Anti-variable MUC1* antibodies and uses thereof

Abstract

The present application discloses an antibody or a fragment thereof for diagnosing, treating or preventing cancer, wherein the antibody specifically binds to a PSMGFR peptide (SEQ ID NO: 2) or a fragment thereof of the peptide.

Description

[Technical Field]

[0001] The present application provides human, humanized and non-human anti-MUC1 * This application relates to 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 present 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 the growth factor receptor that drives the growth of more than 75% of all human cancers. * The truncated form of MUC1, termed the (prominent 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 dimers NME1, NME6, NME7, and NME7. AB , exposing binding sites for activating NME7-X1 or NME8 ligands. It is an ideal target for cancer drugs because it is aberrantly expressed in over 75% of all cancers and is likely overexpressed in a particularly high percentage of metastatic cancers (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 systemic rejection of the foreign antibody. The process of engineering recognition fragments of non-human antibodies into human antibodies is commonly referred to as "humanization." 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 to identify those that bind to the target antigen, rather than immunizing animals with the antigen. Another approach is to engineer the variable regions of antibodies into antibody-like molecules. Another approach involves immunizing humanized animals. The present invention also provides a method for the production of humanized antibodies that the inventors have identified as MUC1. * 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 shown promise 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 a tumor antigen linked to a T cell transmembrane domain, a signaling domain, and one or more costimulatory domains. When the receptor binds to a cancer-associated antigen, a signal is transmitted, leading to 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. When derived from a donor, immune cells can be mutated or engineered so that they do not induce the graft to fight host disease in the recipient. When CAR T cells bind to an antigen on a cancer cell, they attack the cancer cell and then expand the T cell population.

[0006] To date, CAR T therapy has been highly successful in treating hematologic cancers, but its efficacy against human solid tumors has yet to be demonstrated. Because most hematologic cancers are B-cell malignancies, CAR T cells can eliminate virtually all of a patient's B cells without causing serious harm to the patient. Solid tumors lack a B-cell equivalent. Most tumor-associated antigens are also expressed in normal tissues; they are simply expressed at higher levels in cancerous tissues. Therefore, the challenge is to develop antibodies that recognize epitopes of tumor-associated antigens that are somewhat different in the context of normal tissue versus tumor. To further minimize the risk of off-tumor / on-target killing of normal tissue, antibodies should recognize and bind to cancerous tissue at least twice as often as normal tissue. Less cancer-selective antibodies can be used therapeutically if they are inducibly expressed at the tumor site.

[0007] Another cancer therapy incorporating cancer-selective antibodies is a bispecific T cell engager, also known as a BiTE. The BiTE approach attempts to eliminate the CAR T-associated risk of extratumoral / off-target effects. Unlike CAR T, BiTEs are bispecific antibodies and should not pose any greater risk than conventional antibody-based therapies. However, unlike typical anti-cancer antibodies that bind to and block cancer antigens, BiTEs are designed to bind to antigens on tumor cells and simultaneously bind to antigens on 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). The drawback of BiTE technology is that, unlike CAR T cells, they do not proliferate in patients, resulting in limited 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 toxin precursor is linked to the cancer-selective antibody. Unlike CAR T cells, which use the natural killing of CD8+ T cells to kill cancer cells, ADCs deliver a toxic payload to tumors. Disadvantages of ADCs include the potential for delivering a toxic payload to normal cells and the fact that most ADCs require binding to cell surface molecules and then internalization after binding, resulting in cell death requiring approximately 10,000 surface molecules. Summary of the Invention

[0009] In one aspect, the invention provides a non-human, human, or humanized anti-MUC1 antibody that binds to a region in the extracellular domain of a MUC1 isoform or cleavage product that lacks the tandem repeat domain. * Antibodies, 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:

[0010] (i) the PSMGFR region of MUC1;

[0011] (ii) a PSMGFR peptide,

[0012] (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) (SEQ ID NO: 3)

[0013] (iv) a peptide having the following amino acid sequence:

[0014] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19) (SEQ ID NO: 4)

[0015] (v) a peptide having the following amino acid sequence:

[0016] NLTISDVSVSDVPFPFSAQSGA(N-23) (SEQ ID NO: 5)

[0017] (vi) a peptide having the following amino acid sequence:

[0018] ISDVSVSDVPFPFSAQSGA(N-26) (SEQ ID NO: 6)

[0019] (vii) a peptide having the following amino acid sequence:

[0020] SVSDVPFPFSAQSGA(N-30) (SEQ ID NO: 7)

[0021] (viii) a peptide having the following amino acid sequence:

[0022] QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5) (SEQ ID NO: 8)

[0023] (ix) a peptide having the following amino acid sequence:

[0024] ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) (SEQ ID NO: 9)

[0025] (x) a peptide having the following amino acid sequence:

[0026] FPFSAQSGA (SEQ ID NO: 10)

[0027] The non-human, human or humanized antibody may be IgG1, IgG2, IgG3, IgG4 or IgM. The human or humanized antibody fragment or antibody-like protein may be scFv or scFv-Fc.

[0028] The murine, camelid, human or humanized antibody, antibody fragment or antibody-like protein may be derived from murine monoclonal MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 and H11 antibodies and may comprise heavy and light chain variable regions having at least 80%, 90%, 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 with the sequence of the antibody heavy chain variable region specifically set forth herein in the Sequence Listing section, and the light chain variable regions of CDR1 and CDR2 may have at least 90%, 95%, or 98% sequence identity with the sequence of the antibody heavy chain variable region specifically set forth herein in the Sequence Listing section. The heavy chain variable region of CDR3 may have at least 80%, 85%, or 90% sequence identity with the sequence of the antibody heavy chain variable region specifically set forth herein in the Sequence Listing section, and the light chain variable region of CDR3 may have at least 80%, 85%, or 90% sequence identity with the sequence of the antibody heavy chain variable region specifically set forth herein in the Sequence Listing section.

[0029] The above-mentioned murine, camelid, human or humanized antibodies, antibody fragments or antibody-like proteins may comprise complementarity determining regions (CDRs) in the heavy and light chain variable regions that have at least 90%, 95%, or 98% sequence identity with the sequences of the specifically indicated antibody heavy chain CDR1, CDR2 or CDR3 regions and light chain CDR1, CDR2 or CDR3 regions described herein in the Sequence Listing section.

[0030] In another aspect, the invention provides an anti-MUC1 antibody, which can be any of the antibodies described above, that is composed of a sequence represented by a humanized IgG2 heavy chain or a humanized IgG1 heavy chain paired with a humanized kappa light chain or a humanized lambda light chain. *Regarding extracellular domain antibodies or anti-N-10 antibodies, the humanized IgG2 heavy chain can be SEQ ID NO: 55, the humanized IgG1 heavy chain can be SEQ ID NO: 58, the humanized kappa light chain can be SEQ ID NO: 110, and the humanized lambda light chain can be SEQ ID NO: 114, or a sequence having 90%, 95%, or 98% sequence identity thereto.

[0031] In another aspect, the invention provides an anti-MUC1 antibody, which can be any of the antibodies described above, that is composed of a sequence represented by a human IgG2 heavy chain or a human IgG1 heavy chain paired with a human kappa light chain or a human lambda light chain. * Regarding extracellular domain antibodies or anti-N-10 antibodies, the human IgG2 heavy chain can be SEQ ID NO: 55, the human IgG1 heavy chain can be SEQ ID NO: 58, the human kappa light chain can be SEQ ID NO: 110, and the human lambda light chain can be SEQ ID NO: 114, or sequences with 90%, 95%, or 98% sequence identity thereto.

[0032] In another aspect, the present invention provides an anti-MUC1 antibody comprised of a humanized MNC2 sequence represented by a humanized IgG1 heavy chain, a humanized IgG2 heavy chain, paired with a humanized lambda light chain and a humanized kappa light chain. * It relates to extracellular domain antibodies or anti-N-10 antibodies.

[0033] In another aspect, the invention provides an anti-MUC1 antibody comprised 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.

[0034] 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, in which they have the same or very similar binding pattern to a subset of peptides derived from PSMGFR peptides and do not recognize a linear epitope, and are similar to NME1 or NME7. AB MUC1 * competitively inhibits binding to MUC1, recognizes the MUC1 transmembrane cleavage product produced by cleavage by MMP9, or contains CDR sequences at least 80% homologous to the MNE6, MNC2, MN18G12, MN20A10, MN25E6, MN28F9, MN5C6F3, MN3C2B1, and MN1E4 CDR consensus sequences.

[0035] 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 sequences of the heavy and light chain complementarity determining regions of these antibodies are set forth herein in the Sequence Listing section.

[0036] 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 can be human or humanized. The antibody can be murine or camelid. The antibody can be bivalent or monovalent. The antibody can be a fragment containing one single-chain fragment (scFv) of the antibody. The antibody or antibody fragment can be administered directly to the patient or incorporated into a multispecific antibody-like molecule, a bispecific antibody, a bispecific T cell engager (BiTE), or an antibody-drug conjugate (ADC). The antibody or antibody fragment can be incorporated into a T cell receptor (TCR). The antibody or antibody fragment sequence can be incorporated into a chimeric antigen receptor (CAR) or other similar entity, 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 can be T cells or natural killer cells, can be derived from a donor or patient. In one embodiment, the immune cells are derived from stem cells that have been directed to differentiate into that immune cell type in vitro. In another embodiment, the immune cells are derived from stem cells that have been induced to differentiate into that immune cell type in vitro. In another embodiment, the CAR-containing sequence of an antibody is expressed in stem cells, which can then differentiate into immune cells. In one case, the immune cells are T cells. In another case, the immune cells are NK cells. In one embodiment, the antibody or the CAR-containing sequence of the antibody can be expressed from an inducible promoter. In one example, 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, recognition of the CAR on a target tumor cell activates the immune cell, resulting in NFAT-induced expression of a cytokine such as IL-12 or IL-18, or expression of a checkpoint inhibitor such as a PD1 inhibitor or a PDL-1 inhibitor. In yet another embodiment, recognition of the CAR on a target tumor cell activates the immune cell, resulting in NFAT-induced expression of a second CAR containing the sequence of a second antibody.

[0037] In another aspect, the present invention provides a method for the production of MUC1 *Mouse, camelid, human, and humanized anti-MUC1 antibodies that bind to the N-10 peptide described above inhibit the binding of NME protein to MUC1. * It relates to an antibody, an antibody fragment, or an antibody-like protein. NME includes NME1, NME6, NME7 AB , NME7-X1, NME7 or NME8.

[0038] In yet another aspect, the present invention provides a method for the treatment of MUC1 * The invention relates to single-chain variable fragments (scFv) comprising heavy and light chain variable regions connected via a linker, which 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 may have SEQ ID NO: 233, 235 or 237 (MNE6), SEQ ID NO: 238-243, 654-655 or 5017-5020 (MNC2), SEQ ID NO: 1574-1581 or 5001-5012 (20A10), SEQ ID NO: 1573 or 1813 (3C2B1), SEQ ID NO: 1385 or 1815 (5C6F3), SEQ ID NO: 1599 or 1601 (25E6).

[0039] In yet another embodiment, 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 lacking the tandem repeats, a linker molecule, a transmembrane domain, and a cytoplasmic domain.

[0040] (i) the PSMGFR region of MUC1;

[0041] (ii) a PSMGFR peptide,

[0042] (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) (SEQ ID NO: 3)

[0043] (iv) a peptide having the following amino acid sequence:

[0044] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19) (SEQ ID NO: 4)

[0045] (v) a peptide having the following amino acid sequence:

[0046] NLTISDVSVSDVPFPFSAQSGA(N-23) (SEQ ID NO: 5)

[0047] (vi) a peptide having the following amino acid sequence:

[0048] ISDVSVSDVPFPFSAQSGA(N-26) (SEQ ID NO: 6)

[0049] (vii) a peptide having the following amino acid sequence:

[0050] SVSDVPFPFSAQSGA(N-30) (SEQ ID NO: 7)

[0051] (viii) a peptide having the following amino acid sequence:

[0052] QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5) (SEQ ID NO: 8)

[0053] (ix) a peptide having the following amino acid sequence:

[0054] ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) (SEQ ID NO: 9)

[0055] (x) a peptide having the following amino acid sequence:

[0056] FPFSAQSGA(N-36) (SEQ ID NO: 10)

[0057] 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.Other antibodies from which single-chain antibody fragments can be produced include those that do not recognize linear epitopes or that have the same or very similar binding patterns to a subset of peptides derived from PSMGFR peptides, and those that do not recognize MUC1. * NME1 or NME7 AB or recognize the MUC1 transmembrane cleavage products produced by cleavage by MMP9, or contain CDR sequences 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.

[0058] In the above-described CARs, the extracellular domain may comprise a murine, camelid, human, non-human, or humanized single-chain antibody fragment of MNE6 scFv shown as SEQ ID NO: 233, 235, or 237, MNC2 scFv (SEQ ID NO: 239, 241, 243, 655, or 5017-5020), 20A10 scFv shown as SEQ ID NO: 1575, 1577, 1579, 1581, or 5001-5012, 3C2B1 scFv shown as SEQ ID NO: 1573 or 1813, 5C6F3 scFv shown as SEQ ID NO: 1385 or 1815, or 25E6 scFv shown as SEQ ID NO: 1599 or 1601.

[0059] In the process of humanizing an antibody, the sequence must be annotated to identify different functional regions, such as complementarity-determining regions (CDRs), framework regions, and constant regions. Various computer programs are available that assign specific sequences to CDRs, framework regions, and constant regions. Depending on the program used, the exact position where one region ends and the next region begins may vary by a few amino acids. Typically, a humanized single-chain construct, scFv, contains heavy and light chain CDRs supported by intervening framework regions, with the heavy and light chains connected via a flexible linker. Depending on the annotation program used, the sequence assigned to framework region IV may extend into the constant region. In some cases, extending framework region IV may confer greater stability to the scFv. Here, we provide sequences of humanized scFvs of the present invention in which the length of framework region IV of the light chain may vary. For example, in some cases, the C-terminus of framework region IV terminates with amino acids R and T. In other cases, it terminates with only R. In still other cases, both the R and T terminals are omitted. In the CARs described herein, the extracellular domain can comprise a murine, camelid, human, non-human, or humanized single chain antibody fragment with framework region IV having variable lengths shown as MNE6 scFv (SEQ ID NO: 5014 or 5016), MNC2 scFv (SEQ ID NO: 5018 or 5020), or 20A10 scFv (SEQ ID NO: 5002, 5004, 5006, 5008, 5010, or 5012) or 25E6 scFv (SEQ ID NO: 5030 or 5032).

[0060] In any of the CARs described herein, the cytoplasmic tail may be composed of one or more signaling sequence motifs and costimulatory domains, including, but not limited to, CD3-zeta, CD3-zeta-1XX, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7. Furthermore, the sequence of the intracellular signaling domain may contain mutations that attenuate the signal to improve persistence or improve killing of low antigen density tumor cells. The cytoplasmic tail may be composed of one or more signaling sequence motifs and costimulatory sequence motifs: CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7. The transmembrane and extracellular hinge regions of the CAR may or may not be derived from the sequence of the adjacent costimulatory domain. For example, a CAR containing a 4-1BB costimulatory domain can have transmembrane and hinge regions derived from CD8 or CD28. In another example, a CAR containing a CD28 costimulatory domain can have transmembrane and hinge regions derived from CD28. In any of the above CARs, the cytoplasmic tail can contain deletions or mutations that attenuate signaling. Such deletions or mutations in one or more of the three immunoreceptor tyrosine-based activation motifs, also known as ITAMs, increase the persistence of CAR-bearing cells and decrease their differentiation, as measured by an increase in the CD62L+CD45RA- population. Such mutations include, but are not limited to, tyrosines mutated to inhibit phosphorylation and signaling (Salter et al., 2018). In another embodiment, one or two ITAMs are deleted, leaving only one or two ITAMs (Feucht et al., 2019). In another embodiment, the position of one or more included ITAMs is moved closer to the costimulatory domain. Suitable ITAM configurations for increasing CAR persistence include, but are not limited to, 1XX, X2X, XX3, 12X, and 23X, where the numbers 1, 2, or 3 refer to ITAM1, ITAM2, or ITAM3, and X refers to a deletion of the ITAM in question.In a preferred embodiment, ITAM1, also known as 1XX, is the only functional ITAM included in the CAR construct. In any of the above CARs, the ITAMs of CD3-zeta can be deleted or mutated to inhibit or suppress signaling. In any of the above CARs, the CD3 cytoplasmic tail can contain deletions or mutations of ITAMs, including those called 1XX (Feucht et al., 2019; SEQ ID NOs: 1796-1797). In any of the above CARs, T cells can be engineered to overexpress c-Jun as a way to suppress T cell exhaustion (Lynn et al., 2019). The above CAR constructs can be expressed in T cells, NK cells, dendritic cells, or other immune cells, which can be autologous or allogeneic. Allogeneic cells can be derived from human stem cells.

[0061] In any of the above CARs, the CAR may comprise a sequence derived from antibody MNE6, including but not limited to (SEQ ID NOs: 12-13, 65-66, 56-57, 107-108, 341-342, 391-394); a sequence derived from antibody MNC2, including but not limited to (SEQ ID NOs: 118-119, 168-169, 144-145, 194-195, 654-655, 1788-1789); a sequence derived from antibody 20A10, including but not limited to (SEQ ID NOs: 988-989, 1004-1005, 1574-1581, 5001-5012, 1677, 1687); a sequence derived from antibody 3C2B1, including but not limited to (SEQ ID NOs: 1820-1823, 1572-1573, 1812-1813); The CAR may comprise a single chain antibody fragment, scFv, comprising a sequence derived from antibody 5C6F3, including but not limited to (SEQ ID NOs: 1816-1819, 1384-1385, 1814-1815), a sequence derived from antibody 25E6, including but not limited to (SEQ ID NOs: 1020-1021, 1036-1037, 1598-1601), the CAR hinge and transmembrane sequences may be derived from CD8 (SEQ ID NOs: 346 and 364) or CD28 (SEQ ID NOs: 350 and 368), and further comprise a costimulatory domain which may be 41BB (SEQ ID NO: 659) or CD28 (SEQ ID NO: 378), and the CD3-zeta signaling domain may be derived from (SEQ ID NO: 661) or may contain mutations including those referred to as 1XX (SEQ ID NOs: 1796-1797).

[0062] In any of the above CARs, the sequences are CAR MNE6 CD28 / CD3z (SEQ ID NO: 298), CAR MNE6 4-1BB / CD3z (SEQ ID NO: 301), CAR MNE6 OX40 / CD3z (SEQ ID NO: 617), CAR MNE6 CD28 / 4-1BB / CD3z (SEQ ID NO: 304), CAR MNE6 CD28 / OX40 / CD3z (SEQ ID NO: 619), CAR MNC2 CD3z (SEQ ID NO: 607), CAR MNC2 CD8 hinge / transmembrane CD28 / CD3z SEQ ID NO: 609), CAR MNC2 CD8 hinge / transmembrane 4-1BB / CD3z (SEQ ID NO: 611 and SEQ ID NO: 719), CAR MNC2 CD8 hinge / transmembrane 4-1BB / 1XX (SEQ ID NO: 1619 and SEQ ID NO: 1621), CAR MNC2 CD8 hinge / transmembrane CD28 / 1XX (SEQ ID NO: 1623 and SEQ ID NO: 1625), CAR MNC2 CD28 hinge / transmembrane CD28 / 1XX (SEQ ID NO: 5042 and SEQ ID NO: 5044), CAR MNC2 OX40 / CD3z (SEQ ID NO: 613), CAR MNC2 CD28 / 4-1BB / CD3z (SEQ ID NO: 307), CAR MNC2 CD28 / OX40 / CD3z (SEQ ID NO: 615), CAR 20A10 CD8 hinge / transmembrane 4-1BB / CD3z (SEQ ID NO: 1583 and SEQ ID NO: 1585), CAR 20A10 CD8 hinge / transmembrane CD28 / CD3z (SEQ ID NO: 1587 and SEQ ID NO: 1589), CAR 20A10 CD8 hinge / transmembrane 4-1BB / 1XX (SEQ ID NO: 1591 and SEQ ID NO: 1593), CAR 20A10 It can be CD8 hinge / transmembrane CD28 / 1XX (SEQ ID NO: 1595 and SEQ ID NO: 1597), CAR 20A10 CD28 hinge / transmembrane CD28 / CD3z (SEQ ID NO: 5022 and SEQ ID NO: 5024), CAR 20A10 CD28 hinge / transmembrane CD28 / 1XX (SEQ ID NO: 5026 and SEQ ID NO: 5028), or CAR MNC3 4-1BB / CD3z (SEQ ID NO: 601).

[0063] In another aspect, the present invention relates to a composition comprising at least two CARs with different extracellular domain units transfected into the same cell, which may be an immune cell derived from a patient in need of cancer treatment. Expression of the second CAR may be inducible and 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 induce cells to recognize MUC1. * The antibody fragments of the first and second CARs can target MUC1-positive tumors. The antibody fragments of the first and second CARs target MUC1, which is produced 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 in the tumor. * The NFAT protein can be induced upon engagement or binding to the NFAT protein. One way to achieve this is to induce expression of the second CAR when, or shortly 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 for the second CAR. In this way, if 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 transcription, they will also bind to the same promoter engineered 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 upstream transcriptional regulatory region for NFAT2. The recognition unit of the second CAR can be an antibody fragment or a peptide, and the recognition unit can bind to NME7, PD-1, PDL-1, or a checkpoint inhibitor.

[0064] At least two CARs may have one CAR that 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 may lack transmembrane and signal transduction motifs. 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 to 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, an antibody fragment, or an scFv. The other recognition unit may be a CAR or an anti-NME7 antibody.

[0065] 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 a cell comprising a CAR having an extracellular domain that binds to a transfected or transduced cell. 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.

[0066] In another aspect, the invention relates to engineered antibody-like proteins.

[0067] In another aspect, the invention relates to a method of treating a disease in a subject, the method comprising administering an antibody of any of the above claims to a person 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.

[0068] 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 of a MUC1 isoform or cleavage product lacking the tandem repeat domain. In a preferred embodiment, the antibody or antibody fragment binds to the N-10 peptide (SEQ ID NO: 3) but not the C-10 peptide (SEQ ID NO: 825). The variable domain fragment may be derived from the murine monoclonal antibody MNE6 (SEQ ID NOs: 13 and 66) or humanized MNE6 (SEQ ID NOs: 39 and 94) or MNE6 scFv (SEQ ID NOs: 233, 235, and 237). Alternatively, the variable domain fragment may be derived from the murine monoclonal antibody MNC2 (SEQ ID NOs: 119 and 169) or humanized MNC2 (SEQ ID NOs: 145 and 195) or MNC2 scFv (SEQ ID NOs: 239, 241, and 243). Alternatively, the variable domains can be derived from monoclonal antibodies MN18G12, MN20A10, MN25E6, MN28F9, MN5C6F3, MN3C2B1, or MN1E4, the heavy and light chain complementarity determining region sequences of which are also set forth in the sequence listing herein.

[0069] In another aspect, the present invention provides a method for the production of MUC1 or MUC1 * diagnosed with MUC1 or MUC1 positive cancer * suspected of having cancer or have a positive MUC1 or MUC1 * The present invention relates to a method of treating a person at risk of developing a human positive cancer, comprising administering to the person an effective amount of an antibody, antibody fragment or scFv as described above, wherein the species can be murine, camelid, human or humanized.

[0070] In another aspect, the invention relates to a polypeptide comprising at least two different scFv sequences, one of which binds to the extracellular domain of a MUC1 isoform or cleavage product lacking the tandem repeat domain. The polypeptide is capable of binding to:

[0071] (i) the PSMGFR region of MUC1;

[0072] (ii) a PSMGFR peptide,

[0073] (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) (SEQ ID NO: 3)

[0074] (iv) a peptide having the following amino acid sequence:

[0075] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19) (SEQ ID NO: 4)

[0076] (v) a peptide having the following amino acid sequence:

[0077] NLTISDVSVSDVPFPFSAQSGA(N-23) (SEQ ID NO: 5)

[0078] (vi) a peptide having the following amino acid sequence:

[0079] ISDVSVSDVPFPFSAQSGA(N-26) (SEQ ID NO: 6)

[0080] (vii) a peptide having the following amino acid sequence:

[0081] SVSDVPFPFSAQSGA(N-30) (SEQ ID NO: 7)

[0082] (viii) a peptide having the following amino acid sequence:

[0083] QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5) (SEQ ID NO: 8)

[0084] (ix) a peptide having the following amino acid sequence:

[0085] ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) (SEQ ID NO: 9)

[0086] (x) a peptide having the following amino acid sequence:

[0087] FPFSAQSGA(N-36) (SEQ ID NO: 10)

[0088] In another aspect, the present invention provides a method for the production of MUC1 * The present invention relates to a method for detecting the presence of a cell that abnormally expresses an antibody, antibody fragment, or scFv-Fc, the method comprising contacting a cell or tissue sample with the antibody, antibody fragment, or scFv-Fc described above, and detecting the presence of binding of the antibody, antibody fragment, or scFv-Fc to the cell. The cell may be a cancer cell.

[0089] In another aspect, the invention provides a method of testing a subject's cancer for suitability for treatment with an antibody of the invention, which may be a murine, camelid, human, or humanized antibody, or a fragment thereof, or a composition comprising a portion of the variable region of antibody MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, comprising contacting a physical specimen from the patient with the antibody in vitro, ex vivo, or in vivo, and determining whether the patient has MUC1 or MUC2+ / MUC1 ... * and concluding that the patient's cancer will respond beneficially to treatment with an agent comprising the antibody or fragment thereof. Antibodies used for these diagnoses may be conjugated to an imaging agent.

[0090] In another aspect, the present invention provides a method of treating a subject suffering from a disease, comprising inducing immune cells, which may be T cells or NK cells, from the subject or from a donor to express MUC1 * Exposure to peptides allows T cells or NK cells to express MUC1 through various maturation cycles. * expressing specific receptors, generating adoptive T cells or NK cells, and MUC1 * Diagnosed with MUC1 positive cancer * Suspected of having a positive cancer or MUC1 *and expanding and administering the adoptive cells to a donor patient at risk of developing MUC1 positive cancer. * The peptide is selected from the following group:

[0091] (i) the PSMGFR region of MUC1;

[0092] (ii) a PSMGFR peptide,

[0093] (iii) a peptide having the following amino acid sequence: QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10)

[0094] (iv) a peptide having the following amino acid sequence:

[0095] ASRYNLTISDVSVSDVPFPFSAQSGA(N-19)

[0096] (v) a peptide having the following amino acid sequence:

[0097] NLTISDVSVSDVPFPFSAQSGA(N-23)

[0098] (vi) a peptide having the following amino acid sequence:

[0099] ISDVSVSDVPFPFSAQSGA(N-26)

[0100] (vii) a peptide having the following amino acid sequence:

[0101] SVSDVPFPFSAQSGA(N-30)

[0102] (viii) a peptide having the following amino acid sequence:

[0103] QFNQYKTEAASRYNLTISDDVSVSDVPFPFS(N-10 / C-5)

[0104] (ix) a peptide having the following amino acid sequence:

[0105] ASRYNLTISDDVSVSDVPFPFS(N-19 / C-5)

[0106] (x) a peptide having the following amino acid sequence:

[0107] FPFSAQSGA(N-36)

[0108] In one aspect of the invention, MUC1 or MUC1 * Antibodies administered to patients to treat or prevent positive cancers are selected for their ability to bind to the N-10 peptide of PSMGFR. When the scFv or antibody fragment can be incorporated into a CAR, BiTE, or ADC, the antibody can be administered alone as a monovalent antibody.

[0109] In another aspect of the invention, MUC1 or MUC1 * Antibodies administered to patients for the treatment or prevention of HIV-positive cancers are selected for their ability to bind to the QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10) peptide, where the presence of the FPFSAQSGA(N-36) sequence is required for binding. When the scFv or antibody fragment can be incorporated into a CAR, BiTE, or ADC, the antibody can be administered alone as a monovalent antibody.

[0110] In one aspect of the invention, MUC1 or MUC1 * Antibodies administered to patients for the treatment or prevention of MUC1 or MUC1 positive cancers * When the scFv or antibody fragment can be incorporated into a CAR, BiTE, or ADC, the antibody can be administered alone as a monovalent antibody.

[0111] 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. Antibodies can be administered alone as monovalent antibodies, while scFvs, bispecific antibodies, multispecific antibodies, or antibody fragments can be incorporated into BiTEs, ADCs, or CARs that can be expressed in immune cells.

[0112] In one aspect of the invention, MUC1 or MUC1 * The antibody administered to patients for the treatment or prevention of MUC1-positive cancers binds to the extracellular domain of MUC1 lacking the tandem repeats and NME7. AB or selected for its ability to competitively inhibit binding of NME7-X1. The antibody can be administered alone, can be administered as an antibody, a monovalent antibody, an scFv, or a bispecific antibody, a multispecific antibody, or an antibody fragment can be incorporated into a BiTE, an ADC, or a CAR that can be expressed in immune cells.

[0113] In another aspect, the invention features a method of treating cancer in a patient comprising administering to the patient an antibody, antibody fragment, BiTE, ADC, or CAR expressed in any of the above immune cells in combination with a checkpoint inhibitor.

[0114] In the above methods, any of the following antibodies or variable regions thereof may be used: MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 or H11.

[0115] In the above method, any of the following variable regions can be used:

[0116] (i) Anti-MUC1 consisting of the sequence of humanized MN-E6 represented by a humanized IgG2 or IgG1 heavy chain paired with a humanized kappa or lambda light chain. * extracellular domain antibody or anti-N-10 antibody,

[0117] (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;

[0118] (iii) anti-MUC1 consisting of the sequence of humanized MN-C2 represented by a humanized IgG1 heavy chain, a humanized IgG2 heavy chain paired with a humanized lambda light chain and a humanized kappa light chain; * extracellular domain antibody or anti-N-10 antibody,

[0119] (iv) the antibody of (iii), which is a humanized IgG1 heavy chain MN-C2 (SEQ ID NO: 159) or IgG2 heavy chain (SEQ ID NO: 164) paired with a lambda light chain (SEQ ID NO: 219) or a kappa light chain (SEQ ID NO: 213), or a sequence with 90%, 95%, or 98% sequence identity thereto;

[0120] 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 (shown as SEQ ID NO: 233, 235, or 237), MN-C2 scFv (SEQ ID NO: 239, 241, or 243). In CARs, the cytoplasmic tail can be composed of one or more signaling sequence motifs and costimulatory domains, including, but not limited to, CD3-zeta-1XX, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7, and CD3-zeta or variants 1XX, X2X, XX3, 12X, or 23X. Additionally, the sequence of the intracellular signaling domain can contain mutations that attenuate the signal to improve persistence or target cell killing.

[0121] The above method can include 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.

[0122] 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.

[0123] The present invention relates to an antibody or a fragment thereof that specifically binds to a PSMGFR peptide (SEQ ID NO: 2) or a fragment of the peptide for the diagnosis, treatment or prevention of cancer.

[0124] 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).

[0125] The antibody interacts with a peptide containing the conformational epitopes SVSDV (SEQ ID NO: 1751) and FPFSA (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.

[0126] The antibody interacts with a peptide containing the conformational epitopes ASRYNLT (SEQ ID NO: 1745), SVSDV (SEQ ID NO: 1751), and FPFSA (SEQ ID NO: 1747) 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-26 peptide.

[0127] The antibody does not bind to the C-10 peptide (SEQ ID NO: 825).

[0128] The antibody binds to the N-10 peptide (SEQ ID NO:3) but not to the C-10 peptide (SEQ ID NO:825).

[0129] The antibody is NME7 AB and MUC1 * inhibits the interaction between

[0130] The antibody is NME7 AB and the PSMGFR peptide (SEQ ID NO: 2).

[0131] The antibody is NME7 AB and 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).

[0132] The antibody recognizes the MUC1 transmembrane enzyme cleavage product.

[0133] In the above, the cleavage enzyme is the enzyme MMP14 or MMP9 or a catalytically active fragment thereof.

[0134] 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.

[0135] The amino acid sequence of the conformational binding-inducing peptide is present in the N-10 peptide (SEQ ID NO: 3).

[0136] The antibody does not bind to the conformationally bonded derived peptide sequence in its linear form, the linear form of the peptide being the denatured form.

[0137] The conformational binding-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.

[0138] The conformational binding-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.

[0139] The binding-inducing peptide sequence may be located within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), where mutations or deletions within FPFS (SEQ ID NO: 1747) disrupt binding of the antibody or fragment thereof to PSMGFR.

[0140] The antibody may have a consensus sequence.

[0141] the heavy chain CDR1 comprises a consensus sequence at least 90% identical to the 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;

[0142] the heavy chain CDR2 comprises a consensus sequence at least 90% identical to the sequence: 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 DSVKG at positions 13-17;

[0143] the heavy chain CDR3 comprises a consensus sequence at least 90% identical to the 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;

[0144] the light chain CDR1 comprises a consensus sequence at least 90% identical to the 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;

[0145] the light chain CDR2 comprises 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; and

[0146] The light chain CDR3 contains a consensus sequence at least 90% identical to the 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.

[0147] An antibody that binds to a conformational epitope within a peptide having the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), wherein a mutation or deletion within FPFS (SEQ ID NO: 1747), SVSDV (SEQ ID NO: 1751) or ASRYNLT (SEQ ID NO: 1745) disrupts binding of the antibody or fragment thereof to PSMGFR.

[0148] The antibody may have additional consensus sequences, in this case, the heavy chain CDR1 comprises a consensus sequence at least 90% identical to the 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; the heavy chain CDR2 comprises a consensus sequence at least 90% identical to the sequence: 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 sequence: 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 AY at positions 14-15; the light chain CDR1 comprises a consensus sequence at least 90% identical to the sequence: K or R at position 1, A or S at position 2, S or R at position 3, S, Y, I or V at position 8, 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 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 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 light chain CDR3 contains 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.

[0149] The antibody is MNC2, heavy chain CDR1 comprises the consensus sequence FTFSGYAMS; heavy chain CDR2 comprises the consensus sequence TISSGGTYIYYPDSVKG; heavy chain CDR3 comprises the consensus sequence -LGGDNYYEYFDV--; light chain CDR1 comprises the consensus sequence RASKS-VSTSGYSYMH; the light chain CDR2 comprises the consensus sequence LASNLES, and The light chain CDR3 contains the consensus sequence QHSRELPFT.

[0150] The antibody is MNE6, heavy chain CDR1 comprises the consensus sequence FTFSRYGMS; heavy chain CDR2 comprises the consensus sequence TISGGGTYIYYPDSVKG; heavy chain CDR3 comprises the consensus sequence DNYGRNYDYGMDY--; light chain CDR1 comprises the consensus sequence -------SATSSVSYIH; the light chain CDR2 comprises the consensus sequence STSNLAS, and The light chain CDR3 contains the consensus sequence QQRSSSPFT.

[0151] The antibody is B2, heavy chain CDR1 comprises the consensus sequence FAFSTFAMS; heavy chain CDR2 contains the consensus sequence AISNGGGYTYYPDTLKG; heavy chain CDR3 comprises the consensus sequence ----RYYDLYFDL--, light chain CDR1 comprises the consensus sequence RSSQNIV-HSNGNTYLE; the light chain CDR2 comprises the consensus sequence KVSNRFS, and The light chain CDR3 contains the consensus sequence FQDSHVPLT.

[0152] The antibody is B7, heavy chain CDR1 comprises the consensus sequence FTFSRYGMS; heavy chain CDR2 comprises the consensus sequence TISSGGTYIYYPDSVKG; heavy chain CDR3 comprises the consensus sequence DNYGSSYDYAMDY--; light chain CDR1 comprises the consensus sequence RSSQTIV-HSNGNTYLE; the light chain CDR2 comprises the consensus sequence KVSNRFS, and The light chain CDR3 contains the consensus sequence FQDSHVPLT.

[0153] The antibody is B9, heavy chain CDR1 comprises the consensus sequence FTFSRYGMS; heavy chain CDR2 comprises the consensus sequence TISSGGTYIYYPDSVKG; heavy chain CDR3 comprises the consensus sequence DNYGSSYDYAMDY--; light chain CDR1 comprises the consensus sequence -------SASSSVSYMH; the light chain CDR2 comprises the consensus sequence TTSNLAS, and The light chain CDR3 contains the consensus sequence QQRSSYPF-.

[0154] The antibody is 8C7F3, heavy chain CDR1 comprises the consensus sequence FTFSTYAMS; heavy chain CDR2 comprises the consensus sequence AISNGGGYTYYPDSLKG; heavy chain CDR3 comprises the consensus sequence ----RYYDHYFDY--, light chain CDR1 comprises the consensus sequence --RASESVATYGNNFMQ; the light chain CDR2 comprises the consensus sequence LASTLDS, and The light chain CDR3 contains the consensus sequence QQNNEDPPT.

[0155] The antibody is H11, heavy chain CDR1 comprises the consensus sequence FAFSTFAMS; heavy chain CDR2 contains the consensus sequence AISNGGGYTYYPDTLKG; heavy chain CDR3 comprises the consensus sequence ----RYYDLYFDL--, light chain CDR1 comprises the consensus sequence RSSQNIV-HSNGNTYLE; the light chain CDR2 comprises the consensus sequence KVSNRFS, and The light chain CDR3 contains the consensus sequence FQDSHVPLT.

[0156] The antibody is B12, heavy chain CDR1 comprises the consensus sequence SYGVH; heavy chain CDR2 comprises the consensus sequence VIWPGGSTNYNSTLMSRM; the heavy chain CDR3 comprises the consensus sequence DRTPRVGAWFAY, and light chain CDR1 comprises the consensus sequence RASESVATYGNNFMQ; the light chain CDR2 comprises the consensus sequence LASTLDS, and The light chain CDR3 contains the consensus sequence QQNNEDPPT.

[0157] The antibody is 20A10, heavy chain CDR1 comprises the consensus sequence FTFSTYAMS; heavy chain CDR2 comprises the consensus sequence -SIGRAGSTYYSDSVKG; heavy chain CDR3 contains the consensus sequence ---GPIYNDYDEFAY; light chain CDR1 comprising the consensus sequence KSSQSVLYSSNQKNYLA; the light chain CDR2 comprises the consensus sequence WASTRES, and The light chain CDR3 contains the consensus sequence HQYLSSLT.

[0158] The antibody is 3C2B1, heavy chain CDR1 comprises the consensus sequence ITFSTYTMS; heavy chain CDR2 contains the consensus sequence TISTGGDKTYYSDSVKG; heavy chain CDR3 comprises the consensus sequence -GTTAMYYYAMDY; light chain CDR1 containing the consensus sequence RASKS---ISTSDYNYIH; the light chain CDR2 comprises the consensus sequence LASNLES, and The light chain CDR3 contains the consensus sequence QHSRELPLT.

[0159] 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 conformation binding-inducing peptide ASRYNLT (SEQ ID NO: 1745) of PSMGFR (SEQ ID NO: 2). heavy chain CDR1 comprises the consensus sequence FTFSSYGMS; heavy chain CDR2 comprises the consensus sequence TISNGGRHTFYPDSVKG; heavy chain CDR3 comprises the consensus sequence QTGTEGWFAY; light chain CDR1 comprises the consensus sequence KSSQSLLDSDGKTYLN; the light chain CDR2 comprises the consensus sequence LVSKLDS_, and The light chain CDR3 contains the consensus sequence WQGTHFPQT.

[0160] 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 conformation binding derivative peptide SVSDV (SEQ ID NO: 1761) of PSMGFR (SEQ ID NO: 2). heavy chain CDR1 comprises the consensus sequence FTFSTYAMS; heavy chain CDR2 comprises the consensus sequence AISNGGGYTYYPDSLKG; heavy chain CDR3 comprises the consensus sequence RYYDHYFDY; light chain CDR1 comprises the consensus sequence RSSQTIVHSNGNTYLE; the light chain CDR2 comprises the consensus sequence KVSNRFS, and The light chain CDR3 contains the consensus sequence FQDSHVPLT.

[0161] Antibodies or fragments thereof according to all of the above may be murine, camelid, human or humanized. The antibody fragment may be an scFv or scFv-Fc, the variable regions of which may be murine, camelid, human or humanized.

[0162] In another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising the above-described antibody fragment, which may further include a mutation in the costimulatory domain or a mutation or deletion of one or two ITAMs in the CD3-zeta signaling domain. The tyrosine may be mutated in CD28 or 4-1BB. CD3-zeta may contain a single ITAM, such as a sole ITAM1, also known as 1XX, ITAM2, also known as X2X, or ITAM3, also known as XX3. In another aspect, CD3-zeta may contain two ITAMs, and the position of the ITAM may be moved to a more proximal position, such as 12X or 23X (Feucht et al., 2019). In yet another aspect, the tyrosines in one or two ITAMs may be mutated to attenuate signaling. In a preferred embodiment, the CD3-zeta domain is 1XX. Examples of 1XX mutations include those exemplified in SEQ ID NOs: 1796-1797.

[0163] In another aspect, the present invention relates to an immune cell comprising the above-mentioned CAR.The immune cell can be a T cell, a NK cell, a dendritic cell or a mast cell.In one aspect, the immune cell is derived from a stem cell that is directed to differentiate into the immune cell type in vitro.In another aspect, the CAR-containing sequence of an antibody is expressed in a stem cell, which can then be differentiated into an immune cell.

[0164] 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 different specificity from the CAR, the second entity being capable of binding to PD-1, PDL-1 or other checkpoint inhibitors, or NME7, or cytokines such as IL-12 or IL-18, or c-Jun.

[0165] 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 can be expressed from an inducible promoter activated by elements of activated immune cells. The second entity can be expressed from an NFAT inducible promoter. The NFAT can be NFATc1, NFATc3, or NFATc2. The second entity can be a cytokine such as IL-7, IL-15, or IL-18. The nucleic acid encoding the second entity can be inserted into a Foxp3 promoter or enhancer region, wherein the cytokine is IL-18. The cytokine can be expressed from an NFAT inducible promoter.

[0166] In another aspect, the invention relates to a BiTE construct comprising the antibody fragment described above.

[0167] In yet another aspect, the invention relates to an antibody drug conjugate (ADC) comprising the above-described antibody or antibody fragment.

[0168] The present invention particularly relates to an antibody or fragment thereof which is (i) binds to PSMGFR (SEQ ID NO: 2) and N-10 (SEQ ID NO: 3); and Does not bind to full-length MUC1 (ii) does not bind to C-10 (SEQ ID NO: 825); (iii) MUC1 * NME1 or NME7 with extracellular domain or PSMGFR peptide AB competitively inhibits the binding of (iv) MUC1 generated by cleavage with a cleavage enzyme * Recognize, (v) recognizes conformational epitopes and not linear epitopes; or (vi) It is cancer-selective by immunohistochemistry on tissues.

[0169] It may meet four of criteria (i)-(vi). It may meet five of criteria (i)-(vi). It may meet six of criteria (i)-(vi). It may meet at least criterion (vi). The cleavage enzyme may be MMP-9.

[0170] In all of the above, the cancer may be breast cancer, pancreatic cancer, ovarian cancer, lung cancer, colon cancer, stomach cancer or esophageal cancer.

[0171] The present invention also provides a method for the treatment of MUC1-related diseases. * The present invention relates to methods of diagnosing, treating, or preventing cancer by administering the antibodies and fragments disclosed herein, which have been identified as expressing a truncated form of MUC1, such as MUC1, to a cancer patient in need thereof.

[0172] 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]

[0173] 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.

[0174] The present invention will become more fully understood from the detailed description given 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.

[0175] [Figure 1]Figures 1A-1D show cell proliferation 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 proliferation, whereas monovalent Fab inhibits proliferation (Figures 1A-1B). The classic bell-shaped curve indicates that ligand-induced dimerization stimulates proliferation. Dimeric NM23-H1, also known as NME1, stimulates proliferation of MUC1*-positive cancer cells, whereas siRNA inhibiting MUC1 expression abolishes this effect (Figure 1C). NME7-AB also stimulates proliferation of MUC1*-positive cells (Figure 1D). [Figure 2] Figures 2A-2I show the results of ELISA assays. The MUC1* peptide PSMGFR, 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), was immobilized on a plate and assayed for binding to: NME7-AB (Figure 2A), MNC2 monoclonal antibody (Figure 2B), MNE6 monoclonal antibody (Figure 2C), or dimeric NME1 (Figure 2D). These assays demonstrate that NME1, NME7-AB, and monoclonal antibodies MNC2 and MNE6 all require the first membrane-proximal 10 amino acids of the MUC1* extracellular domain for binding. The MUC1* peptide, 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), was immobilized on a plate and assayed for binding to: MNC3 (Figure 2E) and MNC8 (Figure 2F). Figure 2G shows the amino acid sequences of the PSMGFR peptides, Figure 2H shows the amino acid sequences of the N-10 peptide, and Figure 2I shows the amino acid sequences of the C-10 peptide. [Figure 3]Figures 3A-3C show the results of a competitive ELISA assay. 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 MNE6 antibody (Figure 3A). The same experiment was performed in which NM23-H7, NME7-AB, alone or after the addition of MNE6 (Figure 3B). The results show that MNE6 competitively inhibits the binding of the MUC1* activating ligands NME1 and NME7. In a similar experiment (Figure 3C), PSMGFR, or the N-terminal minus 10 amino acids of PSMGFR, also known as N-10, was immobilized on a plate. The dimer NM23-H1 was then added. The anti-MUC1* antibodies MNE6, MNC2, MNC3, or MNC8 were then tested for their ability to compete with NM23-H1. The results show that all three antibodies bind to the PSMGFR peptide, but MNE6 and MNC2 competitively inhibit the binding of MUC1* activating ligands. [Figure 4]Figures 4A–4F show FACS scans of the anti-MUC1* antibody huMNC2 scFv, 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 1.5 μg / ml humanized MNC2 at a 1:2 or 1:10 dilution. After two washes, cells were stained with a secondary antibody, anti-penta-His antibody, conjugated to Alexa 488 (Qiagen) at a dilution of 1:200 (Figure 4A), 1:50 (Figure 4B), or 1:10 (Figure 4C) to detect the 6xHis tag of huMNC2 scFv. Figure 4A shows the binding of huMNC2 to ZR-75-1 breast cancer cells to which the secondary antibody was added at a dilution of 1:200. Figure 4B shows huMNC2 binding to ZR-75-1 breast cancer cells with secondary antibody added at a 1:50 dilution. Figure 4C shows huMNC2 binding to ZR-75-1 breast cancer cells with secondary antibody added at a 1:10 dilution. Flow cytometry analysis revealed a concentration-dependent shift in cell subsets, demonstrating specific binding not seen in the absence of MNC2 scFv (Figures 4A-4C). Figure 4D shows anti-MUC1* antibody MNE6 staining of MUC1-negative HCT-116 colon cancer cells transfected with empty vector, single-cell clone #8. Figure 4E shows anti-MUC1* antibody MNE6 staining of HCT-116 colon cancer cells transfected with MUC1* single-cell clone #10. Figure 4F shows anti-MUC1* antibody MNE6 staining of ZR-75-1, also known as 1500, MUC1*-positive breast cancer cells. As shown by FACS scans, both MNC2 and MNE6 stain only MUC1* positive cells and not MUC1 or MUC1* negative cells. [Figure 5] ELISA graphs are shown in which surfaces are coated with either the MUC1* PSMGFR peptide or a control peptide. Humanized MNC2 scFv is then incubated with the surface, washed, and detected according to standard methods. The ELISA shows that huMNC2 scFv binds to the MUC1* peptide with an EC-50 of approximately 333 nM. [Figure 6]Figures 6A and 6B show graphs of cancer cell growth inhibition by the MUC1* antibody variable region fragment, humanized MNC2 scFv. hMNC2 scFv potently inhibited the growth of ZR-75-1 (also known as 1500) and T47D 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] Figures 7A-7B show graphs of tumor growth in immunocompromised mice implanted with human tumors and then treated with the anti-MUC1* antibody MNE6 Fab or mock therapy. 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 bearing tumors measuring at least 150 mm3 and exhibiting three consecutive increases in tumor volume were selected for treatment. Animals were injected subcutaneously with 80 mg / kg MNE6 Fab 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 bearing tumors measuring at least 150 mm3 and exhibiting three consecutive increases in tumor volume were selected for treatment. Animals were injected subcutaneously with 160 mg / kg of MNE6 Fab 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 researchers. Statistics were calculated blindly by an independent statistician, with a P value of 0.0001 for each. Anti-MUC1* Fab inhibited breast and prostate cancer growth. Treatment did not affect body weight, bone marrow cell type, or number. [Figure 8] Graphs are shown from ELISAs in which the surface was immobilized with either the PSMGFR peptide, PSMGFR minus 10 amino acids from the N-terminus, or minus 10 amino acids from the C-terminus. huMNE6scFv-Fc bound to the PSMGFR peptide and the PSMGFR N-10 peptide, but not to the PSMGFR C-10 peptide. The parental MNE6 antibody and humanized MNE6 require the C-terminal 10 amino acids of PSMGFR for binding. [Figure 9] Figures 9A-9B show 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 C-terminus minus 10 amino acids. The MNC3 antibody variants were then assayed for binding to various MUC1* peptides. Figure 9A shows purified mouse monoclonal MNC3 antibody, and Figure 9B shows humanized MNC3 scFv-Fc. The ELISA demonstrates binding to the PSMGFR peptide as well as specific deletion peptides. [Figure 10] Figures 10A-10J. 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 MNC2, 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-10F are color-coded graphs showing the calculated scores for full-length MUC1 staining in each patient's tissue. Figures 10G-10J are color-coded graphs showing the calculated scores for MUC1* staining in each patient's tissue. [Figure 11] 11A-11J. 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 the mouse monoclonal antibody MNC2, which 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 full-length MUC1 staining in each patient's tissue. Figures 11G-11J are color-coded graphs showing the calculated scores for MUC1* staining in 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 MNE6-scFv-Fc biotinylated anti-MUC1* antibody and then stained with 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 secondary antibody alone. [Figure 13] Figures 13A-13F show photographs of normal breast tissue and breast cancer tissue stained with 2.5 μg / mL of humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody and then stained with 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 alone. [Figure 14] Figures 14A-14H show photographs of breast cancer tissues stained with 10 μg / mL MNE6 anti-MUC1* antibody followed by a rabbit anti-mouse secondary HRP antibody. Figures 14A-14D show breast cancer tissues from patient #300. Figures 14E-14H show breast cancer tissues from metastatic patient #291. [Figure 15] Figures 15A-15F show photographs of normal lung tissue and lung cancer tissue stained with 2.5 μg / mL of humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody and then stained with a secondary streptavidin-HRP antibody. Figure 15A shows normal lung tissue. Figures 15B and 15C show lung cancer tissue from the patient indicated. Figures 15D-15F show photographs of corresponding serial sections stained with the secondary antibody alone. [Figure 16] Figures 16A-16F show photographs of normal lung tissue and lung cancer tissue stained with 2.5 μg / mL of humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody and then stained with a secondary streptavidin-HRP antibody. Figure 16A shows normal lung tissue. Figures 16B and 16C show lung cancer tissue from the patient indicated. Figures 16D-16F show photographs of corresponding serial sections stained with the secondary antibody alone. [Figure 17]Figures 17A-17F show photographs of normal lung tissue and lung cancer tissue stained with 25 μg / mL of humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody and then stained with a secondary streptavidin-HRP antibody. Figure 17A shows normal lung tissue. Figures 17B and 17C show lung cancer tissue from the patient indicated. Figures 17D-17F show photographs of corresponding serial sections stained with the secondary antibody alone. [Figure 18] Figures 18A-18F show photographs of normal lung tissue and lung cancer tissue stained with 25 μg / mL of humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody and then stained with a secondary streptavidin-HRP antibody. Figure 18A shows normal lung tissue. Figures 18B and 18C show lung cancer tissue from the patient indicated. Figures 18D-18F show photographs of corresponding serial sections stained with the secondary antibody alone. [Figure 19] Figures 19A-19D show photographs of normal and cancerous small intestinal tissue stained with 5 μg / mL of humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody and then stained with a secondary streptavidin-HRP antibody. Figure 19A shows normal small intestinal tissue. Figure 19B shows small intestinal cancer from the patient shown. Figures 19C-19D show photographs of corresponding serial sections stained with the secondary antibody alone. [Figure 20] Figures 20A-20H show photographs of normal small intestinal tissue stained with 50 μg / mL of humanized MNE6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody. Figures 20A-20D show normal small intestinal tissue. Figures 20E-20H show corresponding serial sections stained with secondary antibody alone. [Figure 21] Figures 21A-21H show photographs of cancerous small intestine tissue stained with 50 μg / mL of humanized MNE6-scFv-Fc anti-MUC1* antibody followed by a secondary goat anti-human HRP antibody. Figures 21A-21D show cancerous small intestine tissue from the patient indicated. Figures 21E-21H show photographs of corresponding serial sections stained with the secondary antibody alone. [Figure 22]Figures 22A-22H show photographs of cancerous small intestine tissue stained with 50 μg / mL of humanized MNE6-scFv-Fc anti-MUC1* antibody followed by a secondary goat anti-human HRP antibody. Figures 22A-22D show cancerous small intestine tissue from the patient indicated. Figures 22E-22H show photographs of 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 MNE6-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] Figures 24A-24H show photographs of colon cancer tissue stained with 50 μg / mL of humanized MNE6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody. Figures 24A-24D show colon cancer tissue obtained from patients with metastasis as indicated. Figures 24E-24H show photographs of corresponding serial sections stained with secondary antibody alone. [Figure 25] Figures 25A-25H show photographs of colon cancer tissue stained with 50 μg / mL of humanized MNE6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody. Figures 25A-25D show colon cancer tissue from a grade 2 patient as indicated. Figures 25E-25H show photographs of corresponding serial sections stained with secondary antibody alone. [Figure 26] Figures 26A-26H show photographs of colon cancer tissue stained with 50 μg / mL of humanized MNE6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody. Figures 26A-26D show colon cancer tissue obtained from patients with metastasis as indicated. Figures 26E-26H show photographs of corresponding serial sections stained with secondary antibody alone. [Figure 27]Figures 27A-27H show photographs of prostate cancer tissue stained with 50 μg / mL of humanized MNE6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody. Figures 27A-27D show prostate cancer tissue from the patient indicated. Figures 27E-27H show photographs of corresponding serial sections stained with secondary antibody alone. [Figure 28] Figures 28A-28H show photographs of prostate cancer tissue stained with 50 μg / mL of humanized MNE6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody. Figures 28A-28D show prostate cancer tissue from the patient indicated. Figures 28E-28H show photographs of corresponding serial sections stained with secondary antibody alone. [Figure 29] Figures 29A-29H show photographs of prostate cancer tissue stained with 50 μg / mL of humanized MNE6-scFv-Fc anti-MUC1* antibody followed by secondary goat anti-human HRP antibody. Figures 29A-29D show prostate cancer tissue from the patient indicated. Figures 29E-29H show photographs of corresponding serial sections stained with secondary antibody alone.

[0176] [Figure 30-1] Figures 30A-30F 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 score is the tumor grade. The percentage of arrays scored as 0, weak, moderate, or strong is graphed as a pie chart. Figure 30A shows a pie chart of the anti-MUC1* antibody staining scores. Figure 30B shows a photograph of the array stained with the antibody. Figures 30C-30D show enlarged photographs of two of the breast cancer specimens obtained from the array. Figures 30E-30F show closer magnifications of portions of the specimens highlighted by boxes. [Figure 30-2]Figures 30A-30F 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 score is the tumor grade. The percentage of arrays scored as 0, weak, moderate, or strong is graphed as a pie chart. Figure 30A shows a pie chart of the anti-MUC1* antibody staining scores. Figure 30B shows a photograph of the array stained with the antibody. Figures 30C-30D show enlarged photographs of two of the breast cancer specimens obtained from the array. Figures 30E-30F show closer magnifications of portions of the specimens highlighted by boxes. [Figure 31-1] Figures 31A-31F show photographs of ovarian cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second score is the tumor grade. The percentage of arrays scored as 0, 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 the array stained with the antibody. Figures 31C-31D show enlarged photographs of two of the breast cancer specimens obtained from the array. Figures 31E-31F show closer magnifications of portions of the specimens highlighted by boxes. [Figure 31-2] Figures 31A-31F show photographs of ovarian cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second score is the tumor grade. The percentage of arrays scored as 0, 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 the array stained with the antibody. Figures 31C-31D show enlarged photographs of two of the breast cancer specimens obtained from the array. Figures 31E-31F show closer magnifications of portions of the specimens highlighted by boxes. [Figure 32-1]Figures 32A-32F show photographs of pancreatic cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second score is the tumor grade. The percentage of arrays scored as 0, weak, moderate, or strong is graphed as a pie chart. Figure 32A shows a pie chart of the anti-MUC1* antibody staining scores. Figure 32B shows a photograph of the array stained with the antibody. Figures 32C-32D show enlarged photographs of two of the breast cancer specimens obtained from the array. Figures 32E-32F show closer magnifications of portions of the specimens highlighted by boxes. [Figure 32-2] Figures 32A-32F show photographs of pancreatic cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second score is the tumor grade. The percentage of arrays scored as 0, weak, moderate, or strong is graphed as a pie chart. Figure 32A shows a pie chart of the anti-MUC1* antibody staining scores. Figure 32B shows a photograph of the array stained with the antibody. Figures 32C-32D show enlarged photographs of two of the breast cancer specimens obtained from the array. Figures 32E-32F show closer magnifications of portions of the specimens highlighted by boxes. [Figure 33-1] Figures 33A-33F show photographs of lung cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second score is the tumor grade. The percentage of arrays scored as 0, weak, moderate, or strong is graphed as a pie chart. Figure 33A shows a pie chart of the anti-MUC1* antibody staining scores. Figure 33B shows a photograph of the array stained with the antibody. Figures 33C-33D show enlarged photographs of two of the breast cancer specimens obtained from the array. Figures 33E-33F show closer magnifications of portions of the specimens highlighted by boxes. [Figure 33-2]Figures 33A-33F show photographs of lung cancer arrays stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second score is the tumor grade. The percentage of arrays scored as 0, weak, moderate, or strong is graphed as a pie chart. Figure 33A shows a pie chart of the anti-MUC1* antibody staining scores. Figure 33B shows a photograph of the array stained with the antibody. Figures 33C-33D show enlarged photographs of two of the breast cancer specimens obtained from the array. Figures 33E-33F show closer magnifications of portions of the specimens highlighted by boxes. [Figure 34] Figures 34A-34I show photographs of normal tissues stained with the anti-MUC1* antibody huMNC2scFv. [Figure 35] Figures 35A-35D show FACS scans of cells expressing neither MUC1, MUC1*, nor full-length MUC1, in which 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*, in which the extracellular domain contains only the sequence of the PSMGFR peptide, in which the cells were probed with antibody MNC2. Figure 35C shows HCT-MUC1-18 cells, a cleavage-resistant single-cell clone of HCT cells transfected with full-length MUC1, also referred to here as HCT-MUC1-41TR, in which the cells were probed with antibody MNC2. Figure 35D shows HCT-MUC1-18 cells probed with antibody VU4H5, an antibody that recognizes the several hundred tandem repeat epitope in full-length MUC1. As can be seen, MNC2 recognizes an ectopic epitope that is inaccessible to full-length MUC1. [Figure 36]Figures 36A-36D show Western blots 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 36A shows Western blots 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 the cleaved form of MUC1. A visible band at 25-35 kDa is readily visible in lane 6, loaded with HCT-MUC1*, whereas only faint bands are present 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 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 truncated MUC1*. [Figure 37]Figures 37A-37C show Western blots and bar graphs of 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 increased recognition of the cleavage products and decreased recognition of full-length MUC1, including the tandem repeat domain, by the anti-MUC1* antibody MNC2 in a dose-dependent manner. [Figure 38] Shown here are photographs of Western blots of HCT-MUC1-18 cells, a cleavage-resistant single-cell clone of HCT cells transfected with full-length MUC1, labeled HCT-18, treated with varying amounts of catalytically active ADAM17 or MMP14. Selected MUC1 tandem repeat domains from full-length MUC1 were immunoprecipitated from conditioned medium, run on a gel, and then probed with VU4H5, which binds to the tandem repeat epitope. As can be seen, MMP14 also efficiently cleaves MUC1 along its entire length, shedding the tandem repeat-containing extracellular domain into the conditioned medium. The cleavage enzyme ADAM17 did not cleave MUC1. [Figure 39]Figures 39A-39B show fluorescence-activated cell sorting (FACS) measurements of human CD34+ hematopoietic stem cells from human bone marrow stained with the anti-MUC1* monoclonal antibodies MNC3, MNC2, MNE6, or an isotype control antibody. Histograms and bar graphs 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 of these antibodies suggests that MNC3 recognizes a MUC1*-like form produced when MUC1 is cleaved by an enzyme distinct from MMP9. [Figure 40] Figures 40A-40G show details of FACS analysis of hematopoietic stem cells probed with either MNC3 or MNE6. Figure 40A shows a FACS scatter plot of whole bone marrow cells. Figure 40B shows a FACS scatter plot of CD34+ cells. Figure 40C shows a FACS histogram of CD34+ cells. Figure 40D shows a FACS scatter plot of CD34+ / CD38- earliest hematopoietic stem cells stained with either MNC3 or MNE6. Figure 40E shows a histogram of the experiment. Figure 40F shows a histogram overlay of MNC3 binding with CD34+ / CD38- cells versus MNE6. Figure 40G shows a bar graph of the FACS experiment. [Figure 41] Figures 41A-42H show detailed FACS analyses of CD34+ / CD38- / lo hematopoietic stem cells probed with polyclonal anti-PSMGFR antibodies SDIX, MNE6, or MNC2. Figure 41A shows a FACS scatter plot of the CD34+ / CD38- / lo population of cells. 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] Figures 42A-42H show photographs of DU145 prostate cancer cells or T47D breast cancer cells treated with either the Fab of the anti-MUC1* antibodies MNC2, MNE6, MNC3, or MNC8. The images show that the cancer-specific antibodies MNC2 and MNE6 effectively kill prostate and breast cancer cells, while the monoclonal antibodies MNC3 and MNC8 do not. [Figure 43] Figure 43 shows a graph of a PCR experiment comparing the expression of a wide range of cleavage enzymes expressed in different cell lines, with values normalized to those expressed in the breast cancer cell line T47D. The cell lines compared are the prostate cancer cell line DU145, a MUC1-negative colon cancer cell line transfected with MUC1, which cleaves the extracellular domain after 41 tandem repeat units; HCT-MUC1-41TR, a T47D breast cancer cell line that is not cleaved by MUC1*; and CD34+ bone marrow cells. Figure 44 shows a graph of a PCR experiment measuring the expression levels of various cleavage enzymes in DU145 prostate cancer cells, HCT116+MUC1FL (also known as the HCT-MUC1-18 cell line) that expresses full-length MUC1, T47D breast cancer cells, and CD34+ hematopoietic stem cells from bone marrow. Fold expression is calculated based on the expression of each cleavage enzyme in T47D breast cancer cells. [Figure 44] Figure 43 shows a graph of the PCR experiment, with the maximum value of the Y axis set to 5. [Figure 45]Figures 45A-45P show photographs of CAR T co-culture assays 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 an extracellular domain cleaved after 41 tandem repeats and is not cleaved to the MUC1* form by itself. HCT-MUC1-41TR cancer cells were incubated with conditioned medium from cells transfected with MMP9 or ADAM17, and then co-cultured with CAR T cells. Conditioned medium from MMP9- or ADAM17-expressing cells was also incubated with APMA, an activator of the cleavage enzymes. The images shown are 4x bright-field images and their overlays of fluorescent images showing cancer cells stained with a red CMTMR lipophilic dye. Figures 45A, 45E, 45I, and 45M show photographs of cells co-cultured with untransduced human T cells. Figures 45B, 45F, 45J, and 45N show photographs of cells co-cultured with human T cells transduced with anti-MUC1* CAR44 at an MOI of 10. Figures 45C, 45G, 45K, and 45O show photographs of cells co-cultured with human T cells transduced with anti-MUC1* CAR50 at an MOI of 10. Figures 45D, 45H, 45L, and 45P show photographs of cells co-cultured with human T cells transduced with anti-MUC1* CAR44 at an MOI of 50, which increases the efficiency of transduction. Figures 45B, 45C, and 45D show that both CAR44- and CAR50-transduced T cells recognized MUC1* expressed in these cancer cells, bound to these cancer cells, induced cluster formation, and killed many cancer cells. Figures 45F, 45G, and 45H show that neither CAR44-transduced nor CAR50-transduced T cells recognized full-length MUC1 expressed in HCT-MUC1-41TR cancer cells. There was no T cell-induced cluster formation, and the number of cancer cells was not reduced.Figures 45J, 45K, and 45L show that activated MMP9 cleaved full-length MUC1 into the MUC1* form, which is recognized by both CAR44- and CAR50-transduced T cells. There was clearly visible CAR T cell-induced cluster formation and a decrease in the number of cancer cells as they died. 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-transduced nor huMNC2-CAR50-transduced T cells recognized these cancer cells. [Figure 46] Figures 46A-46T show photographs of CAR T co-culture assays in which the targeting antibody fragment of the CAR is MNC2scFv, 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 breast cancer T47D cells that were similarly 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 MMP2- and MMP9-expressing cells was incubated with APMA, an activator of these cleavage enzymes. The images shown are 4x bright-field images and overlays of their fluorescent images 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 truncated MUC1*. [Figure 47]Figures 47A-47I show photographs of cancer cells co-cultured with anti-MUC1* CAR T cells, some of which were pre-incubated with activated MMP9 before co-culture with CAR T cells. The cancer cells shown in Figures 47A-47C are the MUC1-negative colon cancer cell line HCT-116 stably transfected to express MUC1*. The cancer cells shown in Figures 47D-47F are the MUC1-positive breast cancer cell line T47D, which expresses high levels of both full-length MUC1 and MUC1*. The cancer cells shown in Figures 47G-47I are 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 and serve as a control. The cells shown in Figures 47B, 47E, and 47H were cocultured with human T cells transduced with huMNC2-CAR44 at an MOI of 10, where MOI represents the multiplicity of infection; the higher the MOI, the more CAR expressed by the T cells. The cells shown in Figures 47C, 47F, and 47I were cocultured with human T cells transduced with huMNC2-CAR44 at an MOI of 50. As can be seen from the photographs, CAR44 T cells bind to, surround, and kill target MUC1*-positive cancer cells. Comparing the photograph in Figure 47I with the others, it can be seen that cells preincubated with MMP9 are much more susceptible to CAR T killing once the antibody-targeting head of the CAR recognizes MUC1*. This also demonstrates that MUC1 cleaved by MMP9 is recognized by huMNC2scFv. [Figure 48]This graph shows xCelligence of T47D breast cancer cells co-cultured with either control, untransduced T cells, 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, preincubating T47D cells with the cleaving enzyme MMP9 significantly improved the killing of huMNC2-CAR44 T cells. 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] This graph shows xCelligence of DU145 prostate cancer cells co-cultured with either control, untransduced T cells, 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, the killing of huMNC2-CAR44 T cells is not affected by preincubation with the cleavage enzyme MMP9. DU145 cancer cells express significantly lower amounts of MUC1, including the full-length form as well as MUC1*. The lower density of full-length MUC1 does not sterically hinder T cell access to the membrane proximal to 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]Figures 51A-51B show bar graphs of ELISA assays 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] Figures 52A-52B show bar graphs of ELISA assays measuring the amount of interleukin-2, IL-2, secreted by huMNC2-CAR44 human T cells after 72 hours of co-culture with normal 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-53J show bar graphs of FACS analysis of live vs. dead markers and photographs of normal vs. cancer cells after co-culture with huMNC2-CAR44 T cells. Figure 53A.1 shows a bar graph of FACS analysis of live vs. 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 vs. 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 vs. 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 a FACS analysis of live vs. 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 a FACS analysis of live vs. 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 a FACS analysis of live vs. 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 53G.1 shows a bar graph of a FACS analysis of live vs. dead cells after co-culturing 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 a FACS analysis of live vs. dead cells after co-culturing 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 a FACS analysis of live vs. dead cells after co-culturing HBEC-5i normal brain cells with huMNC2-CAR44 T cells.Figures 53I.2 and 53I.3 show photographs of the experiment described in Figure 53I.1. Figure 53J.1 shows a bar graph of a FACS analysis of live versus 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 of the experiment described in Figure 53J.1. [Figure 53-2]Figures 53A-53J show bar graphs of FACS analysis of live vs. dead markers and photographs of normal vs. cancer cells after co-culture with huMNC2-CAR44 T cells. Figure 53A.1 shows a bar graph of FACS analysis of live vs. 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 vs. 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 vs. 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 a FACS analysis of live vs. 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 a FACS analysis of live vs. 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 a FACS analysis of live vs. 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 53G.1 shows a bar graph of a FACS analysis of live vs. dead cells after co-culturing 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 a FACS analysis of live vs. dead cells after co-culturing 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 a FACS analysis of live vs. dead cells after co-culturing HBEC-5i normal brain cells with huMNC2-CAR44 T cells.Figures 53I.2 and 53I.3 show photographs of the experiment described in Figure 53I.1. Figure 53J.1 shows a bar graph of a FACS analysis of live versus 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 of the experiment described in Figure 53J.1. [Figure 54] A photograph of a breast cancer tissue array is shown where, for each patient, there is a specimen from the primary tumor and a specimen from that patient's metastases. As can be seen, in most cases, the metastases express more MUC1* than the primary tumor. [Figure 55] Figures 55A-55H show the cytotoxic effect of huMNC2-CAR44 T cells against 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 was stained with a red fluorescent dye. Figure 55B shows a fusion 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 was stained with a red fluorescent dye. Figure 55D shows a fusion 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 real-time killing of DU145 cancer cells by huMNC2-CAR44 T cells (blue trace), but not by untransduced T cells (green). [Figure 56]Figures 56A-56H show 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 was stained with a red fluorescent dye. Figure 56B shows a fusion 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 was stained with a red fluorescent dye. Figure 56D shows a fusion 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 real-time killing of CAPAN-2 cancer cells by huMNC2-CAR44 T cells (blue trace), but not by untransduced T cells (green). [Figure 57] Figures 57A-57C show xCELLigence scans tracking real-time killing of MUC1*-positive cancer cells, but not MUC1*-negative cells, by huMNC2-CAR44 T cells. 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, MUC1-negative cancer cells stably transfected with full-length MUC1. In this cell line, only approximately 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]Figures 58A-58F show photographs of NOD / SCID / GAMMA mice in an IVIS instrument measuring photon emission from tumor cells after treatment with either PBS, untransduced human T cells, or huMNC2-CAR44 T cells. Mice were subcutaneously injected with luciferase-positive HCT-MUC1* tumor cells. Ten minutes before IVIS photographs were taken, mice were injected intraperitoneally (ip) with 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 curve for the experiment. Figure 58F shows a table detailing the molecular makeup of human T cells isolated from the blood of mice after sacrifice. [Figure 59] Figures 59A-59C show photographs of NOD / SCID / GAMMA mice in 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+MUC1*. This was a mixed population of cells, with 95% of the cells being T47D cells stably transfected with MUC1*. Both T47D-wt and T47D+MUC1* cells were luciferase-positive. Ten minutes before IVIS photographs were taken, the mice were injected intraperitoneally (ip) with luciferase substrate, i.e., luciferin. Figure 59A shows a tumor-bearing mouse treated with phosphate-buffered saline (PBS) alone. Figure 59B shows T47D-wt tumor-bearing mice treated with two doses of huMNC2-CAR44 T cells. Figure T90.1C shows T47D-MUC1* tumor-bearing mice treated with two doses of huMNC2-CAR44 T cells. [Figure 60]Figures 60A-60C show photographs of NOD / SCID / GAMMA mice in an IVIS instrument measuring photon emission from tumor cells after treatment with either PBS, untransduced T cells, or huMNC2-CAR44 T cells. Mice were subcutaneously injected with a mixed population of 70% T47D-wt breast cancer cells and 30% T47D cells transfected with a distinct MUC1*. Both cell types were luciferase-positive. Ten minutes before IVIS photographs were taken, mice were injected intraperitoneally (ip) with luciferase substrate, luciferin. 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]Figures 61A-61J show fluorescent images of mice taken with an IVIS instrument. 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, the animals received a single injection of 10 million human T cells transduced with huMNC2-scFv-CAR44 or untransduced T cells. Five million T cells were injected intratumorally and five million were injected via the tail vein. Ten minutes before IVIS imaging, the mice were IP injected with luciferin, which fluoresces after cleavage by luciferase, thus causing tumor cell fluorescence. Figures 61A, 61D, and 61G show photographs of mice treated with huMNC2-scFv-CAR44 T cells that were pre-stimulated by co-culturing with 4 μm beads conjugated with synthetic MUC1* and PSMGFR peptides for 24 hours prior to administration: Protocol 1. Figures 61B, 61E, and 61H show photographs of mice treated with huMNC2-scFv-CAR44 T cells that were pre-stimulated by co-culturing with MUC1*-positive cancer cells for 24 hours prior to administration: Protocol 2. Figures 61C, 61F, and 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]Figures 62A-62M show fluorescent images of mice taken with an IVIS device. On day 0, NSG (NOD / SCID / GAMMA) immunodeficient mice were intraperitoneally (IP) injected with 500K human SKOV-3 cells, a MUC1*-positive ovarian cancer cell line. The cancer cells were stably transfected with luciferase. Tumors were allowed to engraft. On day 4, the animals were injected into the intraperitoneal space with either 10 M human T cells transduced with huMNC2-scFv-CAR44, 10 M untransduced T cells, or PBS. On day 11, the animals were injected again, except that half the cells were injected via the tail vein and the other half via IP injection. Animals were imaged by IVIS on days 3, 7, 10, and 15. Ten minutes before IVIS photography, mice were IP injected with luciferin, which fluoresces after cleavage by luciferase, thus causing tumor cell fluorescence. Figures 62A, 62D, 62G, and 62J show photographs of mice treated with huMNC2-scFv-CAR44 T cells pre-stimulated by co-culture with 1 μm beads conjugated with 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 3 days before administration of CAR T, T cells, or PBS. Figures 62D, 62E, and 62F show IVIS images of animals on day 7, just 4 days after treatment. Figures 62G, 62H, and 621 show IVIS images of animals at day 10. Figures 62J, 62K, and 62L show IVIS images of animals at day 15. Figure 62M is an IVIS color scale relating fluorescence in photons / second to color. [Figure 63]A graph of an ELISA binding assay is shown 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 when the 10 N-terminal amino acids are deleted, but not when the 10 or 9 C-terminal amino acids are deleted. [Figure 64] Figures 64A-64B show graphs of ELISA binding assays. The antibodies tested were derived from animals immunized with PSMGFR peptides. The first selection criterion was to confirm that the antibodies bound to the immunizing PSMGFR peptide. Figure 64A shows graphs of ELISAs 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, suggesting 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 each antibody bar in the ELISA graph is color-coded to match the predicted cognate sequence or portion of that antibody. [Figure 65]Figures 65A-65B show 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 antibodies tested were derived from animals immunized with the N+20 / C-27 peptide. The initial selection criterion was to confirm that the antibodies bound to the immunizing N+20 / C-27 peptide. Figure 65A 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 except 45C11 still bind to the PSMGFR peptide. Although 45C11's binding is weak, preliminary inference indicates that the cognate epitope must be within the SNIKFRPGSVV sequence. 1E4 was able to bind to the N+20 / C-27 peptide, PSMGFR, and 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 correspond to the predicted cognate sequence or portion thereof for that antibody. [Figure 66] Figures 66A-66B show 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 antibodies tested were derived from animals immunized with the N+9 / C-9 peptide. The first selection criterion was to confirm that the antibodies bound to the immunizing N+9 / C-9 peptide. Figure 66A shows a graph of the ELISA assay. All except 39H5 were able to bind to the immunizing peptide, N+9 / C-9. 39H5 showed very weak binding to the PSMGFR and N-10 peptides, consistent with the notion that at least part of its cognate epitope must lie within the QFNQYKTE sequence. Figure 66B shows the sequences of the various peptides. The color of each antibody bar in the ELISA graph is color-coded to correspond to the antibody's predicted cognate sequence or portion thereof. [Figure 67]Figures 67A-67D show the results of ELISA assays further defining antibody epitopes within the MUC1 or MUC1* extracellular domain. All antibodies shown in this figure were generated by immunizing animals with PSMGFR peptides. In binding assays, antibodies were tested 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. These are all subsets of PSMGFR peptides, and the numbers again refer to PSMGFR peptides. 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 of peptides derived from PSMGFR. Figure 67D shows sequences containing all or part of the epitope essential for antibody recognition. [Figure 68] Figures 68A-68D show the results of ELISA assays further defining antibody epitopes within the MUC1 or MUC1* extracellular domain. All antibodies shown in this figure were generated by immunizing animals with the N+20 / C-27 peptide. In binding assays, antibodies were tested 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. These are all subsets of PSMGFR peptides, and the numbers again refer to PSMGFR peptides. 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 of peptides derived from PSMGFR. Figure 68D shows sequences containing all or part of the epitope essential for antibody recognition. [Figure 69]Figures 69A-69D show the results of ELISA assays further defining antibody epitopes within the MUC1 or MUC1* extracellular domain. All antibodies shown in this figure were generated by immunizing animals with the N+9 / C-9 peptide. In binding assays, antibodies were tested 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. These are all subsets of PSMGFR peptides, and the numbers again refer to PSMGFR peptides. 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 of peptides derived from PSMGFR. Figure 69D shows sequences containing all or part of the epitope essential for antibody recognition. [Figure 70] Figures 70A-70B show graphs of an ELISA displacement assay. In this experiment, a multiwell plate was coated with PSMGFR peptide. In Figure 70A, recombinant NME7AB was bound to the surface-immobilized PSMGFR peptide. Various antibodies were added, followed by a washing step. The amount of NME7AB remaining attached to the PSMGFR-coated plate after antibody competition was measured by detecting the NME7AB tag. As a control, anti-NME7AB antibodies were also tested for their ability to displace NME7AB from PSMGFR. As can be seen from the graphs, antibodies MNC2, MNE6, 20A10, 3C2B1, and 5C6F3 displace NME7AB from its binding to the PSMGFR peptide, indicating that the antibodies are cancer-specific. Figure 70B shows that the epitope within the MUC1* extracellular domain to which these antibodies bind is the sequence SVSDVPFPFSAQSGA; if amino acids FPFS are absent or mutated, binding is abolished for MNC2, MNE6, 20A10, 3C2B1, and if amino acids SVSDV are absent or mutated, binding is abolished for 5C6F3. [Figure 71-1]Figures 71A-71H show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes of 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 HCT-MUC1* engineered to express only the PSMGFR sequence in its extracellular domain. Figures 71E-71H show testing of antibodies for their ability to bind to the 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, which binds to the N-10 variant but not the C-10 variant of the PSMGFR peptide. Figures 71B and 71F show MNE6, a monoclonal antibody raised against the PSMGFR peptide that binds to the N-10 mutant but not the C-10 mutant of the PSMGFR peptide. Figures 71C and 71G show SDIX, a polyclonal antibody raised against and binding 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 the linear epitope of the MUC1 species. [Figure 71-2]Figures 71A-71H show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes of 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 HCT-MUC1* engineered to express only the PSMGFR sequence in its extracellular domain. Figures 71E-71H show testing of antibodies for their ability to bind to the 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, which binds to the N-10 variant but not the C-10 variant of the PSMGFR peptide. Figures 71B and 71F show MNE6, a monoclonal antibody raised against the PSMGFR peptide that binds to the N-10 mutant but not the C-10 mutant of the PSMGFR peptide. Figures 71C and 71G show SDIX, a polyclonal antibody raised against and binding 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 the linear epitope of the MUC1 species. [Figure 72-1]Figures 72A-72P show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes of full-length MUC1 or MUC1*. All of these antibodies were raised against and bind to the PSMGFR peptide. Figures 72A-72H 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 HCT-MUC1* engineered to express only the PSMGFR sequence in its extracellular domain. Figures 72I-72P 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 blots were exposed for a longer time to make the MUC1*-specific band more visible. 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 72A-72P show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes of full-length MUC1 or MUC1*. All of these antibodies were raised against and bind to the PSMGFR peptide. Figures 72A-72H 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 HCT-MUC1* engineered to express only the PSMGFR sequence in its extracellular domain. Figures 72I-72P 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 blots were exposed for a longer time to make the MUC1*-specific band more visible. As can be seen, antibodies 25E6, 18B4, and to some extent 5C6F3 recognize linear epitopes, whereas 20A10, 3C2B1, 18G12, and 28F9 do not. [Figure 73-1]Figures 73A-73J show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes of 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-73E 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 HCT-MUC1* engineered to express only the PSMGFR sequence in its extracellular domain. Figures 73F-73J 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 73-2] Figures 73A-73J show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes of 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-73E 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 HCT-MUC1* engineered to express only the PSMGFR sequence in its extracellular domain. Figures 73F-73J 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-74H show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes of full-length MUC1 or MUC1*. All of these antibodies were generated relative to the N+9 / C-9 variant of the PSMGFR peptide and bind to the N+9 / C-9 peptide. Figures 74A-74D 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 HCT-MUC1* engineered to express only the PSMGFR sequence in its extracellular domain. Figures 74E-74H 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 74-2] Figures 74A-74H show photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes of full-length MUC1 or MUC1*. All of these antibodies were generated relative to the N+9 / C-9 variant of the PSMGFR peptide and bind to the N+9 / C-9 peptide. Figures 74A-74D 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 HCT-MUC1* engineered to express only the PSMGFR sequence in its extracellular domain. Figures 74E-74H 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-75P 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 the MMP9- or MMP2-cleaved forms of MUC1. Note that the first bar in each graph indicates that none of the antibodies bind to full-length MUC1 in the absence of cleavage. Each bar is labeled with both the name of the antibody used in that assay and its cognate epitope. The order of the graphs from right to left corresponds to the distance of the antibody's cognate epitope from the cell surface. 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 75O shows antibody 3C2B1. Figure 75P shows antibody 5C6F3. [Figure 75-2]Figures 75A-75P 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 the MMP9- or MMP2-cleaved forms of MUC1. Note that the first bar in each graph indicates that none of the antibodies bind to full-length MUC1 in the absence of cleavage. Each bar is labeled with both the name of the antibody used in that assay and its cognate epitope. The order of the graphs from right to left corresponds to the distance of the antibody's cognate epitope from the cell surface. 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 75O shows antibody 3C2B1. Figure 75P shows antibody 5C6F3. [Figure 76] Figures 76A-76J show graphs of FACS analysis of reference antibodies MNC2, "C2," and VU4H5 binding to either the MUC1-negative cell line HCT-116, HCT transfected with MUC1*, "HCT-MUC1*," a cleavage-resistant single-cell clone of HCT transfected with full-length MUC1, "HCT-MUC1-18," and breast cancer cell line T47D or breast cancer cell line 1500, also known as ZR-75-1. MNC2 binds to an ectopic binding site in 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 several hundred repeating epitopes in the tandem repeat domain. Figures 76A-76E show the percent binding, and Figures 76F-76J show the mean fluorescence intensity, or MFI. [Figure 77] Figures 77A-77N show graphs of FACS analysis of the reference antibody MNC2 "C2" binding to a panel of cancer cell lines that are MUC1* positive, except for MDA-MB-231, which expresses very low levels of MUC1 and MUC1* and is often used as a negative control. MNC2 binds to an ectopic binding site in 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. Figures 77A-77G show the percentage of binding, and Figures 77H-77N show the mean fluorescence intensity (MFI). Figures 77A and 77H show antibody binding to the lung cancer cell line NCI-H292. Figures 77B and 77I show antibody binding to the lung cancer cell line NCI-H1975. Figures 77C and 77J show 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] Figures 78A-78C show color-coded schematic diagrams 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 shows an aligned schematic diagram of various subsets of peptides. Figure 78B lists the antibodies that bind to each color-coded sequence. Figure 78C shows the cancer cell lines recognized by each antibody. [Figure 79]Figures 79A-79I show 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. 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, or 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. Figures 79A and 79E show that 20A10 recognizes MUC1* present in T47D breast cancer cells. Figures 79D and 79H show that 3C2B1 recognizes MUC1* present in T47D breast cancer cells. Figures 79D and 79H show that 5C6F3 recognizes MUC1* present in T47D breast cancer cells. [Figure 80]Figures 80A-80I show color-coded graphs obtained from FACS analysis of each antibody binding to 1500, also known as ZR-75-1, breast cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence. 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, or 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 the PSMGFR sequence extended by 20 amino acids at the N-terminus. Figures 80A and 80E show that antibody 20A10 recognizes MUC1* present in 1500, also known as ZR-75-1 breast cancer cells. Figures 80D and 80H show that antibody 3C2B1 recognizes MUC1* present in 1500, also known as ZR-75-1 breast cancer cells. Figures 80D and 80H show that antibody 5C6F3 recognizes MUC1* present in 1500, also known as ZR-75-1 breast cancer cells. [Figure 81]Figures 81A-81G show 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. 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, or 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. Figures 81A and 81D show that antibody 20A10 recognizes MUC1* present in H292 lung cancer cells. [Figure 82] Figures 82A-82G show 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. 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, or MFI, of each antibody. Figures 82A and 82D show FACS graphs of antibodies generated by immunization with PSMGFR peptides. 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. Figures 82A and 82D show that antibody 20A10 recognizes MUC1* present in H1975 lung cancer cells. [Figure 83]Figures 83A-83G show 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. 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, or 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. Figures 83A and 83D show that antibody 20A10 recognizes MUC1* present in SKOV-3 ovarian cancer cells. [Figure 84] Figures 84A-84G show 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. 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, or MFI, of each antibody. Figures 84A and 84D show FACS graphs of antibodies generated by immunization with PSMGFR peptides. 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. Figures 84A and 84E show that antibody 20A10 recognizes MUC1* present in DU145 prostate cancer cells. Figures 84D and 84H show that antibody 3C2B1 recognizes MUC1* present in DU145 prostate cancer cells, and Figures 84D and 84H show that antibody 5C6F3 recognizes MUC1* present in DU145 prostate cancer cells. [Figure 85]Figures 85A-85G show 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. 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, or 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. Figures 85A and 85D show that antibody 20A10 recognizes MUC1* present in HPAF II pancreatic cancer cells. [Figure 86] Figures 86A-86G show color-coded graphs obtained from FACS analysis of each antibody binding to Capan-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, or 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]Figures 87A-87G show color-coded graphs obtained from FACS analysis of antibodies binding to mostly MUC1-negative MDA-MB-231 breast cancer cells and their respective cognate sequences within the N-terminally extended PSMGFR sequence. 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, or 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] Figures 88A-88L show photographs of normal liver tissue specimens, each derived from the same donor but stained with a different antibody of the present invention. Figures 88A-88F show the entire tissue core. Figures 88G-88L show 40x magnification of specific regions of the tissue. From right to left, the tissues are ordered such that antibodies that bind most membrane-proximal, i.e., antibodies that bind to the C-most portion of the PSMGFR peptide, are on the right, and antibodies that bind to the N-most portion of the MUC1 extracellular domain, even though they extend beyond the PSMGFR region, are on the left. As can be seen, 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 antibodies that bind to epitopes outside of PSMGFR lose all cancer specificity. As can be seen, Figures 88F and 88L demonstrate that antibody 3C2B1, which binds to a portion of the MUC1* extracellular domain containing all or part of the sequence FPFS or PFPFSAQSGA, does not bind to normal liver. [Figure 89]Figures 89A-89H show photographs of normal cardiac tissue specimens stained with different antibodies of the present invention. Figures 89A-89D show whole tissue cores. Figures 89E-89HL show 40x magnification of specific tissue regions. 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 bind to 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. These antibodies also recognize MUC1 cleavage products when the cleavage enzyme is MMP9. As can be seen from the figures, these antibodies do not bind to normal heart tissue. Figures 89A and 89E show that the reference antibody MNC2 does not bind to normal heart tissue. Figures 89B and 89F show that the reference antibody MNE6 does not bind to normal heart tissue. Figures 89C and 89G show that the antibody 20A10 does not bind to normal heart tissue. Figures 89D and 89H show that the antibody 3C2B1 does not bind to normal heart tissue. [Figure 90] Figures 90A-90D show photographs of normal heart tissue specimens stained with different antibodies of the present invention. Figures 90A-90B show entire tissue cores. Figures 90C-90D show 40x magnification of specific areas of tissue. Figures 90A and 90C show staining with MNC3. Figures 90B and 90D show staining with 25E6. These antibodies bind to epitopes containing all or part of the sequence ASRYNLT. All of these antibodies can bind to the PSMGFR peptide, and they bind to the N-10 peptide, but also to the C-10 peptide. As can be seen, these antibodies are not cancer-specific and show some binding to normal heart tissue. [Figure 91]Figures 91A-91B show photographs of normal heart tissue specimens stained with antibody 1E4 of the present invention. Figure 91A shows the entire tissue core. Figure 91B shows a 40x magnification of a specific area of tissue. Antibody 1E4 binds to an epitope that includes all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to the N-10 peptide, but also binds to the C-10 peptide. As can be seen, 1E4 binds to normal heart tissue. As can be seen, these antibodies are not cancer-specific and show some binding to normal heart tissue. [Figure 92] Figures 92A-92H show photographs of normal heart tissue specimens stained with different antibodies of the present invention. Figures 92A-92D show whole tissue cores. Figures 92E-92HL show 40x magnification of specific areas of tissue. 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 containing all or part of the sequence GTINVHDVET, the most N-terminal portion of the PSMGFR peptide. None of these antibodies are able to bind to the N-10 peptide. As can be seen, all of these antibodies, except for 18B4, show binding to normal heart tissue. [Figure 93] Figures 93A-93D show photographs of normal heart tissue specimens stained with antibodies of the present invention. Figures 93A-93B show entire tissue cores. Figures 93C-93D show 40x magnification of specific areas of tissue. 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]Figures 94A-94B show photographs of normal heart tissue specimens stained with antibody 45C11 of the present invention. Figure 94A shows the entire tissue core. Figure 94B shows a 40x magnification of a specific area of tissue. Antibody 45C11 binds to an epitope outside the PSMGFR region and including all or part of the sequence SNIKFRPGSVV. Antibody 45C11 is unable to bind to the N-10 peptide. As can be seen, 45C11 binds strongly to normal heart tissue. [Figure 95] Figures 95A-95H show photographs of normal liver tissue specimens stained with different antibodies of the present invention. Figures 95A-95D show whole tissue cores. Figures 95E-95HL show 40x magnification of specific areas of tissue. Figures 95A and 95E show staining with the reference antibody MNC2-scFv. Figures 95B and 95F show staining with the reference antibody 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 sequence FPFS or PFPFSAQSGA. All of these antibodies can bind to the PSMGFR peptide and bind to 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. These antibodies also recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen, these antibodies do not exhibit binding to normal liver tissue. [Figure 96]Figures 96A-96D show photographs of normal liver tissue specimens stained with different antibodies of the present invention. Figures 96A-96B show entire tissue cores. Figures 96C-96D show 40x magnification of specific areas of tissue. Figures 96A and 96C show staining with MNC3. Figures 96B and 96D show staining with 25E6. These antibodies bind to epitopes containing all or part of the sequence ASRYNLT. All of these antibodies can bind to PSMGFR peptides and bind to N-10 peptides, but also C-10 peptides. As can be seen, these antibodies are not cancer-specific and show some binding to normal liver tissue. [Figure 97] Figures 97A-97B show photographs of normal liver tissue specimens stained with antibody 1E4 of the present invention. Figure 97A shows an entire tissue core. Figure 97B shows a 40x magnification of a specific area of tissue. Antibody 1E4 binds to an epitope containing all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to the N-10 peptide, but also binds to the C-10 peptide. As can be seen, 1E4 binds to normal liver tissue. [Figure 98] Figures 98A-98H show photographs of normal liver tissue specimens stained with different antibodies of the present invention. Figures 98A-98D show whole tissue cores. Figures 98E-98H show 40x magnification of specific areas of tissue. 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 containing all or part of the sequence GTINVHDVET, the most N-terminal portion of the PSMGFR peptide. None of these antibodies are able to 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-99D show photographs of normal liver tissue specimens stained with antibodies of the present invention. Figures 99A-99B show whole tissue cores. Figures 99C-99D show 40x magnification of specific areas of tissue. 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, 8A9 shows strong binding to normal liver tissue. 17H6 is a weak antibody and may not have been used at a high enough concentration in this study. [Figure 100] Figures 100A-100B show photographs of normal liver tissue specimens stained with antibody 45C11 of the present invention. Figure 100A shows an entire tissue core. Figure 100B shows a 40x magnification of a specific area of tissue. Antibody 45C11 binds to an epitope outside the PSMGFR region and including all or part of the sequence SNIKFRPGSVV. Antibody 45C11 is unable to bind to the N-10 peptide. As can be seen, 45C11 binds strongly to normal liver tissue. [Figure 101] Figures 101A-101H show photographs of normal lung tissue specimens stained with different antibodies of the present invention. Figures 101A-101D show whole tissue cores. Figures 101E-101H show 40x magnification of specific tissue regions. 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 bind to 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. These antibodies also recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen, these antibodies show no binding to normal lung tissue. [Figure 102] Figures 102A-102D show photographs of normal lung tissue specimens stained with different antibodies of the present invention. Figures 102A-102B show whole tissue cores. Figures 102C-102D show 40x magnification of specific areas of tissue. Figures 102A and 102C show staining with MNC3. Figures 102B and 102D show staining with 25E6. These antibodies bind to epitopes containing all or part of the sequence ASRYNLT. All of these antibodies can bind to PSMGFR peptides and bind to N-10 peptides, but also C-10 peptides. As can be seen, these antibodies are not cancer-specific and show some binding to normal lung tissue. [Figure 103] Figures 103A-103B show photographs of normal lung tissue specimens stained with antibody 1E4 of the present invention. Figure 103A shows an entire tissue core. Figure 103B shows a 40x magnification of a specific area of tissue. Antibody 1E4 binds to an epitope containing all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to the N-10 peptide, but also binds to the C-10 peptide. [Figure 104] Figures 104A-104H show photographs of normal lung tissue specimens stained with different antibodies of the present invention. Figures 104A-104D show whole tissue cores. Figures 104E-104H show 40x magnification of specific areas of tissue. 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 containing all or part of the sequence GTINVHDVET, the most N-terminal portion of the PSMGFR peptide. None of these antibodies are able to bind to the N-10 peptide. As can be seen, all of these antibodies show strong binding to normal lung tissue. [Figure 105]Figures 105A-105D show photographs of normal lung tissue specimens stained with antibodies of the present invention. Figures 105A-105B show whole tissue cores. Figures 105C-105D show 40x magnification of specific areas of tissue. 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 may not have been used at a high enough concentration in this study. [Figure 106] Figures 106A-106B show photographs of normal lung tissue specimens stained with antibody 45C11 of the present invention. Figure 106A shows the entire tissue core. Figure 106B shows a 40x magnification of a specific area of tissue. Antibody 45C11 binds to an epitope outside the PSMGFR region and including all or part of the sequence SNIKFRPGSVV. Antibody 45C11 is unable to bind to the N-10 peptide. As can be seen, 45C11 binds to normal lung tissue. [Figure 107] Figures 107A-107H show photographs of normal bone marrow tissue specimens stained with different antibodies of the present invention. Figures 107A-107D show whole tissue cores. Figures 107E-107H show 40x magnification of specific tissue regions. 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 bind to 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. These antibodies also recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen, these antibodies do not exhibit binding to normal bone marrow tissue. [Figure 108] Figures 108A-108D show photographs of normal bone marrow tissue specimens stained with different antibodies of the present invention. Figures 108A-108B show the entire tissue core. Figures 108C-108D show 40x magnification of specific areas of tissue. 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 sequence ASRYNLT. All of these antibodies can bind to the PSMGFR peptide, and bind to the N-10 peptide, but also to the C-10 peptide. [Figure 109] Figures 109A-109B show photographs of normal bone marrow tissue specimens stained with antibody 1E4 of the present invention. Figure 109A shows the entire tissue core. Figure 109B shows a 40x magnification of a specific area of tissue. Antibody 1E4 binds to an epitope containing all or part of the sequence QFNQYKTEA. 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] Figures 110A-110H show photographs of normal bone marrow tissue specimens stained with different antibodies of the present invention. Figures 110A-110D show whole tissue cores. Figures 110E-110H show 40x magnification of specific areas of tissue. 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 containing all or part of the sequence GTINVHDVET, the most N-terminal portion of the PSMGFR peptide. None of these antibodies are able to bind to the N-10 peptide. As can be seen, all of these antibodies show strong binding to normal bone marrow tissue. [Figure 111]Figures 111A-111D show photographs of normal bone marrow tissue specimens stained with antibodies of the present invention. Figures 111A-111B show entire tissue cores. Figures 111C-111D show 40x magnification of specific areas of tissue. 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, 8A9 shows strong binding to normal bone marrow tissue. 17H6 is a weak antibody and may not have been used at a high enough concentration in this study. [Figure 112] Figures 112A-112B show photographs of normal bone marrow tissue specimens stained with antibody 45C11 of the present invention. Figure 112A shows the entire tissue core. Figure 112B shows a 40x magnification of a specific area of tissue. Antibody 45C11 binds to an epitope outside the PSMGFR region and including all or part of the sequence SNIKFRPGSVV. Antibody 45C11 is unable to bind to the N-10 peptide. As can be seen, 45C11 binds to normal bone marrow tissue. [Figure 113] Figures 113A-113C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. Figure 113A shows a photograph of the tissue microarray. Figure 113B shows a map of the array with abbreviated tissue descriptors. Figure 113C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 114-1]Figures 114A-114X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. 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 114A-114X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. 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 114A-114X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. 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] Figures 115A-115C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 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 with abbreviated tissue descriptors. Figure 115C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 116] Figures 116A-116F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. 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] Figures 117A-117C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA805c stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. Figure 117A shows a photograph of the tissue microarray. Figure 117B shows a map of the array with abbreviated tissue descriptors. Figure 117C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 118]Figures 118A-118F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA805c stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. 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] Figures 119A-119C show photographs, array maps, and descriptions 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 with abbreviated tissue descriptors. Figure 119C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 120] Figures 120A-120F show 6x and 20x magnification photographs of selected tissues from esophageal cancer tissue array BC001113 stained with 0.25 μg / mL of anti-PSMGFR antibody 20A10. 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] Figures 121A-121C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1. Figure 121A shows a photograph of the tissue microarray. Figure 121B shows a map of the array with abbreviated tissue descriptors. Figure 121C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 122-1]Figures 122A-122X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1. 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 tissue. Figures 122K and 122O are lung tissue. Figures 122L and 122P are ureters. Figures 122Q and 122U are eyes. Figures 122R and 122V are cerebral cortex tissue. Figures 122S and 122W are bone marrow tissue. Figures 122T and 122X are skeletal muscle tissue. [Figure 122-2] Figures 122A-122X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1. 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 tissue. Figures 122K and 122O are lung tissue. Figures 122L and 122P are ureters. Figures 122Q and 122U are eyes. Figures 122R and 122V are cerebral cortex tissue. Figures 122S and 122W are bone marrow tissue. Figures 122T and 122X are skeletal muscle tissue. [Figure 122-3]Figures 122A-122X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1. 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 tissue. Figures 122K and 122O are lung tissue. Figures 122L and 122P are ureters. Figures 122Q and 122U are eyes. Figures 122R and 122V are cerebral cortex tissue. Figures 122S and 122W are bone marrow tissue. Figures 122T and 122X are skeletal muscle tissue. [Figure 123] Figures 123A-123C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1. Figure 123A shows a photograph of the tissue microarray. Figure 123B shows a map of the array with abbreviated tissue descriptors. Figure 123C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 124] Figures 124A-124F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA1003 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1. 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] Figures 125A-125C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 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 with abbreviated tissue descriptors. Figure 125C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 126]Figures 126A-126F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 20 μg / mL of anti-PSMGFR antibody 3C2B1. 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] Figures 127A-127C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 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 with abbreviated tissue descriptors. Figure 127C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 128-1] Figures 128A-128X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 1 μg / mL of the anti-PSMGFR antibody 5C6F3. 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. Figures 128T and 128X are skeletal muscle. [Figure 128-2]Figures 128A-128X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 1 μg / mL of the anti-PSMGFR antibody 5C6F3. 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. Figures 128T and 128X are skeletal muscle. [Figure 128-3] Figures 128A-128X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 1 μg / mL of the anti-PSMGFR antibody 5C6F3. 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. Figures 128T and 128X are skeletal muscle. [Figure 129] Figures 129A-129C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 1-20 μg / mL of anti-PSMGFR antibody 5C6F3. Figure 129A shows a photograph of the tissue microarray. Figure 129B shows a map of the array with abbreviated tissue descriptors. Figure 129C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 130]Figures 130A-130F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA1003 stained with 1 μg / mL of anti-PSMGFR antibody 5C6F3. 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] Figures 131A-131C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 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 with abbreviated tissue descriptors. Figure 131C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 132] Figures 132A-132F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 1 μg / mL of anti-PSMGFR antibody 5C6F3. 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] Figures 133A-133C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 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 with abbreviated tissue descriptors. Figure 133C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 134-1]Figures 134A-134X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 10 μg / mL of anti-PSMGFR antibody 18B4. 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 muscle. [Figure 134-2] Figures 134A-134X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 10 μg / mL of anti-PSMGFR antibody 18B4. 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 muscle. [Figure 134-3]Figures 134A-134X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 10 μg / mL of anti-PSMGFR antibody 18B4. 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 muscle. [Figure 135] Figures 135A-135C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 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 with abbreviated tissue descriptors. Figure 135C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 136] Figures 136A-136F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 10 μg / mL of anti-PSMGFR antibody 18B4. 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] Figures 137A-137C show photographs, array maps, and descriptions 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 with abbreviated tissue descriptors. Figure 137C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 138]Figures 138A-138F show 6x and 20x magnification photographs of selected tissues from esophageal cancer tissue array BC001113 stained with 10 μg / mL of anti-PSMGFR antibody 18B4. 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] Figures 139A-139C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 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 with abbreviated tissue descriptors. Figure 139C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 140-1] Figures 140A-140X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 10 μg / mL of anti-PSMGFR antibody 18G12. Figures 140A and 140E are adrenal glands. Figures 140B and 140F are breast tissue. 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 tissue. Figures 140K and 140O are lung tissue. Figures 140L and 140P are ureters. Figures 140Q and 140U are eyes. Figures 140R and 140V are cerebral cortex tissue. Figures 140S and 140W are bone marrow tissue. Figure 140T and Figure 140X are skeletal muscles. [Figure 140-2]Figures 140A-140X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 10 μg / mL of anti-PSMGFR antibody 18G12. Figures 140A and 140E are adrenal glands. Figures 140B and 140F are breast tissue. 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 tissue. Figures 140K and 140O are lung tissue. Figures 140L and 140P are ureters. Figures 140Q and 140U are eyes. Figures 140R and 140V are cerebral cortex tissue. Figures 140S and 140W are bone marrow tissue. Figure 140T and Figure 140X are skeletal muscles. [Figure 140-3] Figures 140A-140X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 10 μg / mL of anti-PSMGFR antibody 18G12. Figures 140A and 140E are adrenal glands. Figures 140B and 140F are breast tissue. 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 tissue. Figures 140K and 140O are lung tissue. Figures 140L and 140P are ureters. Figures 140Q and 140U are eyes. Figures 140R and 140V are cerebral cortex tissue. Figures 140S and 140W are bone marrow tissue. Figure 140T and Figure 140X are skeletal muscles. [Figure 141] Figures 141A-141C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 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 with abbreviated tissue descriptors. Figure 141C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 142]Figures 142A-142F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 15 μg / mL of anti-PSMGFR antibody 18G12. 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] Figures 143A-143C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 15 μg / mL of anti-PSMGFR antibody 18G12. Figure 143A shows a photograph of the tissue microarray. Figure 143B shows a map of the array with abbreviated tissue descriptors. Figure 143C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 144] Figures 144A-144F show 6x and 20x magnification photographs of selected tissues from the pancreatic cancer tissue array PA1003 stained with 15 μg / mL of anti-PSMGFR antibody 18G12. 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 involvement. [Figure 145] Figures 145A-145C show photographs, array maps, and descriptions 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 with abbreviated tissue descriptors. Figure 145C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 146] Figures 146A-146F show 6x and 20x magnification photographs of selected tissues from esophageal cancer tissue array BC001113 stained with 30 μg / mL of anti-PSMGFR antibody 18G12. 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] Figures 147A-147C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 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 with abbreviated tissue descriptors. Figure 147C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 148-1] Figures 148A-148X show 6x and 20x magnification photographs of selected tissues from FDA Normal Tissue Array 1021 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6. 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 cardiac muscle. 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. Figures 148T and 148X are skeletal muscle. [Figure 148-2] Figures 148A-148X show 6x and 20x magnification photographs of selected tissues from FDA Normal Tissue Array 1021 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6. 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 cardiac muscle. 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. Figures 148T and 148X are skeletal muscle. [Figure 148-3]Figures 148A-148X show 6x and 20x magnification photographs of selected tissues from FDA Normal Tissue Array 1021 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6. 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 cardiac muscle. 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. Figures 148T and 148X are skeletal muscle. [Figure 149] Figures 149A-149C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 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 with abbreviated tissue descriptors. Figure 149C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 150] Figures 150A-150F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6. 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] Figures 151A-151C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6. Figure 151A shows a photograph of the tissue microarray. Figure 151B shows a map of the array with abbreviated tissue descriptors. Figure 151C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 152]Figures 152A-152F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA1003 stained with 5.0 μg / mL of anti-PSMGFR antibody 25E6. 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] Figures 153A-153C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 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 with abbreviated tissue descriptors. Figure 153C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 154-1] Figures 154A-154X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9. 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. Figures 154T and 154X are skeletal muscle. [Figure 154-2]Figures 154A-154X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9. 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. Figures 154T and 154X are skeletal muscle. [Figure 154-3] Figures 154A-154X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9. 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. Figures 154T and 154X are skeletal muscle. [Figure 155] Figures 155A-155C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 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 with abbreviated tissue descriptors. Figure 155C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 156]Figures 156A-156F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 15.0 μg / mL of anti-PSMGFR antibody 28F9. 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] Figures 157A-157C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 7.5 μg / mL of N+20 / C-27 antibody 1E4. Figure 157A shows a photograph of the tissue microarray. Figure 157B shows a map of the array with abbreviated tissue descriptors. Figure 157C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 158-1] Figures 158A-158X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 7.5 μg / mL of N+20 / C-27 antibody 1E4. 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 158A-158X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 7.5 μg / mL of N+20 / C-27 antibody 1E4. 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 158A-158X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 7.5 μg / mL of N+20 / C-27 antibody 1E4. 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] Figures 159A-159C show photographs, array maps, and descriptions of breast cancer tissue array BR1007 stained with 10.0 μg / mL of N+20 / C-27 antibody 1E4. Figure 159A shows a photograph of the tissue microarray. Figure 159B shows a map of the array with abbreviated tissue descriptors. Figure 159C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 160]Figures 160A-160F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1007 stained with 10.0 μg / mL of N+20 / C-27 antibody 1E4. 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] Figures 161A-161C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1. Figure 161A shows a photograph of the tissue microarray. Figure 161B shows a map of the array with abbreviated tissue descriptors. Figure 161C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 162-1] Figures 162A-162X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1. 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 162A-162X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1. 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 162A-162X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1. 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] Figures 163A-163C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1. Figure 163A shows a photograph of the tissue microarray. Figure 163B shows a map of the array with abbreviated tissue descriptors. Figure 163C shows a detailed description of the tissue microarray including non-specific donor data. [Fig. 164]Figures 164A-164F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1. 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] Figures 165A-165C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1. Figure 165A shows a photograph of the tissue microarray. Figure 165B shows a map of the array with abbreviated tissue descriptors. Figure 165C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 166] Figures 166A-166F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA1003 stained with 0.5 μg / mL of N+20 / C-27 antibody 29H1. 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] Figures 167A-167C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1. Figure 167A shows a photograph of the tissue microarray. Figure 167B shows a map of the array with abbreviated tissue descriptors. Figure 167C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 168-1]Figures 168A-168X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1. 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 168A-168X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1. 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 168A-168X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1. 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] Figures 169A-169C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1. Figure 169A shows a photograph of the tissue microarray. Figure 169B shows a map of the array with abbreviated tissue descriptors. Figure 169C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 170] Figures 170A-170F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1. 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] Figures 171A-171C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1. Figure 171A shows a photograph of the tissue microarray. Figure 171B shows a map of the array with abbreviated tissue descriptors. Figure 171C shows a detailed description of the tissue microarray including non-specific donor data. [Fig. 172]Figures 172A-172F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA1003 stained with 0.5 μg / mL of N+20 / C-27 antibody 31A1. 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] Figures 173A-173C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 0.25 μg / mL of N+20 / C-27 antibody 32C1. Figure 173A shows a photograph of the tissue microarray. Figure 173B shows a map of the array with abbreviated tissue descriptors. Figure 173C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 174-1] Figures 174A-174X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.25 μg / mL of N+20 / C-27 antibody 32C1. 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 174A-174X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.25 μg / mL of N+20 / C-27 antibody 32C1. 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 174A-174X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 0.25 μg / mL of N+20 / C-27 antibody 32C1. 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] Figures 175A-175C show photographs, array maps, and descriptions of breast cancer tissue array BR1141 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 with abbreviated tissue descriptors. Figure 175C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 176]Figures 176A-176F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1141 stained with 5.0 μg / mL of N+20 / C-27 antibody 32C1. 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] Figures 177A-177C show photographs, array maps, and descriptions 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 with abbreviated tissue descriptors. Figure 177C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 178] Figures 178A-178F show 6x and 20x magnification photographs of selected tissues from esophageal cancer tissue array BC001113 stained with 1.0 μg / mL of N+20 / C-27 antibody 32C1. 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] Figures 179A-179C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11. Figure 179A shows a photograph of the tissue microarray. Figure 179B shows a map of the array with abbreviated tissue descriptors. Figure 179C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 180-1]Figures 180A-180X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11. 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. Figure 180T and Figure 180X are skeletal muscles. [Figure 180-2] Figures 180A-180X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11. 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. Figure 180T and Figure 180X are skeletal muscles. [Figure 180-3]Figures 180A-180X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11. 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. Figure 180T and Figure 180X are skeletal muscles. [Figure 181] Figures 181A-181C show photographs, array maps, and descriptions of breast cancer tissue array BR1007 stained with 10.0 μg / mL of N+20 / C-27 antibody 45C11. Figure 181A shows a photograph of the tissue microarray. Figure 181B shows a map of the array with abbreviated tissue descriptors. Figure 181C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 182] Figures 182A-182F show 6x and 20x magnification photographs of selected tissues from breast cancer tissue array BR1007 stained with 10.0 μg / mL of N+20 / C-27 antibody 45C11. 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] Figures 183A-183C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA805c stained with N+20 / C-27 antibody 45C11 at 12.5 μg / mL. Figure 183A shows a photograph of the tissue microarray. Figure 183B shows a map of the array with abbreviated tissue descriptors. Figure 183C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 184]Figures 184A-184F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA805c stained with 12.5 μg / mL of N+20 / C-27 antibody 45C11. 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] Figures 185A-185C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5. Figure 185A shows a photograph of the tissue microarray. Figure 185B shows a map of the array with abbreviated tissue descriptors. Figure 185C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 186-1] Figures 186A-186X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5. 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 186A-186X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5. 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 186A-186X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5. 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] Figures 187A-187C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5. Figure 187A shows a photograph of the tissue microarray. Figure 187B shows a map of the array with abbreviated tissue descriptors. Figure 187C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 188]Figures 188A-188F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA1003 stained with 10.0 μg / mL of N+9 / C-9 antibody 3C5. 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 involvement. [Figure 189] Figures 189A-189C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9. Figure 189A shows a photograph of the tissue microarray. Figure 189B shows a map of the array with abbreviated tissue descriptors. Figure 189C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 190-1] Figures 190A-190X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9. 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. Figure 190T and Figure 190X are skeletal muscles. [Figure 190-2]Figures 190A-190X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9. 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. Figure 190T and Figure 190X are skeletal muscles. [Figure 190-3] Figures 190A-190X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9. 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. Figure 190T and Figure 190X are skeletal muscles. [Figure 191] Figures 191A-191C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9. Figure 191A shows a photograph of the tissue microarray. Figure 191B shows a map of the array with abbreviated tissue descriptors. Figure 191C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 192]Figures 192A-192F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA1003 stained with 15.0 μg / mL of N+9 / C-9 antibody 8A9. 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] Figures 193A-193C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 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 with abbreviated tissue descriptors. Figure 193C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 194-1] Figures 194A-194X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6. 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 194A-194X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6. 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 194A-194X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6. 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] Figures 195A-195C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA805c stained with N+9 / C-9 antibody 17H6 at 30.0 μg / mL. Figure 195A shows a photograph of the tissue microarray. Figure 195B shows a map of the array with abbreviated tissue descriptors. Figure 195C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 196]Figures 196A-196F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA805c stained with 30.0 μg / mL of N+9 / C-9 antibody 17H6. 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 involvement. Figures 196C and 196F are photographs of grade 3 invasive adenocarcinoma. [Figure 197] Figures 197A-197C show photographs, array maps, and descriptions of the FDA normal tissue array MNO1021 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5. Figure 197A shows a photograph of the tissue microarray. Figure 197B shows a map of the array with abbreviated tissue descriptors. Figure 197C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 198-1] Figures 198A-198X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5. 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 198A-198X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5. 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 198A-198X show 6x and 20x magnification photographs of selected tissues from the FDA normal tissue array MNO1021 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5. 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] Figures 199A-199C show photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5. Figure 199A shows a photograph of the tissue microarray. Figure 199B shows a map of the array with abbreviated tissue descriptors. Figure 199C shows a detailed description of the tissue microarray including non-specific donor data. [Figure 200]Figures 200A-200F show 6x and 20x magnification photographs of selected tissues from pancreatic cancer tissue array PA1003 stained with 5.0 μg / mL of N+9 / C-9 antibody 39H5. 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] Figures 201A-201C show graphs of ELISA assays to determine binding of another set of antibodies generated by immunizing animals with PSMGFR peptides. 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] Figures 202A-202C show photographs of pancreatic cancer tissue array PA1003 stained with monoclonal antibody 1E4, monoclonal antibody 18B4, or polyclonal anti-PSMGFR antibody SDIX. 18B4 binds to the GTINVHDVET epitope in the N-terminal extreme of the PSMGFR peptide, while the 1E4 antibody binds to the QFNQYKTEA epitope immediately adjacent to the 18B4 epitope and at the C-terminus. [Figure 203] Figures 203A-203F show magnified images of tissue specimens at position A2 of the pancreatic cancer array PA1003. Figures 203A and 203B show specimens stained with antibody 1E4. Figures 203C and 203D show specimens stained with antibody 18B4. Figures 203E and 203F show specimens stained with polyclonal antibody SDIX. [Figure 204] Figures 204A-204D show magnified images of tissue specimens at position D4 of pancreatic 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]Figures 205A-205D 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] Figures 206A-206D show magnified images of tissue specimens at position C3 of 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] Figures 207A-207D 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 the polyclonal antibody SDIX. [Figure 208] Figures 208A-208C show photographs of pancreatic cancer array PA1003. 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] Figures 209A-209D show photographs of the esophageal cancer array ES1001 stained with various antibodies. 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] Figures 210A-210C show photographs of the pancreatic cancer array PA1003 stained with various antibodies. 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]Figures 211A-211C show graphs of ELISA experiments measuring the amount of IL-18 secreted into the conditioned medium of MUC1*-positive cancer cells co-cultured with huMNC2-CAR44 T cells, which also have NFAT-inducible IL-18. Figure 211A shows a graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with untransduced human T cells. Figure 211B shows a graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T cells that also have an NFAT-inducible IL-18 gene inserted into part of the Foxp3 enhancer. Figure 211C shows a graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T cells that also have an NFAT-inducible IL-18 gene inserted into part of the IL-2 enhancer. [Figure 212] Figures 212A-212X show photographs of T47D breast cancer cells (red) doped with various ratios of T47D cells engineered to express more MUC1* (green). Target cancer cells were co-cultured with huMNC2-CAR44 T cells harboring NFAT-inducible IL-18, in which the IL-18 gene was inserted into either the Foxp3 enhancer / promoter or the IL-2 enhancer / promoter. Figures 212A-212C, 212I-212K, and 212Q-212S show cancer cells co-cultured with untransduced T cells. Figures 212D-212F, 212L-212N, and 212T-212V show cancer cells co-cultured with hiMNC2-CAR44 T cells harboring the NFAT-inducible IL-18 gene inserted into the Foxp3 enhancer / promoter. Figures 212G-212H, 212O-212P, and 212W-212X 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]Figures 213A-213B show graphs of ELISA experiments measuring levels of IL-18 secreted into conditioned medium from huMNC1-CAR44 T cells harboring an NFAT-inducible IL-18 gene inserted into the Foxp3 enhancer or promoter and co-cultured with either MUC1*-positive cancer cells or MUC1-negative non-cancerous cells. Figure 213A shows IL-18 secretion from huMNC2-CAR44 T cells harboring NFAT-inducible IL-18 co-cultured with T47D breast cancer cells, with the population 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 co-cultured with non-cancerous MUC1-negative HEK293 cells, where the population was doped with 5%, 10%, or 30% MUC1*-transfected T47D cells. [Figure 214]Figures 214A-214X show photographs of T47D breast cancer cells (red) or noncancerous HEK293 cells (also red); both cell types have been 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 harboring NFAT-inducible IL-18, in which the IL-18 gene was inserted into the Foxp3 enhancer / promoter. Figures 214A-214F show either T47D cells or HEK293 cells that were not doped with T47D cells engineered to express high MUC1* density. Figures 214G-214L show either T47D cells or HEK293 cells that were doped with 5% T47D cells engineered to express high MUC1* density. Figures 214M-214R show either T47D or HEK293 cells doped with 10% T47D cells engineered to express high MUC1* density. Figures 214S-214X show either T47D or HEK293 cells doped with 30% T47D cells engineered to express high MUC1* density. 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, induced secretion of IL-18 killed low MUC1* density T47D cells but did not induce nonspecific killing of MUC1*-negative HEK293 cells. [Figure 215] Figures 215A-215C show consensus sequences for heavy chain CDRs, where consensus sequences were generated for each group of antibodies binding to the same epitope of PSMGFR and N-terminally extended PSMGFR peptides. Figure 215A shows the consensus sequence for heavy chain CDR1. Figure 215B shows the consensus sequence for heavy chain CDR2. Figure 215C shows the consensus sequence for heavy chain CDR3. [Figure 216]Figures 216A-216C show consensus sequences for the light chain CDRs, where consensus sequences were generated for each group of antibodies binding to the same epitope of PSMGFR and N-terminally extended PSMGFR peptides. Figure 216A shows the consensus sequence for light chain CDR1. Figure 216B shows the consensus sequence for light chain CDR2. Figure 216C shows the consensus sequence for light chain CDR3. [Figure 217] Bispecific antibodies and other alternative bispecific immunotherapy formats are shown subdivided into five major classes: BsIgG, adducted IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. Heavy chains are shown in dark blue, dark pink, and dark green, with the corresponding light chains in lighter shades of the same colors. Connecting peptide linkers are indicated with thin black lines, and engineered disulfide bonds are indicated with thin green lines. Approximate molecular weights are shown, assuming approximately 12.5 kDa per immunoglobulin domain. BsAb formats that have progressed to clinical trials are highlighted (*). To interpret the color references in this figure legend, readers are referred to the web version of the paper, Spiess et al., Molecular Immunology 67, 95–106 (2015), the contents of which are incorporated by reference in their entirety, particularly with regard to the description of Figure 1 in Spiess et al., as well as other descriptions of various methods for making and using bispecific antibody fragments. [Figure 218]This graph shows the volume of tumors measured by an IVIS instrument in which tumor cells were genetically modified to express luciferase. The substrate luciferin was injected 10 minutes before measuring luminescence in sedated animals. On day 1 of the experiment, animals were subcutaneously injected with 250,000 human breast tumor cells. The tumors were heterogeneous; they consisted of two different tumor cell types. The first tumor cell population was T47D-wt, a breast cancer cell line that expresses both full-length MUC1 and the growth factor receptor form MUC1*, which we engineered to express mCherry fluorescence. The second tumor cell population was the same T47D breast cancer cells, except that they were stably transduced to express significantly more MUC1* and GFP fluorescence, referred to as T47D-MUC1*. In this experiment, animals were implanted with T47D-wt + T47D-MUC1*, with the T47D-MUC1* population comprising 30%, 15%, or 7.5% of the tumor population. Animals then received a single injection of either PBS, huMNC2-41BB-3z CAR T cells (4-1BB), huMNC2-CD28-3z CAR T cells (CD28), or huMNC2-CD28-1XX CAR T cells (CD28-1XX or 1XX). CAR T cells were injected into the tail vein at an effector-to-target (E:T) ratio of 10:1, 5:1, or 1:1. [Figure 219] Figures 219A-219B show IVIS photographs and graphs of IVIS tumor volume measurements. Figure 219A shows a photograph of a mouse implanted with a tumor in which 30% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells. Various CAR T cells were administered at a 10:1 CAR T-to-tumor cell ratio, implanting 250,000 tumor cells, and the animals were injected with 2.5 million CAR T cells 5 days later. Figure 219B shows a daily graph of tumor volume measured by IVIS. As can be seen, animals injected with huMNC2-CD28-1XX had much smaller tumors than animals treated with huMNC2-4-1BB-3z or huMNC2-CD28-3z, which are the same CAR Ts except for the lack of the 1XX mutation in the CD3-zeta domain. [Figure 220] Figures 220A-220T show IVIS graphs of 30% of tumors treated with a CAR T to tumor ratio of 10:1, where graphs are shown for individual animals rather than averages for treatment groups. [Figure 221] Figures 221A-221B show IVIS photographs and graphs of IVIS tumor volume measurements. Figure 221A shows a photograph of a mouse implanted with a tumor in which 30% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells. Various CAR T cells were administered at a 1:1 CAR T-to-tumor cell ratio, implanting 250,000 tumor cells, and the animals were injected 5 days later with 250,000 CAR T cells. Figure 221B shows a daily graph of tumor volume measured by IVIS. As can be seen, animals injected with huMNC2-CD28-1XX had much smaller tumors than animals treated with huMNC2-4-1BB-3z or huMNC2-CD28-3z, which are the same CAR Ts except for the lack of the 1XX mutation in the CD3-zeta domain. However, at lower doses of CAR T cells, even tumors in the huMNC2-CD28-1XX treatment group begin to grow again. [Figure 222] Figures 222A-222T show IVIS graphs of 30% of tumors treated with a 1:1 CAR T to tumor ratio, where graphs are shown for individual animals rather than averages for treatment groups. [Figure 223]Figures 223A-223B show IVIS photographs and graphs of IVIS tumor volume measurements. Figure 223A shows a photograph of a mouse implanted with a tumor in which 7.5% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells. Various CAR T cells were administered at a 10:1 CAR T-to-tumor cell ratio, implanting 250,000 tumor cells, and the animals were injected with 2.5 million CAR T cells 5 days later. Figure 223B shows a daily graph of tumor volume measured by IVIS. As can be seen, animals injected with huMNC2-CD28-1XX had much smaller tumors than animals treated with huMNC2-4-1BB-3z or huMNC2-CD28-3z, which are the same CAR Ts except for the lack of the 1XX mutation in the CD3-zeta domain. However, even tumors in the huMNC2-CD28-1XX treatment group began to grow again, consistent with the idea that a small percentage of highly antigen-expressing tumor cells prevents the rest of the tumor from being killed. [Figure 224] Figures 224A-224T show IVIS graphs of 7.5% tumors treated with a CAR T to tumor ratio of 10:1. Here, graphs are shown for individual animals rather than averages for treatment groups. [Figure 225] Figures 225A-225B show IVIS photographs and graphs of IVIS tumor volume measurements. Figure 225A shows a photograph of a mouse implanted with a tumor in which 7.5% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells. Various CAR T cells were administered at a 1:1 CAR T-to-tumor cell ratio, implanting 250,000 tumor cells, and the animals were injected with 250,000 CAR T cells 5 days later. Figure 225B shows a daily graph of tumor volume measured by IVIS. As can be seen, animals injected with huMNC2-CD28-1XX had smaller tumors than animals treated with huMNC2-4-1BB-3z or huMNC2-CD28-3z, which are the same CAR Ts except for the lack of the 1XX mutation in the CD3-zeta domain. However, at low antigen density combined with very low doses of CAR T cells, even tumors in the huMNC2-CD28-1XX treatment group begin to grow again. [Figure 226] Figures 226A-226T show IVIS graphs for 7.5% of tumors treated with a 1:1 CAR T to tumor ratio, where graphs are shown for individual animals rather than averages for treatment groups. [Figure 227-1] The table shows the count of CD3-positive human T cells harvested from the spleens of test animals after sacrifice. In this table, cells isolated from tumor-implanted mice consisted of 30% T47D-MUC1* and were treated with CAR T cells at a 10:1 ratio. As can be seen, huMNC2-CD28-1XX-treated mice, which had smaller tumors, had higher numbers of CAR T cells and CD8-positive killer T cells. TIM3, LAG3, and PD-1 are molecular markers of T cell exhaustion. The table shows that huMNC2-CD28-1XX CAR T cells harvested from animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutation in CD3-zeta increases CAR T cell persistence in vivo. [Figure 227-2] The table shows the count of CD3-positive human T cells harvested from the spleens of test animals after sacrifice. In this table, cells isolated from tumor-implanted mice consisted of 30% T47D-MUC1* and were treated with CAR T cells at a 10:1 ratio. As can be seen, huMNC2-CD28-1XX-treated mice, which had smaller tumors, had higher numbers of CAR T cells and CD8-positive killer T cells. TIM3, LAG3, and PD-1 are molecular markers of T cell exhaustion. The table shows that huMNC2-CD28-1XX CAR T cells harvested from animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutation in CD3-zeta increases CAR T cell persistence in vivo. [Figure 228]The table shows the count of CD3-positive human T cells harvested from the spleens of test animals after sacrifice. In this table, cells isolated from tumor-implanted mice consisted of 30% T47D-MUC1* and were treated with CAR T cells at a 1:1 ratio. As can be seen, huMNC2-CD28-1XX-treated mice, which had smaller tumors, had higher numbers of CAR T cells and CD8-positive killer T cells. TIM3, LAG3, and PD-1 are molecular markers of T cell exhaustion. The table shows that huMNC2-CD28-1XX CAR T cells harvested from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutation in CD3-zeta increases CAR T cell persistence in vivo. [Figure 229] Shown is the count of CD3-positive human T cells collected from the blood of test animals after sacrifice. In this table, cells isolated from tumor-implanted mice consisted of 30% T47D-MUC1* and were treated with CAR T cells at a 1:1 ratio. As can be seen, huMNC2-CD28-1XX-treated mice with smaller tumors had higher numbers of CAR T cells and CD8-positive killer T cells, and lower levels of T-cell exhaustion markers. [Figure 230] The table shows the count of CD3-positive human T cells harvested from the spleens of test animals after sacrifice. In this table, cells isolated from tumor-implanted mice consisted of 7.5% T47D-MUC1* and were treated with CAR T cells at a 10:1 ratio. As can be seen, huMNC2-CD28-1XX-treated mice, which had smaller tumors, had higher numbers of CAR T cells and CD8-positive killer T cells. TIM3, LAG3, and PD-1 are molecular markers of T cell exhaustion. The table shows that huMNC2-CD28-1XX CAR T cells harvested from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutation in CD3-zeta increases CAR T cell persistence in vivo. [Figure 231]The table shows the count of CD3-positive human T cells collected from the blood of test animals after sacrifice. In this table, cells isolated from tumor-implanted mice consisted of 7.5% T47D-MUC1* and were treated with CAR T cells at a 10:1 ratio. As can be seen, huMNC2-CD28-1XX-treated mice, which had smaller tumors, had higher numbers of CAR T cells and CD8-positive killer T cells. TIM3, LAG3, and PD-1 are molecular markers of T cell exhaustion. The table shows that huMNC2-CD28-1XX CAR T cells collected from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutation in CD3-zeta increases CAR T cell persistence in vivo. [Figure 232] The table shows the count of CD3-positive human T cells harvested from the spleens of test animals after sacrifice. In this table, cells isolated from tumor-implanted mice consisted of 7.5% T47D-MUC1* and were treated with CAR T cells at a 1:1 ratio. As can be seen, huMNC2-CD28-1XX-treated mice, which had smaller tumors, had higher numbers of CAR T cells and CD8-positive killer T cells. TIM3, LAG3, and PD-1 are molecular markers of T cell exhaustion. The table shows that huMNC2-CD28-1XX CAR T cells harvested from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutation in CD3-zeta increases CAR T cell persistence in vivo. [Figure 233]The table shows the count of CD3-positive human T cells collected from the blood of test animals after sacrifice. In this table, cells isolated from tumor-implanted mice consisted of 7.5% T47D-MUC1* and were treated with CAR T cells at a 1:1 ratio. As can be seen, huMNC2-CD28-1XX-treated mice, which had smaller tumors, had higher numbers of CAR T cells and CD8-positive killer T cells. TIM3, LAG3, and PD-1 are molecular markers of T cell exhaustion. The table shows that huMNC2-CD28-1XX CAR T cells collected from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutation in CD3-zeta increases CAR T cell persistence in vivo. [Figure 234] Figures 234A-234U show photographs of tumors excised from test animals, and their weights in grams are shown. Tumors were excised from animals implanted with tumors composed of 30% T47D-MUC1* high antigen density cells and 70% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector-to-target ratio of 10:1. [Figure 235] Figures 235A-235N2 show magnified photographs of dissociated tumors excised from animals implanted with tumors composed of 30% T47D-MUC1* high antigen density cells and 70% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector-to-target ratio of 10:1. Overlays of brightfield and fluorescent images are shown, with red fluorescent mCherry indicating low antigen density cells and green fluorescent GFP indicating low antigen density cells. Figures 235A-235J show tumor cells excised from a control animal treated with PBS only. Figures 235K-235T show tumor cells excised from an animal treated with huMNC2-41BB-3z CAR T cells. Figures 235U-235D2 show tumor cells excised from an animal treated with huMNC2-CD28-1XX CAR T cells. Figures 235E2-235N2 show tumor cells excised from animals treated with huMNC2-CD28-3z CAR T cells. [Figure 236]Figures 236A-236U show photographs of tumors excised from test animals, showing their weights in grams. Tumors were excised from animals implanted with tumors composed of 30% T47D-MUC1* high antigen density cells and 70% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector-to-target ratio of 1:1. Figures 236A-236E show tumors excised from animals mock-treated with PBS. Figures 236F-236J show tumors excised from animals treated with huMNC2-41BB-3z. Figures 236K-236O show tumors excised from animals treated with huMNC2-CD28-1XX. Figures 236P-236T show tumors excised from animals treated with huMNC2-CD28-3z. Figure 236U shows a bar graph of the weight of tumors excised from test animals. [Figure 237] Figures 237A-237D2 show magnified photographs of dissociated tumors excised from animals implanted with tumors composed of 30% T47D-MUC1* high antigen density cells and 70% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector-to-target ratio of 1:1. Overlays of brightfield and fluorescent images are shown, with red fluorescent mCherry indicating low antigen density cells and green fluorescent GFP indicating low antigen density cells. Figures 237A-237J show tumor cells excised from a control animal treated with PBS only. Figures 237K-237T show tumor cells excised from an animal treated with huMNC2-41BB-3z CAR T cells. Figures 237U-237D2 show tumor cells excised from an animal treated with huMNC2-CD28-1XX CAR T cells. Figures 237E2-237N2 show tumor cells excised from animals treated with huMNC2-CD28-3z CAR T cells. [Figure 238] Figures 238A-238T show photographs of tumors excised from test animals, and their weights in grams are shown. Tumors were excised from animals implanted with tumors composed of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector-to-target ratio of 10:1. [Figure 239]Figures 239A-239M2 show magnified images of dissociated tumors excised from animals implanted with tumors composed of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector-to-target ratio of 10:1. Overlays of brightfield and fluorescent images are shown, with red fluorescent mCherry indicating low antigen density cells and green fluorescent GFP indicating low antigen density cells. Figures 239A-239J show tumor cells excised from animals mock-treated with PBS. Figures 239K-239T show tumor cells excised from animals treated with huMNC2-41BB-3z CAR T cells. Figures 239U-239C2 show tumor cells excised from animals treated with huMNC2-CD28-1XX CAR T cells. Figures 239D2-239M2 show tumor cells excised from animals treated with huMNC2-CD28-3z CAR T cells. [Figure 240] Figures 240A-240O show photographs of tumors excised from test animals, and their weights in grams are shown. Tumors were excised from animals implanted with tumors composed of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector-to-target ratio of 1:1. [Figure 241]Figures 241A-241D2 show magnified photographs of dissociated tumors excised from animals implanted with tumors composed of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector-to-target ratio of 1:1. Overlays of brightfield and fluorescent images are shown, with red fluorescent mCherry indicating low antigen density cells and green fluorescent GFP indicating low antigen density cells. Figures 241A-241J show tumor cells excised from a control animal treated with PBS only. Figures 241K-241T show tumor cells excised from an animal treated with huMNC2-41BB-3z CAR T cells. Figures 241U-241D2 show tumor cells excised from an animal treated with huMNC2-CD28-1XX CAR T cells. Figures 241E2-241N2 show tumor cells excised from animals treated with huMNC2-CD28-3z CAR T cells. [Figure 242] Figures 242A-242R show photographs of live animals, IVIS measures tumor volume, mCherry detects low-antigen cells within the tumor, and GFP detects high-antigen cells within the tumor. Photographs of excised tumors after sacrifice and a graph of tumor weight are shown. A graph of GFP-positive high-antigen tumor cells is also shown. Both the live GFP photographs and the graph of FACS measurements of high-antigen cells show that huMNC2-CD28-1XX killed all high-antigen cells and most low-antigen cells, even at the lowest levels of high-antigen cells within the tumor and at very low CAR T cell doses. [Figure 243] Figures 243A-243F show photographs taken at two different time points, where the IVIS photographs measure tumor volume, the mCherry fluorescence photographs measure low antigen cells, and the GFP fluorescence photographs measure high antigen cells. In this case, animals were implanted with tumors composed of 30% high antigen density cells (GFP+) and 70% low antigen density cells (mCherry+). Animals received a single dose of CAR T cells at a ratio of 10:1 or 1:1 CAR T to tumor cells. [Figure 244]A graph of IVIS tumor volume measurements over time is shown. Arrows indicate time points when fluorescent photographs, mCherry and GFP, of live animals were taken. In this case, animals were implanted with tumors composed of 30% high antigen density cells (GFP+) and 70% low antigen density cells (mCherry+). Animals received a single dose of CAR T cells at a 10:1 or 1:1 CAR T to tumor cell ratio. [Figure 245] Graphs are shown for IVIS measurements of tumor volume, mCherry measurements of low-antigen cell proliferation rate, and GFP measurements of high-antigen cell proliferation rate at two time points. As can be seen, at high CART doses, the number of high-antigen-density cells (GFP) did not increase over time in mice treated with either huMNC2-41BB-3z or huMNC2-CD28-1XX. However, at low CART doses, some mice treated with huMNC2-41BB-3z showed increased proliferation of high-antigen-density cells, while mice treated with huMNC2-CD28-1XX did not. More importantly, at high or low doses of CAR T, mice treated with huMNC2-41BB-3z showed increased proliferation of low-antigen-density cells, while mice treated with huMNC2-CD28-1XX showed more controlled proliferation of low-antigen-density cells. [Figure 246] Figures 246A-246B show IVIS photographs and graphs of IVIS tumor volume measurements. Figure 246A shows a photograph of a mouse implanted with a tumor in which 15% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells. Various CAR T cells were administered at a 10:1 CAR T-to-tumor cell ratio, implanting 250,000 tumor cells, and the animal was injected 5 days later with 2.5 million CAR T cells. Figure 246B shows a daily graph of tumor volume measured by IVIS. In this experiment, on day 52, the animal outlined in red was implanted with 250,000 additional 100% high antigen density tumor cells. The animal outlined in green received an additional dose of 2.5 million CAR T cells. [Figure 247]Figures 247A-247T show graphs of tumor growth measured by IVIS in each individual animal. Red arrows indicate the injection of more tumor cells, and green arrows indicate the injection of 2.5 million additional CAR T cells. As can be seen, the injection of additional tumor cells does not increase tumor growth in animals treated with huMNC2-CD28-1XX CAR T cells. However, the injection of additional tumor cells does increase tumor growth in animals treated with huMNC2-CD28-3z or huMNC2-41BB-3z CAR T cells. This result is consistent with CAR T cells with a 1XX mutation in CD3z extending CAR T cell persistence in vivo. It can also be seen that the injection of additional CAR T cells suppressed tumor cells in some animals in all groups. This is consistent with the idea that tumor recurrence was not due to tumor escape, as fresh CAR T cells still recognized and killed the tumor cells. [Figure 248] Figures 248A-248D show illustrations of an experimental strategy in which animals were implanted with heterogeneous tumors composed of two cell types with two different fluorescent labels. Animals were implanted with both mCherry and red-fluorescent T47D breast cancer cells, as well as T47D cells that express significantly more MUC1* and are engineered to carry GFP, resulting in green fluorescence. Figure 248A shows an illustration of an animal implanted with a tumor in which 30% of the tumor cells express high levels of MUC1* and fluoresce green. Figure 248B shows an illustration of an animal implanted with a tumor in which 15% of the tumor cells express high levels of MUC1* and fluoresce green. Figure 248C shows an illustration of an animal implanted with a tumor in which 7.5% of the tumor cells express high levels of MUC1* and fluoresce green. The variables used in these experiments are shown in Figure 248D. [Figure 249]Figures 249A-249F show illustrations of the experimental strategy and data. Figure 249A shows an illustration of full-length MUC1 expressed in normal epithelial cells. Figure 249B shows four tissue specimens stained with huMNC2-scFv-Fc. Figure 249C shows an illustration depicting heterogeneous tumors expressing either a high (30%—left) or low (7.5%—right) percentage of high MUC1*-expressing tumor cells. Figure 249D shows flow cytometry verifying the heterogeneous tumor percentage before implantation. Figure 249E shows bar graphs of bioluminescence measured with an IVIS instrument for animals implanted with 30% high MUC1* cells and treated with various CAR T cells at an effector-to-target ratio of 10:1 (top) or 1:1 (bottom). Figure 249F shows a bar graph of bioluminescence measured with an IVIS instrument for animals engrafted with 7.5% MUC1* high cells and treated with various CAR T cells at effector to target ratios of 10:1 (top) or 1:1 (bottom). [Figure 250] Figures 250A-250F show bar graphs of tumor bioluminescence measured by an IVIS instrument for animals implanted with 30% high MUC1* cells or 7.5% high MUC1* tumors and treated with various CAR T cells at various effector-to-target ratios. Figure 250A shows an IVIS graph for an animal implanted with a tumor in which 30% expressed high levels of MUC1* and the animal was treated with CAR T cells at an effector-to-target ratio of 10:1. Figure 250B shows an IVIS graph where the effector-to-target ratio was 5:1. Figure 250C shows an IVIS graph where the effector-to-target ratio was 1:1. Figure 250D shows an IVIS graph for an animal implanted with a tumor in which 7.5% expressed high levels of MUC1* and the animal was treated with CAR T cells at an effector-to-target ratio of 10:1. Figure 250E shows the IVIS graph when the effector to target ratio was 5: 1. Figure 250F shows the IVIS graph when the effector to target ratio was 1: 1. [Figure 251]Figures 251A-251D show photographs of tumor bioluminescence measured by an IVIS instrument for animals implanted with 30% high MUC1* cells or 7.5% high MUC1* tumors and treated with various CAR T cells at effector-to-target ratios of 10:1 or 1:1. Figure 251A shows an IVIS photograph of an animal implanted with a tumor in which 30% expressed high levels of MUC1* and the animal was treated with CAR T cells at an effector-to-target ratio of 10:1. Figure 250B shows an IVIS photograph where the effector-to-target ratio was 1:1. Figure 251C shows an IVIS photograph of an animal implanted with a tumor in which 7.5% expressed high levels of MUC1* and the animal was treated with CAR T cells at an effector-to-target ratio of 10:1. Figure 250D shows an IVIS photograph where the effector-to-target ratio was 1:1. [Figure 252] Figures 252A-252D show magnified fluorescent photographs of dissociated tumors excised from animals implanted with tumors composed of 30% or 7.5% T47D-MUC1* high antigen density cells and the remainder low antigen density cells. Animals were treated with various CAR T cells at an effector-to-target ratio of 10:1 or 1:1. Overlays of brightfield and fluorescent images are shown, with red fluorescent mCherry indicating low antigen density cells and green fluorescent GFP indicating low antigen density cells. Figure 252A shows tumor cells excised from an animal implanted with a 30% high antigen density tumor and treated with various CAR T cells at an effector-to-target ratio of 10:1. Figure 252B shows tumor cells excised from an animal implanted with a 30% high antigen density tumor and treated with various CAR T cells at an effector-to-target ratio of 1:1. Figure 252C shows tumor cells excised from an animal implanted with a 7.5% high antigen density tumor and treated with various CAR T cells at an effector to target ratio of 10: 1. Figure 252D shows tumor cells excised from an animal implanted with a 7.5% high antigen density tumor and treated with various CAR T cells at an effector to target ratio of 1: 1. [Figure 253]Figures 253A-253H show illustrations of the experimental strategy and data. Figure 253A shows an illustration of full-length MUC1 appearing in normal epithelial cells. Figure 253B shows four tissue specimens stained with huMNC2-scFv-Fc. Figure 253C shows an illustration depicting heterogeneous tumors expressing either a high or low percentage of high MUC1*-expressing tumor cells. Figure 253D shows flow cytometry verifying that 15% of tumor cells expressed high levels of MUC1* prior to their implantation. Figure 253E shows a bar graph of bioluminescence measured with an IVIS instrument for animals implanted with 15% high MUC1* cells and treated with various CAR T cells at an effector-to-target ratio of 10:1. Figure 253F shows immunofluorescence photographs of excised tumors from animals implanted with 15% high antigen-density tumors and treated with various CAR T cells at an effector-to-target ratio of 10:1. Figure 253G shows a bar graph of bioluminescence measured on an IVIS instrument for animals implanted with 15% MUC1*-high cells and treated with various CAR T cells at an effector-to-target ratio of 1:1. Figure 253H shows immunofluorescence photographs of excised tumors from animals implanted with 15% antigen-high density tumors and treated with various CAR T cells at an effector-to-target ratio of 1:1. [Figure 254] Figures 254A-254B show bar graphs of bioluminescence measured on an IVIS instrument for animals transplanted with 15% MUC1*-rich cells and treated with various CAR T cells at an effector-to-target ratio of 10:1. Figure 254A shows the graph for an animal treated at a 10:1 effector-to-target ratio. Figure 254B shows the graph for an animal treated at a 1:1 effector-to-target ratio. [Figure 255] Figures 255A-255B show photographs of tumor bioluminescence measured with an IVIS instrument for animals implanted with 15% MUC1*-rich cells and treated with various CAR T cells at an effector-to-target ratio of 10:1. Figure 255A shows a photograph of an animal treated at a 10:1 effector-to-target ratio. Figure 255B shows a photograph of an animal treated at a 1:1 effector-to-target ratio. [Figure 256]Figures 256A-256B show fluorescent photographs of excised tumors, with mCherry-positive low-antigen-density cells shown in red and GFP-positive high-antigen-density cells shown in green. All animals were implanted with a mixture of tumor cells that were 15% high MUC1* expressing cells prior to implantation. Figure 256A shows a photograph of an animal treated with an effector-to-target ratio of 10:1. Figure 256B shows a photograph of an animal treated with an effector-to-target ratio of 1:1. [Figure 257] This table shows the results of flow cytometry, in which cells from excised tumors were assayed for the presence of human T cells and CAR T cells, then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors that were 30% highly antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 10:1. If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 258] This table shows the results of flow cytometry, in which cells from excised tumors were assayed for the presence of human T cells and CAR T cells, then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors that were 15% highly antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 10:1. If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 259] This table shows the results of flow cytometry, in which cells from excised tumors were assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 7.5% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 10:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 260] This table shows the results of flow cytometry, in which cells from excised tumors were assayed for the presence of human T cells and CAR T cells, then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors that were 30% highly antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 5:1. If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 261] This table shows the results of flow cytometry, in which cells from excised tumors were assayed for the presence of human T cells and CAR T cells, then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 7.5% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 5:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 262] This table shows the results of flow cytometry, in which cells from excised tumors were assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 30% high antigen-expressing cells, and the animals were treated with various CAR T cells and a 1:1 effector-to-target ratio. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 263]This table shows the results of flow cytometry, in which the cells of excised tumors are assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion.This shows the analysis of tumors excised from animals implanted with tumors that are 15% high antigen-expressing cells, and the animals are treated with various CAR T cells and an effector-to-target ratio of 1:1.If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 264] This table shows the results of flow cytometry, in which the cells of excised tumors are assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion.This shows the analysis of excised tumors from animals implanted with tumors that were 7.5% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 1:1.If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 265] This table shows the results of flow cytometry, in which cells from the spleens of treated animals were assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 30% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 10:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 266]This table shows the results of flow cytometry, in which cells from the spleens of treated animals were assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 15% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 10:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 267] The table shows the results of flow cytometry, in which cells from the spleens of treated animals were assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 7.5% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 10:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 268] The table shows the results of flow cytometry, in which cells from the spleens of treated animals were assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 30% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 5:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 269]The table shows the results of flow cytometry, in which cells from the spleens of treated animals were assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 15% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 5:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 270] The table shows the results of flow cytometry, in which cells from the spleens of treated animals were assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 7.5% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 5:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 271] The table shows the results of flow cytometry, in which cells from the spleens of treated animals were assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 30% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 1:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Fig. 272]The table shows the results of flow cytometry, in which the cells of the spleen of treated animals are examined for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion.This shows the analysis of tumors excised from animals implanted with tumors that are 15% high antigen-expressing cells, and the animals are treated with various CAR T cells and an effector-to-target ratio of 1:1.If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Fig. 273] The table shows the results of flow cytometry, in which the cells of the spleen of treated animals are examined for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion.This shows the analysis of tumors excised from animals implanted with tumors that were 7.5% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 1:1.If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Fig. 274] The table shows the results of flow cytometry, in which blood from treated animals was assayed for the presence of human T cells and CAR T cells, which were then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors containing 30% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 10:1. If the number of detected CAR T cells is less than 25, the number is marked in red, and further analysis of these few cells is called into question. [Figure 275]The blood from treated animals was assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This table shows the results of flow cytometry. The analysis of tumors excised from animals implanted with tumors containing 15% high antigen-expressing cells was shown here, and the animals were treated with various CAR T cells and an effector-to-target ratio of 10:1. If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 276] The blood from treated animals was assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors that were 7.5% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 10:1. If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 277] The blood from treated animals was assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This table shows the results of flow cytometry. The analysis of tumors excised from animals implanted with tumors containing 30% high antigen-expressing cells was shown here, and the animals were treated with various CAR T cells and an effector-to-target ratio of 5:1. If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Fig. 278]The blood from treated animals was assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This table shows the results of flow cytometry. The analysis of tumors excised from animals implanted with tumors containing 15% high antigen-expressing cells was shown here, and the animals were treated with various CAR T cells and an effector-to-target ratio of 5:1. If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 279] The table shows the results of flow cytometry, in which blood from treated animals was assayed for the presence of human T cells and CAR T cells, which were then enumerated and analyzed for the presence of markers of T cell exhaustion. This shows the analysis of tumors excised from animals implanted with tumors that were 7.5% high antigen-expressing cells, and the animals were treated with various CAR T cells and an effector-to-target ratio of 5:1. If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 280] The blood from treated animals was assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion. This table shows the results of flow cytometry. The analysis of tumors excised from animals implanted with tumors containing 30% high antigen-expressing cells was shown here, and the animals were treated with various CAR T cells and an effector-to-target ratio of 1:1. If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 281]The blood from treated animals is assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion.This shows the analysis of tumors excised from animals transplanted with tumors that are 15% high antigen-expressing cells, and animals are treated with various CAR T cells and an effector-to-target ratio of 1:1.If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 282] The blood from treated animals is assayed for the presence of human T cells and CAR T cells, and then enumerated and analyzed for the presence of markers of T cell exhaustion.This shows the analysis of tumors excised from animals transplanted with tumors that are 7.5% high antigen-expressing cells, and the animals are treated with various CAR T cells and an effector-to-target ratio of 1:1.If the number of detected CAR T cells is less than 25, the number is shown in red, and further analysis of these few cells is called into question. [Figure 283-1]Figures 283A-283L show photographs of MUC1*-positive breast cancer cells T47D cultured with human T cells supplemented with various concentrations of the bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody, and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen in the figures, the addition of the bispecific antibody mediates binding between T cells and cancer cells, seen here as cell clusters. In Figure 283A, the concentration of the bispecific antibody is 1,000 ng / mL. In Figure 283B, the concentration is 333 ng / mL. In Figure 283C, the concentration is 111 ng / mL. In Figure 283D, the concentration is 37 ng / mL. In Figure 283E, the concentration is 12.3 ng / mL. In Figure 283F, the concentration is 4.1 ng / mL. In Figure 283G, the concentration is 1.3 ng / mL. In Figure 283H, the concentration is 0.4 ng / mL. In Figure 283I, the concentration is 0.15 ng / mL. In Figure 283J, the concentration is 0.05 ng / mL. Figure 283K is a control well in which both T cells and cancer cells are present but no bispecific antibody is added. Figure 283L is a control well in which bispecific antibody is added to cancer cells but no T cells are present. [Figure 283-2]Figures 283A-283L show photographs of MUC1*-positive breast cancer cells T47D cultured with human T cells supplemented with various concentrations of the bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody, and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen in the figures, the addition of the bispecific antibody mediates binding between T cells and cancer cells, seen here as cell clusters. In Figure 283A, the concentration of the bispecific antibody is 1,000 ng / mL. In Figure 283B, the concentration is 333 ng / mL. In Figure 283C, the concentration is 111 ng / mL. In Figure 283D, the concentration is 37 ng / mL. In Figure 283E, the concentration is 12.3 ng / mL. In Figure 283F, the concentration is 4.1 ng / mL. In Figure 283G, the concentration is 1.3 ng / mL. In Figure 283H, the concentration is 0.4 ng / mL. In Figure 283I, the concentration is 0.15 ng / mL. In Figure 283J, the concentration is 0.05 ng / mL. Figure 283K is a control well in which both T cells and cancer cells are present but no bispecific antibody is added. Figure 283L is a control well in which bispecific antibody is added to cancer cells but no T cells are present. [Figure 284-1]Figures 284A-284L show photographs of MUC1*-positive breast cancer cells T47D cultured with human T cells supplemented with various concentrations of the bispecific antibody 20A10-12F6-BiTE. 20A10 is a humanized anti-MUC1* antibody, and 12F6 is an antibody that binds to CD3 present on human T cells. As can be seen in the figures, the addition of the bispecific antibody mediates binding between T cells and cancer cells, seen here as cell clusters. In Figure 284A, the concentration of the bispecific antibody is 1,000 ng / mL. In Figure 284B, the concentration is 333 ng / mL. In Figure 284C, the concentration is 111 ng / mL. In Figure 284D, the concentration is 37 ng / mL. In Figure 284E, the concentration is 12.3 ng / mL. In Figure 284F, the concentration is 4.1 ng / mL. In Figure 284G, the concentration is 1.3 ng / mL. In Figure 284H, the concentration is 0.4 ng / mL. In Figure 284I, the concentration is 0.15 ng / mL. In Figure 284J, the concentration is 0.05 ng / mL. Figure 284K is a control well in which both T cells and cancer cells are present but no bispecific antibody is added. Figure 284L is a control well in which bispecific antibody is added to cancer cells but no T cells are present.

Figure 284-2

Figure 285-1

Figure 285-2

Figure 286-1

Figure 286-2

[0177] In this application, "a" and "an" are used to refer to both a singular object and plural objects.

[0178] As used herein, sometimes, for short, a polypeptide may be referred to as being "transduced or transfected" into a cell. In these occurrences, it is understood that a nucleic acid encoding the polypeptide sequence is transduced or transfected into a cell, as it is not possible to transduce or transfect a polypeptide into a cell.

[0179] As used herein, occasionally when referring to the number of cells infused into an animal or in other contexts referring to numbers of cells, "M" refers to millions and "K" refers to thousands.

[0180] As used herein, various monoclonal antibodies are referred to interchangeably. For example, "MNC2" is interchangeable with "C2," "Min-C2," and "MNC2," "MNE6" is interchangeable with "E6," "Min-E6," and "MNE6," "MNC3" is interchangeable with "C3," "Min-C3," and "MNC3," and "MNC8" is interchangeable with "C8," "Min-C8," and "MNC8." The monoclonal antibodies provided herein follow the same convention.

[0181] As used herein, the placement of "h" or "hu" in front of an antibody construct is shorthand for humanized.

[0182] As used herein, the term "antibody-like" refers to a molecule that contains a portion of an antibody but can be engineered to be different from 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 so that the body's immune system is directed to attack a specific target protein or cell. Ylanthia® technology consists of an "antibody-like" library, a collection of synthetic human Fabs that are then screened for binding to peptide epitopes from target proteins. Selected Fab regions can then be engineered into a scaffold or framework to resemble an antibody.

[0183] 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).

[0184] 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 depending on cell type, tissue type, or evolutionary time of the cell, so MUC1 * The exact sequence of the extracellular domain may vary at the N-terminus.

[0185] Other truncated amino acid sequences include SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621) can be mentioned.

[0186] 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, an "N-number," such as "N-10 PSMGFR" or simply "N-10," "N-15 PSMGFR" or simply "N-15," or "N-20 PSMGFR" or simply "N-20," refers to the number of amino acid residues deleted at the N-terminus of PSMGFR. Similarly, a "C-number," such as "C-10 PSMGFR" or simply "C-10," "C-15 PSMGFR" or simply "C-15," or "C-20 PSMGFR" or simply "C-20," refers to the number of amino acid residues deleted at the C-terminus of PSMGFR. Mixed deletions and additions are also possible. For example, N+20 / C-27 refers to a peptide fragment of wild-type MUC1 in which 20 amino acids have been added to the N-terminal PSMGFR and 27 amino acids have been deleted from the C-terminus.

[0187] As used herein, "MUC1 * The "extracellular domain of MUC1" refers to the extracellular portion of the MUC1 protein that lacks the tandem repeat domain. *MUC1 * The MUC1 fragment is a cleavage product consisting of a short extracellular domain lacking the tandem repeats, a transmembrane domain, and a cytoplasmic tail. The exact location of the cleavage in MUC1 is unknown, as it appears that it may be cleaved by more than one enzyme. * The extracellular domain of contains most of the PSMGFR sequence but may have an additional 10-20 N-terminal amino acids.

[0188] As used herein, "sequence identity" refers to the homology in the sequence of a particular polypeptide or nucleic acid to a reference sequence of nucleic acid or amino acid such that the function of the homologous peptide is the same as the reference peptide or nucleic acid. Such homology may be so close to the reference peptide that two sequences may sometimes be 90%, 95%, or 98% identical, yet have the same function in binding or other biological activity.

[0189] 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.

[0190] As used herein, a "MUC1-negative" cell refers to a cell that does not express the MUC1 gene.

[0191] As used herein, "MUC1 * A "positive" cell refers to a cell that expresses 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 transfection or transduction of the cell with a MUC1 protein lacking tandem repeats.

[0192] 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.

[0193] As used herein, "MUC1-positive" cancer cells refer to cancer cells that overexpress the MUC1 gene, express MUC1 in an abnormal pattern, whose expression is not restricted to the apical edge, and / or express MUC1 that lacks tandem repeats.

[0194] 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.

[0195] As used herein, "MUC1 * "Positive" cancer cells refer to cancer cells that overexpress the MUC1 transmembrane protein lacking the tandem repeats.

[0196] 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 tandem repeats.

[0197] As used herein, a "conformational epitope" refers to a peptide sequence that is required to 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 work in a denaturing Western blot but recognizes a natural target expressed, for example, in intact cells, is determined 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 indicates that the antibody binds to MUC1 of cancer cells. *These conformational epitopes are necessary to confer a three-dimensional structure that allows binding by specific antibodies for cancer therapy. Thus, conformational epitopes are essential for the binding of MUC1 to cancer cells. * A conformation-directing peptide sequence is an amino acid sequence that directs antibody binding to a region of the peptide. Thus, the term "conformation-directing peptide sequence" can be used to indicate that the peptide sequence is present within a larger peptide that directs antibody binding to the larger peptide, not as a binding site, but by forming a three-dimensional structure that facilitates antibody binding to the larger peptide.

[0198] MUC1 for treating or preventing cancer * Antibody (anti-PSMGFR)

[0199] The present inventors have discovered that a truncated form of the MUC1 (SEQ ID NO: 1) transmembrane protein is the growth factor receptor that drives the growth of more than 75% of all human solid tumor cancers. * A truncated form of MUC1, termed (prominent muk 1 star), is a potent growth factor receptor. Enzymatic cleavage releases most of the MUC1 extracellular domain. MUC1 * The remaining portion of MUC1, including the truncated extracellular domain, transmembrane domain, and cytoplasmic tail, is termed NME1. Cleavage and release of most of the extracellular domain of MUC1 results in the formation of dimers NME1, NME6, NME8, and NME7. AB This exposes the binding site for activating NME7-X1 or NME7 ligands. Cell proliferation assays show that it is MUC1 that promotes proliferation. * This indicates ligand-induced dimerization of the extracellular domain (Figures 1A-1D). * MUC1 treated with either antibody, monovalent "mv" or Fab, NM23-H1 dimer or NME7-AB * Positive cells. Bivalent anti-MUC1 * Antibodies stimulate cancer cell proliferation, whereas monovalent Fab inhibits proliferation. The classic bell-shaped curve indicates that ligand-induced dimerization stimulates proliferation. Dimer NM23-H1, also known as NME1, binds to MUC1 *NME7-AB stimulates the proliferation of MUC1-positive cancer cells, whereas siRNA that suppresses MUC1 expression abolishes this effect (Fig. 1C). * stimulates the proliferation of positive cells (Figure 1D).

[0200] MUC1 * MUC1 is aberrantly expressed in over 75% of all cancers and is likely overexpressed in a particularly high percentage of metastatic cancers, making it an excellent target for cancer drugs. After cleavage of MUC1, most of its extracellular domain is shed 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, have been identified. AB Antibodies that competitively inhibit the binding of activating ligands such as NME proteins, including NME7-X1 and NME7, are ideal therapeutic agents and can be used as multispecific antibody-like molecules, bispecific antibodies, antibody-drug conjugates, or standalone antibodies, antibody fragments, or variable region fragments thereof, incorporated into chimeric antigen receptors (also known as CARs), MUC1-positive or MUC1-positive antibodies. * These antibodies can then be transfected or transduced into immune cells and then administered to patients to treat or prevent MUC1-positive cancers. * Antibodies can be monoclonal, polyclonal, antibody mimics, engineered antibody-like molecules, complete antibodies, or antibody fragments. 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. Anti-MUC1 antibodies can be monovalent or bispecific. * The antibody is MUC1 * NME1 and NME7 are preferred because their function is activated by ligand-induced dimerization. AB MUC1 *These activating growth factors bind to the PSMGFR peptide portion of MUC1 (Figures 2A and 2D). Furthermore, these activating growth factors do not bind to the PSMGFR peptide when the 10 C-terminal amino acids are deleted, indicating that MUC1 * Similarly, anti-MUC1 * Antibodies MNC2 and MNE6 bind to the PSMGFR peptide only if the C-terminal 10 amino acids are present (Figures 2B and 2C). Antibodies MNC3 and MNC8 bind to a different epitope than MNC2 and MNE6, as they do not depend on the presence of the C-terminal 10 amino acids of the PSMGFR peptide (Figures 2E and 2F). The antibodies MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11, as well as other antibodies of the present invention, or fragments derived therefrom, can be administered to patients as standalone antibodies for the treatment or prevention of cancer, or can be incorporated into BiTEs, ADCs, multispecific antibody-like molecules, bispecific antibodies, with or without Fc or Fc region portions, bi-scFvs, di-scFvs, tandem di-scFvs, diabodies, triabodies, tribodies, tetrabodies, and other antibody-like molecules that are multivalent and multispecific. The antibodies or antibody fragments can be murine, human, humanized, camelid, rabbit, or other non-human species.

[0201] Chimeric antigen receptors, also known as BiTEs or CARs, transduced into immune cells: MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11 antibodies, as well as NME1 and NME7. AB Other anti-MUC1 antibodies that competitively inhibit the binding of * Antibodies are preferred. The antibody or antibody fragment may be murine, human, humanized, camelid, rabbit or other non-human species.

[0202] Therapeutic anti-MUC1 for use as a standalone antibody therapeutic or for incorporation into either a BiTE, CAR, ADC, or multispecific antibody-like molecule * Antibodies can be selected based on certain criteria. Parent antibodies can be produced using typical methods for producing monoclonal antibodies in animals. Alternatively, they can be selected by screening antibody or antibody fragment libraries, including, but not limited to, the strategy described in U.S. Patent No. 9,944,719 B2 to Beckman, which is incorporated herein by reference for a description of how to screen antibodies. Antibodies suitable for therapeutic use can be: MUC1 * Selected based on their ability to bind to the peptide:

[0203] (i) the PSMGFR region of MUC1;

[0204] (ii) a PSMGFR peptide,

[0205] (iii) a peptide having the following amino acid sequence: QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10)

[0206] (iv) a peptide having the following amino acid sequence:

[0207] ASRYNLTISDVSVSDVPFPFSAQSGA(N-19)

[0208] (v) a peptide having the following amino acid sequence:

[0209] NLTISDVSVSDVPFPFSAQSGA(N-23)

[0210] (vi) a peptide having the following amino acid sequence:

[0211] ISDVSVSDVPFPFSAQSGA(N-26)

[0212] (vii) a peptide having the following amino acid sequence:

[0213] SVSDVPFPFSAQSGA(N-30)

[0214] (viii) a peptide having the following amino acid sequence:

[0215] QFNQYKTEAASRYNLTISDDVSVSDVPFPFS(N-10 / C-5)

[0216] (ix) a peptide having the following amino acid sequence:

[0217] ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) or

[0218] (x) a peptide having the following amino acid sequence:

[0219] FPFSAQSGA(N-36).

[0220] The resulting antibodies or antibody fragments thus produced or selected can then be further selected by passing them through additional screening. For example, the antibodies or antibody fragments can be selected from antibodies that bind to MUC1 * The ability of the anti-MUC1 antibody to bind to MUC1-positive cancer cells or tissues, but not to MUC1-negative cancer cells or normal tissues, makes it more preferred. * 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 * It would be more preferable if MNE6 and MNC2 have the ability to competitively inhibit the binding of activating ligands such as NME7AB or NME7-X1 to MUC1. * Figure 1 shows that NF-κB competitively inhibits the binding of activating ligands NME1 and NME7 to NF-κB.

[0221] Anti-MUC1 antibodies for use in treating patients diagnosed with, at risk of developing, or suspected of having MUC1-positive cancer * A process for selecting antibodies, comprising one or more of the following steps: 1) binding to PSMGFR peptides; 2) binding to N-10 PSMGFR peptides; 3) selectively binding to cancer cells; 4) not binding to C-10 PSMGFR peptides; and 5) competitively inhibiting the binding of dimeric NME1 or NME7-AB to PSMGFR peptides. For example, Figures 3A-3C show that monoclonal antibodies MNE6 and MNC2 meet all five criteria, while monoclonal antibodies MNC3 and MNC8 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, when used as a straightforward standalone antibody therapeutic, would be MUC1. * The extracellular domain should not dimerize. For this therapeutic format, suitable antibodies in this regard include monovalent antibodies such as those generated in llamas and camels, Fab, scFv, single domain antibodies (sdAb), scFv-Fc, so long as the Fc portion is constructed so as not to homodimerize.

[0222] FACS scan shows anti-MUC1 * Antibodies MNC2 and MNE6 are MUC1 * Positive solid tumor cancer cells and MUC1 * Specific binding to transfected cells but not to MUC1 * In one example, the humanized MNC2 scFv, ZR-75-1, also known as 1500, does not bind to MUC1-negative or MUC1-negative cells. * MNE6 has been shown to bind to MUC1-positive breast cancer cells (Figures 4A-4C). *MNE6 was shown to bind to MUC1-negative HCT-116 colon cancer cells only when transfected with MUC1. * Positive cancer cells, such as ZR-75-1, aka 1500, MUC1 * MNC2 and MNE6 bound to the PSMGFR peptide and to live MUC1-positive breast cancer cells (Figures 4D-4F). Binding assays, including ELISA and immunofluorescence, all confirmed that MNC2 and MNE6 bound to the PSMGFR peptide and to live MUC1-positive cancer cells. * Antibodies are selected based on their ability to bind to either the PSMGFR peptide or MUC1-positive cancer cells. * Similar to the Fab, the humanized MNC2 scFv binds to the peptide PSMGFR with high affinity, exhibiting an EC-50 of approximately 333 nM. As shown in examples in Figures 6A and 6B, the humanized MNC2 scFv binds to the peptide MUC1. * The humanized scFvs potently inhibit the growth of IgG-positive cancer cells. Similar to the parent antibody, the humanized scFvs show 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). However, murine or humanized MNC3-scFv, which are less suitable for cancer treatment, bind to the PSMGFR peptide, the N-10 peptide, and also to the C-10 peptide (Figure 9), which indicates that they bind to the activating ligand NME7. AB is known to be the epitope to which it binds.

[0223] The Fabs of MNE6 and MNC2 or equivalent single-chain variable regions derived from them inhibit MUC1 in vitro and in vivo. * In some cases, anti-MUC1 * The Fab of the antibody binds to human MUC1 in vivo. * In one case, immunocompromised mice were implanted with human breast tumors and then treated with MNE6 Fab after tumor engraftment. Figure 7A shows that MNE6 Fab inhibited the growth of MUC1-positive tumors. *Female nu / nu mice implanted with estrogen pellets for 90 days were implanted with 6 million T47D human breast canc...

Claims

1. A chimeric antigen receptor (CAR), wherein the CAR is (a) Anti-mucin 1 ＊ (MUC1 ＊ ) an antibody fragment, (i) a heavy chain (HC) variable region comprising a complementarity determining region 1 (HC-CDR1) comprising the amino acid sequence of SEQ ID NO: 123, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 127, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 131; and (ii) an anti-MUC1 comprising a light chain (LC) variable region, the light chain (LC) variable region comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 173, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 177, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 181; ＊ an antibody fragment; (b) a hinge region derived from CD28; and (c) a transmembrane domain derived from CD28; and (d) a cytoplasmic region comprising, from N- to C-terminal, a CD3-zeta intracellular signaling domain comprising three immunoreceptor tyrosine-based activation motifs (ITAMs), i.e., ITAM1, ITAM2, and ITAM3, wherein ITAM2 comprises a substitution of a tyrosine residue with a phenylalanine residue, and wherein ITAM3 comprises a substitution of a tyrosine residue with a phenylalanine residue; and The CAR specifically binds to cancer cells.

2. The anti-MUC1 ＊ The antibody fragment is (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 145; and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 195; The CAR of claim 1, comprising:

3. The anti-MUC1 ＊ The CAR of claim 1, wherein the antibody fragment comprises the amino acid sequence of SEQ ID NO:

239.

4. The CAR of claim 1, wherein the CD3-zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 1797.

5. The CAR according to claim 1, wherein the cytoplasmic region comprises a costimulatory domain comprising a sequence derived from CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7, an Fc receptor gamma domain, or a combination thereof.

6. The CAR of claim 5, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO:

378.

7. The CAR is the anti-MUC1 ＊ having an N-terminal to C-terminal arrangement of an antibody fragment, said hinge region, said transmembrane domain, said costimulatory domain, and said CD3-zeta intracellular signaling domain, (a) the anti-MUC1 ＊ the antibody fragment comprises the amino acid sequence of SEQ ID NO: 239; (b) the hinge region comprises the amino acid sequence of SEQ ID NO: 350; (c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 368; (d) the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 378; (e) the CD3-zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 1797. The CAR according to claim 5.

8. The CAR of claim 1, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 1785.

9. A nucleic acid comprising a nucleic acid sequence encoding the CAR of any one of claims 1 to 8.

10. An expression vector comprising the nucleic acid of claim 9.

11. An immune cell comprising the CAR according to any one of claims 1 to 8.

12. A T cell comprising the CAR of any one of claims 1 to 8.

13. MUC1 ＊ The CAR according to any one of claims 1 to 8, which is used to treat a cancer that is positive for the HIV-1 virus.

14. MUC1 ＊ The nucleic acid of claim 9 for use in the treatment of a human positive cancer.

15. MUC1 ＊ The expression vector of claim 10, which is used to treat a human positive cancer.

16. MUC1 ＊ The immune cells of claim 11, used to treat a cancer that is positive for the immune system.

17. MUC1 ＊ The T cells of claim 12 for use in the treatment of a human immunodeficiency virus (HIV)-positive cancer.

18. MUC1 ＊ 18. The T cell of claim 17, wherein the positive cancer comprises low antigen density cancer cells.

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