUC1* form, which is recognized by both CAR44- and CAR50-transduced T cells. There is clearly visible CAR cell-induced clustering, and a reduction in the number of cancer cells following their death. Figures 45N, 45O, and 45P show that activated ADAM17 either did not cleave MUC1 or cleaved it at a position not recognized by MNC2. Neither huMNC2-CAR44 nor huMNC2-CAR50-transduced T cells recognized these cancer cells. [Figure 46] This image shows a CAR T co-culture assay in which the targeting antibody fragment of the CAR is the MNC2 scFv; CAR44 has the CD8 transmembrane domain followed by 41BB-3 zeta; and CAR50 has the CD4 transmembrane domain followed by 41BB-3 zeta. The target cancer cells were breast cancer T47D cells, which were also incubated with conditioned medium from cells transfected with MMP2, MMP9, or ADAM17 before co-culture with MNC2-CAR T cells. In some cases, conditioned medium from cells expressing MMP2 and MMP9 was also incubated with APMA, an activator of these cleavage enzymes. The image shown is a 4x brightfield image overlaid with the same fluorescent image showing cancer cells stained with the red CMTMR lipophilic dye. As can be seen, MNC2-CAR T cells bind to and attack only target cancer cells expressing the cleaved form of MUC1*. [Figure 47]Photographs of cancer cells co-cultured with anti-MUC1* CAR T cells are shown; some of the cancer cells were pre-incubated with activated MMP9 before co-culture with CAR T cells. The cancer cells shown in Figures 47A-C are the MUC1-negative colon cancer cell line HCT-116 stably transfected to express MUC1*. The cancer cells shown in Figures 47D-F are the MUC1-positive breast cancer cell line T47D, which expresses high levels of both full-length MUC1 and MUC1*. Figures 47G-I show the MUC1-positive breast cancer cell line T47D pre-incubated with activated MMP9. The cells shown in Figures 47A, 47D, and 47G were co-cultured with untransduced human T cells, which serve as controls. The cells shown in Figures 47B, 47E, and 47H were co-cultured with human T cells transduced with huMNC2-CAR44 at an MOI of 10. Here, MOI represents the multiplicity of infection; the higher the MOI, the more CAR is expressed in the T cells. The cells shown in Figures 47C, 47F, and 47I were co-cultured with human T cells transduced with huMNC2-CAR44 at an MOI of 50. As can be seen in the photographs, CAR44 T cells bind to, surround, and kill target MUC1*-positive cancer cells. Comparing the photograph in Figure 47I with the other photographs, it can be seen that cells pre-incubated with MMP9 are much more susceptible to CAR T killing when an antibody targeting the CAR head recognizes MUC1*. It also shows that MUC1 cleaved by MMP9 is recognized by huMNC2scFv. [Figure 48]This figure shows the xCelligence graph of T47D breast cancer cells co-cultured with either non-transduced T cells as a control or huMNC2-CAR44 T cells for 45 hours. After 18 hours of cancer cell growth, the catalytic subunit MMP9 was added to some cells. At 25 hours, T cells were added. As can be seen, killing of huMNC2-CAR44 T cells is significantly improved when T47D cells are preincubated with the cleaving enzyme MMP9. In the xCelligence system, adherent target cancer cells are plated on an electrode array plate. Adherent cells insulate the electrodes, increasing impedance. The number of adherent cancer cells is directly proportional to impedance. T cells are not adherent and do not contribute to impedance. Therefore, an increase in impedance reflects cancer cell growth, and a decrease in impedance reflects cancer cell death. [Figure 49] Shown is an xCelligence graph of DU145 prostate cancer cells co-cultured with either non-transduced T cells as a control or huMNC2-CAR44 T cells for 45 hours. After 18 hours of cancer cell growth, the catalytic subunit MMP9 was added to some cells. At 25 hours, T cells were added. As can be seen, killing of huMNC2-CAR44 T cells is not affected by pre-incubation with the cleavage enzyme MMP9. DU145 cancer cells express significantly lower amounts of MUC1, including MUC1* as well as full-length forms. The low density of full-length MUC1 does not sterically hinder T cell access to membrane-proximal MUC1*. [Figure 50] 1 shows a bar graph of a PCR experiment measuring the amount of MUC1 expressed by a panel of cell lines and primary cells, consisting of normal and cancer cells. [Figure 51]Figure 51A shows bar graphs of an ELISA assay measuring the amount of interferon gamma (IFN-g) secreted by huMNC2-CAR44 human T cells after 72 hours of co-culture with normal or HCT-MUC1* cancer cells. Figure 51A shows the results of an experiment in which the ratio of CAR44 T cells to target cells was 1:1. Figure 51B shows the results of an experiment in which the ratio of CAR44 T cells to target cells was 0.5:1. [Figure 52] Figure 52A shows bar graphs of an ELISA assay measuring the amount of interleukin-2 (IL-2) secreted by huMNC2-CAR44 human T cells after 72 hours of co-culture with normal cells or HCT-MUC1* cancer cells. Figure 52A shows the results of an experiment in which the ratio of CAR44 T cells to target cells was 1:1. Figure 52B shows the results of an experiment in which the ratio of CAR44 T cells to target cells was 0.5:1. [Figure 53-1]Figures 53A-F show bar graphs of FACS analysis of live and dead markers and photographs of normal and cancer cells after co-culture with huMNC2-CAR44 T cells. Figure 53A.1 shows a bar graph of FACS analysis of live and dead cells after co-culture of HCT-MUC1* cancer cells with huMNC2-CAR44 T cells. Figures 53A.2 and 53A.3 show photographs of the experiment described in Figure 53A.1. Figure 53B.1 shows a bar graph of FACS analysis of live and dead cells after co-culture of MCF-12A normal breast cells with huMNC2-CAR44 T cells. Figures 53B.2 and 53B.3 show photographs of the experiment described in Figure 53B.1. Figure 53C.1 shows a bar graph of FACS analysis of live and dead cells after co-culture of THLE-3 normal liver cells with huMNC2-CAR44 T cells. Figures 53C.2 and 53C.3 show photographs of the experiment described in Figure 53C.1. Figure 53D.1 shows a bar graph of FACS analysis of live and dead cells after co-culturing T / G HA-HSMC normal cardiac cells with huMNC2-CAR44T cells. Figures 53D.2 and 53D.3 show photographs of the experiment described in Figure 53D.1. Figure 53E.1 shows a bar graph of FACS analysis of live and dead cells after co-culturing Hs1.Tes normal testicular cells with huMNC2-CAR44T cells. Figures 53E.2 and 53E.3 show photographs of the experiment described in Figure 53E.1. Figure 53F.1 shows a bar graph of FACS analysis of live and dead cells after co-culturing HEK-293 MUC1-negative cells with huMNC2-CAR44T cells. Figures 53F.2 and 53F.3 show photographs of the experiment described in Figure 53F.1. [Figure 53-2]Figures 53G-J show bar graphs of FACS analysis of live and dead markers and photographs of normal and cancer cells after co-culture with huMNC2-CAR44 T cells. Figure 53G.1 shows a bar graph of FACS analysis of live and dead cells after co-culture of HRCE normal kidney cells with huMNC2-CAR44 T cells. Figures 53G.2 and 53G.3 show photographs of the experiment described in Figure 53G.1. Figure 53H.1 shows a bar graph of FACS analysis of live and dead cells after co-culture of CCD-18Lu normal lung cells with huMNC2-CAR44 T cells. Figures 53H.2 and 53H.3 show photographs of the experiment described in Figure 53H.1. Figure 53I.1 shows a bar graph of FACS analysis of live and dead cells after co-culture of HBEC-5i normal brain cells with huMNC2-CAR44 T cells. Figures 53I.2 and 53I.3 show photographs from the experiment described in Figure 53I.1. Figure 53J.1 shows a bar graph of FACS analysis of live and dead cells after co-culturing Hs.738.St / Int normal gastric and intestinal cells with huMNC2-CAR44 T cells. Figures 53J.2 and 53J.3 show photographs from the experiment described in Figure 53J.1. [Figure 54] Shown is a picture of a breast cancer tissue array (CB-insert array number) where for each patient there is a sample of the primary tumor and a sample of that patient's metastasis. As can be seen in the figure, in most cases the metastases express more MUC1* than the primary tumor. [Figure 55]Figure 55A shows the cytotoxic effect of huMNC2-CAR44 T cells on MUC1*-positive DU145 prostate cancer cells, as measured by various assays. Figure 55A is a fluorescent photograph of untransduced T cells co-cultured with prostate cancer cells, where granzyme B is stained with a red fluorophore. Figure 55B shows a merge of DAPI and granzyme B. Figure 55C is a fluorescent photograph of huMNC2-CAR44 T cells co-cultured with prostate cancer cells, where granzyme B is stained with a red fluorophore. Figure 55D shows a merge of DAPI and granzyme B. Figure 55E is a FACS scan of fluorescently labeled granzyme B in untransduced T cells incubated with cancer cells. Figure 55F is a FACS scan showing a positive increase in fluorescently labeled granzyme B for huMNC2-CAR44 T cells incubated with cancer cells. Figure 55G is a graph of mean fluorescence intensity. Figure 55H is an xCELLigence scan tracking in real time the killing of DU145 cancer cells by huMNC2-CAR44 T cells (blue trace) but not by untransduced T cells (green). [Figure 56]Figure 56 shows the cytotoxic effect of huMNC2-CAR44 T cells against MUC1*-positive CAPAN-2 pancreatic cancer cells, as measured by various assays. Figure 56A is a fluorescent photograph of untransduced T cells co-cultured with pancreatic cancer cells, where granzyme B is stained with a red fluorophore. Figure 56B shows a merge of DAPI and granzyme B. Figure 56C is a fluorescent photograph of huMNC2-CAR44 T cells co-cultured with pancreatic cancer cells, where granzyme B is stained with a red fluorophore. Figure 56D shows a merge of DAPI and granzyme B. Figure 56E is a FACS scan of fluorescently labeled granzyme B in untransduced T cells incubated with cancer cells. Figure 56F is a FACS scan showing a positive increase in fluorescently labeled granzyme B for huMNC2-CAR44 T cells incubated with cancer cells. Figure 56G is a graph of mean fluorescence intensity. Figure 56H is an xCELLigence scan tracking in real time the killing of CAPAN-2 cancer cells by huMNC2-CAR44 T cells (blue trace) but not by non-transduced T cells (green). [Figure 57] Figure 57 shows xCELLigence scans tracking the killing of MUC1*-positive cancer cells, but not MUC1*-negative cells, by huMNC2-CAR44 T cells in real time. Figure 57A shows that huMNC2-CAR44 T cells effectively kill HCT colon cancer cells stably transfected with MUC1*. Figure 57B shows that huMNC2-CAR44 T cells have little effect on HCT-MUC1-41TR, a MUC1-negative cancer cell line stably transfected with full-length MUC1. In this cell line, only about 10% of the cells have MUC1 cleaved to MUC1*. Figure 57C shows that huMNC2-CAR44 T cells have no effect on HCT-116 cells, a MUC1-negative colon cancer cell line. [Figure 58]Figure 58 shows photographs of NOD / SCID / GAMMA mice taken with an IVIS instrument measuring photon emission from tumor cells after the mice were left untreated or treated with PBS, untransduced human T cells, or huMNC2-CAR44 T cells. The mice were subcutaneously injected with HCT-MUC1* tumor cells that had been rendered luciferase-positive. Ten minutes before the IVIS photographs were taken, the mice were injected intraperitoneally (ip) with the luciferase substrate luciferin. Figure 58A shows a tumor-bearing mouse treated with phosphate-buffered saline (PBS) alone. Figure 58B shows a tumor-bearing mouse treated with untransduced T cells alone. Figure 58C shows a tumor-bearing mouse treated with a single dose of huMNC2-CAR44 T cells. Figure 58D shows the color scale of the images. Figure 58E shows the Kaplan-Meier survival curves for the experiment. Figure 58F shows a table detailing the molecular makeup of human T cells isolated from the blood of the mice after sacrifice. [Figure 59] Figure 59 shows photographs of NOD / SCID / GAMMA mice taken with an IVIS instrument measuring photon emission from tumor cells after treatment with either PBS or huMNC2-CAR44 T cells. Mice were subcutaneously injected with T47D-wt breast cancer cells or T47D and MUC1*. This is a mixed population of cells, with 95% of the cells being T47D cells stably transfected with MUC1*. In addition to both T47D-wt and T47D, more MUC1* cells became luciferase-positive. Ten minutes before taking the IVIS photograph, the mice were intraperitoneally (ip) injected with luciferin, a luciferase substrate. Figure 59A shows a tumor-bearing mouse treated with phosphate-buffered saline (PBS) alone. Figure 59B shows a T47D-wt tumor-bearing mouse treated with two doses of huMNC2-CAR44 T cells. Figure T90.1C shows a T47D-MUC1* tumor-bearing mouse treated with two doses of huMNC2-CAR44 T cells. [Figure 60]Figure 60A shows a NOD / SCID / GAMMA mouse photographed with an IVIS instrument measuring photon emission from tumor cells after the mouse was left untreated or treated with PBS, untransduced T cells, or huMNC2-CAR44 T cells. The mouse was subcutaneously injected with a mixed population of 70% T47D-wt breast cancer cells transfected with more MUC1* and 30% T47D cells. Both cell types became luciferase-positive. Ten minutes before the IVIS photograph was taken, the mouse was intraperitoneally (ip) injected with luciferin, a luciferase substrate. Figure 60A shows a tumor-bearing mouse treated with phosphate-buffered saline (PBS) alone. Figure 60B shows a tumor-bearing mouse treated with untransduced T cells alone. Figure 60C shows a tumor-bearing mouse treated with two doses of huMNC2-CAR44 T cells. [Figure 61]Fluorescence photographs of mice taken with an IVIS instrument are shown. NSG (NOD / SCID / GAMMA) immunodeficient mice were injected subcutaneously in the flank with 500K human BT-20 cells, a MUC1*-positive triple-negative breast cancer cell line, on day 0. The cancer cells were stably transfected with luciferase. Tumors were allowed to engraft. Six days after IVIS measurements, animals received a single injection of either 10 million huMNC2-scFv-CAR44-transduced or untransduced human T cells. Five million T cells were injected intratumorally and five million were injected via the tail vein. Ten minutes before IVIS photography, mice were IP injected with luciferin, which fluoresces after cleavage by luciferase, allowing tumor cells to fluoresce. Figure 61A, Figure 61D, Figure 61G show photographs of mice treated with huMNC2-scFv-CAR44 T cells pre-stimulated by co-culture with 4 μm beads attached with synthetic MUC1*, PSMGFR peptide for 24 hours prior to administration ("Protocol 1"). Figure 61B, Figure 61E, Figure 61H show photographs of mice treated with huMNC2-scFv-CAR44 T cells pre-stimulated by co-culture with MUC1*-positive cancer cells twice for 24 hours 24 hours prior to administration ("Protocol 2"). Figure 61C, Figure 61F, Figure 61I show photographs of mice treated with untransduced human T cells. Figure 61J is a color scale relating fluorescence in photons / second to color. [Figure 62]Fluorescence photographs of mice taken with an IVIS instrument are shown. NSG (NOD / SCID / GAMMA) immunodeficient mice were injected intraperitoneally (IP) with 500K human SKOV-3 cells, a MUC1*-positive ovarian cancer cell line, on day 0. The cancer cells were stably transfected with luciferase. Tumors were allowed to engraft. On day 4, animals were injected IP with 10 μg of either huMNC2-scFv-CAR44-transduced human T cells, untransduced T cells, or PBS. On day 11, animals were injected again, except half of the cells were injected via the tail vein and the other half were injected IP. Animals were imaged by IVIS on days 3, 7, 10, and 15. Ten minutes prior to IVIS photography, mice were IP injected with luciferin, which fluoresces after cleavage by luciferase, allowing tumor cells to fluoresce. Figures 62A, 62D, 62G, and 62J show photographs of mice treated with huMNC2-scFv-CAR44 T cells that were pre-stimulated by co-culture with 1 μm beads bearing synthetic MUC1* and PSMGFR peptides for 24 hours prior to administration. Figures 62B, 62E, 62H, and 62K show photographs of mice treated with untransduced human T cells. Figures 62C, 62F, 62I, and 62L show photographs of mice treated with PBS. Figures 62A, 62B, and 62C are IVIS images taken three days before administration of CAR T, T cells, or PBS. Figures 62D, 62E, and 62F show IVIS images of animals at day 7, just four days after treatment. Figures 62G, 62H, and 62I show IVIS images of animals at day 10. Figures 62J, 62K, and 62L show IVIS images of animals on day 15. Figure 62M is the IVIS color scale relating fluorescence in photons / second to color. [Figure 63]1 shows a graph of an ELISA binding assay in which various monoclonal antibodies were tested for their ability to bind to the PSMGFR peptide, N-10, C-10, N+20 / C-27, or N+9 / C-9 peptide, with antibody concentrations of 10 μg / mL or 1 μg / mL. Note that the anti-MUC1* monoclonal antibodies C2 and E6, which have been demonstrated to be cancer-specific, bind to the PSMGFR peptide and still bind without the 10 N-terminal amino acids, but not without the 10 or 9 C-terminal amino acids. [Figure 64] Graphs of ELISA binding assays are shown. The antibodies tested were derived from animals immunized with PSMGFR peptides. The initial selection criterion was to confirm that the antibodies bound to the immunizing PSMGFR peptide. Figure 64A shows ELISA graphs of selected antibodies that were further tested to determine their ability to bind to the PSMGFR peptides, N-10, C-10, N+20 / C-27, or N+9 / C-9 peptides. All antibodies except 18B4 were able to bind to the N-10 peptide. 18B4 recognized the N+20 / C-27 but not the N-10 peptide, implying that its cognate epitope is within the GTINVHDVET sequence. All except 20A10 and C2 showed some binding to the C-10 and N+9 / C-9 peptides, indicating that both 20A10 and C2 require 10 membrane-proximal amino acids for binding. C2, which requires 10 membrane-proximal amino acids for binding, has been demonstrated to be cancer-specific. Figure 64B shows the sequences of the various peptides. The color of the bar for each antibody in the ELISA graph is color-coded to correspond to the deduced cognate sequence of that antibody or a portion thereof. [Figure 65]Figure 65A shows graphs of ELISA binding assays in which various monoclonal antibodies were tested for their ability to bind to the PSMGFR peptide, N-10, C-10, N+20 / C-27, or N+9 / C-9 peptide. The test antibodies were derived from animals immunized with the N+20 / C-27 peptide. The first selection criterion was to confirm that the antibodies bound to the immunizing N+20 / C-27 peptide. Figure 65B shows graphs of ELISA binding assays testing each antibody's ability to bind to various peptides. Although these antibodies were raised against the N+20 / C-27 peptide, all but one of the antibodies, 45C11, still bind to the PSMGFR peptide. Although 45C11's binding is weak, a priori inference indicates that the cognate epitope must be within the SNIKFRPGSVV sequence. 1E4 can bind to the N+20 / C-27 peptide, PSMGFR, and the N-10 peptide, consistent with the idea that its epitope must lie within the QFNQYKTE sequence. Figure 65B shows the sequences of the various peptides. The color of each antibody bar in the ELISA graph is color-coded to match the a priori cognate sequence or a portion thereof for that antibody. [Figure 66] Figure 66A shows graphs of ELISA binding assays in which various monoclonal antibodies were tested for their ability to bind to the PSMGFR peptide, N-10, C-10, N+20 / C-27, or N+9 / C-9 peptide. The test antibodies were derived from animals immunized with the N+9 / C-9 peptide. The first selection criterion was to confirm that the antibody bound to the immunizing peptide N+9 / C-9. Figure 66A shows graphs of the ELISA assays. All antibodies except for one, 39H5, were able to bind only to the immunizing peptide N+9 / C-9. 39H5 showed very weak binding to the PSMGFR and N-10 peptides, consistent with the idea that at least a portion of its cognate epitope must lie within the QFNQYKTE sequence. Figure 66B shows the sequences of the various peptides. The color of the bar for each antibody in the ELISA graph is color-coded to match the antibody's deduced cognate sequence or a portion thereof. [Figure 67]Figure 67 shows the results of ELISA assays to further define antibody epitopes within the extracellular domain of MUC1 or MUC1*. All antibodies shown in this figure were generated by immunizing animals with PSMGFR peptides. Binding assays tested antibodies for their ability to bind to peptides N-19, N-26, N-30, N-10 / C-5, N-19 / C-5, PSMGFR, N-10, and C-10, all of which are subsets of PSMGFR peptides, and the numbering refers to the PSMGFR peptide. Figure 67A shows the binding of various antibodies to various peptides. Figure 67B shows the sequence of a PSMGFR peptide extended by 20 amino acids at the N-terminus. Figure 67C shows the sequence of a subset peptide derived from PSMGFR. Figure 67D shows sequences containing all or part of the epitope essential for antibody recognition. [Figure 68] Figure 68A shows the results of ELISA assays to further define antibody epitopes within the extracellular domain of MUC1 or MUC1*. All antibodies shown in this figure were generated by immunizing animals with the N+20 / C-27 peptide. Binding assays tested antibodies for their ability to bind to peptides N-19, N-26, N-30, N-10 / C-5, N-19 / C-5, PSMGFR, N-10, and C-10, all of which are subsets of PSMGFR peptides, and the numbering refers to the PSMGFR peptide. Figure 68A shows the binding of various antibodies to various peptides. Figure 68B shows the sequence of a PSMGFR peptide extended by 20 amino acids at the N-terminus. Figure 68C shows the sequence of a subset peptide derived from PSMGFR. Figure 68D shows sequences containing all or part of the epitope essential for antibody recognition. [Figure 69]Figure 69 shows the results of ELISA assays to further define antibody epitopes within the extracellular domain of MUC1 or MUC1*. All antibodies shown in this figure were generated by immunizing animals with the N+9 / C-9 peptide. Binding assays tested antibodies for their ability to bind to peptides N-19, N-26, N-30, N-10 / C-5, N-19 / C-5, PSMGFR, N-10, and C-10, all of which are subsets of PSMGFR peptides, and the numbering refers to the PSMGFR peptide. Figure 69A shows the binding of various antibodies to various peptides. Figure 69B shows the sequence of a PSMGFR peptide extended by 20 amino acids at the N-terminus. Figure 69C shows the sequence of a subset peptide derived from PSMGFR. Figure 69D shows sequences containing all or part of the epitope essential for antibody recognition. [Figure 70] A graph of an ELISA displacement assay is shown. In this experiment, multiwell plates were coated with PSMGFR peptide. Recombinant NME7AB was bound to the surface-immobilized PSMGFR peptide. Various antibodies were added, followed by a wash step. The amount of NME7AB still attached to the PSMGFR-coated plate after antibody competition was measured by detecting the tag on NME7AB. As a control, anti-NME7AB antibodies were also tested for their ability to displace NME7AB from PSMGFR. [Figure 71-1]Figures 71A-D show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes in full-length MUC1 or MUC1*. Figures 71A-71D show testing of antibodies for their ability to bind to the MUC1-negative cell line, HCT-116, or the engineered cell line, HCT-MUC1-18, a cleavage-resistant clone expressing full-length MUC1, or the engineered cell line, HCT-MUC1*, engineered to express only the PSMGFR sequence in its extracellular domain. Figures 71B and 71F show MNE6, a monoclonal antibody raised against the PSMGFR peptide that binds to the N-10 variant but not the C-10 variant. Figures 71C and 71G show SDIX, a polyclonal antibody raised against and that binds to the PSMGFR peptide. Figures 71D and 71H show VU4H5, a commercially available monoclonal antibody that binds to the tandem repeats of full-length MUC1. As can be seen, neither MNC2 nor MNE6 binds to linear epitopes of the MUC1 species. [Figure 71-2]Figures 71E-H show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes in full-length MUC1 or MUC1*. Figures 71E-71H show testing of antibodies for their ability to bind to breast cancer cell lines T47D or 1500, also known as ZR-75-1. Figures 71A and 71E show MNC2, a monoclonal antibody raised against the PSMGFR peptide that binds to the N-10 variant but not the C-10 variant. Figures 71B and 71F show MNE6, a monoclonal antibody raised against the PSMGFR peptide that binds to the N-10 variant but not the C-10 variant. Figures 71C and 71G show SDIX, a polyclonal antibody raised against and that binds to the PSMGFR peptide. Figures 71D and 71H show VU4H5, a commercially available monoclonal antibody that binds to the tandem repeats of full-length MUC1. As can be seen, neither MNC2 nor MNE6 bind to linear epitopes of the MUC1 species. [Figure 72-1]Figures 72A-H show photographs of Western blots in which antibodies were tested for their ability to bind linear epitopes in full-length MUC1 or MUC1*. All of these antibodies were raised against and bind to the PSMGFR peptide. Figures 72A-H show testing of antibodies for their ability to bind to the MUC1-negative cell line, HCT-116, or the engineered cell line, HCT-MUC1-18, a cleavage-resistant clone expressing full-length MUC1, or the engineered cell line, HCT-MUC1*, engineered to express only the SMGFR sequence in its extracellular domain. Figures 72A and 72I show 20A10. Figures 72B and 72J show 25E6. Figures 72C and 72K show 18B4. Figures 72D and 72L show 18G12. Figures 72E and 72M show 28F9. Figures 72F and 72N show 3C2B1. Figures 72G and 72O show 5C6F3. Figures 72H and 72P show 5C6F3, where the blot was exposed for a longer period to better visualize the MUC1*-specific band. As can be seen, antibodies 25E6, 18B4, and to some extent 5C6F3 recognize linear epitopes, whereas 20A10, 3C2B1, 18G12, and 28F9 do not. [Figure 72-2]Figures 72I-P show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes in full-length MUC1 or MUC1*. All of these antibodies were raised against and bind to the PSMGFR peptide. Figures 72I-P show testing of antibodies for their ability to bind to breast cancer cell lines T47D or 1500, also known as ZR-75-1. Figures 72A and 72I show 20A10. Figures 72B and 72J show 25E6. Figures 72C and 72K show 18B4. Figures 72D and 72L show 18G12. Figures 72E and 72M show 28F9. Figures 72F and 72N show 3C2B1. Figures 72G and 72O show 5C6F3. Figures 72H and 72P show 5C6F3, where the blot was exposed for a longer period to better visualize the MUC1*-specific band. As can be seen, antibodies 25E6, 18B4, and to some extent 5C6F3, but not 20A10, 3C2B1, 18G12, and 28F9, recognize linear epitopes. [Figure 73-1] Figures 73A-E show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes in full-length MUC1 or MUC1*. All of these antibodies were raised against the N+20 / C-27 variant of the PSMGFR peptide and bind to the N+20 / C-27 peptide. Figures 73A-E show testing of antibodies for their ability to bind to the MUC1-negative cell line, HCT-116, or the engineered cell line, HCT-MUC1-18, a cleavage-resistant clone expressing full-length MUC1, or the engineered cell line, HCT-MUC1*, engineered to express only the SMGFR sequence in its extracellular domain. Figures 73A and 73F show 1E4. Figures 73B and 73G show 45C11. Figures 73C and 73H show 31A1. Figures 73D and 73I show 32C1. Figures 73E and 73J show 29H1. As can be seen, antibodies 31A1 and 32C1 recognize linear epitopes. [Figure 73-2]Figures 73F-J show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes in full-length MUC1 or MUC1*. All of these antibodies were raised against the N+20 / C-27 variant of the PSMGFR peptide and bind to the N+20 / C-27 peptide. Figures 73F-J show testing of antibodies for their ability to bind to breast cancer cell lines T47D or 1500, also known as ZR-75-1. Figures 73A and 73F show 1E4. Figures 73B and 73G show 45C11. Figures 73C and 73H show 31A1. Figures 73D and 73I show 32C1. Figures 73E and 73J show 29H1. As can be seen, antibodies 31A1 and 32C1 recognize linear epitopes. [Figure 74-1] Figures 74A-D show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes within full-length MUC1 or MUC1*. All of these antibodies were raised against the N+9 / C-9 variant of the PSMGFR peptide and bind to the N+9 / C-9 peptide. Figures 74A-D show testing of antibodies for their ability to bind to the MUC1-negative cell line, HCT-116, or the engineered cell line HCT-MUC1-18, a cleavage-resistant clone expressing full-length MUC1, or the engineered cell line HCT-MUC1*, which has been engineered to express only the PSMGFR sequence in its extracellular domain. Figures 74A and 74E show 8A9. Figures 74B and 74F show 17H6. Figures 74C and 74G show 3C5. Figures 74D and 74H show 39H5. [Figure 74-2]Figures 74E-H show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes within full-length MUC1 or MUC1*. All of these antibodies were raised against the N+9 / C-9 variant of the PSMGFR peptide and bind to the N+9 / C-9 peptide. Figures 74E-H show testing of antibodies for their ability to bind to breast cancer cell lines T47D or 1500, also known as ZR-75-1. Figures 74A and 74E show 8A9. Figures 74B and 74F show 17H6. Figures 74C and 74G show 3C5. Figures 74D and 74H show 39H5. [Figure 75-1] Figures 75A-N show graphs of FACS analysis. HCT-MUC1-18 cells expressing full-length MUC1 were incubated with catalytically active MMP9 or MMP2 for 24 hours, incubated with antibodies of the invention, and then analyzed by FACS to determine whether the antibodies bound to MMP9 or MMP2-cleaved forms of MUC1. Note that the first bar in each graph indicates that in the absence of cleavage, neither antibody binds to full-length MUC1. Each bar graph displays both the name of the antibody used in that assay and its cognate epitope. Graph order, from right to left, corresponds to the distance from the cell surface of the antibody's cognate epitope. Figure 75A shows antibody 1E4. Figure 75B shows antibody 28F9. Figure 75C shows antibody 18G12. Figure 75D shows antibody 25E6. Figure 75E shows antibody 20A10. Figure 75F shows antibody 3C5. Figure 75G shows antibody 29H1. Figure 75H shows antibody 32C1. Figure 75I shows antibody 31A1. Figure 75J shows antibody 18B4. Figure 75K shows antibody 45C11. Figure 75L shows antibody 8A9. Figure 75M shows antibody 17H6. Figure 75N shows antibody 39H5. [Figure 75-2]Figures 75O-P show graphs of FACS analysis. HCT-MUC1-18 cells expressing full-length MUC1 were incubated with catalytically active MMP9 or MMP2 for 24 hours, incubated with antibodies of the invention, and then analyzed by FACS to determine whether the antibodies bound to MMP9 or MMP2-cleaved forms of MUC1. Note that the first bar in each graph indicates that in the absence of cleavage, neither antibody binds to full-length MUC1. Each bar graph is labeled with both the name of the antibody used in that assay and its cognate epitope. Graph order, from right to left, corresponds to the distance from the cell surface of the antibody's cognate epitope. Figure 75O shows antibody 3C2B1. Figure 75P shows antibody 5C6F3. [Figure 76] This figure shows graphs of FACS analysis of reference antibodies MNC2, "C2," and VU4H5 binding to either the MUC1-negative cell line HCT-116, "HCT-MUC1*," a HCT transfected with MUC1*, or "HCT-MUC1-18," a cleavage-resistant single-cell clone of HCT transfected with full-length MUC1, and MNC2 binding to breast cancer cell lines T47D or 1500 (also known as ZR-75-1). MNC2 binds to an ectopic binding site on the extracellular domain of MUC1* within the membrane-proximal portion of the PSMGFR sequence. The MNC2 binding site is only accessible after cleavage and release of most of the extracellular domain, including the tandem repeat domain. VU4H5 binds to hundreds of repeat epitopes in the tandem repeat domain. 76A to 76E show the binding rate, and 76F to 76J show the mean fluorescence intensity (MFI). [Figure 77]Figures 77A-77G show the binding rate, and Figures 77H-77N show the mean fluorescence intensity (MFI). Figures 77A and 77H show the antibody binding to the lung cancer cell line NCI-H292. Figures 77B and 77I show the antibody binding to the lung cancer cell line NCI-H1975. Figures 77C and 77J show the antibody binding to the ovarian cancer cell line SKOV-3. Figures 77D and 77K show antibodies binding to the pancreatic cancer cell line HPAF-II. Figures 77E and 77L show antibodies binding to the pancreatic cancer cell line Capan-1. Figures 77F and 77M show antibodies binding to the prostate cancer cell line DU145. Figures 77G and 77N show antibodies binding to the breast cancer cell line MDA-MB-231, which is largely MUC1 and MUC1* negative. [Figure 78] Figure 78A shows a color-coded schematic of the basic PSMGFR sequence, extended or deleted at both the N- and C-termini. Antibodies of the invention were tested against this subset of peptides to further refine the epitope bound by each antibody or key amino acids within the epitope bound by each antibody. Figure 78A is a schematic of an alignment of various subsets of peptides. Figure 78B lists the antibodies that bind to each of the color-coded sequences. Figure 78C lists the cancer cell lines recognized by each antibody. [Figure 79]Color-coded graphs obtained from FACS analysis of each antibody binding to T47D breast cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence are shown. Figures 79A-79D are FACS graphs showing the percentage of cells recognized by each antibody. Figures 79E-79H are FACS graphs showing the mean fluorescence intensity (MFI) of each antibody. Figures 79A and 79E show FACS graphs of antibodies generated by immunization with the PSMGFR peptide. Figures 79B and 79F show FACS graphs of antibodies generated by immunization with the N+20 / C-27 peptide. Figures 79C and 79G show FACS graphs of antibodies generated by immunization with the N+9 / C-9 peptide. Figures 79D and 79H also show FACS graphs of antibodies generated by immunization with the PSMGFR peptide. Figure 79I shows the PSMGFR sequence extended by 20 amino acids at the N-terminus. [Figure 80] Color-coded graphs obtained from FACS analysis of each antibody binding to 1500 breast cancer cells, also known as ZR-75-1, and their respective cognate sequences within the N-terminally extended PSMGFR sequence are shown. Figures 80A-80C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 80D-80F are FACS graphs showing the mean fluorescence intensity (MFI) of each antibody. Figures 80A, 80E, 80D, and 80H show FACS graphs of antibodies generated by immunization with PSMGFR peptides. Figures 80B and 80F show FACS graphs of antibodies generated by immunization with the N+20 / C-27 peptide. Figures 80C and 80G show FACS graphs of antibodies generated by immunization with the N+9 / C-9 peptide. Figure 80I shows a PSMGFR sequence extended by 20 amino acids at the N-terminus. [Figure 81]Color-coded graphs obtained from FACS analysis of each antibody binding to NCI-H292 lung cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence are shown. Figures 81A-81C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 81D-81F are FACS graphs showing the mean fluorescence intensity (MFI) of each antibody. Figures 81A and 81D show FACS graphs of antibodies generated by immunization with the PSMGFR peptide. Figures 81B and 81E show FACS graphs of antibodies generated by immunization with the N+20 / C-27 peptide. Figures 81C and 81F show FACS graphs of antibodies generated by immunization with the N+9 / C-9 peptide. Figure 81G shows the PSMGFR sequence extended by 20 amino acids at the N-terminus. [Figure 82] Color-coded graphs obtained from FACS analysis of each antibody binding to NCI-H1975 lung cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence are shown. Figures 82A-82C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 82D-82F are FACS graphs showing the mean fluorescence intensity (MFI) of each antibody. Figures 82A and 82D show FACS graphs of antibodies generated by immunization with the PSMGFR peptide. Figures 82B and 82E show FACS graphs of antibodies generated by immunization with the N+20 / C-27 peptide. Figures 82C and 82F show FACS graphs of antibodies generated by immunization with the N+9 / C-9 peptide. Figure 82G shows the PSMGFR sequence extended by 20 amino acids at the N-terminus. [Figure 83]Color-coded graphs obtained from FACS analysis of each antibody binding to SKOV-3 ovarian cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence are shown. Figures 83A-83C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 83D-83F are FACS graphs showing the mean fluorescence intensity (MFI) of each antibody. Figures 83A and 83D show FACS graphs of antibodies generated by immunization with the PSMGFR peptide. Figures 83B and 83E show FACS graphs of antibodies generated by immunization with the N+20 / C-27 peptide. Figures 83C and 83F show FACS graphs of antibodies generated by immunization with the N+9 / C-9 peptide. Figure 83G shows the PSMGFR sequence extended by 20 amino acids at the N-terminus. [Figure 84] Color-coded graphs obtained from FACS analysis of each antibody binding to DU145 prostate cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence are shown. Figures 84A-84C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 84D-84F are FACS graphs showing the mean fluorescence intensity (MFI) of each antibody. Figures 84A and 84D show FACS graphs of antibodies generated by immunization with the PSMGFR peptide. Figures 84B and 84E show FACS graphs of antibodies generated by immunization with the N+20 / C-27 peptide. Figures 84C and 84F show FACS graphs of antibodies generated by immunization with the N+9 / C-9 peptide. Figure 84G shows the PSMGFR sequence extended by 20 amino acids at the N-terminus. [Figure 85]Color-coded graphs obtained from FACS analysis of each antibody binding to HPAF-II pancreatic cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence are shown. Figures 85A-85C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 85D-85F are FACS graphs showing the mean fluorescence intensity (MFI) of each antibody. Figures 85A and 85D show FACS graphs of antibodies generated by immunization with the PSMGFR peptide. Figures 85B and 85E show FACS graphs of antibodies generated by immunization with the N+20 / C-27 peptide. Figures 85C and 85F show FACS graphs of antibodies generated by immunization with the N+9 / C-9 peptide. Figure 85G shows the PSMGFR sequence extended by 20 amino acids at the N-terminus. [Figure 86] Figures 86A-86C show color-coded graphs of antibody binding to IgG-1 pancreatic cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence. Figures 86A-86C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 86D-86F are FACS graphs showing the mean fluorescence intensity (MFI) of each antibody. Figures 86A and 86D show FACS graphs of antibodies generated by immunization with the PSMGFR peptide. Figures 86B and 86E show FACS graphs of antibodies generated by immunization with the N+20 / C-27 peptide. Figures 86C and 86F show FACS graphs of antibodies generated by immunization with the N+9 / C-9 peptide. Figure 86G shows the PSMGFR sequence extended by 20 amino acids at the N-terminus. [Figure 87]Color-coded graphs obtained from FACS analysis of each antibody binding to mostly MUC1-negative MDA-MB-231 breast cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence are shown. Figures 87A-87C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 87D-87F are FACS graphs showing the mean fluorescence intensity (MFI) of each antibody. Figures 87A and 87D show FACS graphs of antibodies generated by immunization with the PSMGFR peptide. Figures 87B and 87E show FACS graphs of antibodies generated by immunization with the N+20 / C-27 peptide. Figures 87C and 87F show FACS graphs of antibodies generated by immunization with the N+9 / C-9 peptide. Figure 87G shows the PSMGFR sequence extended by 20 amino acids at the N-terminus. [Figure 88] Photographs of normal liver tissue specimens are shown, each from the same donor but stained with a different antibody of the invention. Figures 88A-88F show the entire tissue core. Figures 88G-88L show specific regions of the tissue at 40x magnification. The tissue is arranged from right to left with antibodies that bind most proximal to the membrane, i.e., the most C-terminal portion of the PSMGFR peptide is on the right, and antibodies that bind further beyond the PSMGFR region, to the most N-terminal portion of the MUC1 extracellular domain, are on the left. As can be seen in the figures, the most cancer-specific antibodies are those that bind to the more membrane-proximal portion of the PSMGFR sequence; antibodies that bind to the most distal N-terminal portion lose cancer specificity; and those that bind epitopes outside of the PSMGFR lose all cancer specificity. [Figure 89]Photographs of normal cardiac tissue specimens stained with different antibodies of the present invention are shown. Figures 89A-89D show the entire tissue core. Figures 89E-89HL show specific regions of the tissue at 40x magnification. Figures 89A and 89E show staining with MNC2-scFv. Figures 89B and 89F show staining with MNE6. Figures 89C and 89G show staining with 20A10. Figures 89D and 89H show staining with 3C2B1. These antibodies bind to epitopes containing all or part of the sequence FPFS or PFPFSAQSGA. All of these antibodies can bind to the PSMGFR peptide and the N-10 peptide, but not the C-10 peptide. Furthermore, these antibodies disrupt binding of NME7AB to the MUC1* extracellular domain, as exemplified by the PSMGFR peptide. Furthermore, these antibodies recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen in the figure, these antibodies show no binding to normal cardiac tissue. [Figure 90] Photographs of normal cardiac tissue specimens stained with different antibodies of the present invention are shown. Figures 90A-90B show the entire tissue core. Figures 90C-90D show specific areas of tissue at 40x magnification. Figures 90A and 90C show staining with MNC3. Figures 90B and 90D show staining with 25E6. These antibodies bind to epitopes that include all or part of the ASRYNLT sequence. These antibodies can bind to the PSMGFR peptide, the N-10 peptide, and also the C-10 peptide. [Figure 91] Photographs of normal cardiac tissue specimens stained with antibody 1E4 of the present invention are shown. Figure 91A shows the entire tissue core. Figure 91B shows a specific area of ​​the tissue at 40x magnification. Antibody 1E4 binds to an epitope that includes all or part of the QFNQYKTEA sequence. Antibody 1E4 can bind to the N-10 peptide, but can also bind to the C-10 peptide. As can be seen in the figures, 1E4 binds to normal cardiac tissue. [Figure 92]Photographs of normal heart tissue specimens stained with different antibodies of the present invention are shown. Figures 92A-92D show entire tissue cores. Figures 92E-92HL show specific regions of tissue at 40x magnification. Figures 92A and 92E show staining with 18B4. Figures 92B and 92F show staining with 31A1. Figures 92C and 92G show staining with 32C1. Figures 92D and 92H show staining with 29H1. These antibodies bind to epitopes that include all or part of the GTINVHDVET sequence, the most N-terminal portion of the PSMGFR peptide. None of these antibodies can bind to the N-10 peptide. As can be seen, all of these antibodies, except for 18B4, demonstrate binding to normal heart tissue. [Figure 93] Photographs of normal heart tissue specimens stained with different antibodies of the present invention are shown. Figures 93A-93B show the entire tissue core. Figures 93C-93D show specific areas of the tissue at 40x magnification. Figures 93A and 93C show staining with antibody 8A9. Figures 93B and 93D show staining with antibody 17H6. Both antibodies bind to epitopes outside the PSMGFR region and include all or part of the sequence VQLTLAFRE. As can be seen, both antibodies show strong binding to normal heart tissue. [Figure 94] Photographs of normal cardiac tissue specimens stained with antibody 45C11 of the present invention are shown. Figure 94A shows the entire tissue core. Figure 94B shows a specific area of ​​the tissue at 40x magnification. Antibody 45C11 binds to an epitope outside the PSMGFR region and including all or part of the SNIKFRPGSVV sequence. Antibody 45C11 is unable to bind to the N-10 peptide. As can be seen, 45C11 binds strongly to normal cardiac tissue. [Figure 95]Photographs of normal liver tissue specimens stained with different antibodies of the present invention are shown. Figures 95A-95D show entire tissue cores. Figures 95E-95HL show specific regions of tissue at 40x magnification. Figures 95A and 95E show staining with MNC2-scFv. Figures 95B and 95F show staining with MNE6. Figures 95C and 95G show staining with 20A10. Figures 95D and 95H show staining with 3C2B1. These antibodies bind to epitopes containing all or part of the sequences FPFS or PFPFSAQSGA. All of these antibodies can bind to the PSMGFR peptide and the N-10 peptide, but not the C-10 peptide. Furthermore, these antibodies disrupt binding of NME7AB to the MUC1* extracellular domain, as exemplified by the PSMGFR peptide. Furthermore, these antibodies recognize MUC1 cleavage products when the cleavage enzyme is MMP9. As can be seen in the figure, these antibodies show no binding to normal liver tissue. [Figure 96] Photographs of normal liver tissue specimens stained with different antibodies of the present invention are shown. Figures 96A-96B show the entire tissue core. Figures 96C-96D show specific areas of the tissue at 40x magnification. Figures 96A and 96C show staining with MNC3. Figures 96B and 96D show staining with 25E6. These antibodies bind to epitopes that include all or part of the ASRYNLT sequence. All of these antibodies can bind to the PSMGFR peptide, the N-10 peptide, but also the C-10 peptide. [Figure 97] Photographs of normal liver tissue specimens stained with antibody 1E4 of the present invention are shown. Figure 97A shows an entire tissue core. Figure 97B shows a specific area of ​​tissue at 40x magnification. Antibody 1E4 binds to an epitope that includes all or part of the QFNQYKTEA sequence. Antibody 1E4 can bind to the N-10 peptide, but also to the C-10 peptide. As can be seen in the figures, 1E4 binds to normal liver tissue. [Figure 98]Photographs of normal liver tissue specimens stained with different antibodies of the present invention are shown. Figures 98A-98D show entire tissue cores. Figures 98E-98H show specific regions of tissue at 40x magnification. Figures 98A and 98E show staining with 18B4. Figures 98B and 98F show staining with 31A1. Figures 98C and 98G show staining with 32C1. Figures 98D and 98H show staining with 29H1. These antibodies bind to epitopes that include all or part of the GTINVHDVET sequence, the most N-terminal portion of the PSMGFR peptide. Neither of these antibodies can bind to the N-10 peptide. As can be seen, 32C1 shows some binding to normal liver, while 29H1 shows very strong binding to normal liver tissue. [Figure 99] Figures 99A-99B show photographs of normal liver tissue specimens stained with different antibodies of the present invention. Figures 99A-99D show specific areas of tissue at 40x magnification. Figures 99A and 99C show staining with antibody 8A9. Figures 99B and 99D show staining with antibody 17H6. Both antibodies bind to epitopes outside the PSMGFR region and include all or part of the sequence VQLTLAFRE. As can be seen in the figures, 8A9 shows strong binding to normal liver tissue. 17H6 is a weak antibody, and it may not have been used at a high enough concentration in this study. [Figure 100] Photographs of normal liver tissue specimens stained with antibody 45C11 of the present invention are shown. Figure 100A shows the entire tissue core. Figure 100B shows a specific area of ​​the tissue at 40x magnification. Antibody 45C11 binds to an epitope outside the PSMGFR region and including all or part of the SNIKFRPGSVV sequence. Antibody 45C11 cannot bind to the N-10 peptide. As can be seen, 45C11 binds strongly to normal liver tissue. [Figure 101]Photographs of normal lung tissue specimens stained with different antibodies of the present invention are shown. Figures 101A-101D show entire tissue cores. Figures 101E-101H show specific areas of tissue at 40x magnification. Figures 101A and 101E show staining with MNC2-scFv. Figures 101B and 101F show staining with MNE6. Figures 101C and 101G show staining with 20A10. Figures 101D and 101H show staining with 3C2B1. These antibodies bind to epitopes containing all or part of the sequence FPFS or PFPFSAQSGA. All of these antibodies can bind to the PSMGFR peptide and the N-10 peptide, but not the C-10 peptide. Furthermore, these antibodies disrupt the binding of NME7AB to the MUC1* extracellular domain, as exemplified by the PSMGFR peptide. Furthermore, these antibodies recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen in the figure, these antibodies show no binding to normal lung tissue. [Figure 102] Photographs of normal lung tissue specimens stained with different antibodies of the present invention are shown. Figures 102A-102B show the entire tissue core. Figures 102C-102D show specific areas of tissue at 40x magnification. Figures 102A and 102C show staining with MNC3. Figures 102B and 102D show staining with 25E6. These antibodies bind to epitopes that include all or part of the ASRYNLT sequence. All of these antibodies can bind to the PSMGFR peptide, the N-10 peptide, but also the C-10 peptide. [Figure 103] Photographs of normal lung tissue specimens stained with antibody 1E4 of the present invention are shown. Figure 103A shows the entire tissue core. Figure 103B shows a specific area of ​​the tissue at 40x magnification. Antibody 1E4 binds to an epitope that includes all or part of the QFNQYKTEA sequence. Antibody 1E4 can bind to the N-10 peptide, but also to the C-10 peptide. [Figure 104]Photographs of normal lung tissue specimens stained with different antibodies of the present invention are shown. Figures 104A-104D show whole tissue cores. Figures 104E-104H show specific areas of tissue at 40x magnification. Figures 104A and 104E show staining with 18B4. Figures 104B and 104F show staining with 31A1. Figures 104C and 104G show staining with 32C1. Figures 104D and 104H show staining with 29H1. These antibodies bind to epitopes that include all or part of the GTINVHDVET sequence, the most N-terminal portion of the PSMGFR peptide. None of these antibodies can bind to the N-10 peptide. As can be seen, all of these antibodies demonstrate strong binding to normal lung tissue. [Figure 105] Figures 105A-105B show photographs of normal lung tissue specimens stained with different antibodies of the present invention. Figures 105A-105B show entire tissue cores. Figures 105C-105D show specific areas of tissue at 40x magnification. Figures 105A and 105C show staining with antibody 8A9. Figures 105B and 105D show staining with antibody 17H6. Both antibodies bind to epitopes outside the PSMGFR region and include all or part of the sequence VQLTLAFRE. As can be seen, 8A9 shows strong binding to normal lung tissue. 17H6 is a weak antibody, and it may not have been used at a high enough concentration in this study. [Figure 106] Photographs of normal lung tissue specimens stained with antibody 45C11 of the present invention are shown. Figure 106A shows the entire tissue core. Figure 106B shows a specific area of ​​tissue at 40x magnification. Antibody 45C11 binds to an epitope outside the PSMGFR region and including all or part of the SNIKFRPGSVV sequence. Antibody 45C11 cannot bind to the N-10 peptide. As can be seen, 45C11 binds to normal lung tissue. [Figure 107]Photographs of normal bone marrow tissue specimens stained with different antibodies of the present invention are shown. Figures 107A-107D show the entire tissue core. Figures 107E-107H show specific areas of the tissue at 40x magnification. Figures 107A and 107E show staining with MNC2-scFv. Figures 107B and 107F show staining with MNE6. Figures 107C and 107G show staining with 20A10. Figures 107D and 107H show staining with 3C2B1. These antibodies bind to epitopes containing all or part of the sequence FPFS or PFPFSAQSGA. All of these antibodies can bind to the PSMGFR peptide and the N-10 peptide, but not the C-10 peptide. Furthermore, these antibodies disrupt the binding of NME7AB to the MUC1* extracellular domain, as exemplified by the PSMGFR peptide. Furthermore, these antibodies recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen in the figure, these antibodies show no binding to normal bone marrow tissue. [Figure 108] Photographs of normal bone marrow tissue specimens stained with different antibodies of the present invention are shown. Figures 108A-108B show the entire tissue core. Figures 108C-108D show specific areas of the tissue at 40x magnification. Figures 108A and 108C show staining with MNC3. Figures 108B and 108D show staining with 25E6. These antibodies bind to epitopes that include all or part of the ASRYNLT sequence. All of these antibodies can bind to the PSMGFR peptide, the N-10 peptide, but also the C-10 peptide. [Figure 109] Photographs of normal bone marrow tissue specimens stained with antibody 1E4 of the present invention are shown. Figure 109A shows the entire tissue core. Figure 109B shows a specific area of ​​the tissue at 40x magnification. Antibody 1E4 binds to an epitope that includes all or part of the QFNQYKTEA sequence. Antibody 1E4 can bind to the N-10 peptide, but also binds to the C-10 peptide. 1E4 binds to normal bone marrow. [Figure 110]Photographs of normal bone marrow tissue specimens stained with different antibodies of the present invention are shown. Figures 110A-110D show entire tissue cores. Figures 110E-110H show specific regions of tissue at 40x magnification. Figures 110A and 110E show staining with 18B4. Figures 110B and 110F show staining with 31A1. Figures 110C and 110G show staining with 32C1. Figures 110D and 110H show staining with 29H1. These antibodies bind to epitopes that include all or part of the GTINVHDVET sequence, the most N-terminal portion of the PSMGFR peptide. None of these antibodies can bind to the N-10 peptide. As can be seen, all of these antibodies demonstrate strong binding to normal bone marrow tissue. [Figure 111] Photographs of normal bone marrow tissue specimens stained with antibodies of the present invention are shown. Figures 111A-111B show the entire tissue core. Figures 111C-111D show specific areas of the tissue at 40x magnification. Figures 111A and 111C show staining with antibody 8A9. Figures 111B and 111D show staining with antibody 17H6. Both antibodies bind to epitopes outside the PSMGFR region and include all or part of the sequence VQLTLAFRE. As can be seen in the figures, 8A9 shows strong binding to normal bone marrow tissue. 17H6 is a weak antibody, and it may not have been used at a high enough concentration in this study. [Figure 112] Figure 112A shows a photograph of a normal bone marrow tissue specimen stained with antibody 45C11 of the present invention. Figure 112A shows the entire tissue core. Figure 112B shows a specific area of ​​the tissue at 40x magnification. Antibody 45C11 binds to an epitope outside the PSMGFR region and including all or part of the SNIKFRPGSVV sequence. Antibody 45C11 cannot bind to the N-10 peptide. As can be seen, 45C11 binds to normal bone marrow tissue. [Figure 113]Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10 are shown. Figure 113A shows a photograph of the tissue microarray. Figure 113B shows a map of the array including abbreviated tissue descriptors. Figure 113C details the tissue microarray including de-identified donor data. [Figure 114-1] Figures 114A-H show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10 at 6x and 20x magnification. Figures 114A and 114E are adrenal glands. Figures 114B and 114F are breast. Figures 114C and 114G are fallopian tubes. Figures 114D and 114H are kidneys. Figures 114I and 114M are myocardium. Figures 114J and 114N are liver. Figures 114K and 114O are lungs. Figures 114L and 114P are ureters. Figures 114Q and 114U are eyes. Figures 114R and 114V are cerebral cortex. Figures 114S and 114W are bone marrow. Figure 114T and Figure 114X are skeletal muscles. [Figure 114-2] Figures 114I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10 at 6x and 20x magnification. Figures 114A and 114E are adrenal glands. Figures 114B and 114F are breast. Figures 114C and 114G are fallopian tubes. Figures 114D and 114H are kidneys. Figures 114I and 114M are myocardium. Figures 114J and 114N are liver. Figures 114K and 114O are lungs. Figures 114L and 114P are ureters. Figures 114Q and 114U are eyes. Figures 114R and 114V are cerebral cortex. Figures 114S and 114W are bone marrow. Figure 114T and Figure 114X are skeletal muscles. [Figure 114-3]Figures 114Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10 at 6x and 20x magnification. Figures 114A and 114E are adrenal glands. Figures 114B and 114F are breast. Figures 114C and 114G are fallopian tubes. Figures 114D and 114H are kidneys. Figures 114I and 114M are myocardium. Figures 114J and 114N are liver. Figures 114K and 114O are lungs. Figures 114L and 114P are ureters. Figures 114Q and 114U are eyes. Figures 114R and 114V are cerebral cortex. Figures 114S and 114W are bone marrow. Figure 114T and Figure 114X are skeletal muscles. [Figure 115] Figure 115A shows a photograph, array map, and description of a breast cancer tissue array 1141 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. Figure 115A shows a photograph of the tissue microarray. Figure 115B shows a map of the array including abbreviated tissue descriptors. Figure 115C details the tissue microarray including de-identified donor data. [Figure 116] Photographs of specific tissues from breast cancer tissue array 1141 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10 are shown at 6x and 20x magnification. Figures 116A and 116D are photographs of grade 2 invasive ductal carcinoma. Figures 116B and 116E are photographs of grade 2 invasive ductal carcinoma. Figures 116C and 116F are photographs of grade 2 invasive ductal carcinoma. [Figure 117] Photographs, array maps, and descriptions of the pancreatic cancer tissue array PA805c stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10 are shown. Figure 117A shows a photograph of the tissue microarray. Figure 117B shows a map of the array including abbreviated tissue descriptors. Figure 117C details the tissue microarray including de-identified donor data. [Figure 118]Photographs of selected tissues from the pancreatic cancer tissue array PA805c stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10 are shown at 6x and 20x magnifications. Figures 118A and 118D are photographs of grade 2 papillary adenocarcinoma. Figures 118B and 118E are photographs of grade 2-3 ductal carcinoma. Figures 118C and 118F are photographs of grade 3 invasive adenocarcinoma. [Figure 119] Figure 119A shows a photograph, array map, and description of the esophageal cancer tissue array BC001113 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. Figure 119A shows a photograph of the tissue microarray. Figure 119B shows a map of the array including abbreviated tissue descriptors. Figure 119C details the tissue microarray including de-identified donor data. [Figure 120] Photographs of specific tissues from the esophageal cancer tissue array BC001113 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10 are shown at 6x and 20x magnification. Figures 120A and 120D are photographs of specimens at position A1. Figures 120B and 120E are photographs of specimens at position A7. Figures 120C and 120F are photographs of specimens at position A8. [Figure 121] Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1 are shown. Figure 121A shows a photograph of the tissue microarray. Figure 121B shows a map of the array including abbreviated tissue descriptors. Figure 121C details the tissue microarray including de-identified donor data. [Figure 122-1]Figures 122A-H show photographs of specific tissues from the FDA normal tissue array 1021 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1 at 6x and 20x magnification. Figures 122A and 122E are adrenal glands. Figures 122B and 122F are breast tissue. Figures 122C and 122G are fallopian tubes. Figures 122D and 122H are kidneys. Figures 122I and 122M are myocardium. Figures 122J and 122N are liver. Figures 122K and 122O are lungs. Figures 122L and 122P are ureters. Figures 122Q and 122U are eyes. Figures 122R and 122V are cerebral cortex. Figures 122S and 122W are bone marrow. Figures 122T and 122X are skeletal muscles. [Figure 122-2] Figures 122I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1 at 6x and 20x magnification. Figures 122A and 122E are adrenal glands. Figures 122B and 122F are breast. Figures 122C and 122G are fallopian tubes. Figures 122D and 122H are kidneys. Figures 122I and 122M are myocardium. Figures 122J and 122N are liver. Figures 122K and 122O are lungs. Figures 122L and 122P are ureters. Figures 122Q and 122U are eyes. Figures 122R and 122V are cerebral cortex. Figures 122S and 122W are bone marrow. Figures 122T and 122X are skeletal muscles. [Figure 122-3]Figures 122Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1 at 6x and 20x magnification. Figures 122A and 122E are adrenal glands. Figures 122B and 122F are breast tissue. Figures 122C and 122G are fallopian tubes. Figures 122D and 122H are kidneys. Figures 122I and 122M are myocardium. Figures 122J and 122N are liver. Figures 122K and 122O are lungs. Figures 122L and 122P are ureters. Figures 122Q and 122U are eyes. Figures 122R and 122V are cerebral cortex. Figures 122S and 122W are bone marrow. Figures 122T and 122X are skeletal muscles. [Figure 123] Photographs, array maps, and descriptions of the pancreatic cancer tissue array PA1003 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1 are shown. Figure 123A shows a photograph of the tissue microarray. Figure 123B shows a map of the array including abbreviated tissue descriptors. Figure 123C details the tissue microarray including de-identified donor data. [Figure 124] Photographs of specific tissues from the pancreatic cancer tissue array PA1003 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1 are shown at 6x and 20x magnification. Figures 124A and 124D are photographs of grade 2 adenocarcinoma. Figures 124B and 124E are photographs of grade 2 adenocarcinoma. Figures 124C and 124F are photographs of grade 2 adenocarcinoma. [Figure 125] Figure 125A shows a photograph, array map, and description of a breast cancer tissue array 1141 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1. Figure 125A shows a photograph of the tissue microarray. Figure 125B shows a map of the array including abbreviated tissue descriptors. Figure 125C details the tissue microarray including de-identified donor data. [Figure 126]Photographs of specific tissues from breast cancer tissue array 1141 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1 are shown at 6x and 20x magnification. Figures 126A and 126D are photographs of grade 2 invasive ductal carcinoma. Figures 126B and 126E are photographs of grade 2 invasive ductal carcinoma. Figures 126C and 126F are photographs of grade 2 invasive carcinoma. [Figure 127] Figure 127A shows a photograph, array map, and description of the FDA normal tissue array 1021 stained with 1 μg / mL of anti-PSMGFR antibody 5C6F3. Figure 127A shows a photograph of the tissue microarray. Figure 127B shows a map of the array including abbreviated tissue descriptors. Figure 127C details the tissue microarray including de-identified donor data. [Figure 128-1] Figures 128A-H show photographs of specific tissues from the FDA normal tissue array 1021 stained with 1 μg / mL of anti-PSMGFR antibody 5C6F3 at 6x and 20x magnification. Figures 128A and 128E are adrenal glands. Figures 128B and 128F are breast. Figures 128C and 128G are fallopian tubes. Figures 128D and 128H are kidneys. Figures 128I and 128M are myocardium. Figures 128J and 128N are liver. Figures 128K and 128O are lungs. Figures 128L and 128P are ureters. Figures 128Q and 128U are eyes. Figures 128R and 128V are cerebral cortex. Figures 128S and 128W are bone marrow. Figure 128T and Figure 128X are skeletal muscles. [Figure 128-2]Figures 128I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 1 μg / mL of anti-PSMGFR antibody 5C6F3 at 6x and 20x magnification. Figures 128A and 128E are adrenal glands. Figures 128B and 128F are breast. Figures 128C and 128G are fallopian tubes. Figures 128D and 128H are kidneys. Figures 128I and 128M are myocardium. Figures 128J and 128N are liver. Figures 128K and 128O are lungs. Figures 128L and 128P are ureters. Figures 128Q and 128U are eyes. Figures 128R and 128V are cerebral cortex. Figures 128S and 128W are bone marrow. Figure 128T and Figure 128X are skeletal muscles. [Figure 128-3] Figures 128Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 1 μg / mL of anti-PSMGFR antibody 5C6F3 at 6x and 20x magnification. Figures 128A and 128E are adrenal glands. Figures 128B and 128F are breast. Figures 128C and 128G are fallopian tubes. Figures 128D and 128H are kidneys. Figures 128I and 128M are myocardium. Figures 128J and 128N are liver. Figures 128K and 128O are lungs. Figures 128L and 128P are ureters. Figures 128Q and 128U are eyes. Figures 128R and 128V are cerebral cortex. Figures 128S and 128W are bone marrow. Figure 128T and Figure 128X are skeletal muscles. [Figure 129] Photographs, array maps, and descriptions of the pancreatic cancer tissue array PA1003 stained with 1-20 μg / mL of the anti-PSMGFR antibody 5C6F3 are shown. Figure 129A shows a photograph of the tissue microarray. Figure 129B shows a map of the array including abbreviated tissue descriptors. Figure 129C details the tissue microarray including de-identified donor data. [Figure 130]Photographs of specific tissues from the pancreatic cancer tissue array PA1003 stained with 1 μg / mL of anti-PSMGFR antibody 5C6F3 are shown at 6x and 20x magnifications. Figures 130A and 130D are photographs of grade 2 adenocarcinoma. Figures 130B and 130E are photographs of grade 2 adenocarcinoma. Figures 130C and 130F are photographs of grade 2 adenocarcinoma. [Figure 131] Figure 131A shows a photograph, array map, and description of a breast cancer tissue array 1141 stained with 1 μg / mL of anti-PSMGFR antibody 5C6F3. Figure 131A shows a photograph of the tissue microarray. Figure 131B shows a map of the array including abbreviated tissue descriptors. Figure 131C details the tissue microarray including de-identified donor data. [Figure 132] Photographs of specific tissues from breast cancer tissue array 1141 stained with 1 μg / mL of anti-PSMGFR antibody 5C6F3 are shown at 6x and 20x magnification. Figures 132A and 132D are photographs of grade 2 invasive ductal carcinoma. Figures 132B and 132E are photographs of grade 2 invasive ductal carcinoma. Figures 132C and 132F are photographs of grade 2 invasive carcinoma. [Figure 133] Figure 133A shows a photograph, array map, and description of the FDA normal tissue array 1021 stained with 10 μg / mL of anti-PSMGFR antibody 18B4. Figure 133A shows a photograph of the tissue microarray. Figure 133B shows a map of the array including abbreviated tissue descriptors. Figure 133C details the tissue microarray including de-identified donor data. [Figure 134-1]Figures 134A-H show photographs of specific tissues from the FDA normal tissue array 1021 stained with 10 μg / mL of anti-PSMGFR antibody 18B4 at 6x and 20x magnification. Figures 134A and 134E are adrenal glands. Figures 134B and 134F are breast. Figures 134C and 134G are fallopian tubes. Figures 134D and 134H are kidneys. Figures 134I and 134M are myocardium. Figures 134J and 134N are liver. Figures 134K and 134O are lungs. Figures 134L and 134P are ureters. Figures 134Q and 134U are eyes. Figures 134R and 134V are cerebral cortex. Figures 134S and 134W are bone marrow. Figures 134T and 134X are skeletal muscles. [Figure 134-2] Figures 134I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 10 μg / mL of anti-PSMGFR antibody 18B4 at 6x and 20x magnification. Figures 134A and 134E are adrenal glands. Figures 134B and 134F are breast. Figures 134C and 134G are fallopian tubes. Figures 134D and 134H are kidneys. Figures 134I and 134M are myocardium. Figures 134J and 134N are liver. Figures 134K and 134O are lungs. Figures 134L and 134P are ureters. Figures 134Q and 134U are eyes. Figures 134R and 134V are cerebral cortex. Figures 134S and 134W are bone marrow. Figures 134T and 134X are skeletal muscles. [Figure 134-3]Figures 134Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 10 μg / mL of anti-PSMGFR antibody 18B4 at 6x and 20x magnification. Figures 134A and 134E are adrenal glands. Figures 134B and 134F are breast. Figures 134C and 134G are fallopian tubes. Figures 134D and 134H are kidneys. Figures 134I and 134M are myocardium. Figures 134J and 134N are liver. Figures 134K and 134O are lungs. Figures 134L and 134P are ureters. Figures 134Q and 134U are eyes. Figures 134R and 134V are cerebral cortex. Figures 134S and 134W are bone marrow. Figures 134T and 134X are skeletal muscles. [Figure 135] Figure 135A shows a photograph, array map, and description of a breast cancer tissue array 1141 stained with 10 μg / mL of anti-PSMGFR antibody 18B4. Figure 135A shows a photograph of the tissue microarray. Figure 135B shows a map of the array including abbreviated tissue descriptors. Figure 135C details the tissue microarray including de-identified donor data. [Figure 136] Photographs of specific tissues from breast cancer tissue array 1141 stained with 10 μg / mL of anti-PSMGFR antibody 18B4 are shown at 6x and 20x magnification. Figures 136A and 136D are photographs of grade 2 invasive ductal carcinoma. Figures 136B and 136E are photographs of grade 2 invasive ductal carcinoma. Figures 136C and 136F are photographs of grade 2 invasive ductal carcinoma. [Figure 137] Figure 137 shows a photograph, array map, and description of the esophageal cancer tissue array BC001113 stained with 10 μg / mL of anti-PSMGFR antibody 18B4. Figure 137A shows a photograph of the tissue microarray. Figure 137B shows a map of the array including abbreviated tissue descriptors. Figure 137C details the tissue microarray including de-identified donor data. [Figure 138]Photographs of specific tissues from the esophageal cancer tissue array BC001113 stained with 10 μg / mL of anti-PSMGFR antibody 18B4 are shown at 6x and 20x magnification. Figures 138A and 138D are photographs of specimens at position A1. Figures 138B and 138E are photographs of specimens at position A7. Figures 138C and 138F are photographs of specimens at position A8. [Figure 139] Figure 139A shows a photograph, array map, and description of the FDA normal tissue array 1021 stained with 10 μg / mL of anti-PSMGFR antibody 18G12. Figure 139A shows a photograph of the tissue microarray. Figure 139B shows a map of the array including abbreviated tissue descriptors. Figure 139C details the tissue microarray including de-identified donor data. [Figure 140-1] Figures 140A-H show photographs of specific tissues from the FDA normal tissue array 1021 stained with 10 μg / mL of anti-PSMGFR antibody 18G12 at 6x and 20x magnification. Figures 140A and 140E are adrenal glands. Figures 140B and 140F are breast. Figures 140C and 140G are fallopian tubes. Figures 140D and 140H are kidneys. Figures 140I and 140M are myocardium. Figures 140J and 140N are liver. Figures 140K and 140O are lungs. Figures 140L and 140P are ureters. Figures 140Q and 140U are eyes. Figures 140R and 140V are cerebral cortex. Figures 140S and 140W are bone marrow. Figure 140T and Figure 140X are skeletal muscles. [Figure 140-2]Figures 140I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 10 μg / mL of anti-PSMGFR antibody 18G12 at 6x and 20x magnification. Figures 140A and 140E are adrenal glands. Figures 140B and 140F are breast. Figures 140C and 140G are fallopian tubes. Figures 140D and 140H are kidneys. Figures 140I and 140M are myocardium. Figures 140J and 140N are liver. Figures 140K and 140O are lungs. Figures 140L and 140P are ureters. Figures 140Q and 140U are eyes. Figures 140R and 140V are cerebral cortex. Figures 140S and 140W are bone marrow. Figure 140T and Figure 140X are skeletal muscles. [Figure 140-3] Figures 140Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 10 μg / mL of anti-PSMGFR antibody 18G12 at 6x and 20x magnification. Figures 140A and 140E are adrenal glands. Figures 140B and 140F are breast. Figures 140C and 140G are fallopian tubes. Figures 140D and 140H are kidneys. Figures 140I and 140M are myocardium. Figures 140J and 140N are liver. Figures 140K and 140O are lungs. Figures 140L and 140P are ureters. Figures 140Q and 140U are eyes. Figures 140R and 140V are cerebral cortex. Figures 140S and 140W are bone marrow. Figure 140T and Figure 140X are skeletal muscles. [Figure 141] Figure 141A shows a photograph, array map, and description of a breast cancer tissue array 1141 stained with 15 μg / mL of anti-PSMGFR antibody 18G12. Figure 141A shows a photograph of the tissue microarray. Figure 141B shows a map of the array including abbreviated tissue descriptors. Figure 141C details the tissue microarray including de-identified donor data. [Figure 142]Photographs of selected tissues from breast cancer tissue array 1141 stained with 15 μg / mL of anti-PSMGFR antibody 18G12 are shown at 6x and 20x magnification. Figures 142A and 142D are photographs of grade 2 invasive ductal carcinoma. Figures 142B and 142E are photographs of grade 2 invasive ductal carcinoma. Figures 142C and 142F are photographs of grade 2 invasive ductal carcinoma. [Figure 143] Photographs, array maps, and descriptions of the pancreatic cancer tissue array PA1003 stained with 15 μg / mL of anti-PSMGFR antibody 18G12 are shown. Figure 143A shows a photograph of the tissue microarray. Figure 143B shows a map of the array including abbreviated tissue descriptors. Figure 143C details the tissue microarray including de-identified donor data. [Figure 144] Photographs of specific tissues from the pancreatic cancer tissue array PA1003 stained with 15 μg / mL of anti-PSMGFR antibody 18G12 are shown at 6x and 20x magnifications. Figures 144A and 144D are photographs of grade 2 adenocarcinoma. Figures 144B and 144E are photographs of grade 2 adenocarcinoma. Figures 144C and 144F are photographs of grade 2-3 adenocarcinoma with lymph node metastasis. [Figure 145] Figure 145 shows a photograph, array map, and description of the esophageal cancer tissue array BC001113 stained with 30 μg / mL of anti-PSMGFR antibody 18G12. Figure 145A shows a photograph of the tissue microarray. Figure 145B shows a map of the array including abbreviated tissue descriptors. Figure 145C details the tissue microarray including de-identified donor data. [Figure 146] Photographs of specific tissues from esophageal cancer tissue array BC001113 stained with 30 μg / mL of anti-PSMGFR antibody 18G12 are shown at 6x and 20x magnification. Figures 146A and 146D are photographs of specimens at position A1. Figures 146B and 146E are photographs of specimens at position A7. Figures 146C and 146F are photographs of specimens at position A8. [Figure 147]Figure 147A shows a photograph, array map, and description of FDA normal tissue array 1021 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6. Figure 147A shows a photograph of the tissue microarray. Figure 147B shows a map of the array including abbreviated tissue descriptors. Figure 147C details the tissue microarray including de-identified donor data. [Figure 148-1] Figures 148A-H show photographs of selected tissues from the FDA normal tissue array 1021 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6 at 6x and 20x magnification. Figures 148A and 148E are adrenal glands. Figures 148B and 148F are breast tissue. Figures 148C and 148G are fallopian tubes. Figures 148D and 148H are kidneys. Figures 148I and 148M are myocardium. Figures 148J and 148N are liver. Figures 148K and 148O are lungs. Figures 148L and 148P are ureters. Figures 148Q and 148U are eyes. Figures 148R and 148V are cerebral cortex. Figures 148S and 148W are bone marrow. Figure 148T and Figure 148X are skeletal muscles. [Figure 148-2] Figures 148I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6 at 6x and 20x magnification. Figures 148A and 148E are adrenal glands. Figures 148B and 148F are breast. Figures 148C and 148G are fallopian tubes. Figures 148D and 148H are kidneys. Figures 148I and 148M are myocardium. Figures 148J and 148N are liver. Figures 148K and 148O are lungs. Figures 148L and 148P are ureters. Figures 148Q and 148U are eyes. Figures 148R and 148V are cerebral cortex. Figures 148S and 148W are bone marrow. Figure 148T and Figure 148X are skeletal muscles. [Figure 148-3]Figures 148Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6 at 6x and 20x magnification. Figures 148A and 148E are adrenal glands. Figures 148B and 148F are breast tissue. Figures 148C and 148G are fallopian tubes. Figures 148D and 148H are kidneys. Figures 148I and 148M are myocardium. Figures 148J and 148N are livers. Figures 148K and 148O are lungs. Figures 148L and 148P are ureters. Figures 148Q and 148U are eyes. Figures 148R and 148V are cerebral cortex. Figures 148S and 148W are bone marrow. Figure 148T and Figure 148X are skeletal muscles. [Figure 149] Figure 149A shows a photograph, array map, and description of a breast cancer tissue array 1141 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6. Figure 149A shows a photograph of the tissue microarray. Figure 149B shows a map of the array including abbreviated tissue descriptors. Figure 149C details the tissue microarray including de-identified donor data. [Figure 150] Photographs of specific tissues from breast cancer tissue array 1141 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6 are shown at 6x and 20x magnification. Figures 150A and 150D are photographs of grade 2 invasive ductal carcinoma. Figures 150B and 150E are photographs of grade 2 invasive ductal carcinoma. Figures 150C and 150F are photographs of grade 2 invasive ductal carcinoma. [Figure 151] Photographs, array maps, and descriptions of the pancreatic cancer tissue array PA1003 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6 are shown. Figure 151A shows a photograph of the tissue microarray. Figure 151B shows a map of the array including abbreviated tissue descriptors. Figure 151C details the tissue microarray including de-identified donor data. [Figure 152]Photographs of specific tissues from the pancreatic cancer tissue array PA1003 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6 are shown at 6x and 20x magnification. Figures 152A and 152D are photographs of grade 2 adenocarcinoma. Figures 152B and 152E are photographs of grade 1 adenocarcinoma. Figures 152C and 152F are photographs of grade 1 adenocarcinoma. [Figure 153] Figure 153A shows a photograph, array map, and description of the FDA normal tissue array 1021 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9. Figure 153A shows a photograph of the tissue microarray. Figure 153B shows a map of the array including abbreviated tissue descriptors. Figure 153C details the tissue microarray including de-identified donor data. [Figure 154-1] Figures 154A-H show photographs of selected tissues from the FDA normal tissue array 1021 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9 at 6x and 20x magnification. Figures 154A and 154E are adrenal glands. Figures 154B and 154F are breast. Figures 154C and 154G are fallopian tubes. Figures 154D and 154H are kidneys. Figures 154I and 154M are myocardium. Figures 154J and 154N are liver. Figures 154K and 154O are lungs. Figures 154L and 154P are ureters. Figures 154Q and 154U are eyes. Figures 154R and 154V are cerebral cortex. Figures 154S and 154W are bone marrow. Figure 154T and Figure 154X are skeletal muscles. [Figure 154-2]Figures 154I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9 at 6x and 20x magnification. Figures 154A and 154E are adrenal glands. Figures 154B and 154F are breast. Figures 154C and 154G are fallopian tubes. Figures 154D and 154H are kidneys. Figures 154I and 154M are myocardium. Figures 154J and 154N are liver. Figures 154K and 154O are lungs. Figures 154L and 154P are ureters. Figures 154Q and 154U are eyes. Figures 154R and 154V are cerebral cortex. Figures 154S and 154W are bone marrow. Figure 154T and Figure 154X are skeletal muscles. [Figure 154-3] Figures 154Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9 at 6x and 20x magnification. Figures 154A and 154E are adrenal glands. Figures 154B and 154F are breast. Figures 154C and 154G are fallopian tubes. Figures 154D and 154H are kidneys. Figures 154I and 154M are myocardium. Figures 154J and 154N are liver. Figures 154K and 154O are lungs. Figures 154L and 154P are ureters. Figures 154Q and 154U are eyes. Figures 154R and 154V are cerebral cortex. Figures 154S and 154W are bone marrow. Figure 154T and Figure 154X are skeletal muscles. [Figure 155] Figure 155A shows a photograph, array map, and description of a breast cancer tissue array 1141 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9. Figure 155A shows a photograph of the tissue microarray. Figure 155B shows a map of the array including abbreviated tissue descriptors. Figure 155C details the tissue microarray including de-identified donor data. [Figure 156]Photographs of specific tissues from breast cancer tissue array 1141 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9 are shown at 6x and 20x magnification. Figures 156A and 156D are photographs of grade 2 invasive ductal carcinoma. Figures 156B and 156E are photographs of grade 2 invasive ductal carcinoma. Figures 156C and 156F are photographs of grade 2 invasive ductal carcinoma. [Figure 157] Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 7.5 μg / mL of N+20 / C-27 antibody 1E4 are shown. Figure 157A shows a photograph of the tissue microarray. Figure 157B shows a map of the array including abbreviated tissue descriptors. Figure 157C details the tissue microarray including de-identified donor data. [Figure 158-1] Figures 158A-H show photographs of selected tissues from the FDA normal tissue array 1021 stained with 7.5 μg / mL of N+20 / C-27 antibody 1E4 at 6x and 20x magnification. Figures 158A and 158E are adrenal glands. Figures 158B and 158F are breast. Figures 158C and 158G are fallopian tubes. Figures 158D and 158H are kidneys. Figures 158I and 158M are myocardium. Figures 158J and 158N are liver. Figures 158K and 158O are lungs. Figures 158L and 158P are ureters. Figures 158Q and 158U are eyes. Figures 158R and 158V are cerebral cortex. Figures 158S and 158W are bone marrow. Figure 158T and Figure 158X are skeletal muscles. [Figure 158-2]Figures 158I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 7.5 μg / mL of N+20 / C-27 antibody 1E4 at 6x and 20x magnification. Figures 158A and 158E are adrenal glands. Figures 158B and 158F are breast. Figures 158C and 158G are fallopian tubes. Figures 158D and 158H are kidneys. Figures 158I and 158M are myocardium. Figures 158J and 158N are liver. Figures 158K and 158O are lungs. Figures 158L and 158P are ureters. Figures 158Q and 158U are eyes. Figures 158R and 158V are cerebral cortex. Figures 158S and 158W are bone marrow. Figure 158T and Figure 158X are skeletal muscles. [Figure 158-3] Figures 158Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 7.5 μg / mL of N+20 / C-27 antibody 1E4 at 6x and 20x magnification. Figures 158A and 158E are adrenal glands. Figures 158B and 158F are breast. Figures 158C and 158G are fallopian tubes. Figures 158D and 158H are kidneys. Figures 158I and 158M are myocardium. Figures 158J and 158N are liver. Figures 158K and 158O are lungs. Figures 158L and 158P are ureters. Figures 158Q and 158U are eyes. Figures 158R and 158V are cerebral cortex. Figures 158S and 158W are bone marrow. Figure 158T and Figure 158X are skeletal muscles. [Figure 159] Photographs, array maps, and descriptions of breast cancer tissue array BR1007 stained with 10.0 μg / mL of N+20 / C-27 antibody 1E4 are shown. Figure 159A shows a photograph of the tissue microarray. Figure 159B shows a map of the array including abbreviated tissue descriptors. Figure 159C details the tissue microarray including de-identified donor data. [Figure 160]Photographs of selected tissues from breast cancer tissue array BR1007 stained with 10.0 μg / mL of N+20 / C-27 antibody 1E4 are shown at 6x and 20x magnification. Figures 160A and 160D are photographs of grade 2 invasive ductal carcinoma with positive lymph nodes. Figures 160B and 160E are photographs of grade 2 invasive ductal carcinoma. Figures 160C and 160F are photographs of grade 2 invasive ductal carcinoma. [Figure 161] Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1 are shown. Figure 161A shows a photograph of the tissue microarray. Figure 161B shows a map of the array including abbreviated tissue descriptors. Figure 161C details the tissue microarray including de-identified donor data. [Figure 162-1] Figures 162A-H show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1 at 6x and 20x magnification. Figures 162A and 162E are adrenal glands. Figures 162B and 162F are breast. Figures 162C and 162G are fallopian tubes. Figures 162D and 162H are kidneys. Figures 162I and 162M are myocardium. Figures 162J and 162N are liver. Figures 162K and 162O are lungs. Figures 162L and 162P are ureters. Figures 162Q and 162U are eyes. Figures 162R and 162V are cerebral cortex. Figures 162S and 162W are bone marrow. Figure 162T and Figure 162X are skeletal muscles. [Figure 162-2]Figures 162I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1 at 6x and 20x magnification. Figures 162A and 162E are adrenal glands. Figures 162B and 162F are breast. Figures 162C and 162G are fallopian tubes. Figures 162D and 162H are kidneys. Figures 162I and 162M are myocardium. Figures 162J and 162N are liver. Figures 162K and 162O are lungs. Figures 162L and 162P are ureters. Figures 162Q and 162U are eyes. Figures 162R and 162V are cerebral cortex. Figures 162S and 162W are bone marrow. Figure 162T and Figure 162X are skeletal muscles. [Figure 162-3] Figures 162Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1 at 6x and 20x magnification. Figures 162A and 162E are adrenal glands. Figures 162B and 162F are breast. Figures 162C and 162G are fallopian tubes. Figures 162D and 162H are kidneys. Figures 162I and 162M are myocardium. Figures 162J and 162N are liver. Figures 162K and 162O are lungs. Figures 162L and 162P are ureters. Figures 162Q and 162U are eyes. Figures 162R and 162V are cerebral cortex. Figures 162S and 162W are bone marrow. Figure 162T and Figure 162X are skeletal muscles. [Figure 163] Photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1 are shown. Figure 163A shows a photograph of the tissue microarray. Figure 163B shows a map of the array including abbreviated tissue descriptors. Figure 163C details the tissue microarray including de-identified donor data. [Fig. 164]Photographs of specific tissues from breast cancer tissue array 1141 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1 are shown at 6x and 20x magnification. Figures 164A and 164D are photographs of grade 2 invasive ductal carcinoma. Figures 164B and 164E are photographs of grade 2 invasive ductal carcinoma. Figures 164C and 164F are photographs of grade 2 invasive ductal carcinoma. [Figure 165] Photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1 are shown. Figure 165A shows a photograph of the tissue microarray. Figure 165B shows a map of the array including abbreviated tissue descriptors. Figure 165C details the tissue microarray including de-identified donor data. [Figure 166] Photographs of selected tissues from the pancreatic cancer tissue array PA1003 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1 are shown at 6x and 20x magnification. Figures 166A and 166D are photographs of grade 2 adenocarcinoma. Figures 166B and 166E are photographs of grade 2 adenocarcinoma. Figures 166C and 166F are photographs of grade 3 adenocarcinoma. [Figure 167] Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1 are shown. Figure 167A shows a photograph of the tissue microarray. Figure 167B shows a map of the array including abbreviated tissue descriptors. Figure 167C details the tissue microarray including de-identified donor data. [Figure 168-1]Figures 168A-H show photographs of selected tissues from the FDA normal tissue array 1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1 at 6x and 20x magnification. Figures 168A and 168E are adrenal glands. Figures 168B and 168F are breast. Figures 168C and 168G are fallopian tubes. Figures 168D and 168H are kidneys. Figures 168I and 168M are myocardium. Figures 168J and 168N are liver. Figures 168K and 168O are lungs. Figures 168L and 168P are ureters. Figures 168Q and 168U are eyes. Figures 168R and 168V are cerebral cortex. Figures 168S and 168W are bone marrow. Figure 168T and Figure 168X are skeletal muscles. [Figure 168-2] Figures 168I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1 at 6x and 20x magnification. Figures 168A and 168E are adrenal glands. Figures 168B and 168F are breast. Figures 168C and 168G are fallopian tubes. Figures 168D and 168H are kidneys. Figures 168I and 168M are myocardium. Figures 168J and 168N are liver. Figures 168K and 168O are lungs. Figures 168L and 168P are ureters. Figures 168Q and 168U are eyes. Figures 168R and 168V are cerebral cortex. Figures 168S and 168W are bone marrow. Figure 168T and Figure 168X are skeletal muscles. [Figure 168-3]Figures 168Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1 at 6x and 20x magnification. Figures 168A and 168E are adrenal glands. Figures 168B and 168F are breast. Figures 168C and 168G are fallopian tubes. Figures 168D and 168H are kidneys. Figures 168I and 168M are myocardium. Figures 168J and 168N are liver. Figures 168K and 168O are lungs. Figures 168L and 168P are ureters. Figures 168Q and 168U are eyes. Figures 168R and 168V are cerebral cortex. Figures 168S and 168W are bone marrow. Figure 168T and Figure 168X are skeletal muscles. [Figure 169] Photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1 are shown. Figure 169A shows a photograph of the tissue microarray. Figure 169B shows a map of the array including abbreviated tissue descriptors. Figure 169C details the tissue microarray including de-identified donor data. [Figure 170] Photographs of specific tissues from breast cancer tissue array 1141 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1 are shown at 6x and 20x magnification. Figures 170A and 170D are photographs of grade 2 invasive ductal carcinoma. Figures 170B and 170E are photographs of grade 2 invasive ductal carcinoma. Figures 170C and 170F are photographs of grade 2 invasive ductal carcinoma. [Figure 171] Photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1 are shown. Figure 171A shows a photograph of the tissue microarray. Figure 171B shows a map of the array including abbreviated tissue descriptors. Figure 171C details the tissue microarray including de-identified donor data. [Fig. 172]Photographs of selected tissues from pancreatic cancer tissue array PA1003 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1 are shown at 6x and 20x magnification. Figures 172A and 172D are photographs of grade 1 adenocarcinoma. Figures 172B and 172E are photographs of grade 2 adenocarcinoma. Figures 172C and 172F are photographs of grade 3 adenocarcinoma. [Figure 173] Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 0.25 μg / mL of N+20 / C-27 antibody 32C1 are shown. Figure 173A shows a photograph of the tissue microarray. Figure 173B shows a map of the array including abbreviated tissue descriptors. Figure 173C details the tissue microarray including de-identified donor data. [Figure 174-1] Figures 174A-H show photographs of selected tissues from the FDA normal tissue array 1021 stained with 0.25 μg / mL of N+20 / C-27 antibody 32C1 at 6x and 20x magnification. Figures 174A and 174E are adrenal glands. Figures 174B and 174F are breast. Figures 174C and 174G are fallopian tubes. Figures 174D and 174H are kidneys. Figures 174I and 174M are myocardium. Figures 174J and 174N are liver. Figures 174K and 174O are lungs. Figures 174L and 174P are ureters. Figures 174Q and 174U are eyes. Figures 174R and 174V are cerebral cortex. Figures 174S and 174W are bone marrow. Figure 174T and Figure 174X are skeletal muscles. [Figure 174-2]Figures 174I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.25 μg / mL of N+20 / C-27 antibody 32C1 at 6x and 20x magnification. Figures 174A and 174E are adrenal glands. Figures 174B and 174F are breast. Figures 174C and 174G are fallopian tubes. Figures 174D and 174H are kidneys. Figures 174I and 174M are myocardium. Figures 174J and 174N are liver. Figures 174K and 174O are lungs. Figures 174L and 174P are ureters. Figures 174Q and 174U are eyes. Figures 174R and 174V are cerebral cortex. Figures 174S and 174W are bone marrow. Figure 174T and Figure 174X are skeletal muscles. [Figure 174-3] Figures 174Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 0.25 μg / mL of N+20 / C-27 antibody 32C1 at 6x and 20x magnification. Figures 174A and 174E are adrenal glands. Figures 174B and 174F are breast. Figures 174C and 174G are fallopian tubes. Figures 174D and 174H are kidneys. Figures 174I and 174M are myocardium. Figures 174J and 174N are liver. Figures 174K and 174O are lungs. Figures 174L and 174P are ureters. Figures 174Q and 174U are eyes. Figures 174R and 174V are cerebral cortex. Figures 174S and 174W are bone marrow. Figure 174T and Figure 174X are skeletal muscles. [Figure 175] Figure 175A shows a photograph, array map, and description of a breast cancer tissue array 1141 stained with 5.0 μg / mL of N+20 / C-27 antibody 32C1. Figure 175A shows a photograph of the tissue microarray. Figure 175B shows a map of the array including abbreviated tissue descriptors. Figure 175C details the tissue microarray including de-identified donor data. [Figure 176]Photographs of selected tissues from breast cancer tissue array 1141 stained with 5.0 μg / mL of N+20 / C-27 antibody 32C1 are shown at 6x and 20x magnification. Figures 176A and 176D are photographs of grade 2 invasive ductal carcinoma. Figures 176B and 176E are photographs of grade 2 invasive ductal carcinoma. Figures 176C and 176F are photographs of grade 2 invasive ductal carcinoma. [Figure 177] Figure 177A shows a photograph, array map, and description of the esophageal cancer tissue array ES1001 stained with 1.0 μg / mL of N+20 / C-27 antibody 32C1. Figure 177A shows a photograph of the tissue microarray. Figure 177B shows a map of the array including abbreviated tissue descriptors. Figure 177C details the tissue microarray including de-identified donor data. [Figure 178] Photographs of specific tissues from the esophageal cancer tissue array BC001113 stained with 1.0 μg / mL of N+20 / C-27 antibody 32C1 are shown at 6x and 20x magnification. Figures 178A and 178D are photographs of squamous cell carcinoma. Figures 178B and 178E are photographs of adenocarcinoma. Figures 178C and 178F are photographs of squamous cell carcinoma. [Figure 179] Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11 are shown. Figure 179A shows a photograph of the tissue microarray. Figure 179B shows a map of the array including abbreviated tissue descriptors. Figure 179C details the tissue microarray including de-identified donor data. [Figure 180-1]Figures 180A-H show photographs of specific tissues from the FDA normal tissue array 1021 stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11 at 6x and 20x magnification. Figures 180A and 180E are adrenal glands. Figures 180B and 180F are breast. Figures 180C and 180G are fallopian tubes. Figures 180D and 180H are kidneys. Figures 180I and 180M are myocardium. Figures 180J and 180N are liver. Figures 180K and 180O are lungs. Figures 180L and 180P are ureters. Figures 180Q and 180U are eyes. Figures 180R and 180V are cerebral cortex. Figures 180S and 180W are bone marrow. Figures 180T and 180X are skeletal muscles. [Figure 180-2] Figures 180I-P show photographs of specific tissues from the FDA normal tissue array 1021 stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11 at 6x and 20x magnification. Figures 180A and 180E are adrenal glands. Figures 180B and 180F are breast. Figures 180C and 180G are fallopian tubes. Figures 180D and 180H are kidneys. Figures 180I and 180M are myocardium. Figures 180J and 180N are liver. Figures 180K and 180O are lungs. Figures 180L and 180P are ureters. Figures 180Q and 180U are eyes. Figures 180R and 180V are cerebral cortex. Figures 180S and 180W are bone marrow. Figures 180T and 180X are skeletal muscles. [Figure 180-3]Figures 180Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11 at 6x and 20x magnification. Figures 180A and 180E are adrenal glands. Figures 180B and 180F are breast. Figures 180C and 180G are fallopian tubes. Figures 180D and 180H are kidneys. Figures 180I and 180M are myocardium. Figures 180J and 180N are liver. Figures 180K and 180O are lungs. Figures 180L and 180P are ureters. Figures 180Q and 180U are eyes. Figures 180R and 180V are cerebral cortex. Figures 180S and 180W are bone marrow. Figures 180T and 180X are skeletal muscles. [Figure 181] Photographs, array maps, and descriptions of breast cancer tissue array BR1007 stained with 10.0 μg / mL of N+20 / C-27 antibody 45C11 are shown. Figure 181A shows a photograph of the tissue microarray. Figure 181B shows a map of the array including abbreviated tissue descriptors. Figure 181C details the tissue microarray including de-identified donor data. [Figure 182] Photographs of selected tissues from breast cancer tissue array BR1007 stained with 10.0 μg / mL of N+20 / C-27 antibody 45C11 are shown at 6x and 20x magnification. Figures 182A and 182D are photographs of grade 2 invasive ductal carcinoma with positive lymph nodes. Figures 182B and 182E are photographs of grade 2 invasive ductal carcinoma. Figures 182C and 182F are photographs of grade 2 invasive ductal carcinoma. [Figure 183] Photographs, array maps, and descriptions of pancreatic cancer tissue array PA805c stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11 are shown. Figure 183A shows a photograph of the tissue microarray. Figure 183B shows a map of the array including abbreviated tissue descriptors. Figure 183C details the tissue microarray including de-identified donor data. [Figure 184]Photographs of selected tissues from the pancreatic cancer tissue array PA805c stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11 are shown at 6x and 20x magnification. Figures 184A and 184D are photographs of grade 2 papillary adenocarcinoma. Figures 184B and 184E are photographs of grade 2-3 ductal carcinoma. Figures 184C and 184F are photographs of grade 3 invasive adenocarcinoma. [Figure 185] Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5 are shown. Figure 185A shows a photograph of the tissue microarray. Figure 185B shows a map of the array including abbreviated tissue descriptors. Figure 185C details the tissue microarray including de-identified donor data. [Figure 186-1] Figures 186A-H show photographs of selected tissues from the FDA normal tissue array 1021 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5 at 6x and 20x magnification. Figures 186A and 186E are adrenal glands. Figures 186B and 186F are breast. Figures 186C and 186G are fallopian tubes. Figures 186D and 186H are kidneys. Figures 186I and 186M are myocardium. Figures 186J and 186N are liver. Figures 186K and 186O are lungs. Figures 186L and 186P are ureters. Figures 186Q and 186U are eyes. Figures 186R and 186V are cerebral cortex. Figures 186S and 186W are bone marrow. Figure 186T and Figure 186X are skeletal muscles. [Figure 186-2]Figures 186I-P show photographs of selected tissues from the FDA normal tissue array 1021 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5 at 6x and 20x magnification. Figures 186A and 186E are adrenal glands. Figures 186B and 186F are breast. Figures 186C and 186G are fallopian tubes. Figures 186D and 186H are kidneys. Figures 186I and 186M are myocardium. Figures 186J and 186N are liver. Figures 186K and 186O are lungs. Figures 186L and 186P are ureters. Figures 186Q and 186U are eyes. Figures 186R and 186V are cerebral cortex. Figures 186S and 186W are bone marrow. Figure 186T and Figure 186X are skeletal muscles. [Figure 186-3] Figures 186Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5 at 6x and 20x magnification. Figures 186A and 186E are adrenal glands. Figures 186B and 186F are breast. Figures 186C and 186G are fallopian tubes. Figures 186D and 186H are kidneys. Figures 186I and 186M are myocardium. Figures 186J and 186N are liver. Figures 186K and 186O are lungs. Figures 186L and 186P are ureters. Figures 186Q and 186U are eyes. Figures 186R and 186V are cerebral cortex. Figures 186S and 186W are bone marrow. Figure 186T and Figure 186X are skeletal muscles. [Figure 187] Photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5 are shown. Figure 187A shows a photograph of the tissue microarray. Figure 187B shows a map of the array including abbreviated tissue descriptors. Figure 187C details the tissue microarray including de-identified donor data. [Figure 188]Photographs of specific tissues from the pancreatic cancer tissue array PA1003 stained with 10.0 μg / mL of the N+9 / C-9 antibody 3C5 are shown at 6x and 20x magnifications. Figures 188A and 188D are photographs of grade 2 adenocarcinoma. Figures 188B and 188E are photographs of grade 2 adenocarcinoma. Figures 188C and 188F are photographs of grade 2-3 adenocarcinoma with lymph node metastasis. [Figure 189] Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9 are shown. Figure 189A shows a photograph of the tissue microarray. Figure 189B shows a map of the array including abbreviated tissue descriptors. Figure 189C details the tissue microarray including de-identified donor data. [Figure 190-1] Figures 190A-H show photographs of selected tissues from the FDA normal tissue array 1021 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9 at 6x and 20x magnification. Figures 190A and 190E are adrenal glands. Figures 190B and 190F are breast. Figures 190C and 190G are fallopian tubes. Figures 190D and 190H are kidneys. Figures 190I and 190M are myocardium. Figures 190J and 190N are liver. Figures 190K and 190O are lungs. Figures 190L and 190P are ureters. Figures 190Q and 190U are eyes. Figures 190R and 190V are cerebral cortex. Figures 190S and 190W are bone marrow. Figures 190T and 190X are skeletal muscles. [Figure 190-2]Figures 190I-P show photographs of selected tissues from the FDA normal tissue array 1021 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9 at 6x and 20x magnification. Figures 190A and 190E are adrenal glands. Figures 190B and 190F are breast. Figures 190C and 190G are fallopian tubes. Figures 190D and 190H are kidneys. Figures 190I and 190M are myocardium. Figures 190J and 190N are liver. Figures 190K and 190O are lungs. Figures 190L and 190P are ureters. Figures 190Q and 190U are eyes. Figures 190R and 190V are cerebral cortex. Figures 190S and 190W are bone marrow. Figures 190T and 190X are skeletal muscles. [Figure 190-3] Figures 190Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9 at 6x and 20x magnification. Figures 190A and 190E are adrenal glands. Figures 190B and 190F are breast. Figures 190C and 190G are fallopian tubes. Figures 190D and 190H are kidneys. Figures 190I and 190M are myocardium. Figures 190J and 190N are liver. Figures 190K and 190O are lungs. Figures 190L and 190P are ureters. Figures 190Q and 190U are eyes. Figures 190R and 190V are cerebral cortex. Figures 190S and 190W are bone marrow. Figures 190T and 190X are skeletal muscles. [Figure 191] Photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9 are shown. Figure 191A shows a photograph of the tissue microarray. Figure 191B shows a map of the array including abbreviated tissue descriptors. Figure 191C details the tissue microarray including de-identified donor data. [Figure 192]Photographs of specific tissues from the pancreatic cancer tissue array PA1003 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9 are shown at 6x and 20x magnification. Figures 192A and 192D are photographs of grade 2 adenocarcinoma. Figures 192B and 192E are photographs of grade 2 adenocarcinoma. Figures 192C and 192F are photographs of grade 2 adenocarcinoma. [Figure 193] Figure 193A shows a photograph, array map, and description of FDA normal tissue array 1021 stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6. Figure 193A shows a photograph of the tissue microarray. Figure 193B shows a map of the array including abbreviated tissue descriptors. Figure 193C details the tissue microarray including de-identified donor data. [Figure 194-1] Figures 194A-H show photographs of selected tissues from the FDA normal tissue array 1021 stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6 at 6x and 20x magnification. Figures 194A and 194E are adrenal glands. Figures 194B and 194F are breast. Figures 194C and 194G are fallopian tubes. Figures 194D and 194H are kidneys. Figures 194I and 194M are myocardium. Figures 194J and 194N are liver. Figures 194K and 194O are lungs. Figures 194L and 194P are ureters. Figures 194Q and 194U are eyes. Figures 194R and 194V are cerebral cortex. Figures 194S and 194W are bone marrow. Figure 194T and Figure 194X are skeletal muscles. [Figure 194-2]Figures 194I-P show photographs of selected tissues from the FDA normal tissue array 1021 stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6 at 6x and 20x magnification. Figures 194A and 194E are adrenal glands. Figures 194B and 194F are breast. Figures 194C and 194G are fallopian tubes. Figures 194D and 194H are kidneys. Figures 194I and 194M are myocardium. Figures 194J and 194N are liver. Figures 194K and 194O are lungs. Figures 194L and 194P are ureters. Figures 194Q and 194U are eyes. Figures 194R and 194V are cerebral cortex. Figures 194S and 194W are bone marrow. Figure 194T and Figure 194X are skeletal muscles. [Figure 194-3] Figures 194Q-X show photographs of specific tissues from the FDA normal tissue array 1021 stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6 at 6x and 20x magnification. Figures 194A and 194E are adrenal glands. Figures 194B and 194F are breast. Figures 194C and 194G are fallopian tubes. Figures 194D and 194H are kidneys. Figures 194I and 194M are myocardium. Figures 194J and 194N are liver. Figures 194K and 194O are lungs. Figures 194L and 194P are ureters. Figures 194Q and 194U are eyes. Figures 194R and 194V are cerebral cortex. Figures 194S and 194W are bone marrow. Figure 194T and Figure 194X are skeletal muscles. [Figure 195] Photographs, array maps, and descriptions of pancreatic cancer tissue array PA805c stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6 are shown. Figure 195A shows a photograph of the tissue microarray. Figure 195B shows a map of the array including abbreviated tissue descriptors. Figure 195C details the tissue microarray including de-identified donor data. [Figure 196]Photographs of specific tissues from the pancreatic cancer tissue array PA805c stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6 are shown at 6x and 20x magnifications. Figures 196A and 196D are photographs of grade 2 papillary adenocarcinoma. Figures 196B and 196E are photographs of grade 2-3 ductal carcinoma with lymph node metastasis. Figures 196C and 196F are photographs of grade 3 invasive adenocarcinoma. [Figure 197] Photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5 are shown. Figure 197A shows a photograph of the tissue microarray. Figure 197B shows a map of the array including abbreviated tissue descriptors. Figure 197C details the tissue microarray including de-identified donor data. [Figure 198-1] Figures 198A-H show photographs of selected tissues from the FDA normal tissue array 1021 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5 at 6x and 20x magnification. Figures 198A and 198E are adrenal glands. Figures 198B and 198F are breast. Figures 198C and 198G are fallopian tubes. Figures 198D and 198H are kidneys. Figures 198I and 198M are myocardium. Figures 198J and 198N are liver. Figures 198K and 198O are lungs. Figures 198L and 198P are ureters. Figures 198Q and 198U are eyes. Figures 198R and 198V are cerebral cortex. Figures 198S and 198W are bone marrow. Figure 198T and Figure 198X are skeletal muscles. [Figure 198-2]Figures 198I-P show photographs of selected tissues from the FDA normal tissue array 1021 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5 at 6x and 20x magnification. Figures 198A and 198E are adrenal glands. Figures 198B and 198F are breast. Figures 198C and 198G are fallopian tubes. Figures 198D and 198H are kidneys. Figures 198I and 198M are myocardium. Figures 198J and 198N are liver. Figures 198K and 198O are lungs. Figures 198L and 198P are ureters. Figures 198Q and 198U are eyes. Figures 198R and 198V are cerebral cortex. Figures 198S and 198W are bone marrow. Figure 198T and Figure 198X are skeletal muscles. [Figure 198-3] Figures 198Q-X show photographs of selected tissues from the FDA normal tissue array 1021 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5 at 6x and 20x magnification. Figures 198A and 198E are adrenal glands. Figures 198B and 198F are breast. Figures 198C and 198G are fallopian tubes. Figures 198D and 198H are kidneys. Figures 198I and 198M are myocardium. Figures 198J and 198N are liver. Figures 198K and 198O are lungs. Figures 198L and 198P are ureters. Figures 198Q and 198U are eyes. Figures 198R and 198V are cerebral cortex. Figures 198S and 198W are bone marrow. Figure 198T and Figure 198X are skeletal muscles. [Figure 199] Photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5 are shown. Figure 199A shows a photograph of the tissue microarray. Figure 199B shows a map of the array including abbreviated tissue descriptors. Figure 199C details the tissue microarray including de-identified donor data. [Figure 200]Photographs of specific tissues from the pancreatic cancer tissue array PA1003 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5 are shown at 6x and 20x magnification. Figures 200A and 200D are photographs of grade 2 adenocarcinoma. Figures 200B and 200E are photographs of grade 2 adenocarcinoma. Figures 200C and 200F are photographs of grade 2 adenocarcinoma. [Figure 201] 201A and 201B show graphs of an ELISA assay to determine binding of another set of antibodies generated by immunizing animals with PSMGFR peptide. Figure 201A shows binding to the PSMGFR peptide. Figure 201B shows binding to the N-10 peptide. Figure 201C shows binding to the C-10 peptide. As can be seen, none of the antibodies bound to the C-10 peptide. F3, B12, B2, B7, B9, 8C7F3, and H11 all bound to the PSMGFR peptide and the N-10 peptide. [Figure 202] Photographs of the pancreatic cancer tissue array PA1003 stained with monoclonal antibody 1E4, monoclonal antibody 18B4, or polyclonal anti-PSMGFR antibody SDIX are shown. 18B4 binds to the GTINVHDVET epitope in the most N-terminal part of the PSMGFR peptide, while the 1E4 antibody binds to the QFNQYKTEA epitope immediately adjacent to and C-terminal to the 18B4 epitope. [Figure 203] 203A and 203B show magnified images of tissue specimens at position A2 of the pancreatic cancer array PA1003. 203A and 203B show specimens stained with antibody 1E4. 203C and 203D show specimens stained with antibody 18B4. 203E and 203F show specimens stained with polyclonal antibody SDIX. [Figure 204] 204A and 204B show magnified images of tissue specimens at position D4 of the pancreatic cancer array PA1003. Figures 204A and 204B show specimens stained with antibody 18B4. Figures 204C and 204D show specimens stained with polyclonal antibody SDIX. [Figure 205]205A and 205B show magnified images of tissue specimens at position E1 of the pancreatic cancer array PA1003. Figures 205A and 205B show specimens stained with antibody 18B4. Figures 205C and 205D show specimens stained with polyclonal antibody SDIX. [Figure 206] 206A and 206B show magnified images of tissue specimens at position C3 of the pancreatic cancer array PA1003. Figures 206A and 206B show specimens stained with antibody 1E4. Figures 206C and 206D show specimens stained with polyclonal antibody SDIX. [Figure 207] 207A and 207B show magnified images of tissue specimens at position D1 of the pancreatic cancer array PA1003. Figures 207A and 207B show specimens stained with antibody 1E4. Figures 207C and 207D show specimens stained with polyclonal antibody SDIX. [Figure 208] Photographs of the pancreatic cancer array PA1003 are shown. Figure 208A shows a specimen stained with polyclonal antibody SDIX. Figure 208B shows a specimen stained with antibody 20A10. Figure 208C shows a specimen stained with antibody 29H1. [Figure 209] Photographs of the esophageal cancer array ES1001 stained with various antibodies are shown. Figure 209A shows an array stained with polyclonal antibody SDIX. Figure 209B shows an array stained with antibody 20A10. Figure 209C shows an array stained with antibody 29H1. Figure 209D shows an array stained with antibody 31A1. [Figure 210] Photographs of the pancreatic cancer array PA1003 stained with various antibodies are shown. Figure 210A shows the array stained with polyclonal antibody SDIX. Figure 210B shows the array stained with antibody 20A10. Figure 210C shows the array stained with antibody 29H1. [Figure 211]Figure 211A shows graphs of IL-18 secreted into the conditioned medium of MUC1*-positive cancer cells co-cultured with huMNC2-CAR44 T cells, where the cells also harbor NFAT-inducible IL-18. Figure 211A shows graphs of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with untransduced human T cells. Figure 211B shows graphs of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T cells that also harbor an NFAT-inducible IL-18 gene inserted into a portion of the Foxp3 enhancer. Figure 211C shows graphs of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T cells that also harbor an NFAT-inducible IL-18 gene inserted into a portion of the IL-2 enhancer. [Figure 212] Figures 212A-C, 212I-K, and 212Q-S show cancer cells cocultured with untransduced T cells. Figures 212D-F, 212L-N, and 212T-V show cancer cells cocultured with hiMNC2-CAR44 T cells harboring the NFAT-inducible IL-18 gene inserted into either the Foxp3 enhancer / promoter or the IL-2 enhancer / promoter. Figures 212G-H, 212O-P, and 212W-X show cancer cells co-cultured with hiMNC2-CAR44 T cells harboring the NFAT-inducible IL-18 gene inserted into the IL-2 enhancer / promoter. [Figure 213]Figure 213A shows graphs of ELISA experiments in which the levels of IL-18 secreted into the conditioned medium were measured for huMNC1-CAR44 T cells harboring an NFAT-inducible IL-18 gene inserted into the Foxp3 enhancer or promoter, co-cultured with either MUC1*-positive cancer cells or MUC1-negative non-cancer cells. Figure 213B shows IL-18 secretion from huMNC2-CAR44 T cells harboring NFAT-inducible IL-18 in co-culture with T47D breast cancer cells, where the population was doped with 5%, 10%, or 30% MUC1*-transfected T47D cells. Figure 213B shows IL-18 secretion from huMNC2-CAR44 T cells with NFAT-induced IL-18 in co-culture with non-cancerous MUC1-negative HEK293 cells, where the cell population was doped with 5%, 10%, or 30% T47D cells transfected with more MUC1*. [Figure 214]Photographs of T47D breast cancer cells (red) or noncancerous HEK293 cells (red) are shown. Here, both cell types were doped with varying percentages of T47D cells engineered to express more MUC1* (green). These target cancer cells were co-cultured with huMNC2-CAR44 T cells, which carry NFAT-inducible IL-18, in which the IL-18 gene has been inserted into the Foxp3 enhancer / promoter. Figures 214A-F show either T47D cells or HEK293 cells that were not doped with T47D cells engineered to express high-density MUC1*. Figures 214G-L show either T47D cells or HEK293 cells that were doped with 5% T47D cells engineered to express high-density MUC1*. Figures 214M-R show either T47D or HEK293 cells doped with 10% T47D cells engineered to express high-density MUC1*. Figures 214S-X show either T47D or HEK293 cells doped with 30% T47D cells engineered to express high-density MUC1*. Figures 214A-B, G-H, M-N, and S-T show T47D breast cancer cells. Figures 214C-F, I-L, O-R, and U-X show HEK293 cells. As can be seen in the figures, induced secretion of IL-18 resulted in the killing of low-MUC1* density T47D cells but did not induce nonspecific killing of MUC1*-negative HEK293 cells. [Figure 215] Figure 215 shows the consensus sequences of heavy chain CDRs generated for each group of antibodies that bind to the same epitope in PSMGFR and N-terminally extended PSMGFR peptides. Figure 215A shows the consensus sequence of heavy chain CDR1. Figure 215B shows the consensus sequence of heavy chain CDR2. Figure 215C shows the consensus sequence of heavy chain CDR3. [Figure 216]Figure 216A shows the consensus sequence of the light chain CDR generated for each group of antibodies that bind to the same epitope in PSMGFR and N-terminally extended PSMGFR peptides. Figure 216A shows the consensus sequence of the light chain CDR1. Figure 216B shows the consensus sequence of the light chain CDR2. Figure 216C shows the consensus sequence of the light chain CDR3. DETAILED DESCRIPTION OF THE INVENTION

[0091] In this application, "a" and "an" are used to refer to both a single object and multiple objects.

[0092] As used herein, sometimes for shorthand, a polypeptide is referred to as being "transduced or transfected" into a cell. In these occurrences, it is understood that a nucleic acid encoding a polypeptide sequence is transduced or transfected into a cell, as it is not possible for a polypeptide to be transduced or transfected into a cell.

[0093] As used herein, when referring to the number of cells injected into an animal, or in contexts where the number of cells is mentioned, "M" may refer to millions and "K" may refer to thousands.

[0094] As used herein, interchangeable designations of various monoclonal antibodies are used, as follows: "MNC2" interchangeable with "C2," "Min-C2," and "MNC2"; "MNE6" interchangeable with "E6," "Min-E6," and "MNE6"; "MN-C3" interchangeable with "C3," "Min-C3," and "MNC3"; "MN-C8" interchangeable with "C8," "Min-C8," and "MNC8," etc. The monoclonal antibodies provided herein follow the same convention.

[0095] As used herein, "h" or "hu" placed before an antibody construct is an abbreviation for humanized.

[0096] As used herein, the term "antibody-like" refers to a molecule that can be engineered to contain portions of an antibody but is not a naturally occurring antibody. Examples include, but are not limited to, CAR (chimeric antigen receptor) T cell technology and Ylanthia® technology. CAR technology uses an antibody epitope fused to a portion of a T cell, which directs the body's immune system to attack a specific target protein or cell. Ylanthia® technology consists of an "antibody-like" library, a collection of synthetic human Fabs, which are then screened for binding to peptide epitopes of target proteins. Selected Fab regions can then be engineered into a scaffold or framework to resemble an antibody.

[0097] As used herein, "PSMGFR" is an abbreviation for the Primary Sequence of the MUC1 Growth Factor Receptor, identified by SEQ ID NO: 2, and therefore should not be confused with the six amino acid sequence. A "PSMGFR peptide" or "PSMGFR region" refers to a peptide or region incorporating the primary sequence of the MUC1 growth factor receptor (SEQ ID NO: 2).

[0098] As used herein, "MUC1 * The extracellular domain is primarily defined by the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 2)). The exact site of MUC1 cleavage depends on the enzyme that cleaves it, and cleavage enzymes vary across cell types, tissue types, or cellular evolutionary time; therefore, MUC1 * The exact sequence of the extracellular domain of may vary at the N-terminus.

[0099] Other truncated amino acid sequences include SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); or SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621) may be mentioned.

[0100] As used herein, the term "PSMGFR" is an acronym for the primary sequence of the MUC1 growth factor receptor, which is set forth as GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 2). In this regard, "N-10 PSMGFR" or simply "N-10," "N-15 PSMGFR" or simply "N-15," or "N-20 PSMGFR" or simply "N-20" refer to the number of amino acid residues deleted at the N-terminus of PSMGFR. Similarly, "C-10 PSMGFR" or simply "C-10," "C-15 PSMGFR" or simply "C-15," or "C-20 PSMGFR" or simply "C-20" refer to the number of amino acid residues deleted at the N-terminus of PSMGFR. A mixture of deletions and additions is also possible. For example, N+20 / C-27 refers to a peptide fragment of wild-type MUC1 in which 20 amino acids are added to PSMGFR at the N-terminus and 27 amino acids are deleted from the C-terminus.

[0101] As used herein, "MUC1 * The "extracellular domain of MUC1" refers to the extracellular portion of the MUC1 protein that lacks the tandem repeat domain. * is a cleavage product, and MUC1 * The MUC1 fragment consists of a short extracellular domain lacking tandem repeats, a transmembrane domain, and a cytoplasmic tail. The exact location of the cleavage site in MUC1 is unknown, as it can likely be cleaved by multiple enzymes. * The extracellular domain of will contain most of the PSMGFR sequence but may have an additional 10-20 N-terminal amino acids.

[0102] As used herein, "sequence identity" means the homology in a particular polypeptide or nucleic acid sequence to a reference sequence of nucleic acids or amino acids such that the function of the homologous peptide is the same as the reference peptide or nucleic acid. Such homology can sometimes be so close to the reference peptide that two sequences can be 90%, 95%, or 98% identical and still retain the same function in binding or other biological activity.

[0103] As used herein, a "MUC1-positive" cell refers to a cell that expresses the gene for MUC1, MUC1-Y or MUC1-Z or other MUC1 variants.

[0104] As used herein, a "MUC1-negative" cell refers to a cell that does not express the MUC1 gene.

[0105] As used herein, "MUC1 * "Positive" cells refer to cells that express the gene for MUC1, the expressed protein of which is a transmembrane protein lacking tandem repeats, which may be the result of post-translational modification, truncation, alternative splicing, or transfecting or transducing the cells with a MUC1 protein lacking tandem repeats.

[0106] As used herein, "MUC1 * "Negative" cells refer to cells that may or may not express the gene for MUC1, but do not express the MUC1 transmembrane protein lacking the tandem repeats.

[0107] As used herein, "MUC1-positive" cancer cells refer to cancer cells that overexpress the MUC1 gene and express MUC1 in an abnormal pattern, where the expression is not limited to the apical border and / or express MUC1 that lacks tandem repeats.

[0108] As used herein, "MUC1-negative" cancer cells refer to cancer cells that may or may not express the MUC1 gene, but do not overexpress MUC1 or do not overexpress the MUC1 transmembrane protein lacking tandem repeats.

[0109] As used herein, "MUC1 * "Positive" cancer cells refer to cancer cells that overexpress the MUC1 transmembrane protein lacking the tandem repeats.

[0110] As used herein, "MUC1 * "Negative" cancer cells refer to cancer cells that may or may not express the gene for MUC1, but do not overexpress the MUC1 transmembrane protein lacking the tandem repeats.

[0111] As used herein, a "conformational epitope" refers to a peptide sequence that must exist in a specific three-dimensional structure or conformation for an antibody to bind. However, antibodies bind when the peptide sequence is in a three-dimensional structure or conformation, but not when it is linear. A common technique for determining whether an antibody binds to a linear stretch or conformational epitope is to use the antibody to probe a denaturing Western blot. Proteins and peptides become linear when passed through a denaturing gel. An antibody that does not function in a denaturing Western blot but recognizes a natural target expressed, for example, in intact cells, is considered to recognize a conformational epitope. As used herein, an antibody may or may not actually bind to a "conformational epitope," but the presence of a "conformational epitope" sequence is required for the three-dimensional structure, thereby allowing the MUC1 protein on cancer cells to bind. * The conformational epitope is a region that can be bound by an antibody specific for cancer therapy. *The term "conformation-inducing peptide sequence" may therefore be used to indicate that the peptide sequence is present within a larger peptide rather than as a binding site, but induces antibody binding to the larger peptide by constraining the three-dimensional structure to a configuration that promotes antibody binding to the larger peptide. MUC1 for the treatment or prevention of cancer * Antibody (anti-PSMGFR)

[0112] The present inventors have discovered that a truncated form of the MUC1 (SEQ ID NO: 1) transmembrane protein is a growth factor receptor that drives the growth of more than 75% of all human solid tumor cancers. * A truncated form of MUC1, called MUC1 (pronounced mu-k-1-star), is a potent growth factor receptor. Enzymatic cleavage releases most of the MUC1 extracellular domain. This is due to the MUC1 * The remaining part of the extracellular domain of MUC1, called NME1, contains a truncated extracellular domain, a transmembrane domain, and a cytoplasmic tail. Cleavage and release of most of the extracellular domain of MUC1 results in the formation of dimeric ligands NME1, NME6, NME8, and NME7. AB , NME7-X1, or NME7-activating binding sites. Cell growth assays show that this activates the growth-promoting MUC1 * This indicates that ligand-induced dimerization of the extracellular domain of MUC1 occurs (Figure 1A-D). * Positive cells were screened with bivalent "bv" anti-MUC1 * Treatment with either monovalent "mv" or Fab, NM23-H1 dimer, or NME7-AB. * The antibody stimulates cancer cell growth, whereas the monovalent Fab inhibits growth. The classic bell-shaped curve indicates that ligand-induced dimerization stimulates growth. The dimer NM23-H1, also known as NME1, is a complex with MUC1. * NME7-AB stimulates the growth of MUC1-positive cancer cells, whereas siRNA to suppress MUC1 expression abolishes this effect (Figure 1C). *stimulate the growth of positive cells (Figure 1D).

[0113] MUC1 * MUC1 is a prime target for anti-cancer drugs because it is aberrantly expressed in over 75% of all cancers and may be overexpressed in an even higher percentage of metastatic cancers. After MUC1 cleavage, most of its extracellular domain is released from the cell surface. The remaining portion has a truncated extracellular domain that contains at least the primary growth factor receptor sequence, PSMGFR (SEQ ID NO: 2). Antibodies that bind to the PSMGFR sequence, particularly NME1, NME6, NME8, and NME7, are available. AB Antibodies that competitively inhibit the binding of activating ligands such as NME proteins, including NME7-X1 and NME7, are ideal therapeutics and can be used as stand-alone antibodies, antibody fragments or their variable region fragments incorporated into bispecific antibodies, or chimeric antigen receptors (CARs), to treat MUC1-positive cancers or MUC1-positive cancers. * These can then be transfected or transduced into immune cells, which are then administered to the patient.

[0114] Therapeutic anti-MUC1 * The antibody can be monoclonal, polyclonal, antibody mimic, engineered antibody-like molecule, complete antibody, or antibody fragment. Examples of antibody fragments include, but are not limited to, Fab, scFv, and scFv-Fc. Human or humanized antibodies are preferred for use in cancer treatment or prevention. Mutations can be introduced into any of these antibody-like molecules to prevent or minimize dimer formation. MUC1 * Anti-MUC1 antibodies that are monovalent or bispecific, as their function is activated by ligand-induced dimerization * Antibodies are preferred. Exemplary binding assays involve the use of NME1 and NME7 antibodies. AB MUC1 * They further demonstrate that these activating growth factors bind to the PSMGFR peptide portion of MUC1 (Figure 2A, 2D). Furthermore, they demonstrate that these activating growth factors do not bind to the PSMGFR peptide if the 10 C-terminal amino acids are missing, thus inhibiting MUC1. *Similarly, anti-MUC1 * Antibodies MN-C2 and MN-E6 bind to the PSMGFR peptide only when the 10 C-terminal amino acids are present (Figure 2B, Figure 2C). Antibodies MN-C3 and MN-C8 bind to a different epitope than MN-C2 and MN-E6 because they do not depend on the presence of the 10 C-terminal amino acids of the PSMGFR peptide (Figure 2E, 2F). Antibodies MN-C2, MN-E6, or fragments derived from them can be administered to patients as standalone antibodies or incorporated into bispecific antibodies, BiTEs, or chimeric antigen receptors (also known as CARs) transduced into immune cells to treat or prevent cancer. NME1 and NME7 AB MNC2 and MNE6 competitively inhibit the binding of MUC1 and other anti- * Antibodies are preferred for use as stand-alone antibody therapeutics.

[0115] Therapeutic anti-MUC1 for use as a stand-alone antibody therapeutic or for integration in a BiTE or CAR * Antibodies can be selected based on specific criteria. Parent antibodies can be generated using typical methods for generating monoclonal antibodies in animals. Alternatively, they can be used to generate antibodies against MUC1. * Selection can be made by screening antibody and antibody fragment libraries for the ability to bind to the peptide, which may be: (I) PSMGFR region of MUC1; (ii) PSMGFR peptide; (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10); (iv) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFSAQSGA(N-19); (v) a peptide having the amino acid sequence NLTISDVSVSDVPFPFSAQSGA(N-23); (vi) a peptide having the amino acid sequence ISDVSVSDVPFPFSAQSGA(N-26); (vii) a peptide having the amino acid sequence SVSDVPFPFSAQSGA(N-30); (viii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5); (ix) a peptide having the amino acid sequence ASRYNLTISDVSVSDVPFPFS(N-19 / C-5), or (x) A peptide having the amino acid sequence FPFSAQSGA(N-36).

[0116] The resulting antibodies or antibody fragments generated or selected in this manner can be further selected by passing them through additional screens. For example, the antibodies or antibody fragments can be selected to target MUC1 * These antibodies are more preferred due to their ability to bind to MUC1-positive cancer cells or tissues but not to MUC1-negative cancer cells or normal tissues. * Antibodies or antibody fragments may be deselected as anti-cancer therapeutics if they bind to stem or progenitor cells. * The antibody or antibody fragment may be * 3A-3C show that MN-E6 and MN-C2 inhibit the binding of activating ligands NME1 and NME7 to MUC1. * This indicates that it competitively inhibits binding to

[0117] Anti-MUC1 for use in treating patients diagnosed with, at risk of developing, or suspected of having MUC1-positive cancer *The antibody selection process involves selecting antibodies or antibody fragments that: 1) bind to the PSMGFR peptide; 2) bind to the N-10 PSMGFR peptide; 3) bind to cancer cells; 4) do not bind to stem or progenitor cells; or 5) competitively inhibit the binding of dimeric NME1 or NME7-AB to the PSMGFR peptide. For example, Figures 3A-3C show that monoclonal antibodies MN-E6 and MN-C2 meet all five criteria, while monoclonal antibodies MN-C3 and MN-C8 do not competitively inhibit the binding of the activating ligands NME1 and NME7 (Figure 3C). * Recall that growth factor receptors are activated by ligand-induced dimerization of their extracellular domains. Therefore, an ideal antibody therapeutic would target MUC1. * The extracellular domain should not dimerize. Preferably, suitable antibodies in this regard include monovalent antibodies such as those produced in llamas and camels, Fabs, scFvs, single domain antibodies (sdAbs), scFv-Fc, etc., so long as the Fc portion is constructed so as not to homodimerize.

[0118] FACS scan shows anti-MUC1 * Antibodies MN-C2 and MN-E6 inhibit MUC1 * Positive solid tumor cancer cells and MUC1 * Specific binding to transfected cells but not to MUC1 * In one example, humanized MN-C2 scFv binds to ZR-75-1, also known as 1500, MUC1 * MN-E6 has been shown to bind to MUC1-positive breast cancer cells (Figures 4A-4C). * MN-E6 was shown to bind to MUC1-negative HCT-116 colon cancer cells only when transfected with ZR-75-1, also known as 1500, a MUC1 * MUC1-positive breast cancer cells *MN-C2 and MN-E6 also bound to PSMGFR peptides and live MUC1-positive breast cancer cells (Figures 4D-4F). Binding assays, including ELISA and immunofluorescence, all confirmed that MN-C2 and MN-E6 bound to the PSMGFR peptide and live MUC1-positive cancer cells. * Antibodies are selected based on their ability to bind to PSMGFR peptides or MUC1-positive cancer cells. * The humanized MN-C2 scFv binds to the peptide PSMGFR with high affinity, with an EC-50 of approximately 333 nM. Similar to the Fab, the humanized MN-C2 scFv binds to MUC1 as shown in the examples in Figures 6A and 6B. * These antibodies potently inhibit the growth of IgG-positive cancer cells. Similar to the parent antibodies, the humanized scFvs exhibit the same binding pattern. huMNE6-scFv binds to the PSMGFR peptide and the N-10 peptide, but not to the C-10 peptide (SEQ ID NO: 825) (Figure 8). Mouse or humanized MNC3-scFv binds to the PSMGFR peptide, the N-10 peptide, and the C-10 peptide (Figure 9).

[0119] The Fabs of MN-E6 and MN-C2, or equivalent single-chain variable regions derived from them, bind to MUC1 in vitro and in vivo. * In some instances, anti-MUC1 * The Fab fragment of the antibody binds to human MUC1 in vivo. * In one example, immune-compromised mice were implanted with human breast tumors and then treated with MN-E6 Fab after tumor engraftment. Figure 7A shows that MN-E6 Fab inhibited the growth of MUC1-positive cancers. * Female nu / nu mice implanted with estrogen pellets for 90 days were implanted with 6 million T47D human breast cancer cells mixed 50 / 50 with Matrigel. Tumors with a volume of at least 150 mm were grown. 3Mice with tumors that were ≥ 100% and had three consecutive tumors were selected for treatment. The animals were injected subcutaneously twice weekly with 80 mg / kg of MN-E6Fab, and an equal number of mice meeting the same selection criteria were injected with vehicle alone (Fig. 7A).

[0120] In another embodiment, MN-E6 was shown to halt prostate cancer growth. Figure 7B shows that MN-E6 Fab inhibits MUC1 * The results show that anti-MUC1 antibody potently inhibited the growth of anti-MUC1 positive prostate cancer cells. Male NOD / SCID mice were implanted with 6 million DU-145 human prostate cancer cells mixed 50 / 50 with Matrigel. Mice with tumor volumes of at least 150 mm3 and tumors that grew three consecutive times were selected for treatment. Animals were injected subcutaneously with 160 mg / kg of MN-E6Fab every 48 hours, and an equal number of mice meeting the same selection criteria were injected with vehicle alone (Figure 7B). Tumors were measured and recorded independently twice weekly by two researchers. Statistics were calculated blindly by independent statisticians, with a P value of 0.0001 for each. * The Fab inhibited breast and prostate cancer growth. Treatment had no effect on body weight, bone marrow cell types, or numbers. MN-E6 Fab effectively inhibited tumor growth, while tumors in the control group continued to grow until sacrifice. No side effects from the treatment were observed or detected.

[0121] Recombinant forms of MN-E6 and MNC2 were constructed to be monomeric, like Fab. In this case, MN-E6 was humanized and MN-C2 was humanized. There are several methods known to those skilled in the art for humanizing antibodies. In addition to humanization, libraries of human antibodies can be screened to identify other fully human antibodies that bind to PSMGFR.

[0122] A single chain of the humanized MN-E6 variable region, called scFv, was engineered to be connected to the Fc portion of an antibody (SEQ ID NOs: 256 and 257). The Fc portion confers certain advantages to antibody fragments for use as therapeutics. The Fc portion of an antibody recruits complement, which generally means that it can recruit other aspects of the immune system, not only inhibiting the target but also amplifying the anti-tumor response. The addition of an Fc portion also increases the half-life of the antibody fragment (Czajkowsky DM, Hu J, Shao ZandPleass RJ. (2012) Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med. 4(10):1015-1028). However, the Fc portion of an antibody homodimerizes, which is a drawback for anti-MUC1 antibodies. * For antibody-based therapies, MUC1 * Ligand-induced dimerization of the receptor stimulates growth and is therefore suboptimal. Therefore, mutations in the Fc region that resist dimerization are useful for anti-MUC1 * Deletions of the hinge region and other mutations in the Fc region that render the Fc variants resistant to dimerization could be made and used as therapeutic agents, which are desirable for anti-cancer therapy.

[0123] Human or humanized MN-E6 antibody or antibody fragment, Fab, MN-E6 scFv or huMN-E6 scFv-Fc mut MUC1 or MUC1 * Diagnosed with MUC1 or MUC1 positive cancer * suspected of having cancer or have a positive MUC1 or MUC1 * It is an effective anti-cancer agent that can be administered to people at risk of developing positive cancer. Humanization

[0124] -MUC1 *Humanized antibodies or antibody fragments or fully human antibodies that bind to the extracellular domain of are preferred for therapeutic use. The techniques described herein for humanizing antibodies are some of the various methods known to those skilled in the art. The present invention is not meant to be limited by the technique used to humanize the antibody.

[0125] Humanization is the process of replacing non-human regions of therapeutic antibodies (usually murine monoclonal antibodies) with human regions without altering their binding specificity and affinity. The primary goal of humanization is to reduce the immunogenicity of therapeutic monoclonal antibodies when administered to humans. Three different types of humanization are possible. First, chimeric antibodies are created by replacing the non-human constant regions of an antibody with human constant regions. Such antibodies contain the murine Fab region and approximately 80–90% human sequences. Second, humanized antibodies are created by grafting murine CDR regions (responsible for binding specificity) into the variable regions of a human antibody and replacing the human CDRs (CDR grafting). Such antibodies contain approximately 90–95% human sequences. Finally, fully human antibodies (100% human sequences) can be created by phage display, which screens human antibody libraries to select antigen-specific human antibodies, or by immunizing transgenic mice expressing human antibodies.

[0126] A typical technique for humanizing antibodies is roughly as follows: Monoclonal antibodies are generated in a host animal, typically a mouse. The monoclonal antibodies are then screened for binding affinity and specificity to the target. Once a monoclonal antibody with the desired effect and desired properties is identified, it is sequenced. The sequence of the animal-generated antibody is then aligned with the sequences of many human antibodies to find the human antibody with the most homologous sequence to the animal antibody. Biochemical techniques are used to paste the human and animal antibody sequences together. Typically, non-human CDRs are grafted onto the human antibody with the most homology to the non-human antibody. This process can generate many candidate humanized antibodies that need to be tested to identify which antibody(ies) have the desired affinity and specificity.

[0127] Once a human or humanized antibody is generated, it can be further modified for use as a Fab fragment, as a complete antibody, or as an antibody-like entity, such as a single chain molecule containing the variable region, such as scFv or scFv-Fc. In some cases, it is desirable to mutate the Fc region of the antibody or antibody-like molecule to prevent it from dimerizing.

[0128] In addition to introducing human sequences into antibodies generated in non-human species, fully human antibodies can be obtained by screening human antibody libraries with peptide fragments of the antigen. Fully human antibodies that function like MN-E6 or MN-C2 are generated by screening human antibody libraries with peptides having the sequence of the PSMGFRN-10 peptide. Humanized anti-MUC1 * The antibodies were generated based on the sequences of the mouse monoclonal antibodies MN-E6 and MN-C2. *In another aspect of the invention, patients diagnosed with MUC1 positive cancer are treated with an effective amount of murine or camel MNC2, MNE6, 20A10 (SEQ ID NOS: 1574-1581), 3C2B1 (SEQ ID NOS: 1572-1573), 5C6F3, 25E6 (SEQ ID NOS: 1598-1601), 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. * Patients diagnosed with MUC1-positive cancer are treated with an effective amount of humanized MN-E6 or MN-C2. * In another aspect of the invention, patients diagnosed with MUC1 positive cancer are treated with an effective amount of humanized MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. * Patients diagnosed with MUC1*-positive cancers are treated with an effective amount of humanized monovalent MNC2, MNE6, 20A10 (SEQ ID NOS: 1574-1581), 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, where monovalent refers to the corresponding Fab fragment, corresponding scFv, or corresponding scFv-Fc fusion. In a preferred embodiment, patients diagnosed with MUC1*-positive cancers are treated with an effective amount of humanized scFv or monomeric humanized scFv-Fc of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. * Because growth factor receptors are activated by ligand-induced dimerization of their extracellular domains, and because the Fc portion of antibodies homodimerizes, constructs containing Fc portions preferably use mutated Fc regions that prevent or minimize dimerization.

[0129] Antibodies that bind to the PSMGFR (SEQ ID NO: 2) peptide of the extracellular domain of the MUC1* receptor, more specifically the N-10 peptide, are * These are potent anti-cancer therapeutic agents that are effective in treating or preventing cytotoxic and cytotoxic cancers. They are the activating ligands, dimeric NME1 (SEQ ID NO: 1781) and NME7.AB MUC1 (SEQ ID NO: 827) * It has been shown that anti-MUC1 binds to the PSMGFR sequence and inhibits binding to the extracellular domain of * Antibodies inhibit MUC1 receptor activity, particularly if they inhibit ligand-induced receptor dimerization. * Inhibits the growth of anti-MUC1 positive cancer cells. * The Fab of the antibody has been demonstrated to inhibit tumor growth in animals. * Antibodies or antibody fragments that bind to the extracellular domain of MUC1 * These compounds may be beneficial in treating cancers that express IL-1.

[0130] MUC1 * Antibodies that bind to the PSMGFR region of MUC1 or to synthetic PSMGFR peptides are preferred. *Several monoclonal antibodies that bind to the extracellular domain of IgG1 were identified. Among these were the murine monoclonal antibodies MNC2 (SEQ ID NOs: 118-131, 144-158, 163-164, 168-181, 194-209), MNE6 (SEQ ID NOs: 12-25, 39-59, 65-78, 93-114), 20A10 (SEQ ID NOs: 988-1019, 1574-1597, 1659-1666); 3C2B1 (SEQ ID NOs: 1386-1413, 1572-1573), 5C6F3 (SEQ ID NOs: 1356-1385), 25E6 (SEQ ID NOs: 1020-1051, 1598-1617, 1667-1674), and 18G12 (SEQ ID NOs: 1060-1070). These antibodies include 28F9 (SEQ ID NOs: 1052-1083), 1E4 (SEQ ID NOs: 1116-1227), B12 (SEQ ID NOs: 1414-1431, 1733-1742), B2 (SEQ ID NOs: 1432-1459), B7 (SEQ ID NOs: 1460-1487), B9 (SEQ ID NOs: 1544-1571), 8C7F3 (SEQ ID NOs: 1488-1515), and H11 (SEQ ID NOs: 1516-1543), whose variable regions were sequenced and given as MN-E6 (SEQ ID NOs: 12-13 and 65-66), and MN-C2 (SEQ ID NOs: 118-119 and 168-169). The CDRs of these antibodies constitute the recognition units of the antibody and are the most important parts of the mouse antibody that must be preserved when grafting it into a human antibody. The CDR sequences of each mouse monoclonal are as follows, with the heavy chain sequence followed by the light chain: MN~E6 CDR1 (SEQ ID NOS: 16-17 and 69-70), CDR2 (SEQ ID NOS: 20-21 and 73-74), CDR3 (SEQ ID NOS: 24-25 and 77-78), MN~C2 CDR1 (SEQ ID NOS: 122-123 and 172-173), CDR2 (SEQ ID NOS: 126-127 and 176-177), CDR3 (SEQ ID NOS: 130-131 and 180-181). In some cases, portions of the framework regions deemed important for the three-dimensional structure of the CDRs by modeling were also imported from the mouse sequence.

[0131] The monoclonal antibodies MN-E6 and MN-C2 inhibit MUC1 as found in cancer cells. *Monoclonal antibodies MN-C3 and MN-C8 show higher affinity for MUC1, as found in stem cells. * It shows higher affinity for

[0132] All four antibodies were humanized, a process that resulted in several humanized forms of each antibody. CDRs from the variable regions of the murine antibodies were biochemically grafted onto homologous human antibody variable region sequences. The humanized variable regions of MN~E6 (SEQ ID NOS: 38-39 and 93-94), MN~C2 (SEQ ID NOS: 144-145 and 194-195), MN~C3 (SEQ ID NOS: 439-440 and 486-487), and MN~C8 (SEQ ID NOS: 525-526 and 543-544) were generated by grafting the murine CDRs onto the variable regions of the homologous human antibodies. The humanized heavy chain variable constructs are then fused to either the human IgG1 heavy chain constant region (SEQ ID NOs: 58-59) or the human IgG2 heavy chain constant region (SEQ ID NOs: 54-55), which are then paired with either the human kappa chain (SEQ ID NOs: 109-110) or the human lambda chain (SEQ ID NOs: 113-114) constant region. Other IgG isotypes, including IgG3 or IgG4, can be used as constant regions.

[0133] Examples of antibodies produced included humanized MN-E6 variable regions in IgG2 heavy chains (SEQ ID NOS: 52-53) and IgG1 heavy chains (SEQ ID NOS: 56-57) paired with either lambda light chains (SEQ ID NOS: 111-112 and 216-219) or kappa chains (SEQ ID NOS: 107-108 and 210-213), humanized MN-C2 variable regions in IgG1 heavy chains (SEQ ID NOS: 157-158) or IgG2 heavy chains (SEQ ID NOS: 163-164), and humanized MN-C3 (SEQ ID NOS: 455-456, 453-454, 500-501, 502-503) and MN-C8 (SEQ ID NOS: 541-542, 539-540, 579-580, 581-582) antibodies. Which IgG constant region is fused to the humanized variable region depends on the desired effect, as each isotype has its own characteristic activity. The isotype of the human constant region is selected based on factors such as whether antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) is desired, but may also depend on the yield of antibody produced in a cell-based protein expression system. In a preferred embodiment, a humanized anti-MUC1 * The antibody or antibody fragment is administered to a human who has been diagnosed with or is at risk of developing a MUC1-positive cancer.

[0134] Humanized anti-MUC1 may be most useful in treating cancer patients or those at risk of developing cancer * One method for testing and selecting antibodies is to use MUC1 * The goal is to test them for their ability to inhibit the binding of activating ligands to the extracellular domain. * It can bind to the extracellular domain and dimerize it, thereby stimulating the growth of cancer cells. * Antibodies and antibody fragments that compete with NME1 for binding to the extracellular domain are therefore anti-cancer agents. AB MUC1 * In some cases, MUC1 is another activating ligand for * NME7 to the extracellular domain, or NME7 AB Preferably, antibodies that block binding of truncated or cleaved products of NME7-X1 are identified.* Antibodies and antibody fragments that compete with NME7 and NME7 variants for binding to the extracellular domain are effective as anti-cancer therapeutic agents.These antibodies include but are not limited to MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, and single-chain versions of these antibodies and their humanized scFvs.Other NME proteins, including NME6 and NME8, can also bind to MUC1 or MUC1. * Binds to MUC1 * Antibodies that compete with these proteins for binding to MUC1 may also be useful as therapeutic agents. In a preferred embodiment, murine, camelid, human, or humanized anti-MUC1 * The antibody or antibody fragment is administered to a human diagnosed with or at risk of developing a MUC1-positive cancer. In a more preferred embodiment, a single-chain antibody fragment or a monomeric scFv-Fc fusion derived from the humanized sequence of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 is administered to a human diagnosed with or at risk of developing a MUC1-positive cancer.

[0135] Single chain variable fragments, scFv, or other formats resulting in monovalent antibodies or antibody-like proteins are also useful. * It is desirable to prevent dimerization of the extracellular domains. Single-chain variable fragments, Fabs, and other monovalent antibody-like proteins are capable of binding to MUC1. * and binding to the extracellular domain of MUC1 * These single-chain variable fragments, Fabs, and other monovalent antibody-like molecules have been shown to be effective in blocking dimerization of MUC1. These single-chain variable fragments, Fabs, and other monovalent antibody-like molecules have effectively inhibited cancer growth in vitro and in animals xenografted with human MUC1-positive cancer cells. * Antibodies or antibody-like molecules may therefore be highly effective as anti-cancer therapeutics. *Such humanized single-chain antibodies, Fabs, and other monovalent antibody-like molecules that bind to the extracellular domain or PSMGFR peptides are useful as anti-cancer therapeutics. * The single chain variable fragment is MUC1 * These antibodies are generated by grafting the non-human CDRs of an antibody that binds to the extracellular domain of PSMGFR or the PSMGFR peptide onto the framework of a homologous human antibody. The resulting humanized heavy and light chain variable regions are then connected to each other via a suitable linker. The linker must be flexible and long enough to allow binding of the heavy chain to the light chain but prevent binding of the heavy chain of one molecule to the light chain of another molecule. For example, a linker of approximately 10-15 residues is used. Preferably, the linker contains [(glycine)4(serine)1]3 (SEQ ID NOs: 401-402), but is not limited to this sequence, as other sequences are possible.

[0136] In one embodiment, the humanized variable regions of MN-E6 (SEQ ID NOS: 38-39 and 93-94), MN-C2 (SEQ ID NOS: 144-145 and 194-195), or other antibodies of the invention are biochemically grafted into a construct that connects the heavy and light chains via a linker. Humanized single-chain anti-MUC1 antibodies containing humanized sequences from the variable regions of MN-E6 and MN-C2 are also included. * Exemplary antibodies have been produced: Several humanized MN-E6 single chain proteins have been produced (SEQ ID NOS: 232-237); Several humanized MN-C2 single chain proteins have been produced (SEQ ID NOS: 238-243); In a preferred embodiment, a humanized anti-MUC1 * An antibody fragment, e.g., a variable fragment, scFv antibody fragment MN-E6 scFv, MN-C2 scFv, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 scFv, is administered to a human diagnosed with or at risk of developing a MUC1-positive cancer.

[0137] One aspect of the present invention is directed to MUC1-positive cancer or MUC1 *A method for treating a patient diagnosed with, suspected of developing, or at risk of developing a positive cancer, wherein the patient is administered an effective amount of monomeric MN-E6 scFv, MN-C2 scFv, or MN-E6 scFv-Fc, MN-C2 scFv-Fc, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, wherein the antibody variable fragment portion is human or humanized, and the Fc portion of the antibody-like protein is mutated to make it resistant to dimer formation. CAR T and Cancer Immunotherapy Technologies

[0138] In another aspect of the invention, anti-MUC1 *Some or all of the single-chain portions of antibody fragments have been biochemically fused to immune system molecules using several different chimeric antigen receptor ("CAR") strategies. The idea is to fuse the recognition portion of an antibody, typically as a single-chain variable fragment, to an immune system molecule that has a transmembrane domain and a cytoplasmic tail capable of transmitting signals that activate the immune system. The recognition unit can be an antibody fragment, a single-chain variable fragment, an scFv, or a peptide. In one embodiment, the recognition portion of the extracellular domain of the CAR is composed of sequences from the humanized variable regions of MN-E6 (SEQ ID NOS: 38-39 and 93-94), MN-C2 (SEQ ID NOS: 144-145 and 194-195), 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. Examples of murine or humanized antibodies of the present invention, or their single-chain fragments, scFvs, that can be incorporated into CARs, BiTEs, or ADCs, are provided as 3C2B1 (SEQ ID NOs: 1572-1573), 20A10 (SEQ ID NOs: 1574-1581), and 25E6 (SEQ ID NOs: 1598-1601). In another embodiment, it is composed of sequences from single-chain variable fragments. Examples of single-chain constructs are shown. Several humanized MN-E6 single-chain protein scFvs have been generated (SEQ ID NOs: 232-237). Several humanized MN-C2 single-chain protein scFvs have been generated (SEQ ID NOs: 238-243). The transmembrane region of the CAR can be derived from other transmembrane regions, including those of CD8, CD4, antibody domains, or proximal cytoplasmic costimulatory domains, such as CD28, 4-1BB, and others. The cytoplasmic tail of the CAR can be composed of one or more motifs that signal immune system activation. This group of cytoplasmic signaling motifs, sometimes referred to as costimulatory cytoplasmic domains, includes, but is not limited to, CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7, and Fc receptor gamma domain. Minimal CARs have a CD3-zeta or Fc receptor gamma domain followed by one or two of the above domains in tandem in the cytoplasmic tail.In one embodiment, the cytoplasmic tail comprises CD3-zeta, CD28, 4-1BB and / or OX40.

[0139] MUC1 * The extracellular domain recognition unit of the targeting CAR comprises the variable region of any non-human, humanized, or human antibody capable of binding to at least 12 consecutive amino acids of the PSMGFR peptide (SEQ ID NO: 2) or the N-10 peptide. * The targeting moiety comprises variable regions from non-human, humanized, or human MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. Examples of some antibodies of the present invention incorporated into CARs, either murine or humanized, are provided as 20A10 (SEQ ID NOs: 1582-1597) and 25E6 (SEQ ID NOs: 1602-1617). In the humanization process, antibody CDRs can be inserted into multiple different framework regions. As a demonstration, three versions of humanized 20A10 were generated that differ only in the framework regions. These were incorporated into CARs (SEQ ID NOs: 1675, 1678, 1685) and, when transduced into human T cells, targeted MUC1. * It can recognize and kill expressing cells. In one embodiment, the extracellular domain recognition unit of the CAR consists essentially of humanized MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 single-chain variable fragment scFv. The transmembrane region of the CAR can be derived from CD8 (SEQ ID NOs: 363-364) or the transmembrane domain of CD3-zeta, CD28, 41bb, OX40, or other transmembrane region (SEQ ID NOs: 361-372), and can be a transmembrane domain of MUC1. *The cytoplasmic domain of a CAR carrying an antibody fragment targeting the extracellular domain can be composed of one or more immune system co-stimulatory cytoplasmic domains, including, but not limited to, CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7, and Fc receptor gamma domains (SEQ ID NOs: 373-382).

[0140] The described CARs can be transfected or transduced into cells of the immune system. In a preferred embodiment, MUC1 * The target CAR is transfected or transduced into T cells. In one embodiment, the T cells are CD3+ / CD28+ T cells. In another, the T cells are dendritic cells. In another, the T cells are B cells. In another, the T cells are mast cells. In yet another, the T cells are natural killer (NK) cells. The recipient cells can be derived from the patient or donor. If derived from the donor, they may be engineered to remove molecules that would cause rejection. The cells transfected or transduced with the CAR of the present invention can be expanded ex vivo or in vitro and then administered to the patient. The route of administration is selected from the group including, but not limited to, bone marrow transplantation, intravenous injection, in situ injection or transplantation. In a preferred embodiment, the MUC1 * The targeted CAR is administered to people who have been diagnosed with or are at risk of developing MUC1-positive cancer.

[0141] MUC1 * Many possible anti-MUC1 antibodies that can be transduced into T cells or other immune cells to treat or prevent MUC1-positive cancers * There are CAR constructs: CARs are composed of modules, and the identity of some modules is relatively unimportant, while the identity of other modules is very important.

[0142] The present inventors have shown that intracellular signaling modules such as CD3-zeta (SEQ ID NOs: 373-376), CD28 (SEQ ID NOs: 377-378), and 41BB (SEQ ID NOs: 379-380), alone or in combination, stimulate immune cell expansion, cytokine secretion, and immune cell-mediated killing of target tumor cells (Pule MA, Straathof KC, Dotti G, Heslop HE, Rooney CM and Brenner MK (2005). A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells. Mol Ther. 12(5):933-941; Hombach AA, Heiders J, Foppe M, Chmielewski Mand Abken H (2012). OX40 costimulation by a chimeric antigen receptor abrogates CD28 and IL-2 induced IL-10 secretion by redirected CD4(+) T cells). cells.Oncoimmunology.1(4):458-466;Kowolik CM,Topp MS,Gonzalez S,Pfeiffer T,Olivares S,Gonzalez N,Smith DD,Forman SJ,Jensen MCandCooper LJ(2006)CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells.Cancer Res.66(22):10995-11004;Loskog A, Giandomenico V, Rossig C, Pule M, Dotti GandBrenner MK.(2006) Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells. Leukemia. 20(10):1819 - 1828; Milone MC, Fish JD, Carpenito C, Carroll RG, Binder GK, Teachey D, Samanta M, Lakhal M, Gloss B, Danet-Desnoyers G, Campana D, Riley JL, Grupp SA and June CH (2009) Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther. 17(8):1453 - 1464; Song DG, Ye Q, Carpenito C, Poussin M, Wang LP, Ji C, Figini M, June CH, Coukos G, Powell DJ Jr. (2011) In vivo persistence, tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137(4-1BB). Cancer Res.71(13):4617-4627). Antibodies of the present invention, including but not limited to fragments of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, may also be incorporated into CARs with mutant cytoplasmic tails, such as mutant tyrosines or ITAMs. In any of the above CARs, the cytoplasmic tail may contain mutations that attenuate signaling. Such mutations include, but are not limited to, tyrosines mutated to inhibit phosphorylation and signaling (Salter et al., 2018). In any of the above CARs, the ITAM of CD3-zeta may be mutated to inhibit or attenuate signaling (Feucht et al., 2019). In any of the above CARs, the CD3 in the cytoplasmic tail may contain mutations in the ITAM, including those referred to as 1XX. Examples of antibodies of the present invention incorporated into CARs with 1XX mutations in the ITAM of CD3-zeta are provided in the following sequences: MNC2 (SEQ ID NOs: 1618-1625), MNE6 (SEQ ID NOs: 1626-1633), 20A10 (SEQ ID NOs: 1590-1595), and 25E6 (SEQ ID NOs: 1610-1617). Note that antibody CDRs can be inserted into multiple, different framework region backgrounds. As an example, the 20A10 CDRs were inserted into three different sets of framework regions (SEQ ID NOs: 1692, 1699, and 1706), and all were functional when transduced into T cells. In any of the above CARs, T cells can be engineered to overexpress c-Jun as a way to inhibit T cell exhaustion (Lynn et al., 2019). Various promoters can be used upstream of the genes for CAR and other compositions of the invention, including insertion into naturally occurring promoters in cells, such as the TRAC locus, using CRISPR, Sleeping Beauty, or similar techniques for site-specific insertion of genes. Among commonly used promoters are the CMV promoter, or mini-CMV (SEQ ID NO: 1634), the minimal IL-2 promoter (SEQ ID NO: 1635), or the minimal promoter mini-P (SEQ ID NO: 1636).

[0143] Monoclonal anti-MUC1, designated MN-E6 or MN-C2 * Single-chain antibody fragments containing the variable domains of antibodies were engineered into a panel of CARs. * The targeting CARs were then transduced into immune cells, either separately or in combination. * Peptide, MUC1 * Antigen-presenting cells transfected with, or MUC1 * When challenged with surfaces presenting MUC1 positive cancer cells * Immune cells transduced with targeted CARs elicited immune responses, including cytokine release, killing of target cells, and expansion of immune cells.

[0144] For example, the genes encoding the CAR and activated T cell induction genes described herein can be transduced into immune cells using viruses or inserted into the downstream region of one of the cellular promoters or enhancers, such as the TRAC (T cell receptor alpha chain) locus.Viral delivery systems and viral vectors can be used, including but not limited to, retroviruses such as gammaretroviruses, lentiviruses, adenoviruses, adeno-associated viruses, baculoviruses, poxviruses, herpes simplex viruses, oncolytic viruses, HF10, and T-Vec.In addition to viral transduction, the CAR and activated T cell induction genes described herein can be directly spliced ​​into the genome of recipient cells using methods such as CRISPR technology, CRISPR-Cas9 and -CPF1, TALEN, Sleeping Beauty transposon system, and SB100X.

[0145] Similarly, the identity of the molecules that make up the non-targeting portion of CAR, such as the extracellular domain, transmembrane domain, and membrane-proximal portion of the cytoplasmic domain, is important for determining whether MUC1 *The targeting portion of the CAR is not essential for its function. For example, the extracellular domain, transmembrane domain, and membrane-proximal portion of the cytoplasmic domain can be composed of portions of common antibody domains such as CD8, CD4, CD28, or Fc, CH2CH3, or CH3. Furthermore, the non-targeting portion of the CAR can be a composite of one or more portions of these molecules or other family members.

[0146] One aspect of the present invention is directed to MUC1-positive cancer or MUC1 * diagnosed with MUC1-positive cancer or MUC1 * suspected of having MUC1-positive cancer or MUC1-positive cancer or MUC1 * A method for treating a patient at risk of developing a MUC1 positive cancer, wherein the patient is * In another embodiment of the invention, the immune cells are T cells isolated from the patient, which are then transduced with a CAR, wherein the targeting head of the CAR is a T cell that encodes a targeting molecule that encodes a CAR encoding ... targeting molecule that encodes a CAR encoding a targeting molecule that encodes a targeting molecule that encodes a targeting molecule that encode * and after expansion of the transduced T cells, the CAR T cells are administered to the patient in an effective amount. In yet another embodiment of the invention, the immune cells are T cells isolated from the patient, which are then transduced with a CAR, wherein the targeting head of the CAR comprises a portion of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, and after optional expansion of the transduced T cells, the CAR T cells are administered to the patient in an effective amount. anti-MUC1 * Targeting antibody specificity

[0147] As these experiments demonstrate, the key part of a CAR is the antibody fragment that directs immune cells to tumor cells. As shown in the next section, MN-E6 and MN-C2 target MUC1, an antibody fragment expressed on tumor cells. *The next most important part of the CAR is the cytoplasmic tail, which carries immune system costimulatory domains. The identity of these domains regulates the magnitude of the immune response but does not affect its specificity. As shown, the identity of the transmembrane portion of the CAR is the least important. As long as the transmembrane portion has some flexibility and is long enough for the antibody fragment to reach its cognate receptor on the tumor cell, this is considered sufficient. CARs with a variety of different extracellular, transmembrane, and short cytoplasmic tails, including MN-E6-targeting antibody fragments, intracellular costimulatory domain 41BB, and CD3-zeta, all function to specifically kill target cells while stimulating the expansion of host T cells.

[0148] The most accurate way to demonstrate antibody specificity is to test the antibody on normal human tissue samples compared to cancerous tissue samples. MN-C2 and MN-E6 bind to MUC1 or MUC1 * Several breast tumor arrays were tested with several anti-MUC1 or MUC1 antibodies, which were shown to specifically bind to MUC1-positive cancer cells. * The study, which included serial sections of breast cancer tissue samples from over 1,200 different breast cancer patients, showed that very little full-length MUC1 remained on breast cancer tissue. The majority of expressed MUC1 was expressed in the MUC1 * and stained by MN-C2. Analysis was performed by Clarient Diagnostics, and tissue staining was scored using the Allred method. For example, Figure 10 shows the results of VU4H5, a commercially available anti-MUC1 antibody that binds to the tandem repeats, or MUC1 * Figures 10 and 11 show serial sections of breast cancer tissue arrays stained with either VU4H5, which recognizes MUC1-FL (full length), or MN-C2, which binds to cancerous MUC1. *These images show breast cancer tissue arrays stained with either VU4H5 or MN-C2, which recognizes MUC1. Tissue staining was scored using the Allred scoring method, which combines intensity and distribution scores. Below the tissue array photograph is a color-coded graph displaying the results. As can be seen, arrays stained with VU4H5 are very bright, whereas many tissues show no staining at all, despite published reports that MUC1 is aberrantly expressed in over 96% of all breast cancers, as evidenced by nucleic acid-based diagnostics. In contrast, arrays stained with MN-C2 are very dark (red vs. yellow or white in the graph). Furthermore, many tissues showed no staining at all with anti-full-length MUC1 but very dark staining with MN-C2 (see green boxes in the graph). Similarly, normal or cancerous breast tissues were stained with humanized MN-E6scFv-Fc. The antibody fragment was biotinylated, allowing it to be visualized with a secondary streptavidin-based antibody. As can be seen in Figure 12, hMN-E6 scFv-Fc does not stain normal breast tissue but does stain cancerous breast tissue. Furthermore, the intensity and uniformity of staining increases with the patient's tumor grade and / or metastasis grade (Figures 12-13). Similarly, hMN-E6 scFv-Fc did not stain normal lung tissue but did stain lung cancer tissue (Figures 14-18), with the intensity and distribution of staining increasing as the tumor grade or metastasis grade increased. Figure 19 shows the results of 5 μg / mL humanized MN-E6-scFv-Fc biotinylated anti-MUC1 * Photographs of normal and cancerous small intestinal tissues stained with the antibody and then with a secondary streptavidin-HRP antibody are shown. A) Normal small intestinal tissue. B) Small intestinal cancer from the patient as indicated. C, D are photographs of corresponding serial sections stained with the secondary antibody alone. Figure 20 shows the results of staining with 50 μg / mL of humanized MN-E6-scFv-Fc anti-MUC1. * Figure 21 shows photographs of normal small intestinal tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. A-D are normal small intestinal tissues. E-H are photographs of corresponding serial sections stained with secondary antibody alone. Figure 21 shows photographs of normal small intestinal tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody.* Figure 22 shows photographs of cancerous small intestine tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. A-D are cancerous small intestine tissues from patients as indicated. E-H are photographs of corresponding serial sections stained with secondary antibody alone. Figure 22 shows photographs of cancerous small intestine tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. * Figure 23 shows photographs of cancerous small intestine tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. A-D are cancerous small intestine tissues from patients as indicated. E-H are photographs of corresponding serial sections stained with secondary antibody alone. Figure 23 shows photographs of cancerous small intestine tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. * Figure 24 shows photographs of normal colon tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. A-D are normal colons. E-H are photographs of corresponding serial sections stained with secondary antibody alone. Figure 24 shows photographs of normal colon tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. * Figure 25 shows photographs of colon cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. A-D are colon tissues from metastatic patients, as indicated. E-H are photographs of corresponding serial sections stained with secondary antibody alone. Figure 25 shows photographs of colon cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. * Figure 26 shows photographs of colon cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. A-D are colon cancer tissues from a grade 2 patient, as indicated. E-H are photographs of corresponding serial sections stained with secondary antibody only. Figure 26 shows photographs of colon cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. * Figure 27 shows photographs of colon cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. A-D are colon tissues from metastatic patients, as indicated. E-H are photographs of corresponding serial sections stained with secondary antibody alone. Figure 27 shows photographs of colon cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. *Figure 28 shows photographs of prostate cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. A-D are prostate cancer tissues from patients as indicated. E-H are photographs of corresponding serial sections stained with secondary antibody only. Figure 28 shows photographs of prostate cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. * Figure 29 shows photographs of prostate cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. A-D are prostate cancer tissues from patients as indicated. E-H are photographs of corresponding serial sections stained with secondary antibody alone. Figure 29 shows photographs of prostate cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1 antibody followed by secondary goat anti-human HRP antibody. * Photographs of prostate cancer tissue stained with antibody and then with a secondary goat anti-human HRP antibody are shown. A-D are prostate cancer tissues from patients as indicated. E-H are photographs of corresponding serial sections stained with secondary antibody only.

[0149] One aspect of the present invention is directed to MUC1-positive cancer or MUC1 *

[0013] A method for treating a patient diagnosed with, suspected of developing, or at risk of developing a MUC1-positive cancer, wherein a specimen is obtained from the patient's cancer and tested for reactivity with an antibody that binds to PSMGFR SEQ ID NO:2, more specifically the N-10 peptide. The patient is then treated with an scFv, scFv-Fc, or CAR T that contains an antibody variable fragment from an antibody that reacted with the cancer specimen, or which may be selected from among MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. Another aspect of the invention relates to a method for treating a patient diagnosed with, suspected of developing, or at risk of developing a MUC1-positive cancer. * A method for treating a patient diagnosed with, suspected of having, or at risk for a positive cancer, wherein a specimen is obtained from the patient's cancer and tested for reactivity with MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, and the patient is then treated with an antibody, antibody fragment, scFv, scFv-Fc-mut, BiTE, or CART comprising a portion of an antibody that reacted with the cancer specimen.

[0150] As previously reported, it is not the full-length MUC1, but the transmembrane truncation product, MUC1, that is the growth factor receptor that promotes tumor growth. * MUC1 * Growth factors that activate MUC1 bind to an ectopic site that is exposed only after cleavage and release of the tandem repeat portion of MUC1. The antibodies of the present invention cannot bind to full-length MUC1, as activated growth factors can. FACS analysis shows that anti-MUC1 * The antibody MNC2 was unable to bind to HCT-116, MUC1-negative cells (Fig. 35A), indicating that they were MUC1 * These results clearly show that VU4H5 binds strongly to these cells when transfected with MUC1 (Figure 35B), but not to HCT cells transfected with full-length MUC1 (Figure 35C). The commercially available anti-tandem repeat antibody VU4H5 clearly recognizes full-length MUC1 (Figure 35D).

[0151] The present inventors have demonstrated that MUC1 is cleaved by multiple cleavage enzymes. * and the cleavage site affects its folding, so that any monoclonal antibody may bind to its MUC1 * We found that the morphology of MUC1 affects the ability to recognize it. Different cancer cells or cancerous tissues express different cleavage enzymes. We tested various cleavage enzyme inhibitors on various cancer cell lines and found that inhibitors that inhibit MUC1 cleavage in one cancer cell line do not inhibit cleavage in another cancer cell line. Similarly, PCR experiments showed that cleavage enzymes are expressed at different levels in different cells or cell lines. For example, hematopoietic stem cells in the bone marrow express MUC1, which is recognized by the monoclonal antibody MNC3 but not by MNE6 or MNC2. * The growth of DU145 prostate cancer cells and T47D breast cancer cells was inhibited by Fabs of MNC2 and MNE6, but not by Fabs of MNC3 or MNC8, indicating that the cancer cell lines express MUC1, which is recognized by MNE6 and MNC2, but not by MNC3 or MNC8. *These results suggest that bone marrow CD34-positive cells express approximately 2,500 times more MMP2 and approximately 350 times more ADAM28 than T47D breast cancer cells, whereas DU145 prostate cancer cells express approximately 2,000 times more ADAM TS16, approximately 400 times more MMP14, and approximately 100 times more MMP1 than T47D breast cancer cells (Figures 43 and 44). Conversely, T47D breast cancer cells express approximately 80 times more MMP9 than bone marrow cells and approximately twice as much MMP9 as DU145 prostate cancer cells. Various cleavage enzyme inhibitors were tested for their ability to inhibit cleavage of various types of cancer cells. MUC1 * General strategies for using antibodies, antibody fragments, and CARs targeting the extracellular domain of

[0152] In one embodiment of the present invention, a second factor, which may be a cleavage enzyme, an antibody, a cytokine, or a second CAR, and a CAR are transduced into the same T cell. In another embodiment of the present invention, the second factor is on an inducible promoter so that its expression is activated when the CAR engages with the target cancer cell. In some cases, the expression of the second factor is controlled by an inducible promoter. In one embodiment of the present invention, the expression of the second factor is induced when the immune cell is activated, for example, when it recognizes or engages its target. In one example, the T cell is transfected or transduced with a second factor, whose expression is induced when the T cell recognizes the target cancer cell. One way to do this is to induce the expression of the second factor when, or immediately after, the NFAT protein is expressed or translocated to the nucleus. For example, a sequence derived from the NFAT promoter region is placed upstream of the gene of the second factor. Thus, when a transcription factor that binds to the promoter of an NFAT protein is present at a concentration sufficient to bind to and induce transcription of the NFAT protein, it also binds to the same promoter that is operated in front of the sequence for transcription of a second factor.The NFAT protein can be NFAT1, also known as NFATc2, NFAT2, also known as NFATc or NFATc1, NFAT3, also known as NFATc4, NFAT4, also known as NFATc3, or NFAT5.In one embodiment of the present invention, the NFAT is NFATc1, NFATc3, or NFATc2.In one embodiment of the present invention, the NFAT is NFAT2, also known as NFATc1.SEQ ID NO: 646 shows the nucleic acid sequence of the transcriptional regulatory region upstream of NFAT2. The promoter sequence of the NFAT gene may include, for example, the nucleic acid sequence of SEQ ID NOs: 781-783 or 815, but it can be confirmed that the optimal or minimal sequence for expression of the second factor can be obtained by creating a fragment, extension, or mutation of the promoter and testing the strength of the promoter with respect to expression of the second factor. In one embodiment of the present invention, the transcriptional regulatory region of NFAT2 is engineered upstream of the gene encoding the second factor, in the case of the cleavage enzyme MMP9 (SEQ ID NO: 647) or the catalytic subunit of MMP9 (SEQ ID NO: 648).In one embodiment of the present invention, the NFAT is NFATc3, and the promoter sequence of NFATc3 comprises the nucleic acid sequence of SEQ ID NO: 816. In one embodiment of the present invention, the transcriptional regulatory region of NFATc3 is engineered upstream of a gene encoding a second factor, here by way of example only, MMP9. In another embodiment of the present invention, the NFAT is NFATc2. SEQ ID NOs: 817-818 show the nucleic acid sequences of the transcriptional regulatory region upstream of NFATc2. In one embodiment of the present invention, the transcriptional regulatory region of NFATc2 is engineered upstream of a gene encoding a second factor, which may be the cleavage enzyme MMP9 (SEQ ID NO: 647) or the catalytic subunit of MMP9 (SEQ ID NO: 648).

[0153] Another method for inducing the expression of a second factor when T cells or CAR T cells are activated is to have the gene for the second factor on an inducible promoter, through which the NFAT protein itself binds to the second factor and induces transcription. In this case, an NFAT response element (NFAT RE) can be located upstream of the gene for the second factor or a fragment of the second factor. NFAT, alone or as part of a complex, can bind to its response element upstream of the second factor. The NFAT protein can be NFATc1, NFATc2, NFATc3, NFATc4, or NFAT5. In a preferred embodiment, the NFAT protein is NFAT2, also known as NFATc1, also known as NFATc. The gene for the second factor or a fragment thereof is cloned downstream of an NFAT response element (SEQ ID NO: 649), which can be a repeat of the response element (SEQ ID NO: 650) and the CMV minimal promoter (mCMV) (SEQ ID NO: 651), to induce the expression of the second factor by the NFAT protein. The NFAT response element may include the nucleic acid sequence of the NFAT consensus sequence (SEQ ID NO: 804). The NFAT response element may include, for example, the nucleic acid sequences of SEQ ID NOs: 805-814, but it can be confirmed that the optimal or minimal sequence for expression of the second factor can be obtained by creating a fragment, extension, or mutation of the response element nucleic acid and testing the strength of the response element for expression of the second factor. The Foxp3 enhancer region also includes an NFAT response element within 120 bp from 2079 to 2098 (SEQ ID NO: 821). The NFAT response element can include the nucleic acid NFAT consensus sequence (5'-cattttttccat-3') (SEQ ID NO: 819) or (5'-tttttcca-3') (SEQ ID NO: 820) to which NFATc1 specifically binds (Xu et al., Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15, Blood 2014;123:3750-3759), or repeats thereof.NFAT response elements may also be separated by a nucleic acid spacer sequence. Other NFAT response elements may exist and may be discovered, and if directed to determine an NFAT response element, those skilled in the art would do so by performing molecular biological assays to obtain it, given the guidance of at least the response elements set forth, by way of example only, as set forth in SEQ ID NOS: 804-814. In one embodiment of the present invention, the cleavage enzyme downstream of the NFAT response element and the CMV minimal promoter is MMP9 (SEQ ID NO: 652). In another embodiment of the present invention, the cleavage enzyme is the catalytic subunit of MMP9 (SEQ ID NO: 653).

[0154] Because NFAT1-4 are regulated by the calcineurin pathway, potential toxicity in patients can be halted by treatment with immunosuppressants such as FK506, cyclosporine, cyclosporine A, and tacrolimus, which block calcineurin activity and inhibit NFAT translocation to the nucleus. T cells transduced or transfected with a cleavage enzyme on an inducible promoter can also be transduced or transfected with a CAR that recognizes a protein or molecule on cancer cells. In a specific example, the cleavage enzyme is an enzyme that can cleave full-length MUC1, and the CAR binds it to MUC1 on the surface of cancer cells. * It carries an antibody fragment directed against

[0155] To determine which cleavage enzyme cleaves MUC1 on cancer cells, a series of MMP and ADAM enzyme inhibitors were tested. These experiments showed that MMP9 is the key cleavage enzyme in cancer cells. To confirm that MMP9 cleaves MUC1 on cancer cells, HCT-116 MUC1-negative colon cancer cells were transfected with a mimic of full-length MUC1 with 41 tandem repeat domains: HCT-MUC1-41TR. Single cell cloning confirmed that MUC1 cleaves MUC1. *Figures 36A-36D show that HCT-MUC1-41TR was 95% positive for full-length MUC1 and expressed only minimally cleaved forms, such as MUC1. * Western blot and FACS analysis show that only 5-10% of cells were positive for MUC1. HCT-MUC1-41TR cells were incubated with various concentrations of MMP9 and assayed by immunofluorescence to measure binding of the resulting MNC2 monoclonal antibody to the cells. As can be seen in Figures 37A-37C, MNC2 binding increased as the concentration of MMP9 added to the cells increased. These experiments demonstrate that MMP9 cleaves MUC1 into a form recognized by MNC2. Human cancer tissue array studies conducted by the inventors (Figures 30A-30F, Figures 31A-31F, Figures 32A-32F, Figures 33A-33F) show that MNC2 recognizes a cleaved form of MUC1 that is present in cancerous tissue but not in healthy cells or tissues (Figures 34A-34I). Importantly, MNC2 does not recognize the cleaved form of MUC1 expressed on healthy hematopoietic stem cells in the bone marrow (Figures 39-41).

[0156] In one embodiment of the present invention, immune cells are transduced with both a CAR for targeting immune cells to tumors and a cleavage enzyme. The CAR and the cleavage enzyme can be encoded on the same plasmid or on two different plasmids. In one embodiment, the cleavage enzyme is on an inducible promoter. In another embodiment, the expression of the cleavage enzyme is induced by a protein that is expressed when immune cells are activated. In some cases, the expression of the cleavage enzyme is induced by an NFAT protein. In another embodiment, the expression of the cleavage enzyme is induced by NFATc1. In another embodiment, the expression of the cleavage enzyme is induced when one of the NFAT proteins binds to an NFAT response element inserted upstream of the gene of the cleavage enzyme or its catalytically active fragment. In one embodiment, the cleavage enzyme is MMP9 or a catalytically active fragment of MMP9.

[0157] In one embodiment of the present invention, the cleavage enzyme is MMP9 (SEQ ID NO: 643). Some cleavage enzymes are naturally expressed as proenzymes that need to be activated. This can be achieved by biochemical means, by expressing a coenzyme that activates the cleavage enzyme, or by engineering the activated form of the enzyme. The present invention anticipates overcoming this problem by co-expressing the cleavage enzyme with its activator. In one embodiment of the present invention, the cleavage enzyme is MMP9 and the co-activator is MMP3. In another embodiment of the present invention, the cleavage enzyme is expressed in an already active form, for example, by expressing a fragment of the cleavage enzyme that still has catalytic function. In some cases, the cleavage enzyme is a catalytically active MMP9 fragment. An example of an MMP9 catalytic fragment is provided as SEQ ID NO: 645.

[0158] MMP9, which requires activation by MMP3, is overexpressed in the majority of solid tumors. * Monoclonal antibodies are known to recognize MUC1 after it has been cleaved by MMP9. Various breast, ovarian, pancreatic, and lung cancer tissue arrays shown in Figures 30-33 were probed with MNC2-scFv, further demonstrating that MUC1 in these cancers is cleaved by MMP9. To confirm whether tumor cleavage by MMP9 increases T cell access to tumors, a series of experiments were performed using full-length MUC1, HCT-MUC1-41TR, full-length MUC1, and MUC1. * Breast cancer cell lines highly expressing both MUC1 and MUC2 * 45 This was done using a cell line expressing MMP9 and a MUC1-negative cell line transfected with MMP3. The cells were transfected with MMP9 and MMP3, which activates MMP9. The supernatant of these cells, which contained activated MMP9, was collected and added to various cells, which were then transfected with anti-MUC1. * These cells were co-cultured with T cells transduced with CAR:huMNC2-CAR44. The results showed that the target MUC1 / MUC1 expression was significantly increased compared to control cells that were not incubated with the MUC1 cleaving enzyme. *CART cell killing of positive cancer cells was significantly increased.

[0159] APMA is a biochemical that activates MMPs. We used APMA with conditioned medium from cells transfected with either MMP9 or ADAM17 to determine whether either of these cleavage enzymes cleaves MUC1 in the HCT-MUC1-41TR cell line, which expresses only full-length MUC1. As a control, we used HCT-MUC1 * The enzymes on the cells were also tested: MUC1 and MUC1 * Expressing cells were stained with the red dye CMTMR. * Human T cells transduced with CAR, CAR44, or CAR50 were co-cultured with cancer cells. Non-transduced T cells were used as a control (Figures 45A-45P). As can be seen in Figures 45B, 45C, and 45D, anti-MUC1 * CAR T cells express HCT-MUC1, a signaling agent for T cell activation and killing. * CAR T cells efficiently recognized and clustered cancer cells. However, clustering induced by CAR T cells was not observed in wells containing full-length MUC1-expressing cells, HCT-MUC1-41TR (Figure 45F, Figure 45G, and Figure 45H). However, cells incubated with activated MMP9 showed a dramatic increase in CAR T cell-induced clustering (Figure 45J, Figure 45K, and Figure 45L), demonstrating that MMP9 inhibits full-length MUC1 and MUC1 recognized by the MNC2 monoclonal antibody, more specifically, huMNC2-scFv. * ADAM17 had no apparent effect. ADAM17 either did not cleave MUC1 or likely cleaved it at a position not recognized by MNC2 (Figures 45N-P).

[0160] We performed the same experiment and this time, we found that they were probably MUC1 * Not only MUC1 but also full-length MUC1 is expressed at high levels. *T47D breast cancer cells were used, which are difficult to kill using CAR T cells (Figures 46A-46T). As can be seen in Figures 46B, 46C, and 46D, anti-MUC1 * CAR44 and CAR50 have little effect on T47D cancer cells. Only in Figure 46D, where CAR44 is the highest level of CAR expression in T cells, is a small amount of CAR T cell-induced clustering observed. However, the presence of activated MMP2 (Figures 46J, 46K, 46L) or activated MMP9 (Figures 46R, 46S, 46T) dramatically increases CAR T cell recognition, clustering, and killing, indicating that cleavage of full-length MUC1 increases T cell access to cancer cells. The addition of APMA may increase cleavage by some other mechanism or anti-MUC1. * To ensure that it did not induce CAR T recognition, we generated a catalytically active form of MMP9, added it to T47D cells, and co-cultured it with MNC2-CAR44 T cells (Figures 47A-47I). As can be seen, MNC2-CAR T cells were able to recognize MUC1 * The IL-11 receptor mediated the expression of MUC1 and MUC1 in the IL-11 receptor. * MNC2 clusters poorly on T47D breast cancer cells expressing both MUC1 and MMP9 (Figures 47E-47F), but binds and clusters tightly to T47D cells after the addition of catalytically active MMP9 (Figures 47H-47I). This result suggests that MNC2 does not recognize full-length MUC1, but rather binds and clusters T47D cells expressing the growth factor receptor MUC1. * Note that full-length MUC1 expressed in this cell line may sterically hinder binding of CART cells near the cell membrane.

[0161] In another example, T47D MUC1-positive tumor cells were incubated with the recombinant catalytic domain of MMP9 (Enzo Life Sciences, Inc., Farmingdale, NY) at either 100 ng / mL or 500 ng / mL. Western blot analysis demonstrated a MUC1 / MUC1 * Positive cancer cells are MUC1 to MUC1 *In another example, T47D breast cancer cells were preincubated with human recombinant MMP9 catalytic domain protein and then treated with anti-MUC1 * T47D cells were co-cultured with CAR44 T cells. The specific killing of T47D cells by CAR44 T cells was monitored in real time using an xCelligence instrument, which measures impedance over time. This analysis uses an electrode array onto which cancer cells are seeded. Adherent cancer cells insulate the electrodes, causing an increase in impedance as they grow. Conversely, T cells, being unadherent and remaining free-floating, do not increase or decrease impedance. However, when T cells or CAR T cells kill cancer cells on the electrode plate, the cancer cells swell and float as they die, which decreases impedance. Addition of the MMP9 catalytic domain dramatically increased killing of T47D cancer cells. Figure 48 shows xCelligence graphs of T47D breast cancer cells co-cultured with either non-transduced T cells as a control or huMNC2-CAR44 T cells over a 45-hour period. After 18 hours of cancer cell growth, the catalytic subunit MMP9 was added to some cells. At 25 hours, T cells were added. As can be seen, killing of huMNC2-CAR44 T cells is significantly improved when T47D cells are preincubated with the cleaving enzyme MMP9. In the xCelligence system, adherent target cancer cells are plated on an electrode array plate. The adherent cells insulate the electrodes and increase impedance. The number of adherent cancer cells is directly proportional to the impedance. T cells are not adherent and do not contribute to impedance. Therefore, an increase in impedance reflects cancer cell growth, and a decrease in impedance reflects cancer cell death. The prostate cancer cell line DU145 expresses MUC1 and MUC1 * DU145 cells express both anti-MUC1 and anti-MUC1 cleavage enzymes, but at much lower levels than T47D cells. * It is efficiently killed by CART cells.

[0162] Figure 49 shows the xCelligence graph of DU145 prostate cancer cells co-cultured with either non-transduced T cells as a control or huMNC2-CAR44 T cells over a 45-hour period. After 18 hours of cancer cell growth, the catalytic subunit MMP9 was added to some cells. At 25 hours, T cells were added. As can be seen, low-density MUC1 / MUC1 * Killing of huMNC2-CAR44 T cells from MUC1-positive cancer cells is not affected by pre-incubation with the cleavage enzyme MMP9. * The low density of full-length MUC1 is due to the membrane-proximal MUC1. * DU145 cells represent an early stage cancer that expresses both full-length and truncated MUC1, but at low levels that do not sterically hinder T cell access. T47D cells express both MUC1 and MUC1. * HCT-MUC1 represents a type of intermediate-stage cancer that expresses high levels of both MUC1 and MUC1, and the density of full-length MUC1 sterically restricts T cell access to the tumor. * The cells were MUC1 * 45 This is a MUC1-negative cell line stably transfected with MMP9, representing late-stage cancer cells. * MUC1 is cleaved into anti-MUC1, the targeting head of CAR. *It is important that tumor antigens on the surface of cancer cells are recognized by the antibody MNC2. Access of immune cells to tumor antigens on the surface of cancer cells can be sterically hindered by the presence of large extracellular domain proteins or other interfering elements, also known as the tumor microenvironment. The foregoing serves as an example that can be extended to improve the efficacy of CART therapy targeting other tumor antigens. In one embodiment of the present invention, immune cells are transfected or transduced with both a CAR comprising an antibody fragment targeting a tumor antigen and a cleavage enzyme. In another embodiment of the present invention, immune cells are transfected or transduced with both a CAR comprising an antibody fragment targeting a tumor antigen and a cleavage enzyme that cleaves the tumor antigen into a form recognized by the antibody fragment of the CAR. In one embodiment, immune cells are transfected or transduced with both a CAR comprising an antibody fragment targeting a tumor antigen and a cleavage enzyme that cleaves the tumor antigen into a form recognized by the antibody fragment of the CAR, and the antibody fragment of the CAR is MUC1. * The cleavage enzyme recognizes the extracellular domain of MUC1 and cleaves it into MUC1 * In one embodiment, immune cells, which may be T cells or NK cells, are transfected or transduced with a CAR comprising an antibody fragment derived from MNC2, MNE6, MNC3, or MNC8 and a cleavage enzyme selected from the group comprising MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP11, MMP12, MMP13, MMP14, MMP16, ADAM9, ADAM10, ADAM17, ADAM19, ADAMTS16, ADAM28, or catalytically active fragments thereof.

[0163] In one embodiment of the present invention, a sufficient amount of immune cells transduced with both a CAR and a cleaving enzyme are administered to a person diagnosed with cancer or at risk of developing cancer. In another embodiment of the present invention, a sufficient amount of immune cells transduced with both a CAR and a cleaving enzyme are administered to a person diagnosed with cancer or at risk of developing cancer, wherein the cleaving enzyme is on an inducible promoter that is activated by a protein expressed when the immune cells are activated. In another embodiment of the present invention, a sufficient amount of immune cells transduced with both a CAR and a cleaving enzyme are administered to a person diagnosed with cancer or at risk of developing cancer, wherein the cleaving enzyme is on an inducible promoter that is activated by one or more NFATs. In some cases, the NFAT is NFATc1. In another embodiment, the NFAT is NFATc3. In another embodiment, the NFAT is NFATc2. In any of the above cases, the extracellular domain of the CAR comprises a fragment of an anti-MUC1* antibody. In one embodiment, the anti-MUC1 * The antibody is MNC2scFv or a humanized form of MNC2scFv. * The antibody is MNE6scFv or a humanized form of MNE6scFv. In any of the above cases, the immune cell can be a T cell, NK cell, mast cell, or dendritic cell.

[0164] This is not intended to limit the present invention to one or two specific methods of induced expression of a cleavage enzyme by activated T cells. The inventors demonstrated specific expression of a cleavage enzyme only upon T cell activation by constructing a plasmid containing a cleavage enzyme gene downstream of the NFAT promoter sequence or downstream of one or more repeats of an NFAT response element. In another embodiment of the present invention, cleavage enzyme expression is induced by constructing a plasmid in which the cleavage enzyme gene is inserted downstream of the IL-2 promoter sequence or downstream of the IL-2 response element, and then inserting the plasmid into immune cells. In another embodiment of the present invention, cleavage enzyme expression is induced by constructing a plasmid in which the cleavage enzyme gene is inserted downstream of the calcineurin promoter sequence or downstream of the calcineurin response element, then inserting the plasmid into immune cells, and then administering the plasmid to a patient to treat or prevent cancer. Drug-inducible plasmids also exist that can be used to induce cleavage enzyme expression or to silence expression induced by elements in activated T cells. These drug inducible systems may include tetracycline inducible systems, Tet-on, Tet-off, tetracycline response elements, doxycycline, tamoxifen inducible systems, ecdysone inducible systems, and the like.

[0165] This is not intended to limit the present invention to one or two specific promoters used in the plasmid encoding the CAR or inducible cleavage enzyme. As known to those skilled in the art, many promoters are interchangeable, including SV40, PGK1, Ubc, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, and U6. Another solution to the steric hindrance of CAR cell access caused by large cell surface proteins such as MUC1-FL is to increase the length of the linker region of the CAR expressed by T cells. In standard CAR designs, the extracellular linker region between the transmembrane portion and the antibody fragment is approximately 45-50 amino acids long. Long-arm CARs were created in which the extracellular linker length was extended from approximately 50 amino acids to 217-290 amino acids. Co-culture assays demonstrated that CARs containing longer extracellular linkers improved access to tumor-associated antigens on target cancer cells. BiTE

[0166] Divalent (or bivalent) single-chain variable fragments (di-scFv, bi-scFv) can be engineered by linking two scFvs. H Area and two V LThis can be achieved by generating a single peptide chain containing the variable regions, generating tandem scFvs. Another possibility is to create scFvs with a linker peptide (approximately five amino acids) that is too short for the two variable regions to fold together, forcing the scFv to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40-fold lower than their scFv counterparts, meaning they have much higher affinity for their targets. As a result, diabody drugs can be administered at much lower doses than other therapeutic antibodies and can target tumors highly specifically in vivo. Even shorter linkers (one or two amino acids) result in the formation of trimers, so-called tribodies or tribodies. Tetrabodies have also been produced; they exhibit even higher affinity for their targets than diabodies.

[0167] All of these formats can be composed of variable fragments with specificity for two different antigens, in this case they are a type of bispecific antibody. The most developed of these is the bispecific tandem di-scFv, known as bispecific T cell engaging (BiTE) antibody construct. BiTE is a fusion protein consisting of two scFvs of different antibodies on a single peptide chain of approximately 55 kilodaltons. One of the scFvs binds to T cells via, for example, the CD3 receptor, and the other binds to an abnormally expressed MUC1 receptor. * The antibody may bind to tumor cells via tumor-specific molecules such as:

[0168] Another aspect of the present invention is directed to MUC1-positive cancer or MUC1 * A method for treating a patient diagnosed with, suspected of developing, or at risk of developing a positive cancer, wherein the patient is administered an effective amount of a BiTE, wherein one antibody variable fragment of the BiTE binds to a T cell surface antigen and another antibody variable fragment of the BiTE binds to PSMGFR (SEQ ID NO: 2), more specifically the N-10 peptide. *Antibody variable fragments of BiTEs that bind to include a portion of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11.

[0169] In another embodiment of the present invention, a MUC1 comprising PSMGFR (SEQ ID NO: 2) * The peptide, or most or all of the N-10 peptide, is used in an adoptive T cell approach, in which the patient's T cells express MUC1 * Upon exposure to peptides and through various rounds of maturation, T cells express MUC1 * The adapted T cells are then expanded and develop specific receptors for MUC1. * It is administered to donor patients who have been diagnosed with, are suspected of having, or are at risk of developing positive cancer.

[0170] A series of CARs with MNC2 and humanized MNC2 as the extracellular targeting head were also produced. These CAR constructs were inserted into plasmids, which were then inserted into lentiviral vectors. Human T cells were then transduced with lentiviral vectors carrying MNC2CAR and huMNC2CAR. MNC2-scFv-CARs, either murine or humanized, were generated. In one embodiment of the present invention, the CAR comprised huMNC2-scFv-short hinge region-CD8-derived transmembrane domain-short intracellular fragment-4-1BB-3 zeta. In another embodiment, the transmembrane domain was derived from the CD4 transmembrane sequence. In another embodiment, the intracellular costimulatory domain was CD28-3 zeta. In yet another embodiment, the intracellular costimulatory domain was CD28-4-1BB-3 zeta.

[0171] There are various methods to assess whether T cells recognize target cells and are in the process of initiating an immune response. T cells cluster when they recognize target or foreign cells. This can be easily seen with the naked eye or at low magnification. The appearance of CART cell clusters when co-cultured with target cancer cells is one measure of: a) whether they recognize the cells as target cells; and b) whether they are activated to attack the target cells (cancer cells in this case). Figures 45-47 show the results of CART cell clusters stably transfected with mCherry or stained with CMTMR, hence the red, MUC1. * Photographs of positive T47D breast cancer cells are shown. These were co-cultured with either CAR-free human T cells or human T cells transduced with huMNC2-scFv-CAR44 or huMNC2-scFv-CAR50. CAR T cells are transparent. As can be seen, when the T cells do not carry a CAR, there is no T cell-induced clustering of cancer cells. However, when the T cells express MUC1 * MUC1 if carrying the target CAR * There is a dramatic clustering of positive cancer cells.

[0172] After T cells recognize and cluster target cells, they overexpress perforin and granzyme B. Together, these two molecules activate the cell death pathway of the target cell. Perforin is thought to create holes in the target cell into which the T cell injects granzyme B, which then activates apoptotic proteases and lyses the target cell. Figures 55 and 56 show that huMNC2-scFV-CAR44 T cells can target MUC1 * It has been shown that granzyme B binds to positive prostate and pancreatic cancer cells and is injected.

[0173] Another measure of whether T cells recognize target cells and are activated to kill them is the upregulation and secretion by the T cells of the cytokines interferon gamma (IFN-g) and interleukin-2 (IL-2). CAR T cell activation can be easily measured in vitro, as evidenced by IFN-g and IL-2 secretion. CAR T cells are co-cultured with target cells, and after an incubation period, the conditioned medium is assayed by ELISA to detect secreted IFN-g and IL-2. To determine the cancer specificity of CAR T cells, where the targeting head of the CAR is either huMNC2 or huMNE6, these experiments were performed using CAR T cells expressing either huMNC2 or huMNE6. * This study was performed using huMNC2-CAR44T cells and huMNE6-CAR44T cells co-cultured with MUC1-positive cancer cells and normal cells. Table 1 shows the details of the MUC1-positive normal or primary cells tested.

[0174] [Table 1-1]

[0175] [Table 1-2]

[0176] Figure 50 is a graph of PCR measurements of various cell lines tested to measure MUC1 mRNA levels. The cancer cell lines tested in these assays were HCT-MUC1 *and T47D breast cancer cells. These cells were cocultured with huMNC2-CAR44 human T cells. Coculture of huMNC2-CAR44 T cells with cancer cells induced the CAR T cells to secrete large amounts of IFN-γ and IL-2 into the surrounding medium, whereas coculture with MUC1-positive normal cells did not induce cytokine secretion (Figures 51 and 52). In addition to testing for IFN-γ and IL-2 secretion by CAR T cells, normal cells were assayed for signs of cell death, which may be induced by CAR T cells if the head-targeting antibodies are not extremely cancer-specific. After coculture with huMNC2-CAR44 T cells, the cells were incubated with cell death markers and then assayed by FACS. huMNC2-CAR44 T cells did not induce cell death in normal cells (Figures 53A-J).

[0177] In addition to FACS analysis, many researchers now use the xCELLigence instrument to measure CAR T killing of cancer cells. FACS is not the best method for tracking T cell-induced cell death because T cells lyse target cells. Dead cells are difficult to measure with FACS because they are excluded as cellular debris. Therefore, the amount of cell death must be estimated and various methods must be used to determine whether the lost cells are T cells or cancer cells.

[0178] The xCELLigence device uses an electrode array onto which cancer cells are seeded. The attached cancer cells insulate the electrodes, causing an increase in impedance as they grow. Conversely, T cells do not adhere and remain suspended, so they do not contribute to the insulation of the electrodes, which would increase impedance. However, when T cells or CAR T cells kill cancer cells on the electrode plate, the cancer cells swell and rise to the surface as they die, which reduces impedance. The xCELLigence device measures impedance as a function of time, which correlates with cancer cell death. Additionally, the electrode plate also has a viewing window. When CAR T cells effectively kill the attached target cancer cells, the impedance drops, but it can also be confirmed that no cancer cells remain on the plate surface.

[0179] Figures 55A-55H show the activity of MUC1 as measured by various assays. * Figure 55A shows the cytotoxic effect of huMNC2-CAR44 T cells on positive DU145 prostate cancer cells. Figure 55A is a fluorescent photograph of untransduced T cells co-cultured with prostate cancer cells, where granzyme B is stained with a red fluorophore. Figure 55C is a fluorescent photograph of huMNC2-CAR44 T cells co-cultured with prostate cancer cells, where granzyme B is stained with a red fluorophore. Figure 55D is a merge of DAPI and granzyme B. Figure 55E is a FACS scan of fluorescently labeled granzyme B for untransduced T cells incubated with cancer cells. Figure 55F is a FACS scan showing a positive increase in fluorescently labeled granzyme B for huMNC2-CAR44 T cells incubated with cancer cells. Figure 55G is a graph of mean fluorescence intensity. Figure 55H is an xCELLigence scan tracking in real time the killing of DU145 cancer cells by huMNC2-CAR44 T cells (blue trace), but not by non-transduced T cells (green). Figures 56A-56H show the MUC1 activity measured by various assays. *Figure 56A shows the cytotoxic effect of huMNC2-CAR44 T cells against positive CAPAN-2 pancreatic cancer cells. Figure 56A is a fluorescent photograph of untransduced T cells co-cultured with pancreatic cancer cells, where granzyme B is stained with a red fluorophore. Figure 56B is a merged image of DAPI and granzyme B. Figure 56C is a fluorescent photograph of huMNC2-CAR44 T cells co-cultured with pancreatic cancer cells, where granzyme B is stained with a red fluorophore. Figure 56D is a merged image of DAPI and granzyme B. Figure 56E is a FACS scan of fluorescently labeled granzyme B in untransduced T cells incubated with cancer cells. Figure 56F is a FACS scan showing a positive increase in fluorescently labeled granzyme B for huMNC2-CAR44 T cells incubated with cancer cells. Figure 56G is a graph of mean fluorescence intensity. Figure 56H is an xCELLigence scan tracking in real time the killing of CAPAN-2 cancer cells by huMNC2-CAR44 T cells (blue trace), but not by non-transduced T cells (green). Figures 57A-57C show the upregulation of MUC1 by huMNC2-CAR44 T cells. * MUC1 negative cells, but not * Figure 57A shows xCELLigence scans tracking the real-time killing of positive cancer cells. * Figure 57B shows that huMNC2-CAR44 T cells effectively kill HCT colon cancer cells stably transfected with MUC1. Figure 57B shows that huMNC2-CAR44 T cells have little effect on HCT-MUC1-41TR, MUC1-negative cancer cells stably transfected with MUC1 full-length. In this cell line, only about 10% of the cells express MUC1. * Figure 57C shows that huMNC2-CAR44 T cells have no effect on HCT-116 cells, ...

Claims

1. An antibody or a fragment thereof for the diagnosis, treatment or prevention of cancer, wherein the antibody specifically binds to PSMGFR peptide (SEQ ID NO: 2) or a fragment thereof of said peptide.

2. The antibody or fragment thereof of claim 1, which binds to N-10 peptide (SEQ ID NO: 3), N-19 peptide (SEQ ID NO: 4), N-23 peptide (SEQ ID NO: 5), N-26 peptide (SEQ ID NO: 6), N-30 peptide (SEQ ID NO: 7), N-10 / C-5 peptide (SEQ ID NO: 8), N-19 / C-5 peptide (SEQ ID NO: 9), or C-5 peptide (SEQ ID NO: 825).

3. 2. The antibody or fragment thereof of claim 1, which interacts with a peptide containing the conformational epitopes SVSDV (SEQ ID NO: 1751) and FPSA (SEQ ID NO: 1747) within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), and wherein mutation or deletion of FPFS (SEQ ID NO: 1747) disrupts binding of the antibody or fragment thereof to the N-26 peptide.

4. 2. The antibody or fragment thereof of claim 1, which interacts with a peptide comprising the conformational epitopes ASRYNLT (SEQ ID NO: 1745), SVSDV (SEQ ID NO: 1751), and FPSA (SEQ ID NO: 1747) within the N-19 sequence ASRYNLT ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4), and wherein mutation or deletion of ASRYNLT (SEQ ID NO: 1745) disrupts binding of the antibody or fragment thereof to the N-26 peptide.

5. The antibody or fragment thereof of claim 1, which does not bind to the C-10 peptide (sequence number 825).

6. The antibody or fragment thereof according to claim 5, which binds to the N-10 peptide (SEQ ID NO: 3) but does not bind to the C-10 peptide (SEQ ID NO: 825).

7. NME7 ＡＢ and MUC1 ＊ The antibody or fragment thereof according to claim 1, which inhibits the interaction between

8. NME7 ＡＢ and PSMGFR peptide (SEQ ID NO: 2).

9. NME7 ＡＢ The antibody or fragment thereof according to claim 2, which inhibits the interaction of the antibody or fragment thereof with N-10 peptide (SEQ ID NO: 3), N-19 peptide (SEQ ID NO: 4), N-23 peptide (SEQ ID NO: 5), N-26 peptide (SEQ ID NO: 6), N-30 peptide (SEQ ID NO: 7), N-10 / C-5 peptide (SEQ ID NO: 8), N-19 / C-5 peptide (SEQ ID NO: 9), or C-5 peptide (SEQ ID NO: 825).

10. The antibody or fragment thereof according to claim 1, wherein the antibody recognizes a MUC1 transmembrane enzymatic cleavage product.

11. The antibody or fragment thereof of claim 10, wherein the cleaving enzyme is MMP14 or MMP9 or a catalytically active fragment thereof of said enzyme.

12. 2. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof binds to PSMGFR (SEQ ID NO: 2) or a fragment thereof and the presence of an amino acid sequence within PSMGFR (SEQ ID NO: 2) induces binding of the antibody to the PSMGFR.

13. The antibody of claim 12, wherein the amino acid sequence of the binding conformation-inducing peptide is present in the N-10 peptide (SEQ ID NO: 3).

14. 13. The antibody or fragment thereof of claim 12, which does not bind to the linear form of the bound conformation-guiding peptide sequence, and wherein the linear form of the peptide is a denatured form.

15. 15. The antibody or fragment thereof of claim 14, wherein the binding conformation-inducing peptide sequence is in the N-26 peptide sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), and wherein a mutation or deletion of FPFS (SEQ ID NO: 1747) disrupts binding of the antibody or fragment thereof to the N-26 peptide.

16. 15. The antibody or fragment thereof of claim 14, wherein the binding conformation-inducing peptide sequence is located within the N-19 peptide sequence ASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4), and wherein a mutation or deletion of ASRYNLT (SEQ ID NO: 1745) disrupts binding of the antibody or fragment thereof to the N-19 peptide.

17. An antibody or fragment thereof for the diagnosis, treatment or prevention of cancer, wherein a binding-inducing peptide sequence is located within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6) and a mutation or deletion within FPFS (SEQ ID NO: 1747) disrupts binding of the antibody or fragment thereof to PSMGFR.

18. heavy chain CDR1 comprises a consensus sequence at least 90% identical to the following sequence: F or I at position 1, T at position 2, F at position 3, S at position 4, T, G, or R at position 5, Y at position 6, A, G, or T at position 7, M at position 8, and S at position 9; heavy chain CDR2 comprises T at position 1, I or S at position 2, I or S at position 3, G or R at position 5, G or A at position 6, T or I at position 9, Y at position 10, Y at position 11, P or S at position 12, and a consensus sequence at positions 13-17 that is at least 90% identical to the sequence DSVKG; the heavy chain CDR3 comprises a consensus sequence at least 90% identical to the following sequence: G, L, or N at position 2, G or T at position 4, Y at position 7, D or E at position 12, A at position 14, and Y at position 15; light chain CDR1 comprises a consensus sequence at least 90% identical to the following sequence: K or R at position 1, A or S at position 2, S at position 3, K or Q at position 4, S at position 5, L or V at position 6, L at position 7, T or S at position 10, Y at position 15, and I, L, or M at position 16; the light chain CDR2 comprises a consensus sequence at least 90% identical to the following sequence: L or W or S at position 1, A or T at position 2, S at position 3, N or T at position 4, L or R at position 5, E or A at position 6, and S at position 7; and The antibody or fragment thereof of claim 17, wherein the light chain CDR3 comprises a consensus sequence at least 90% identical to the sequence of Q at position 1, H or Q at position 2, S, Q or R at position 3, R, S or Y at position 4, E, L, or S at position 5, L or S at position 6, P or S at position 7, F or L at position 8 and T at position 9.

19. 1. An antibody or fragment thereof for the diagnosis, treatment or prevention of cancer, wherein a binding conformation-guiding peptide sequence is within said N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6) and wherein mutations or deletions within FPFS (SEQ ID NO: 1747), SVSDV (SEQ ID NO: 1751), or ASRYNLT (SEQ ID NO: 1745) disrupt binding of said antibody or fragment thereof to PSMGFR.

20. heavy chain CDR1 comprises a consensus sequence at least 90% identical to the following sequence: F or I at position 1, T or A at position 2, F at position 3, S at position 4, T, G, or R at position 5, Y or F at position 6, A, G, or T at position 7, M at position 8, and S at position 9; heavy chain CDR2 comprises a consensus sequence at least 90% identical to the sequence of T or A at position 1, I or S at position 2, I or S at position 3, N, S, T or G at position 4, G or R at position 5, G or A at position 6, G, T, or D at position 7, Y, K, H or S at position 8, T or I at position 9, Y or F at position 10, Y at position 11, P or S at position 12, and D at position 13, S or T at position 14, V or L at position 15, and KG at positions 16-17; the heavy chain CDR3 comprises a consensus sequence at least 90% identical to the following sequence at position 2: G, L, or N; at position 3: G, T, or Y; at position 4: G or T; at position 7: Y; at position 10: Y, A, or G; at position 11: M, D, or F; at position 12: D or E; and at positions 14-15: AY; light chain CDR1 comprises a consensus sequence at least 90% identical to the following sequence at position 1: K or R, at position 2: A or S, at position 3: S or R, at position 8: S, Y, I, or V, at position 10: T or S, at position 10: G, S, D, or Q, at position 12: V, Y, K, or N, at position 13: N, S, or T, at position 14: Y or F, and at position 15: I, L, or M; the light chain CDR2 comprises a consensus sequence at least 90% identical to the following sequence: A, T, or V at position 2, S at position 3, N, T, or K at position 4, L or R at position 5, E, A, F, or D at position 6, and S at position 7; and The antibody or fragment thereof of claim 17, wherein the light chain CDR3 comprises a consensus sequence at least 90% identical to the sequence: Q, F or W at position 1, H or Q at position 2, R, S, T, Y or N at position 4, E, L, S or H at position 5, L, S, V, D or Y at position 6, P or S at position 7, and T at position 9.

21. MNC2, heavy chain CDR1 containing the consensus sequence FTFSGYAMS; heavy chain CDR2 containing the consensus sequence TISSGGTYIYYPDSVKG; a heavy chain CDR3 containing the consensus sequence -LGGDNYYEYFDV--; Light chain CDR1 containing the consensus sequence RASKS--VSTSGYSYMH; A light chain CDR2 comprising the consensus sequence LASNLES; and Light chain CDR3 containing the consensus sequence QHSRELPFT The antibody or fragment thereof of claim 17, having the following structure:

22. MNE6, heavy chain CDR1 containing the consensus sequence FTFSRYGMS; heavy chain CDR2 containing the consensus sequence TISGGGTYIYYPDSVKG; a heavy chain CDR3 containing the consensus sequence DNYGRNYDYGMDY--; Light chain CDR1 containing the consensus sequence ---SATSSVSYIH; A light chain CDR2 comprising the consensus sequence STSNLAS; and Light chain CDR3 containing the consensus sequence QQRSSSPFT The antibody or fragment thereof of claim 17, having the following structure:

23. B2, heavy chain CDR1 containing the consensus sequence FAFSTTFAMS; heavy chain CDR2 containing the consensus sequence AISNGGGYTYYPDTLKG; Heavy chain CDR3 containing the consensus sequence ---RYYDLYFDL--; light chain CDR1 containing the consensus sequence RSSQNIV-HSNGNTYLE; A light chain CDR2 comprising the consensus sequence KVSNRFS; and Light chain CDR3 containing the consensus sequence FQDSHVPLT The antibody or fragment thereof of claim 17, having the following structure:

24. B7, heavy chain CDR1 containing the consensus sequence FTFSRYGMS; heavy chain CDR2 containing the consensus sequence TISSGGTYIYYPDSVKG; a heavy chain CDR3 containing the consensus sequence DNYGSSYDYAMDY--; Light chain CDR1 containing the consensus sequence RSSQTIV-HSNGNTYLE; A light chain CDR2 comprising the consensus sequence KVSNRFS; and Light chain CDR3 containing the consensus sequence FQDSHVPLT The antibody or fragment thereof of claim 17, having the following structure:

25. B9, heavy chain CDR1 containing the consensus sequence FTFSRYGMS; heavy chain CDR2 containing the consensus sequence TISSGGTYIYYPDSVKG; a heavy chain CDR3 containing the consensus sequence DNYGSSYDYAMDY--; Light chain CDR1 containing the consensus sequence ---SASSSVSYMH; A light chain CDR2 comprising the consensus sequence TTSNLAS; and Light chain CDR3 containing the consensus sequence QQRSSYPF- The antibody or fragment thereof of claim 17, having the following structure:

26. 8C7F3, heavy chain CDR1 containing the consensus sequence FTFSTYAMS; heavy chain CDR2 containing the consensus sequence AISNGGGYTYYPDSLKG; a heavy chain CDR3 containing the consensus sequence ---RYYDHYFDY--; Light chain CDR1 containing the consensus sequence—RASESVATYGNNFMQ; A light chain CDR2 comprising the consensus sequence LASTLDS; and Light chain CDR3 containing the consensus sequence QQNNEDPPT The antibody or fragment thereof of claim 17, having the following structure:

27. H11, heavy chain CDR1 containing the consensus sequence FAFSTTFAMS; heavy chain CDR2 containing the consensus sequence AISNGGGYTYYPDTLKG; Heavy chain CDR3 containing the consensus sequence ---RYYDLYFDL--; light chain CDR1 containing the consensus sequence RSSQNIV-HSNGNTYLE; A light chain CDR2 comprising the consensus sequence KVSNRFS; and Light chain CDR3 containing the consensus sequence FQDSHVPLT The antibody or fragment thereof of claim 17, having the following structure:

28. B12, heavy chain CDR1 containing the consensus sequence SYGVH; Heavy chain CDR2 containing the consensus sequence VIWPGGSTNYNSTLMSRM; A heavy chain CDR3 comprising the consensus sequence DRTPRVGAWFAY; and light chain CDR1 containing the consensus sequence RASESVATYGNNFMQ; A light chain CDR2 comprising the consensus sequence LASTLDS; and Light chain CDR2 containing the consensus sequence QQNNEDPPT The antibody or fragment thereof of claim 17, having the following structure:

29. 20A10, heavy chain CDR1 containing the consensus sequence FTFSTYAMS; Heavy chain CDR2 containing the consensus sequence -SIGRAGSTYYSDSVKG; Heavy chain CDR3 containing the consensus sequence ---GPIYNDYDEFAY; light chain CDR1 containing the consensus sequence KSSQSVLYSSNQKNYLA; A light chain CDR2 comprising the consensus sequence WASTRES; and Light chain CDR3 containing the consensus sequence HQYLSSLT The antibody or fragment thereof of claim 17, having the following structure:

30. 3C2B1, heavy chain CDR1 containing the consensus sequence ITFSTYTMS; heavy chain CDR2 containing the consensus sequence TISTGGDKTYYSDSVKG; Heavy chain CDR3 containing the consensus sequence -GTTAMYYYAMDY; Light chain CDR1 containing the consensus sequence RASKS---ISTSDYNYIH; A light chain CDR2 comprising the consensus sequence LASNLES; and a light chain CDR3 comprising the consensus sequence QHSRELPLT; The antibody or fragment thereof of claim 17, having the following structure:

31. An antibody or fragment thereof for the diagnosis, treatment or prevention of cancers which require the presence of the antibody-binding conformation-guiding peptide ASRYNLT (SEQ ID NO: 1745) of PSMGFR (SEQ ID NO: 2).

32. 25E6, heavy chain CDR1 containing the consensus sequence FTFSSYGMS; heavy chain CDR2 containing the consensus sequence TISNGGRHTFYPDSVKG; heavy chain CDR3 containing the consensus sequence QTGTEGWFAY; light chain CDR1 containing the consensus sequence KSSQSLLDSDGKTYLN; Light chain CDR2 containing the consensus sequence LVSKLDS_; Light chain CDR3 containing the consensus sequence WQGTHFPQT 32. The antibody or fragment thereof of claim 31 , having the following structure:

33. An antibody or fragment thereof for the diagnosis, treatment or prevention of cancer which requires the presence of the antibody-binding conformation-inducing peptide SVSDV (SEQ ID NO: 1761) of PSMGFR (SEQ ID NO: 2).

34. 5C6F3, heavy chain CDR1 containing the consensus sequence FTFSTYAMS; heavy chain CDR2 containing the consensus sequence AISNGGGYTYYPDSLKG; heavy chain CDR3 containing the consensus sequence RYYDHYFDY; Light chain CDR1 containing the consensus sequence RSSQTIVHSNGNTYLE; A light chain CDR2 comprising the consensus sequence KVSNRFS; and Light chain CDR3 containing the consensus sequence FQDSHVPLT 34. The antibody of claim 33, having the following structure:

35. 35. The antibody or fragment thereof of any one of claims 1 to 34, which is murine, camelid, human or humanized.

36. The antibody or fragment thereof according to any one of claims 1 to 34, wherein the antibody fragment is an scFv or scFv-Fc, the variable region of which may be murine, camel, human or humanized.

37. A chimeric antigen receptor (CAR) comprising the antibody fragment of claim 36.

38. 37. A chimeric antigen receptor (CAR) comprising the antibody fragment of claim 36, further comprising a mutation in the costimulatory domain or the CD3-zeta signaling domain.

39. The CAR of claim 38, wherein the tyrosine is mutated in CD28 or 4-1BB.

40. The CAR of claim 38, wherein the CD3-zeta comprises a 1XX mutation.

41. An immune cell comprising the CAR of claim 37.

42. 42. The immune cell of claim 41, which is a T cell, NK cell, dendritic cell, or mast cell.

43. A cell composition expressed in a cell comprising the CAR of claim 37 and a second entity having a biological recognition unit with different specificity from the CAR.

44. 44. The composition of claim 43, wherein the second entity binds to PD-1, PDL-1, or other checkpoint inhibitors.

45. 44. The composition of claim 43, wherein the second entity binds to NME7.

46. 44. The composition of claim 43, wherein the second entity is a cytokine.

47. 47. The composition of claim 46, wherein the cytokine is IL-12.

48. 47. The composition of claim 46, wherein the cytokine is IL-18.

49. 44. The composition of claim 43, wherein the second entity is c-Jun.

50. 50. An immune cell engineered to express a nucleic acid encoding a CAR of claim 37 and a nucleic acid encoding a second entity of any one of claims 1 to 49, wherein said second entity is expressed from an inducible promoter.

51. 51. The immune cell of claim 50, wherein the second entity is expressed from an inducible promoter that is activated by elements of an activated immune cell.

52. 52. The immune cell of claim 51, wherein the second entity is expressed from an NFAT-inducible promoter.

53. The immune cell of claim 52, wherein the NFAT is NFATc1, NFATc3 or NFATc2.

54. 51. The immune cell of claim 50, wherein the second entity is a cytokine.

55. The immune cell of claim 51, wherein the cytokine is IL-7, IL-15, or IL-18.

56. 51. The immune cell of claim 50, wherein the nucleic acid encoding the second entity is inserted into a Foxp3 promoter or enhancer region.

57. The immune cell of claim 56, wherein the cytokine is IL-18.

58. 57. The immune cell of claim 56, wherein the cytokine is expressed from an NFAT-inducible promoter.

59. A BiTE construct comprising the antibody fragment of claim 36.

60. 36. An antibody-drug conjugate (ADC) comprising the antibody or antibody fragment of claim 35.

61. specifically binds to PSMGFR (SEQ ID NO: 2) and N-10 (SEQ ID NO: 3); and (i) does not bind to full-length MUC1; (ii) does not bind to C-10 (SEQ ID NO: 825); (iii) MUC1 ＊ NME1 or NME7 to the extracellular domain or PSMGFR peptide ＡＢ competitively inhibits the binding of (iv) MUC1 generated by cleavage with a cleavage enzyme ＊ Recognize; (v) recognizes a conformational epitope and does not recognize a linear epitope; or (vi) is cancer-selective by tissue immunohistochemistry; An antibody or fragment thereof.

62. 62. The antibody or fragment thereof of claim 61, wherein four of the criteria (i) to (vi) are met.

63. 62. The antibody or fragment thereof of claim 61, wherein five of the criteria (i) to (vi) are met.

64. 62. The antibody or fragment thereof of claim 61, wherein six of the criteria (i) to (vi) are met.

65. 62. The antibody or fragment thereof of claim 61, wherein at least criterion (vi) is met.

66. The antibody or fragment thereof according to claim 61, wherein the cleaving enzyme is MMP-9.

67. The antibody or fragment thereof according to any one of claims 1 to 66, wherein the cancer is breast cancer, pancreatic cancer, ovarian cancer, lung cancer, colon cancer, gastric cancer or esophageal cancer.

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