Anti-variable MUC1* antibodies and uses thereof

Abstract

The present application discloses anti-variable MUC1 * antibodies and uses thereof. Specifically, 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 of the peptide.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of January 13, 2020, application number 202080020073.0, and invention name “Anti-variable MUC1* antibodies and their uses” (the corresponding PCT application with an application date of January 13, 2020, and application number PCT / US2020 / 013410). Background of the Invention 1. Technical field:

[0002] This application relates to human, humanized and non-human anti-MUC1* antibodies and methods of making and using them. This application also relates to the use of immune cells transfected or transduced with a cleavage enzyme to treat cancer. This invention also relates to the use of immune cells transfected or transduced with a CAR and another protein to treat cancer. 2. Background technology:

[0003] We previously discovered that the cleaved form of the MUC1 (SEQ ID NO: 1) transmembrane protein is a growth factor receptor that drives the growth of more than 75% of all human cancers. The cleaved form of MUC1, which we call MUC1* (pronounced muk 1 asterisk), is a potent growth factor receptor. Cleavage and release of the majority of the extracellular domain of MUC1 exposes the activating ligands NME1, NME6, NME7, and NME7. AB , NME7-X1, or NME8. It is an ideal target for cancer drugs because it is aberrantly expressed in more than 75% of all cancers and may be overexpressed in an even higher proportion 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* is a determinant of trastuzumab (Herceptin) resistance in breast cancer cells,” Breast Cancer Res Treat. 118(1):113-124). After MUC1 is cleaved, most of its extracellular domain is shed from the cell surface. The remaining part has a truncated extracellular domain that contains most or all of the primary growth factor receptor sequence called PSMGFR (SEQ ID NO: 2).

[0004] Antibodies are increasingly used to treat human diseases. Antibodies produced in non-human species have historically been used as therapeutic agents for humans, such as equine antibodies. Recently, antibodies have been engineered or selected so that they contain mostly or entirely human sequences to avoid the general rejection of foreign antibodies. The process of engineering the recognition fragments of non-human antibodies into human antibodies is commonly referred to as 'humanization'. The amount of non-human sequences used to replace human antibody sequences determines whether they are called chimeric, humanized, or fully human.

[0005] There are alternative technologies that can produce humanized or fully human antibodies. These strategies involve screening libraries of human antibodies or antibody fragments and identifying those that bind to the target antigen, rather than immunizing animals with 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 is also intended to encompass these methods when used with recognition fragments of antibodies that the inventors have determined bind to the extracellular domain of MUC1*.

[0006] In addition to treating patients with antibodies, cancer immunotherapy has recently been shown to be effective in treating blood cancers. A cancer immunotherapy, called CAR T (chimeric antigen receptor T cell) therapy, engineers 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). This receptor consists of a single-chain antibody fragment (scFv) that recognizes tumor antigens, which is connected to a T cell transmembrane domain, a signaling domain, and a costimulatory domain. After the receptor binds to the cancer-associated antigen, a signal is transmitted, resulting in T cell activation, proliferation, and targeted killing of cancer cells. In practice, T cells are isolated from a patient or donor and transduced with CAR, amplified, and then injected back into the patient. If from a donor, the immune cells can be mutated or engineered so that they do not induce graft-versus-host disease in the recipient. When the CAR T cells bind to antigens on cancer cells, they attack the cancer cells and then expand the T cell population.

[0007] To date, CAR T therapy has been very successful in treating blood cancers, but has not yet shown efficacy against human solid tumors. Because most blood cancers are B-cell malignancies, CAR-T cells can just eliminate all of a patient's B cells without causing serious harm to the patient. There is no B cell equivalent in solid tumors. Most tumor-associated antigens are also expressed on normal tissues; they are just expressed at higher levels in cancerous tissues. Therefore, the challenge is to develop antibodies that recognize epitopes on tumor-associated antigens that are somewhat different in the tumor environment compared to normal tissue. To further minimize the risk of off-tumor / on-target killing of normal tissue, the antibody should recognize cancerous tissue and the binding to cancerous tissue should be at least two times greater than to normal tissue. If antibodies that are not as cancer-selective are inducibly expressed at the tumor site, they can be used for treatment.

[0008] Another cancer therapy that incorporates cancer-selective antibodies is bispecific T Cell-engaging agents, also known as BiTEs. The BiTE approach attempts to eliminate the risk of off-tumor / on-target effects associated with CAR T. Unlike CAR T, BiTEs are bispecific antibodies that do not pose any greater risks 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-ε). BiTEs are antibodies made by genetically connecting the scFv of an antibody that binds to a T cell antigen (like anti-CD3-ε) and the scFv of a therapeutic monoclonal antibody that binds to a cancer antigen (Patrick A. Baeuerle and Carsten Reinhardt (2009) Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res. 69 (12): 4941-4944). A drawback of BiTE technology is that, unlike CAR T cells, they do not expand within the patient and so have limited persistence.

[0009] Another cancer therapy that incorporates cancer selective antibodies is antibody drug conjugate (also referred to as ADC) technology. In this case, a toxin or a precursor of a toxin is connected to a cancer selective antibody. Unlike CAR T cells that use the natural killer of CD8 positive T cells to kill cancer cells, ADC brings toxic payloads to tumors. The shortcomings of ADC include the possibility of delivering toxic payloads to normal cells, and most ADCs need to be combined with cell surface molecules, which are then internalized after binding, wherein about 10,000 surface molecules are needed to cause cell death. Summary of the Invention

[0010] In one aspect, the present invention relates to a non-human, human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein that binds to a region on the extracellular domain of a MUC1 isoform or cleavage product that lacks a tandem repeat domain. The non-human, human or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein can specifically bind to

[0011] (i) PSMGFR region of MUC1;

[0012] (ii) PSMGFR peptide;

[0013] (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10) (SEQ ID NO: 3)

[0014] (iv) having an amino acid sequence

[0015] Peptide of ASRYNLTISDVSVSDVPFPFSAQSGA(N-19) (SEQ ID NO: 4)

[0016] (v) having an amino acid sequence

[0017] Peptide of NLTISDVSVSDVPFPFSAQSGA(N-23) (SEQ ID NO: 5)

[0018] (vi) having an amino acid sequence

[0019] Peptide of ISDVSVSDVPFPFSAQSGA(N-26) (SEQ ID NO: 6)

[0020] (vii) having an amino acid sequence

[0021] Peptide of SVSDVPFPFSAQSGA(N-30) (SEQ ID NO: 7)

[0022] (viii) having an amino acid sequence

[0023] Peptide of QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5)(SEQ ID NO:8)

[0024] (ix) having an amino acid sequence

[0025] Peptide of ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) (SEQ ID NO: 9)

[0026] (x) has an amino acid sequence

[0027] Peptide FPFSAQSGA (SEQ ID NO: 10)

[0028] 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.

[0029] The murine, camelid, human or humanized antibody, antibody fragment or antibody-like protein as described above may comprise a heavy chain variable region and a light chain variable region derived from mouse monoclonal MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 and H11 antibodies, and have at least 80%, 90% or 95% or 98% sequence identity with mouse monoclonal MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 and H11. The heavy chain variable regions of CDR1 and CDR2 may have at least 90%, 95%, or 98% sequence identity with the specifically designated antibody heavy chain variable region sequences shown in the sequence listing of the present application, and the light chain variable regions of CDR1 and CDR2 may have at least 90%, 95%, or 98% sequence identity with the specifically designated antibody heavy chain variable region sequences shown in the sequence listing of the present application. The heavy chain variable region of CDR3 may have at least 80%, 85%, or 90% sequence identity with the specifically designated antibody heavy chain variable region sequences shown in the sequence listing of the present application, and the light chain variable region of CDR3 may have at least 80%, 85%, or 90% sequence identity with the specifically designated antibody heavy chain variable region sequences shown in the sequence listing of the present application.

[0030] According to the above, the mouse, camel, human or humanized antibody, antibody fragment or antibody-like protein may contain a complementarity determining region (CDR) in the heavy chain variable region and the light chain variable region, which has at least 90% or 95% or 98% sequence identity with the specifically specified antibody heavy chain CDR1, CDR2 or CDR3 region and light chain CDR1, CDR2 or CDR3 region sequences shown in the sequence listing part of the present application.

[0031] In another aspect, the present invention relates to an anti-MUC1* extracellular domain antibody or an anti-N-10 antibody, which may be any of the antibodies described above, consisting 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. The humanized IgG2 heavy chain may be SEQ ID NO: 53, the humanized IgG1 heavy chain may be SEQ ID NO: 57, the humanized kappa light chain may be SEQ ID NO: 108, and the humanized lambda light chain may be SEQ ID NO: 112, or a sequence having 90%, 95%, or 98% sequence identity thereto.

[0032] In another aspect, the present invention relates to an anti-MUC1* extracellular domain antibody or anti-N-10 antibody consisting of the sequence of humanized MN-C2 represented by a humanized IgG1 heavy chain or a humanized IgG2 heavy chain paired with a humanized λ light chain and a humanized κ light chain.

[0033] In another aspect, the invention relates to an anti-MUC1* extracellular domain antibody or anti-N-10 antibody consisting of a sequence of humanized MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 represented by a humanized IgG1 heavy chain or a humanized IgG2 heavy chain paired with a humanized λ light chain or a humanized κ light chain.

[0034] In another aspect, the present invention relates to an antibody that is "like" MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 in that they have the same or very similar pattern of binding to a subset of peptides derived from PSMGFR peptides, do not recognize a linear epitope, and competitively inhibit NME1 or NME7. AB Binds to MUC1* and recognizes the MUC1 transmembrane cleavage product resulting from MMP9 cleavage or contains a CDR sequence that is at least 80% homologous to the MN-E6, MN-C2, MN-18G12, MN-20A10, MN-25E6, MN-28F9, MN-5C6F3, MN-3C2B1, and MN-1E4 CDR consensus sequence.

[0035] In another aspect, the invention relates to an antibody that binds to the extracellular domain of MUC1 (which may be a cleavage product) lacking a tandem repeat domain. In one aspect of the invention, the antibody binds to a peptide having the sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10). In one aspect of the invention, the antibody binds to a peptide having the sequence ASRYNLTISDVSVSDVPFPFSAQSGA (N-19). In one aspect of the invention, the antibody binds to a peptide having the sequence SVSDVPFPFSAQSGA (N-30). In one aspect of the invention, the antibody binds to a peptide having the sequence FPFSAQSGA (N-36). Examples of such antibodies include, but are not limited to, monoclonal antibodies MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11. The heavy and light chain complementarity determining region sequences of these antibodies are shown in the sequence listing section of this application.

[0036] In one aspect of the invention, one or more of these antibodies are administered to a patient diagnosed with or at risk of developing cancer. The antibodies can be human or humanized. The antibodies can be murine or camelid. The antibodies can be bivalent or monovalent. The antibodies can be fragments of one of the antibodies, including single chain fragments (scFv). The antibodies or antibody fragments can be administered directly to the patient or incorporated into bispecific antibodies, bispecific T In cell binder BiTE or antibody drug conjugate ADC. Antibodies or antibody fragments can be incorporated into T cell receptor TCR. The sequence of the antibody or antibody fragment can be incorporated into chimeric antigen receptor "CAR" or other similar entities, then introduced into immune cells in vitro, and then administered to patients diagnosed with cancer or at risk of developing cancer. Immune cells (which can be T cells or natural killer cells) can be derived from donors or patients. In one aspect, immune cells are derived from stem cells, which have been guided to differentiate into the immune cell types in vitro. In one aspect, antibodies or CARs comprising the sequence of antibodies can be expressed from an inducible promoter. In one case, antibodies or CARs are expressed after T cells or other immune cells are activated. In one case, antibodies or CARs of the present invention are expressed from NFAT response elements. In another case, the CAR recognition of target tumor cells activates immune cells, resulting in the expression of NFAT inducible expression or checkpoint inhibitors (such as PD1 inhibitors or PDL-1 inhibitors) of cytokines (such as IL-12 or IL-18). In yet another aspect, CAR recognition of target tumor cells activates immune cells, resulting in NFAT-inducible expression of a second CAR comprising the sequence of a second antibody.

[0037] In another aspect, the present invention relates to a mouse, camel, human, or humanized anti-MUC1* antibody or antibody fragment or antibody-like protein that binds to the N-10 peptide according to the above, which inhibits the binding of NME protein to MUC1*. NME can be NME1, NME6, NME7 AB , NME7-X1, NME7 or NME8.

[0038] In another aspect, the present invention relates to a single-chain variable fragment (scFv) comprising heavy and light chain variable regions connected by a linker, and further comprising the CDRs of an antibody that binds to the extracellular domain of MUC1*. The CDRs can be derived from MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11. The scFv can be an scFv having SEQ ID NOs: 233, 235, and 237 (MN-E6); or SEQ ID NOs: 239, 241, and 243 (MN-C2).

[0039] In yet another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising a scFv or a humanized variable region bound to the extracellular domain of MUC1 lacking tandem repeats, a linker molecule, a transmembrane domain, and a cytoplasmic domain.

[0040] (i) PSMGFR region of MUC1;

[0041] (ii) PSMGFR peptide;

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

[0043] (iv) having an amino acid sequence

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

[0045] (v) having an amino acid sequence

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

[0047] (vi) having an amino acid sequence

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

[0049] (vii) having an amino acid sequence

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

[0051] (viii) having an amino acid sequence

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

[0053] (ix) having an amino acid sequence

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

[0055] (x) has an amino acid sequence

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

[0057] In CAR as described above, any part or combination of the variable regions shown and described above can be used in the extracellular domain of CAR. CAR also includes a transmembrane region and a cytoplasmic tail comprising a sequence motif that signals activation of the immune system. The extracellular domain can be composed of mouse, camel, human, non-human or humanized single-chain antibody fragments of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 and H11. Additional antibodies from which single-chain antibody fragments can be prepared include, but are not limited to, monoclonal antibodies such as MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11, because they have the same or very similar pattern of binding to a subset of peptides derived from PSMGFR peptides, do not recognize a linear epitope, or competitively inhibit NME1 or NME7. AB Binds to MUC1*, or recognizes a MUC1 transmembrane cleavage product produced by MMP9 cleavage, or comprises a CDR sequence that is at least 80% homologous to the MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11 CDR consensus sequence.

[0058] In the CAR as described above, the extracellular domain may comprise a mouse, camel, human, non-human or humanized single-chain antibody fragment of MN-E6 scFv (as shown in SEQ ID NO: 233, 235 or 237), MN-C2 scFv (SEQ ID NO: 239, 241 or 243), or 20A10 scFv (as shown in SEQ ID NO: 1574-1575), 25E6 scFv (as shown in SEQ ID NO: 1598-1599).

[0059] In any one of the CARs described above, the cytoplasmic tail may be composed of one or more of the signaling sequence motifs CD3-ζ, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5 or CD7. In any one of the CARs described above, the cytoplasmic tail may include a mutation that weakens signal transduction. Such mutations include, but are not limited to, mutations into tyrosine that inhibits phosphorylation and signal transduction (Salter et al., 2018). In any one of the CARs described above, the ITAM of CD3-ζ may mutate into inhibition or weaken signal transduction (Feucht et al. 2019). In any one of the CARs described above, the CD3 of the cytoplasmic tail may include mutations in ITAM, including those mutations referred to as 1XX. In any one of the CARs described above, T cells may be engineered to overexpress c-Jun as a method for suppressing T cell exhaustion (Lynn et al. 2019).

[0060] In any of the CARs described above, the sequence can be CAR MN-E6 CD28 / CD3z (SEQ ID NO: 298); CAR MN-E6 4-1BB / CD3z (SEQ ID NO: 301); CAR MN-E6 OX40 / CD3z (SEQ ID NO: 617); CAR MN-E6 CD28 / 4-1BB / CD3z (SEQ ID NO: 304); CAR MN-E6 CD28 / OX40 / CD3z (SEQ ID NO: 619); CAR MN-C2 CD3z (SEQ ID NO: 607); CAR MN-C2 CD28 / CD3z SEQ ID NO: 609); CAR MN-C2 4-1BB / CD3z (SEQ ID NO: 611 and SEQ ID NO: 719); CAR MN-C2 OX40 / CD3z (SEQ ID NO: 613); CAR MN-C2 CD28 / 4-1BB / CD3z (SEQ ID NO:307); CAR MN-C2CD28 / OX40 / CD3z (SEQ ID NO:615) or CAR MN-C3 4-1BB / CD3z (SEQ ID NO:601).

[0061] On the other hand, the present invention relates to a composition comprising at least two CARs with different extracellular domain units transfected into the same cell, wherein the cell may be an immune cell that may be derived from a patient in need of cancer treatment. The expression of the second CAR can be induced and driven by the recognition of the target by the first CAR. The nucleic acid encoding the second CAR can be connected to an inducible promoter. The expression of the second CAR can be induced by an event that specifically occurs when the immune cell produces an immune response to the target tumor cell. The antibody fragment of one or both of the CARs can guide the cell to a MUC1*-positive tumor. The antibody fragments of the first CAR and the second CAR can bind to the MUC1* produced when MUC1 is cut by two different cutting enzymes. When the antibody fragment of the first CAR engages or binds to MUC1 or MUC1* on the tumor, the expression of the second CAR through the inducible promoter can be induced. One way is to induce the 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 second CAR gene. In this way, when the transcription factor bound to the promoter of the NFAT protein is present at a concentration sufficient to bind to the NFAT protein and induce the transcription of the NFAT protein, they will also bind to the same promoter engineered in front of the sequence for the second CAR transcription. 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 aspect of the present invention, NFAT is NFATc1, NFATc3 or NFATc2. In one aspect of the present invention, NFAT is NFAT2, also known as NFATc1. SEQ ID NO: 646 shows the nucleic acid sequence of the upstream transcriptional regulatory region of NFAT2. The recognition unit of the second CAR can be an antibody fragment or a peptide, wherein the recognition unit can be combined with NME7, PD-1, PDL-1 or a checkpoint inhibitor.

[0062] At least two CARs may have one CAR without a tumor antigen targeting recognition unit and another CAR with a tumor antigen targeting recognition unit. In another aspect of the present invention, one of the extracellular domain recognition units may bind to the MUC1* extracellular domain. In another aspect of the present invention, one of the extracellular domain recognition units may be an antibody fragment, and the other may be a peptide that lacks transmembrane and signaling motifs; the peptide may be a single-chain antibody fragment or an antibody. In another aspect of the present invention, one of the recognition units may bind to PD-1 or PDL-1. In another aspect of the present invention, one extracellular domain recognition unit is an anti-MUC1* antibody, antibody fragment, or scFv 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 a CAR or may be an anti-NME7 antibody.

[0063] In another aspect, the present invention relates to a cell comprising a CAR having an extracellular domain that binds to an extracellular domain of a MUC1 molecule lacking tandem repeats. In another aspect, the present invention relates to a cell comprising a CAR having an extracellular domain that binds to a cell transfected or transduced with MUC1*. The cell comprising the CAR can be an immune system cell, preferably a T cell, a natural killer cell (NK), a dendritic cell, or a mast cell.

[0064] In another aspect, the present invention relates to an engineered antibody-like protein.

[0065] In another aspect, the present invention relates to a method for treating a disease in a subject, comprising administering an antibody according to any one of the preceding claims to a human suffering from the disease, wherein the subject aberrantly expresses MUC1. The disease may be cancer, such as breast cancer, ovarian cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, or esophageal cancer.

[0066] 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. The variable domain fragment can be derived from the mouse monoclonal antibody MN-E6 (SEQ ID NOs: 13 and 66), or from humanized MN-E6 (SEQ ID NOs: 39 and 94), or from a MN-E6 scFv (SEQ ID NOs: 233, 235, and 237). Alternatively, the variable domain fragment can be derived from the mouse monoclonal antibody MN-C2 (SEQ ID NOs: 119 and 169), or from a humanized MN-C2 (SEQ ID NOs: 145 and 195), or from a MN-C2 scFv (SEQ ID NOs: 239, 241, and 243). Alternatively, the variable domains may be derived from monoclonal antibodies MN-18G12, MN-20A10, MN-25E6, MN-28F9, MN-5C6F3, MN-3C2B1 or MN-1E4. The heavy and light chain complementary determining region sequences of these antibodies are also shown in the sequence listing herein.

[0067] In another aspect, the invention relates to a method for treating a human diagnosed with, suspected of having, or at risk of developing a MUC1 or MUC1*-positive cancer, which involves administering to the human an effective amount of the antibody, antibody fragment, or scFv described above, wherein the agent can be murine, camelid, human, or humanized.

[0068] In another aspect, the invention relates to a polypeptide comprising at least two different scFv sequences, wherein one of the scFv sequences is a sequence that binds to the extracellular domain of a MUC1 isoform or cleavage product lacking a tandem repeat domain.

[0069] (i) PSMGFR region of MUC1;

[0070] (ii) PSMGFR peptide;

[0071] (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(N-10) (SEQ ID NO: 3)

[0072] (iv) having an amino acid sequence

[0073] Peptide of ASRYNLTISDVSVSDVPFPFSAQSGA(N-19) (SEQ ID NO: 4)

[0074] (v) having an amino acid sequence

[0075] Peptide of NLTISDVSVSDVPFPFSAQSGA(N-23) (SEQ ID NO: 5)

[0076] (vi) having an amino acid sequence

[0077] Peptide of ISDVSVSDVPFPFSAQSGA(N-26) (SEQ ID NO: 6)

[0078] (vii) having an amino acid sequence

[0079] Peptide of SVSDVPFPFSAQSGA(N-30) (SEQ ID NO: 7)

[0080] (viii) having an amino acid sequence

[0081] Peptide of QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5)(SEQ ID NO:8)

[0082] (ix) having an amino acid sequence

[0083] Peptide of ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) (SEQ ID NO: 9)

[0084] (x) has an amino acid sequence

[0085] Peptide of FPFSAQSGA(N-36) (SEQ ID NO: 10)

[0086] The polypeptide can bind to a receptor on an immune cell, such as a T cell, and in particular CD3 on a T cell.

[0087] In another aspect, the present invention relates to a method for detecting the presence of cells that aberrantly express MUC1*, comprising contacting a cell sample with the scFv-Fc described above and detecting the presence of binding of the scFv-Fc to the cells. The cells may be cancer cells.

[0088] In another aspect, the present invention relates to a method for testing a subject's suitability for treatment of cancer with a composition comprising an antibody of the invention (which may be murine, camelid, human, or humanized), or a fragment thereof, or a portion of the variable region of antibody MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, the method comprising the steps of: contacting a body sample from the patient with the antibody in vitro, ex vivo, or in vivo; and determining that the patient exhibits abnormal MUC1* expression compared to normal tissue or sample. The antibodies used in these diagnostics may be conjugated to an imaging agent.

[0089] In another aspect, the present invention relates to a method of treating a subject suffering from a disease, comprising: exposing T cells from the subject or donor to a MUC1* peptide, wherein the T cells develop MUC1*-specific receptors through multiple rounds of maturation to produce adapted T cells; and expanding and administering the adapted T cells to a donor patient diagnosed with, suspected of having, or at risk of developing a MUC1*-positive cancer. The MUC1* peptide is selected from the group consisting of:

[0090] (i) PSMGFR region of MUC1;

[0091] (ii) PSMGFR peptide;

[0092] (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10)

[0093] (iv) having an amino acid sequence

[0094] ASRYNLTISDVSVSDVPFPFSAQSGA(N-19) peptide

[0095] (v) having an amino acid sequence

[0096] NLTISDVSVSDVPFPFSAQSGA(N-23) peptide

[0097] (vi) having an amino acid sequence

[0098] ISDVSVSDVPFPFSAQSGA(N-26) peptide

[0099] (vii) having an amino acid sequence

[0100] SVSDVPFPFSAQSGA(N-30) peptide

[0101] (viii) having an amino acid sequence

[0102] Peptide of QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5)

[0103] (ix) having an amino acid sequence

[0104] ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) peptide

[0105] (x) has an amino acid sequence

[0106] FPFSAQSGA(N-36) peptide

[0107] In one aspect of the invention, antibodies administered to patients for the treatment or prevention of MUC1 or MUC1*-positive cancers are selected for their ability to bind to the N-10 peptide of PSMGFR. The antibodies can be administered alone as monovalent antibodies, as scFvs, or fragments of the antibodies can be incorporated into CARs, BiTEs, or ADCs.

[0108] In one aspect of the invention, the antibodies administered to patients for the treatment or prevention of MUC1 or MUC1*-positive cancers are selected for their inability to recognize linear epitopes of MUC1 or MUC1*. The antibodies can be administered alone as monovalent antibodies, as scFvs, or fragments of the antibodies can be incorporated into CARs, BiTEs, or ADCs.

[0109] In one aspect of the invention, antibodies administered to patients for the treatment or prevention of MUC1 or MUC1*-positive cancers are selected for their ability to recognize the transmembrane cleavage products of MUC1 following MMP9 cleavage. The antibodies can be administered alone as monovalent antibodies, as scFvs, or fragments of the antibodies can be incorporated into CARs, BiTEs, or ADCs.

[0110] In one aspect of the invention, the antibody administered to a patient for the treatment or prevention of MUC1 or MUC1* positive cancer is directed against a protein that competitively inhibits NME7. AB The antibodies were selected based on their ability to bind to the extracellular domain of MUC1 lacking tandem repeats. The antibodies can be administered alone as monovalent antibodies, as scFvs, or fragments of the antibodies can be incorporated into CARs, BiTEs, or ADCs.

[0111] In another aspect, the invention relates to a method of treating cancer in a patient, comprising administering to the patient a combination of any of the above-described immune cells and a checkpoint inhibitor.

[0112] In the above methods, any of the following antibodies or variable regions thereof can be used: MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11.

[0113] In the above method, any of the variable regions shown below can be used:

[0114] (i) an anti-MUC1* extracellular domain antibody or an anti-N-10 antibody consisting of the sequence of humanized MN-E6 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;

[0115] (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;

[0116] (iii) an anti-MUC1* extracellular domain antibody or an anti-N-10 antibody consisting of the sequence of humanized MN-C2 represented by a humanized IgG1 heavy chain, a humanized IgG2 heavy chain paired with a humanized λ light chain and a humanized κ light chain;

[0117] (iv) The antibody of (iii), wherein the humanized IgG1 heavy chain MN-C2 (SEQ ID NO: 159) or IgG2 heavy chain (SEQ ID NO: 164) is paired with a lambda light chain (SEQ ID NO: 219) or a kappa light chain (SEQ ID NO: 213), or a sequence having 90%, 95% or 98% sequence identity thereto;

[0118] In the above method, in 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 (as shown in SEQ ID NO: 233, 235 or 237), MN-C2 scFv (SEQ ID NO: 239, 241 or 243). In CAR, the cytoplasmic tail may be composed of one or more of the signaling sequence motifs CD3-ζ, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5 or CD7.

[0119] The above method may include transfecting at least two CARs having different extracellular domain units into the same cell. One of the extracellular domain recognition units can bind to the MUC1* extracellular domain. One of the extracellular domain recognition units can bind to PD-1. One of the extracellular domain recognition units can be an antibody fragment, and the other can be a peptide or anti-MUC1* antibody fragment.

[0120] The method may include transfecting or transducing immune cells with a plasmid encoding a CAR and a plasmid encoding a non-CAR substance expressed from an inducible promoter. The non-CAR substance may be expressed from an inducible promoter activated by an element of the activated immune cell. The non-CAR substance may be expressed from an NFAT inducible promoter. The NFAT may be NFATc1, NFATc3, or NFATc2. The cleavage enzyme may be MMP2, MMP3, MMP9, MMP13, MMP14, MMP16, ADAM10, ADAM17, or ADAM28, or a catalytically active fragment thereof. The non-CAR substance may be a cytokine. The cytokine may be IL-7, IL-12, IL-15, or IL-18.

[0121] The present invention relates to an antibody or a fragment thereof for diagnosing, treating or preventing cancer, wherein the antibody specifically binds to PSMGFR peptide (SEQ ID NO: 2) or a fragment of the peptide.

[0122] The antibody binds to the following: N-10 peptide (SEQ ID NO:3), N-19 peptide (SEQ ID NO:4), N-23 peptide (SEQ ID NO:5), N-26 peptide (SEQ ID NO:6), N-30 peptide (SEQ ID NO:7), N-10 / C-5 peptide (SEQ ID NO:8), N-19 / C-5 peptide (SEQ ID NO:9) or C-5 peptide (SEQ ID NO:825).

[0123] The antibody interacts with a peptide comprising the conformational epitopes SVSDV (SEQ ID NO: 1751) and FPSA (SEQ ID NO: 1747) within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), wherein mutation or deletion of FPFS (SEQ ID NO: 1747) abolishes binding of the antibody or fragment thereof to the N-26 peptide.

[0124] The antibody interacts with a peptide comprising the conformational epitopes ASRYNLT (SEQ ID NO: 1745), SVSDV (SEQ ID NO: 1751) and FPSA (SEQ ID NO: 1747) within the N-19 sequence ASRYNLT ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4), wherein mutation or deletion of ASRYNLT (SEQ ID NO: 1745) abolishes binding of the antibody or fragment thereof to the N-26 peptide.

[0125] The antibody did not bind to the C-10 peptide (SEQ ID NO: 825).

[0126] The antibody bound to the N-10 peptide (SEQ ID NO: 3) but not to the C-10 peptide (SEQ ID NO: 825).

[0127] The antibody inhibits NME7 AB Interaction with MUC1*.

[0128] The antibody inhibits NME7 AB Interaction with PSMGFR peptide (SEQ ID NO: 2).

[0129] The antibody inhibits NME7 AB Interaction with the following: N-10 peptide (SEQ ID NO:3), N-19 peptide (SEQ ID NO:4), N-23 peptide (SEQ ID NO:5), N-26 peptide (SEQ ID NO:6), N-30 peptide (SEQ ID NO:7), N-10 / C-5 peptide (SEQ ID NO:8), N-19 / C-5 peptide (SEQ ID NO:9) or C-5 peptide (SEQ ID NO:825).

[0130] The antibody recognizes the MUCl transmembrane enzymatic cleavage product.

[0131] In the above, the cleaving enzyme is MMP14 or MMP9 or a catalytically active fragment of said enzyme.

[0132] The antibody binds to PSMGFR (SEQ ID NO: 2) or a fragment thereof, wherein the presence of an amino acid sequence within PSMGFR (SEQ ID NO: 2) induces binding of the antibody to PSMGFR.

[0133] The amino acid sequence that binds to the conformation-inducing peptide is present in the N-10 peptide (SEQ ID NO: 3).

[0134] The antibody does not bind to the linear form of the bound conformation-inducing peptide sequence, wherein the linear form of the peptide is a denatured form.

[0135] The binding conformation-inducing peptide sequence is within the N-26 peptide sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), wherein mutation or deletion of FPFS (SEQ ID NO: 1747) disrupts binding of the antibody or fragment thereof to the N-26 peptide.

[0136] The binding conformation-inducing peptide sequence is located within the N-19 sequence ASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4), wherein mutation or deletion of ASRYNLT (SEQ ID NO: 1745) disrupts binding of the antibody or fragment thereof to the N-19 peptide.

[0137] The binding inducing peptide sequence may be located within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), wherein a mutation or deletion within FPFS (SEQ ID NO: 1747) disrupts binding of the antibody or fragment thereof to PSMGFR.

[0138] Antibodies may have consensus sequences.

[0139] the heavy chain CDR1 comprises a consensus sequence at least 90% identical to the following: 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;

[0140] the heavy chain CDR2 comprises a consensus sequence at least 90% identical to the following: 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;

[0141] the heavy chain CDR3 comprises a consensus sequence at least 90% identical to the following: 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;

[0142] the light chain CDR1 comprises a consensus sequence at least 90% identical to the following: 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;

[0143] the light chain CDR2 comprises a consensus sequence at least 90% identical to: 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

[0144] The light chain CDR3 comprises a consensus sequence at least 90% identical to the following sequences: 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.

[0145] The antibody binds to the conformation-inducing peptide within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), wherein mutations or deletions within FPFS (SEQ ID NO: 1747), SVSDV (SEQ ID NO: 1751), or ASRYNLT (SEQ ID NO: 1745) disrupt binding of the antibody or fragment thereof to PSMGFR.

[0146] The antibody may have another consensus sequence,

[0147] in

[0148] the heavy chain CDR1 comprises a consensus sequence at least 90% identical to the following: 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;

[0149] the heavy chain CDR2 comprises a consensus sequence at least 90% identical to the following: 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;

[0150] The heavy chain CDR3 comprises a consensus sequence that is at least 90% identical to the following sequences: G, L, or N at position 2, G, T, or Y , 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;

[0151] The light chain CDR1 comprises a consensus sequence that is at least 90% identical to the following sequences: 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 , N, S or T at position 14, Y or F at position 15, and I, L or M at position 16;

[0152] the light chain CDR2 comprises a consensus sequence at least 90% identical to: 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

[0153] The light chain CDR3 comprises a consensus sequence at least 90% identical to the following sequences: 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.

[0154] The above-mentioned antibody may be MNC2, which has

[0155] The heavy chain CDR1 contains the consensus sequence FTFSGYAMS;

[0156] The heavy chain CDR2 contains the consensus sequence TISSGGTYIYYPDSVKG;

[0157] The heavy chain CDR3 contains the consensus sequence -LGGDNYYEYFDV--;

[0158] The light chain CDR1 contains the consensus sequence RASKS--VSTSGYSYMH;

[0159] The light chain CDR2 comprises the consensus sequence LASNLES; and

[0160] The light chain CDR3 contains the consensus sequence QHSRELPFT.

[0161] It can be MNE6, which has

[0162] The heavy chain CDR1 contains the consensus sequence FTFSRYGMS;

[0163] The heavy chain CDR2 contains the consensus sequence TISGGGTYIYYPDSVKG;

[0164] The heavy chain CDR3 contains the consensus sequence DNYGRNYDYGMDY--;

[0165] The light chain CDR1 contains the consensus sequence -------SATSSVSYIH;

[0166] The light chain CDR2 comprises the consensus sequence STSNLAS; and

[0167] The light chain CDR3 comprises the consensus sequence QQRSSSPFT.

[0168] It can be B2, which has

[0169] The heavy chain CDR1 contains the consensus sequence FAFSTFAMS;

[0170] The heavy chain CDR2 contains the consensus sequence AISNGGGYTYYPDTLKG;

[0171] The heavy chain CDR3 contains the consensus sequence ----RYYDLYFDL--;

[0172] The light chain CDR1 contains the consensus sequence RSSQNIV-HSNGNTYLE;

[0173] The light chain CDR2 comprises the consensus sequence KVSNRFS; and

[0174] The light chain CDR3 comprises the consensus sequence FQDSHVPLT.

[0175] It can be B7, which has

[0176] The heavy chain CDR1 contains the consensus sequence FTFSRYGMS;

[0177] The heavy chain CDR2 contains the consensus sequence TISSGGTYIYYPDSVKG;

[0178] The heavy chain CDR3 contains the consensus sequence DNYGSSYDYAMDY--;

[0179] The light chain CDR1 contains the consensus sequence RSSQTIV-HSNGNTYLE;

[0180] The light chain CDR2 comprises the consensus sequence KVSNRFS; and

[0181] The light chain CDR3 comprises the consensus sequence FQDSHVPLT.

[0182] It can be B9, which has

[0183] The heavy chain CDR1 contains the consensus sequence FTFSRYGMS;

[0184] The heavy chain CDR2 contains the consensus sequence TISSGGTYIYYPDSVKG;

[0185] The heavy chain CDR3 contains the consensus sequence DNYGSSYDYAMDY--;

[0186] Light chain CDR1 contains the consensus sequence -------SASSSVSYMH;

[0187] The light chain CDR2 comprises the consensus sequence TTSNLAS; and

[0188] The light chain CDR3 comprises the consensus sequence QQRSSYPF-.

[0189] It can be 8C7F3, which has

[0190] The heavy chain CDR1 contains the consensus sequence FTFSTYAMS;

[0191] The heavy chain CDR2 contains the consensus sequence AISNGGGYTYYPDSLKG;

[0192] The heavy chain CDR3 contains the consensus sequence ----RYYDHYFDY--;

[0193] The light chain CDR1 contains the consensus sequence--RASESVATYGNNFMQ;

[0194] The light chain CDR2 comprises the consensus sequence LASTLDS; and

[0195] The light chain CDR3 comprises the consensus sequence QQNNEDPPT.

[0196] It can be H11, which has

[0197] The heavy chain CDR1 contains the consensus sequence FAFSTFAMS;

[0198] The heavy chain CDR2 contains the consensus sequence AISNGGGYTYYPDTLKG;

[0199] The heavy chain CDR3 contains the consensus sequence ----RYYDLYFDL--;

[0200] The light chain CDR1 contains the consensus sequence RSSQNIV-HSNGNTYLE;

[0201] The light chain CDR2 comprises the consensus sequence KVSNRFS; and

[0202] The light chain CDR3 comprises the consensus sequence FQDSHVPLT.

[0203] It can be B12, which has

[0204] The heavy chain CDR1 contains the consensus sequence SYGVH;

[0205] The heavy chain CDR2 contains the consensus sequence VIWPGGSTNYNSTLMSRM;

[0206] The heavy chain CDR3 comprises the consensus sequence DRTPRVGAWFAY; and

[0207] The light chain CDR1 contains the consensus sequence RASESVATYGNNFMQ;

[0208] The light chain CDR2 comprises the consensus sequence LASTLDS; and

[0209] The light chain CDR3 comprises the consensus sequence QQNNEDPPT.

[0210] It can be 20A10, which has

[0211] The heavy chain CDR1 contains the consensus sequence FTFSTYAMS;

[0212] The heavy chain CDR2 contains the consensus sequence -SIGRAGSTYYSDSVKG;

[0213] The heavy chain CDR3 contains the consensus sequence ---GPIYNDYDEFAY;

[0214] The light chain CDR1 contains the consensus sequence KSSQSVLYSSNQKNYLA;

[0215] The light chain CDR2 comprises the consensus sequence WASTRES; and

[0216] The light chain CDR3 comprises the consensus sequence HQYLSSLT.

[0217] It can be 3C2B1, which has

[0218] The heavy chain CDR1 contains the consensus sequence ITFSTYTMS;

[0219] The heavy chain CDR2 contains the consensus sequence TISTGGDKTYYSDSVKG;

[0220] The heavy chain CDR3 contains the consensus sequence -GTTAMYYYAMDY;

[0221] The light chain CDR1 contains the consensus sequence RASKS---ISTSDYNYIH;

[0222] The light chain CDR2 comprises the consensus sequence LASNLES; and

[0223] The light chain CDR3 contains the consensus sequence QHSRELPLT.

[0224] In another aspect, the present invention relates to an antibody or fragment thereof for use in diagnosing, treating or preventing cancer, which requires the presence of an antibody binding conformation-inducing peptide ASRYNLT (SEQ ID NO: 1745) of PSMGFR (SEQ ID NO: 2). The antibody may be 25E6, which has

[0225] The heavy chain CDR1 contains the consensus sequence FTFSSYGMS;

[0226] The heavy chain CDR2 contains the consensus sequence TISNGGRHTFYPDSVKG;

[0227] The heavy chain CDR3 contains the consensus sequence QTGTEGWFAY;

[0228] The light chain CDR1 contains the consensus sequence KSSQSLLDSDGKTYLN;

[0229] The light chain CDR2 comprises the consensus sequence LVSKLDS_; and

[0230] The light chain CDR3 comprises the consensus sequence WQGTHFPQT.

[0231] In another aspect, the present invention relates to an antibody or fragment thereof for use in diagnosing, treating or preventing cancer, which requires the presence of an antibody binding conformation-inducing peptide SVSDV (SEQ ID NO: 1761) of PSMGFR (SEQ ID NO: 2). The antibody may be 5C6F3, which has

[0232] The heavy chain CDR1 contains the consensus sequence FTFSTYAMS;

[0233] The heavy chain CDR2 contains the consensus sequence AISNGGGYTYYPDSLKG;

[0234] The heavy chain CDR3 contains the consensus sequence RYYDHYFDY;

[0235] The light chain CDR1 contains the consensus sequence RSSQTIVHSNGNTYLE;

[0236] The light chain CDR2 comprises the consensus sequence KVSNRFS; and

[0237] The light chain CDR3 comprises the consensus sequence FQDSHVPLT.

[0238] According to all of the above, the antibody or its fragment can be murine, camelid, human or humanized. The antibody fragment can be scFv or scFv-Fc, wherein the variable region thereof can be murine, camelid, human or humanized.

[0239] In another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising the above-mentioned antibody fragment, and may further comprise a mutation in the co-stimulatory domain or the CD3-ζ signaling domain. Tyrosine may be mutated in CD28 or 4-1BB. CD3-ζ may comprise an 1XX mutation.

[0240] 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.

[0241] In another aspect, the present invention relates to a cell composition expressed in a cell, comprising the above-mentioned CAR and a second entity of a biological recognition unit having a specificity different from that of the CAR. The second entity can bind to PD-1, PDL-1 or other checkpoint inhibitors; or NME7; or cytokines such as IL-12 or IL-18; or c-Jun.

[0242] In 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 as claimed in any one of the preceding claims, wherein the second entity is expressed from an inducible promoter. The second entity may be expressed from an inducible promoter activated by an element of an activated immune cell. The second entity may be expressed from an NFAT inducible promoter. NFAT may be NFATc1, NFATc3 or NFATc2. The second entity may be a cytokine, such as IL-7, IL-15 or IL-18. The nucleic acid encoding the second entity may be inserted into a Foxp3 promoter or enhancer region, wherein the cytokine is IL-18. The cytokine may be expressed from an NFAT inducible promoter.

[0243] In another aspect, the present invention relates to a BiTE construct comprising the above-mentioned antibody fragment.

[0244] In yet another aspect, the present invention relates to an antibody drug conjugate (ADC) comprising the above-mentioned antibody or antibody fragment.

[0245] The present invention relates to an antibody or a fragment thereof, which specifically binds to PSMGFR (SEQ ID NO: 2) and N-10 (SEQ ID NO: 3); and

[0246] Does not bind to full-length MUC1;

[0247] Does not bind to C-10 (SEQ ID NO: 825);

[0248] Competitive inhibition of NME1 or NME7 AB Binding to the MUC1* extracellular domain or PSMGFR peptide;

[0249] Recognizes MUC1* produced by cleavage by the cleavage enzyme;

[0250] recognizes conformational epitopes and not linear epitopes; or

[0251] Cancer selectivity by immunohistochemistry of tissues.

[0252] Four of criteria (i)-(vi) may be met. Five of criteria (i)-(vi) may be met. Six of criteria (i)-(vi) may be met. At least criterion (vi) may be met. The cleaving enzyme may be MMP-9.

[0253] In all of the above, the cancer may be breast cancer, pancreatic cancer, ovarian cancer, lung cancer, colon cancer, stomach cancer, or esophageal cancer.

[0254] The present invention also relates to a method of diagnosing, treating or preventing cancer by administering the antibodies and fragments disclosed herein to a cancer patient in need thereof who has been identified as aberrantly expressing MUCl and expressing a truncated MUCl, such as MUCl*.

[0255] These and other objects of the present invention will be more fully understood from the following description of the invention, the accompanying reference drawings and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0256] The patent or application file contains at least one drawing drawn 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.

[0257] The present invention will become more fully understood from the detailed description given hereinafter and from the accompanying drawings, which are given by way of illustration only and therefore are not limiting of the present invention, and in which;

[0258] Figures 1A to 1D show cell growth assays of MUC1*-positive cells treated with bivalent 'bv' anti-MUC1* antibodies, monovalent 'mv' or Fab, NM23-H1 dimer, or NME7-AB. Bivalent anti-MUC1* antibodies stimulated the growth of cancer cells, while monovalent Fab inhibited growth (Figures 1A to 1B). The classic bell-shaped curve indicates that ligand-induced dimerization stimulates growth. Dimeric NM23-H1, also known as NME1, stimulated the growth of MUC1*-positive cancer cells, but siRNA that inhibited MUC1 expression abolished its effect (Figure 1C). NME7-AB also stimulated the growth of MUC1*-positive cells (Figure 1D).

[0259] Figure 2 A to Figure 2I shows the results of ELISA assay. 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 the binding of the following substances was measured: NME7-AB ( Figure 2 A), MN-C2 monoclonal antibody ( Figure 2 B), MN-E6 monoclonal antibody ( Figure 2 C) or dimeric NME1 ( Figure 2 D). These assays showed that NME1, NME7-AB, and the monoclonal antibodies MN-C2 and MN-E6 all require the first 10 membrane-proximal amino acids of the MUC1* extracellular domain for binding. MUC1* peptides minus 10 amino acids from the N-terminus of PSMGFR (also known as N-10) or minus 10 amino acids from the C-terminus of PSMGFR (also known as C-10) were immobilized on the plate and the binding of the following substances was measured: MN-C3 ( Figure 2 E) and MN-C8( Figure 2 F). Figure 2 G shows the amino acid sequence of the PSMGFR peptide. Figure 2 H shows the amino acid sequence of the N-10 peptide. Figure 2 I shows the amino acid sequence of the C-10 peptide.

[0260] Figures 3A to 3C The results of competitive ELISA assay are shown. PSMGFR MUC1* peptide was immobilized on the plate and dimeric NM23-H1 (also known as NME1) was added alone or after addition of MN-E6 antibody ( Figure 3A The same experiment was performed, in which NM23-H7, NME7-AB ( Figure 3B ). The results showed that MN-E6 competitively inhibited the binding of MUC1* activating ligands NME1 and NME7. In similar experiments ( Figure 3C In a 2017 study, researchers immobilized PSMGFR or a PSMGFR with 10 amino acids removed from the N-terminus (also known as N-10) on a plate. Dimeric NM23-H1 was then added. The anti-MUC1* antibodies MN-E6, MN-C2, MN-C3, or MN-C8 were then tested for their ability to compete with NM23-H1. The results showed that while all three antibodies bound to the PSMGFR peptide, MN-E6 and MN-C2 competitively inhibited binding of the MUC1*-activating ligand.

[0261] Figure 4 A to Figure 4F shows a FACS scan of the anti-MUC1* antibody huMN-C2 scFv that specifically binds to MUC1*-positive cancer cells and MUC1*-transfected cells but not to MUC1*- or MUC1-negative cells. ZR-75-1 (also known as 1500) MUC1*-positive breast cancer cells were stained with 1:2 or 1:10 dilutions of 1.5 ug / ml humanized MN-C2. After two washes, the cells were incubated with a secondary antibody anti-Penta-His antibody conjugated to Alexa 488 (Qiagen) at a dilution of 1:200 ( Figure 4 A) 1:50( Figure 4 B) or 1:10( Figure 4 C) Dilutions were stained to detect the 6x His tag on huMN-C2scFv. Figure 4 A shows the binding of huMN-C2 to ZR-75-1 breast cancer cells with the secondary antibody added at a 1:200 dilution. Figure 4 B shows binding of huMN-C2 to ZR-75-1 breast cancer cells with secondary antibody added at a 1:50 dilution. Figure 4 C shows the binding of huMN-C2 to ZR-75-1 breast cancer cells in the presence of secondary antibody added at a 1:10 dilution. Flow cytometric analysis revealed a concentration-dependent shift in cell subsets, indicating specific binding, which was not observed in the absence of MN-C2 scFv ( Figure 4 A to Figure 4 C). Figure 4 D shows anti-MUCl* antibody MN-E6 staining of MUCl-negative HCT-116 colon cancer cells (single cell clone #8) transfected with empty vector. Figure 4 E shows anti-MUCl* antibody MN-E6 staining of HCT-116 colon cancer cells (single cell clone #10) transfected with MUCl*. Figure 4 F shows anti-MUCl* antibody MN-E6 staining of ZR-75-1 (aka 1500) MUC1*-positive breast cancer cells. As shown in FACS scans, both MN-C2 and MN-E6 only stained MUC1*-positive cells, but not MUC1 or MUC1*-negative cells.

[0262] Figure 5 Figure 1 shows an ELISA in which a surface was coated with either a MUC1*PSMGFR peptide or a control peptide. Humanized MN-C2 scFv was then incubated with the surface, washed, and detected according to standard methods. The ELISA showed that huMN-C2 scFv bound to the MUC1* peptide with an EC-50 of approximately 333 nM.

[0263] Figures 6A to 6BThe figure shows that the humanized MN-C2 scFv, a fragment of the variable region of the MUC1* antibody, inhibits the growth of cancer cells. hMN-C2 scFv effectively inhibits the growth of ZR-75-1 (also known as 1500) MUC1* positive breast cancer cells ( Figure 6A ) and T47D MUC1*-positive breast cancer cells ( Figure 6B ) growth, where the EC-50 was roughly the same as in vitro ELISA.

[0264] 7A to 7B Figure 2 shows tumor growth in immunocompromised mice that had been implanted with human tumors and then treated with the anti-MUC1* antibody MN-E6 Fab or mock treatment. Female nu / nu mice that had been implanted with estrogen pellets for 90 days were implanted with 6 million T47D human breast cancer cells mixed with Matrigel 50 / 50. Tumors of at least 150 mm were selected. 3 Mice with three consecutive increases in tumor volume were used for treatment. Animals were injected subcutaneously with 80 mg / kg MN-E6Fab twice a week, and the same 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 with Matrigel 50 / 50. The mice were selected to carry tumors of at least 150 mm. 3 Mice with three consecutive increases in tumor volume were used for treatment. Animals were injected subcutaneously with 160 mg / kg MN-E6 Fab every 48 hours, and the same number of mice meeting the same selection criteria were injected with vehicle alone ( Figure 7B Tumors were independently measured and recorded twice weekly by two investigators. Statistics were calculated blindly by an independent statistician, yielding a P value of 0.0001 for each comparison. Anti-MUC1* Fab inhibited breast cancer and prostate cancer growth. Treatment had no effect on body weight or bone marrow cell type or number.

[0265] Figure 8 Shown are graphs of ELISAs in which the surface was immobilized with PSMGFR peptide, PSMGFR minus 10 amino acids from the N-terminus, or PSMGFR minus 10 amino acids from the C-terminus. huMN-E6 scFv-Fc bound to the PSMGFR peptide and PSMGFR N-10 peptide, but not to the PSMGFR C-10 peptide. The parental MN-E6 antibody and humanized MN-E6 required the C-terminal 10 amino acids of PSMGFR for binding.

[0266] Figures 9A to 9BA diagram of an ELISA is shown in which the assay plate surface was immobilized with PSMGFR peptide, PSMGFR minus 10 amino acids from the N-terminus, or PSMGFR minus 10 amino acids from the C-terminus. The MN-C3 antibody variants were then assayed for binding to various MUC1* peptides. Figure 9A Purified mouse monoclonal MN-C3 antibody is shown; and Figure 9B Humanized MN-C3 scFv-Fc is shown. ELISA showed binding to PSMGFR peptide as well as to certain deletion peptides.

[0267] Figure 10 A to Figure 10 J. Figure 10 A10B is a photograph of a breast cancer tissue array. Figure 10 A was stained with VU4H5 that recognizes MUC1-FL (full length); Figure 10 B Staining with the mouse monoclonal antibody MN-C2 that recognizes cancerous MUC1*. Following automated staining (Clarient Diagnostics), tissue staining was scored using the Allred scoring method that combines intensity and distribution scores. Figure 10 C10F is a color-coded graph showing the score calculated for full-length MUC1 staining for each patient tissue. Figure 10 G10J is a color-coded graph showing the score calculated for MUC1* staining for each patient tissue.

[0268] Figure 11 A to Figure 11 J. Figure 11 A11B is a photograph of a breast cancer tissue array. Figure 11 A was stained with VU4H5 that recognizes MUC1-FL (full length); Figure 11 B Staining with the mouse monoclonal antibody MN-C2 that recognizes cancerous MUC1*. Following automated staining (Clarient Diagnostics), tissue staining was scored using the Allred scoring method that combines intensity and distribution scores. Figure 11 C to Figure 11 F is a color-coded graph showing the fraction calculated for full-length MUC1 staining for each patient tissue. Figure 11 G to Figure 11 J is a color-coded graph showing the score calculated for MUC1* staining for each patient tissue.

[0269] Figure 12 A to Figure 12 H shows photographs of normal breast and breast cancer tissues stained with 2.5 ug / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody, followed by streptavidin-HRP secondary antibody staining. Figure 12A is normal breast tissue. Figure 12 B to Figure 12 D is breast cancer tissue from the patient shown in the figure. Figure 12 E to Figure 12 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0270] Figure 13 A to Figure 13 F shows photographs of normal breast and breast cancer tissues stained with 2.5 ug / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody, followed by streptavidin-HRP secondary antibody staining. Figure 13 A is normal breast tissue. Figure 13 B to Figure 13 C is breast cancer tissue from the patient shown in the figure. Figure 13 D to Figure 13 F is a photograph of the corresponding series of sections stained with the secondary antibody alone.

[0271] Figure 14 A to Figure 14 H shows a photograph of breast cancer tissue stained with 10 ug / mL MN-E6 anti-MUCl* antibody followed by rabbit anti-mouse HRP secondary antibody. Figure 14 A to Figure 14 D is breast cancer tissue from patient #300. Figure 14 E to Figure 14 H is breast cancer tissue from metastatic patient #291.

[0272] Figure 15 A to Figure 15 F shows photographs of normal lung and lung cancer tissues stained with 2.5 ug / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody, followed by streptavidin-HRP secondary antibody staining. Figure 15 A is normal lung tissue. Figure 15 B15C is a lung cancer tissue from the patient shown in the figure. Figure 15 D to Figure 15 F is a photograph of the corresponding series of sections stained with the secondary antibody alone.

[0273] Figure 16 A to Figure 16 F shows photographs of normal lung and lung cancer tissues stained with 2.5 ug / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody, followed by streptavidin-HRP secondary antibody staining. Figure 16 A is normal lung tissue. Figure 16 B16C is a lung cancer tissue from the patient shown in the figure. Figure 16 D to Figure 16F is a photograph of the corresponding series of sections stained with the secondary antibody alone.

[0274] Figure 17 A to Figure 17 F shows photographs of normal lung and lung cancer tissues stained with 25 ug / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody, followed by streptavidin-HRP secondary antibody staining. Figure 17 A is normal lung tissue. Figure 17 B to Figure 17 C is lung cancer tissue from the patient shown in the figure. Figure 17 D to Figure 17 F is a photograph of the corresponding series of sections stained with the secondary antibody alone.

[0275] Figure 18 A to Figure 18 F shows photographs of normal lung and lung cancer tissues stained with 25 ug / mL humanized MN-E6-scFv-Fc biotinylated anti-MUCl* antibody, followed by streptavidin-HRP secondary antibody staining. Figure 18 A is normal lung tissue. Figure 18 B to Figure 18 C is lung cancer tissue from the patient shown in the figure. Figure 18 D to Figure 18 F is a photograph of the corresponding series of sections stained with the secondary antibody alone.

[0276] Figure 19 A to Figure 19 D shows photographs of normal small intestine and cancerous small intestine tissue stained with 5 ug / mL humanized MN-E6-scFv-Fc biotinylated anti-MUC1* antibody, followed by streptavidin-HRP secondary antibody staining. Figure 19 A is normal small intestinal tissue. Figure 19 B is a small bowel cancer from the patient shown. Figure 19 C to Figure 19 D is a photograph of the corresponding series of sections stained with the secondary antibody alone.

[0277] Figure 20 A to Figure 20 H shows a photograph of normal small intestine tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody, followed by goat anti-human HRP secondary antibody. Figure 20 A to Figure 20 D is normal small intestinal tissue. Figure 20 E to Figure 20 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0278] Figure 21 A to Figure 21H shows a photograph of cancerous small intestine tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody, followed by goat anti-human HRP secondary antibody. Figure 21 A to Figure 21 D is cancerous small intestinal tissue from the patient shown in the figure. Figure 21 E to Figure 21 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0279] Figure 22 A to Figure 22 H shows a photograph of cancerous small intestine tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody, followed by goat anti-human HRP secondary antibody. Figure 22 A to Figure 22 D is cancerous small intestinal tissue from the patient shown in the figure. Figure 22 E to Figure 22 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0280] Figure 23 A to Figure 23 H shows a photograph of normal colon tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody, followed by goat anti-human HRP secondary antibody. Figure 23 A to Figure 23 D is a normal colon. Figure 23 E to Figure 23 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0281] Figure 24 A to Figure 24 H shows a photograph of colon cancer tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody, followed by goat anti-human HRP secondary antibody. Figure 24 A to Figure 24 D is colon cancer tissue from the metastatic patient shown in the figure. Figure 24 E to Figure 24 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0282] Figure 25 A to Figure 25 H shows a photograph of colon cancer tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody, followed by goat anti-human HRP secondary antibody. Figure 25 A to Figure 25 D is colon cancer tissue from the grade 2 patient shown in the figure. Figure 25 E to Figure 25 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0283] Figure 26 A to Figure 26 H shows a photograph of colon cancer tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody, followed by goat anti-human HRP secondary antibody. Figure 26 A to Figure 26 D is colon cancer tissue from the metastatic patient shown in the figure. Figure 26 E to Figure 26 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0284] Figure 27 A to Figure 27 H shows a photograph of prostate cancer tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody, followed by goat anti-human HRP secondary antibody. Figure 27 A to Figure 27 D is prostate cancer tissue from the patient shown in the figure. Figure 27 E to Figure 27 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0285] Figure 28 A to Figure 28 H shows a photograph of prostate cancer tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody, followed by goat anti-human HRP secondary antibody. Figure 28 A to Figure 28 D is prostate cancer tissue from the patient shown in the figure. Figure 28 E to Figure 28 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0286] Figure 29 A to Figure 29 H shows a photograph of prostate cancer tissue stained with 50 ug / mL humanized MN-E6-scFv-Fc anti-MUCl* antibody, followed by goat anti-human HRP secondary antibody. Figure 29 A to Figure 29 D is prostate cancer tissue from the patient shown in the figure. Figure 29 E to Figure 29 H is a photograph of the corresponding series of sections stained with individual secondary antibodies.

[0287] Figure 30 A to Figure 30 F shows a photograph of a triple-negative breast cancer array stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of arrays with a score of zero, weak, moderate, or strong is plotted in the form of a pie chart. Figure 30 A shows a pie chart of the fraction stained with anti-MUCl* antibody. Figure 30 B shows a photograph of the array stained with antibodies. Figure 30 C to Figure 30 D shows magnified photographs of two breast cancer samples from the array. Figure 30 E to Figure 30 F shows a more magnified photograph of the portion of the sample indicated by the box.

[0288] Figure 31 A to Figure 31 F shows a photograph of an ovarian cancer array stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of arrays with a score of zero, weak, moderate, or strong is plotted in the form of a pie chart. Figure 31 A shows a pie chart of the fraction stained with anti-MUCl* antibody. Figure 31 B shows a photograph of the array stained with antibodies. Figure 31 C to Figure 31 D shows magnified photographs of two breast cancer samples from the array. Figure 31 E to Figure 31 F shows a more magnified photograph of the portion of the sample indicated by the box.

[0289] Figure 32 A to Figure 32 F shows a photograph of a pancreatic cancer array stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of arrays with a score of zero, weak, moderate, or strong is plotted in the form of a pie chart. Figure 32 A shows a pie chart of the fraction stained with anti-MUCl* antibody. Figure 32 B shows a photograph of the array stained with antibodies. Figure 32 C to Figure 32 D shows magnified photographs of two breast cancer samples from the array. Figure 32 E to Figure 32 F shows a more magnified photograph of the portion of the sample indicated by the box.

[0290] Figure 33 A to Figure 33 F shows a photograph of a lung cancer array stained with the anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score, and the second is the tumor grade. The percentage of arrays with a score of zero, weak, moderate, or strong is plotted in the form of a pie chart. Figure 33 A shows a pie chart of the fraction stained with anti-MUCl* antibody. Figure 33 B shows a photograph of the array stained with antibodies. Figure 33 C to Figure 33 D shows magnified photographs of two breast cancer samples from the array. Figure 33 E to Figure 33 F shows a more magnified photograph of the portion of the sample indicated by the box.

[0291] Figure 34 A to Figure 34 I shows a photograph of normal tissue stained with anti-MUCl* antibody huMNC2scFv.

[0292] Figure 35 A to Figure 35 D shows FACS scans of cells not expressing MUCl, MUCl*, or full-length MUCl, where the cells were probed with MNC2 or VU4H5. Figure 35 A shows MUCl negative HCT-116 colon cancer cells probed with antibody MNC2. Figure 35 B shows HCT cells that have been transfected with MUCl*, in which the extracellular domain is simply the sequence of the PSMGFR peptide, where the cells were probed with the antibody MNC2. Figure 35 C shows HCT-MUCl-18 cells, which are cleavage-resistant single-cell clones of HCT cells transfected with full-length MUCl, also referred to herein as HCT-MUCl-41TR, and the cells were probed with antibody MNC2. Figure 35 D shows HCT-MUCl-18 cells probed with antibody VU4H5, which recognizes hundreds of tandemly repeated epitopes in full-length MUCl. As can be seen from the figure, MNC2 recognizes an ectopic epitope that is inaccessible in full-length MUCl.

[0293] Figures 36A to 36D Western blot and corresponding FACs analysis of HCT-116 cells, a MUC1-negative colon cancer cell line, stably transfected with MUC1* or full-length MUC1. Single cell clones shown are HCT-MUC1-41TR and HCT-MUC1*. Figure 36A Western blots of the parental cell lines HCT-116, HCT-MUC1-41TR, and HCT-MUC1* are shown, where the gel has been probed with the rabbit polyclonal antibody SDIX, which recognizes only cleaved MUC1. A visible band between 25 and 35 kDa can be readily seen in lane 6 loaded with HCT-MUC1*, while only a faint band is 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 36Bis a Western blot probed with mouse monoclonal antibody VU4H5, which recognizes tandem repeats of full-length MUCl. As can be seen, only HCT-MUCl-41TR contains full-length MUCl. Figure 36C A FACS scan is shown, which shows that HCT-MUCl* is 95.7% positive for SDIX, which binds only to MUCl* and essentially no binding to full-length MUCl. Figure 36D Shown is a FACS scan showing that HCT-MUCl-41TR cells were 95% positive for full-length MUCl but only about 11% positive for the cleaved form MUCl*.

[0294] Figures 37A to 37C Shown are bar graphs of Western blot and FACS analyses assessing the ability of MNC2 to recognize full-length MUCl after it has been cleaved by MMP9. Figure 37A Shown is 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 image shows that MMP9 cleaves MUC1 into MUC1*, a species of approximately 25 kDa, in a dose-dependent manner. Figure 37B Shown is a Western blot of conditioned medium from the same experiment. The photograph shows that the addition of the cleavage enzyme MMP9 increases the release of the tandem repeat domain into the conditioned medium in a dose-dependent manner. Figure 37C FACS analysis of the experiment is shown. The graph shows that addition of MMP9 increases recognition of the cleavage products by the anti-MUCl* antibody MNC2 in a dose-dependent manner and decreases recognition of the full-length MUCl containing the tandem repeat domain.

[0295] Figure 38 Shown are photographs of Western blots in which HCT-MUC1-18 cells (labeled here as HCT-18), a cleavage-resistant single-cell clone of HCT cells transfected with full-length MUC1, were treated with varying amounts of catalytically active ADAM17 or MMP14. The shed MUC1 tandem repeat domain of full-length MUC1 was immunoprecipitated from conditioned medium and run on a gel, then probed with VU4H5, which binds to the tandem repeat epitope. As can be seen, MMP14 also effectively cleaves full-length MUC1 and sheds the extracellular domain containing the tandem repeats into the conditioned medium. The cleavage enzyme ADAM17 does not cleave MUC1.

[0296] Figures 39A to 39BFigure 2 shows fluorescence-activated cell sorting (FACS) measurements of human CD34+ hematopoietic stem cells from human bone marrow stained with anti-MUC1* monoclonal antibodies MNC3, MNC2, MNE6, or an isotype control antibody. The histogram of the FACS assay and the bar graph showing the data show that MUC1*-positive cells of the 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 dramatic differences in the specificity of these antibodies suggest that MNC3 recognizes a MUC1*-like form produced when MUC1 is cleaved by an enzyme distinct from MMP9.

[0297] Figure 40 A to Figure 40 G shows details of FACS analysis of hematopoietic stem cells probed with MNC3 or MNE6. Figure 40 A shows a FACS dot plot of total bone marrow cells. Figure 40 B shows the FACS dot plot of CD34+ cells. Figure 40 C shows the FACS histogram of CD34+ cells. Figure 40 D shows FACS dot plots of the earliest hematopoietic stem cells stained with MNC3 or MNE6, which are CD34+ / CD38-. Figure 40 E shows the histogram of the experiment. Figure 40 F shows a histogram overlay of MNC3 relative to MNE6 binding to CD34+ / CD38- cells. Figure 40 G shows a bar graph of the FACS experiment.

[0298] Figure 41 A to Figure 41 H shows CD34+ / CD38 probed with polyclonal anti-PSMGFR antibodies SDIX, MNE6, or MNC2. - / lo Details of FACS analysis of hematopoietic stem cells. Figure 41 B shows CD34+ / CD38 - / lo FACS dot plots of cell populations. Figure 41 E shows a table of detailed analysis. Figure 41 B shows CD34+ / CD38 probed with anti-PSMGFR polyclonal antibody SDIX. - / lo FACS dot plots of cell populations. Figure 41 F shows a table of detailed analysis. Figure 41 C shows CD34+ / CD38 detected by MNE6. - / lo FACS dot plots of cell populations. Figure 41 G shows a table of detailed analysis. Figure 41 D shows CD34+ / CD38 detected by MNC2 - / loFACS dot plots of cell populations. Figure 41 H shows a table of detailed analysis.

[0299] Figure 42 A to Figure 42 H shows photographs of DU145 prostate cancer cells or T47D breast cancer cells that have been treated with Fabs of the anti-MUCl* antibodies MNC2, MNE6, MNC3, or MNC8. The images show that cancer-specific antibodies MNC2 and MNE6 effectively kill prostate and breast cancer cells, while monoclonal antibodies MNC3 and MNC8 do not.

[0300] Figure 43 A graph is shown of a PCR experiment comparing the expression of various cleavage enzymes expressed in different cell lines, where the values have been normalized to those expressed in the breast cancer cell line T47D. The cell lines compared are the prostate cancer cell line DU145, HCT-MUC1-41TR (a MUC1-negative colon cancer cell line transfected with MUC1 whose extracellular domain is truncated after 41 tandem repeats and is not cleaved into the MUC1* form), the T47D breast cancer cell line, and CD34+ bone marrow cells.

[0301] Figure 43 Shown is a graph of a PCR experiment in which the expression levels of various cleavage enzymes were measured in DU145 prostate cancer cells, HCT116+MUC1FL (also known as HCT-MUC1-18, a cell line expressing full-length MUCl), T47D breast cancer cells, and CD34+ bone marrow hematopoietic stem cells. Fold expression is relative to the expression of each cleavage enzyme in T47D breast cancer cells, which was set to 1.

[0302] Figure 44 Shown Figure 43 Figure 2. Graph of a PCR experiment with the Y-axis maximum value set to 5.

[0303] Figure 45 A to Figure 45 P shows a photo of a CAR T co-culture assay in which the CAR targeting antibody fragment was huMNC2scFv, where CAR44 had a CD8 transmembrane domain followed by 41BB-3ζ, and CAR50 had a CD4 transmembrane domain followed by 41BB-3ζ. The target cancer cells were HCT-FLR, which was expressed with MUC1*. 45Transfected HCT-116 cells; and HCT-MUC1-41TR, which is a stable single-cell clone HCT-116 cell line expressing MUC1 whose extracellular domain is truncated after 41 tandem repeats and is not cleaved into the MUC1* form. HCT-MUC1-41TR cancer cells were also incubated with conditioned medium from cells transfected with MMP9 or ADAM17 and then co-cultured with CAR T cells. Conditioned medium from cells expressing MMP9 or ADAM17 was also incubated with APMA, an activator of those cleavage enzymes. The image shown is a superposition of a 4X bright field image and its fluorescent image, showing cancer cells stained with red CMTMR lipophilic dye. Figure 45 A. Figure 45 E. Figure 45 I. Figure 45 M shows a photograph of cells co-cultured with untransduced human T cells. Figure 45 B. Figure 45 F. Figure 45 J. Figure 45 N shows a photograph of cells co-cultured with human T cells transduced with anti-MUC1*CAR44 at an MOI of 10. Figure 45 C. Figure 45 G. Figure 45 K. Figure 45 O shows a photograph of cells co-cultured with human T cells transduced with anti-MUC1*CAR50 at an MOI of 10. Figure 45 D. Figure 45 H. Figure 45 L. Figure 45 P shows a photograph of cells co-cultured with human T cells transduced with anti-MUC1*CAR44 at MOI 50, which increases transduction efficiency. Figure 45 B. Figure 45 C. Figure 45 D shows that T cells transduced with both CAR44 and CAR50 recognized MUC1* expressed in these cancer cells, bound to them, induced aggregation, and killed many cancer cells. Figure 45 F. Figure 45 G. Figure 45 H shows that neither CAR44 nor CAR50 transduced T cells recognized full-length MUC1 expressed in HCT-MUC1-41TR cancer cells. There was no T cell-induced aggregation, and the number of cancer cells was not reduced. Figure 45 J. Figure 45 K. Figure 45 L shows that activated MMP9 has cleaved full-length MUC1 into the MUC1* form, which is recognized by T cells transduced with both CAR44 and CAR50. There is clearly visible CAR T cell-induced aggregation, and the number of cancer cells is reduced as they are killed. Figure 45N. Figure 45 O. Figure 45 P shows that activated ADAM17 does not cleave MUC1 or cleaves MUC1 at a site not recognized by MNC2. Neither huMNC2-CAR44 nor huMNC2-CAR50 transduced T cells recognize these cancer cells.

[0304] Figure 46 A to Figure 46 T shows a photograph of a CAR T co-culture assay in which the targeting antibody fragment of the CAR is an MNC2 scFv, wherein CAR44 has a CD8 transmembrane domain followed by 41BB-3ζ, and CAR50 has a CD4 transmembrane domain followed by 41BB-3ζ. The target cancer cells are breast cancer T47D cells, which are also incubated with conditioned medium from cells transfected with MMP2, MMP9, or ADAM17 and then co-cultured with MNC2-CAR T cells. In some cases, conditioned medium from cells expressing MMP2 and MMP9 is also incubated with APMA, which is one of these cleavage enzymes. The image shown is a superposition of a 4X bright field image and its fluorescent image, showing cancer cells stained with a red CMTMR lipophilic dye. As can be seen, MNC2-CAR T cells only bind to and attack target cancer cells expressing the cleaved form of MUC1*.

[0305] Figure 47 A to Figure 47 I shows a photograph 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. Figure 47 A to Figure 47 The cancer cell shown in C is the MUCl -negative colon cancer cell line HCT-116, which has been stably transfected to express MUCl*. Figure 47 D to Figure 47 The cancer cell shown in F is the MUC1-positive breast cancer cell line T47D, which expresses high levels of full-length and MUC1*. Figure 47 G to Figure 47 The cancer cell shown in FIG1 is the MUC1-positive breast cancer cell line T47D, which was pre-incubated with activated MMP9. Figure 47 A. Figure 47 D and Figure 47 The cells shown in G were co-cultured with untransduced human T cells and are controls. Figure 47 B. Figure 47 E and Figure 47 The cells shown in H were co-cultured with human T cells transduced with huMNC2-CAR44 at an MOI of 10, where MOI stands for multiplicity of infection, and the higher the MOI, the more CAR is expressed on the T cells. Figure 47 C. Figure 47 F and Figure 47 The cells shown in Figure 1 were co-cultured with human T cells transduced with huMNC2-CAR44 at an MOI of 50. As can be seen from the photograph, the CAR44 T cells bound to the target MUC1*-positive cancer cells, surrounding and killing them. Figure 47 Comparison with other images shows that when the antibody targeting the CAR head recognizes MUC1*, cells pre-incubated with MMP9 become more sensitive to CAR T cell killing. It also shows that MUC1 cleaved by MMP9 is recognized by huMNC2scFv.

[0306] Figure 48 The xCelligence graph of T47D breast cancer cells co-cultured with untransduced T cells (as a control) or huMNC2-CAR44 T cells for a period of 45 hours is shown. 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, when T47D cells were pre-incubated with the cutting enzyme MMP9, the killing of huMNC2-CAR44 T cells was greatly improved. In the xCelligence system, adherent target cancer cells are plated on the electrode array plate. Adherent cells insulate the electrodes and increase impedance. The number of adherent cancer cells is proportional to the impedance. T cells do not adhere and do not generate impedance. Therefore, increasing impedance reflects the growth of cancer cells, and decreasing impedance reflects the killing of cancer cells.

[0307] Figure 49 The xCelligence graph shows DU145 prostate cancer cells co-cultured with untransduced T cells (as a control) or huMNC2-CAR44 T cells for a period of 45 hours. After 18 hours of cancer cell growth, the catalytic subunit MMP9 was added to some cells. At 25 hours, T cells were added. As can be seen, huMNC2-CAR44 T cell killing was not affected by pre-incubation 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 cells from accessing the membrane-proximal MUC1*.

[0308] Figure 50 Shown are bar graphs of PCR experiments measuring the amount of MUCl expressed by a panel of cell lines and primary cells, including normal and cancer cells.

[0309] Figures 51A to 51BShown is a bar graph of an ELISA assay measuring the amount of interferon gamma (IFN-g) secreted by huMNC2-CAR44 human T cells after 72 hours of co-culture with normal cells or HCT-MUC1* cancer cells. Figure 51A Shown are the results of an experiment in which the ratio of CAR44 T cells to target cells was 1:1. Figure 51B Shown are the results of an experiment in which the ratio of CAR44 T cells to target cells was 0.5:1.

[0310] Figures 52A to 52B Shown is a bar graph of an ELISA assay measuring the amount of interleukin-2 (IL-2) secreted by huMNC2-CAR44 human T cells after 72 hours of co-culture with normal cells or HCT-MUC1* cancer cells. Figure 52A Shown are the results of an experiment in which the ratio of CAR44 T cells to target cells was 1:1. Figure 52B Shown are the results of an experiment in which the ratio of CAR44 T cells to target cells was 0.5:1.

[0311] Figures 53A to 53J Shown are bar graphs of FACS analysis of live versus dead markers and photographs of normal versus cancer cells after co-culture with huMNC2-CAR44 T cells. Figure 53A .1 shows a bar graph of FACS analysis of live versus dead cells after co-culture of HCT-MUC1* cancer cells with huMNC2-CAR44 T cells. Figure 53A .2 and Figure 53A .3 shows Figure 53A Photographs of the experiments described in .1. Figure 53B .1 shows a bar graph of FACS analysis of live versus dead cells after co-culture of MCF-12A normal breast cells with huMNC2-CAR44 T cells. Figure 53B .2 and Figure 53B .3 shows Figure 53B Photographs of the experiments described in .1. Figure 53C .1 shows a bar graph of FACS analysis of live cells versus dead cells after co-culture of THLE-3 normal liver cells with huMNC2-CAR44 T cells. Figure 53C .2 and Figure 53C .3 shows Figure 53C Photographs of the experiments described in .1. Figure 53D .1 shows a bar graph of FACS analysis of live cells versus dead cells after co-culture of T / G HA-HSMC normal cardiac cells with huMNC2-CAR44 T cells. Figure 53D .2 and Figure 53D.3 shows Figure 53D Photographs of the experiments described in .1. Figure 53E .1 shows a bar graph of FACS analysis of live versus dead cells after co-culture of Hs1.Tes normal testicular cells with huMNC2-CAR44 T cells. Figure 53E .2 and Figure 53E .3 shows Figure 53E Photographs of the experiments described in .1. Figure 53F .1 shows a bar graph of FACS analysis of live versus dead cells after co-culture of HEK-293MUC1-negative cells with huMNC2-CAR44 T cells. Figure 53F .2 and Figure 53F .3 shows Figure 53F Photographs of the experiments described in .1. Figure 53G .1 shows a bar graph of FACS analysis of live versus dead cells after co-culture of HRCE normal kidney cells with huMNC2-CAR44 T cells. Figure 53G .2 and Figure 53G .3 shows Figure 53G Photographs of the experiments described in .1. Figure 53H .1 shows a bar graph of FACS analysis of live cells versus dead cells after co-culture of CCD-18Lu normal lung cells with huMNC2-CAR44 T cells. Figure 53H .2 and Figure 53H .3 shows Figure 53H Photographs of the experiments described in .1. Figure 53I .1 shows a bar graph of FACS analysis of live cells versus dead cells after co-culture of HBEC-5i normal brain cells with huMNC2-CAR44 T cells. Figure 53I .2 and Figure 53I .3 shows Figure 53I Photographs of the experiments described in .1. Figure 53J .1 shows a bar graph of FACS analysis of live cells versus dead cells after co-culture of Hs.738.St / Int normal gastric and intestinal cells with huMNC2-CAR44 T cells. Figure 53J .2 and Figure 53J .3 shows Figure 53J Photographs of the experiments described in .1.

[0312] Figure 54 Shown is a photograph of a breast cancer tissue array (CB - insert array number) in which for each patient there is a sample from the primary tumor plus a sample from the patient's metastasis. As can be seen from the figure, metastases often express more MUC1* than primary tumors.

[0313] Figure 55 A to Figure 55 H shows the cytotoxic effect of huMNC2-CAR44T cells on MUC1*-positive DU145 prostate cancer cells as measured by various assays. Figure 55 A is a fluorescent photograph of untransduced T cells co-cultured with prostate cancer cells in which granzyme B was stained with a red fluorophore. Figure 55 B shows the incorporation of DAPI and granzyme B. Figure 55 C is a fluorescent image of huMNC2-CAR44 T cells co-cultured with prostate cancer cells, in which granzyme B was stained with a red fluorophore. Figure 55 D shows the incorporation of DAPI and granzyme B. Figure 55 E is a FACS scan of fluorescently labeled granzyme B against untransduced T cells incubated with cancer cells. Figure 55 F is a FACS scan showing the positive increase of fluorescently labeled granzyme B in huMNC2-CAR44 T cells incubated with cancer cells. Figure 55 G is a graph of mean fluorescence intensity. Figure 55 H 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).

[0314] Figure 56 A to Figure 56 H shows the cytotoxic effect of huMNC2-CAR44T cells on MUC1*-positive CAPAN-2 pancreatic cancer cells as measured by various assays. Figure 56 A is a fluorescent photograph of untransduced T cells co-cultured with pancreatic cancer cells in which granzyme B was stained with a red fluorophore. Figure 56 B shows the incorporation of DAPI and granzyme B. Figure 56 C is a fluorescent photograph of huMNC2-CAR44 T cells co-cultured with pancreatic cancer cells, in which granzyme B was stained with a red fluorophore. Figure 56 D shows the incorporation of DAPI and granzyme B. Figure 56 E is a FACS scan of fluorescently labeled granzyme B against untransduced T cells incubated with cancer cells. Figure 56 F is a FACS scan showing the positive increase of fluorescently labeled granzyme B in huMNC2-CAR44 T cells incubated with cancer cells. Figure 56 G is a graph of mean fluorescence intensity. Figure 56 H 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).

[0315] Figures 57A to 57C Shown is an xCELLigence scan tracking real-time killing of MUC1*-positive cancer cells but not MUC1*-negative cells by huMNC2-CAR44 T cells. Figure 57A huMNC2-CAR44 T cells were shown to effectively kill HCT colon cancer cells that had been stably transfected with MUC1*. Figure 57B The results showed that huMNC2-CAR44T cells had little effect on HCT-MUC1-41TR, a MUC1-negative cancer cell line that had been stably transfected with full-length MUC1. In this cell line, only about 10% of cells had MUC1 cleaved into MUC1*. Figure 57C It was shown that huMNC2-CAR44 T cells had no effect on HCT-116 cells, a MUC1 negative colon cancer cell line.

[0316] Figures 58A to 58F Shown are photos of NOD / SCID / GAMMA mice in an IVIS instrument measuring photon emission from tumor cells after the mice were untreated, treated with PBS, untransduced human T cells, or huMNC2-CAR44 T cells. The mice were subcutaneously injected with luciferase-positive HCT-MUC1* tumor cells. Ten (10) minutes before the IVIS photos were taken, the luciferase substrate luciferin was injected into the intraperitoneal (ip) space of the mice. Figure 58A Shown are tumor-bearing mice treated with phosphate-buffered saline (PBS) alone. Figure 58B Shown are tumor-bearing mice treated with untransduced T cells alone. Figure 58C Shown are tumor-bearing mice treated with a single dose of huMNC2-CAR44 T cells. Figure 58D The color scale of the image is shown. Figure 58E Kaplan-Meier survival curves for the experiments are shown. Figure 58F Shown is a table detailing the molecular composition of human T cells isolated from mouse blood after sacrifice.

[0317] Figures 59A to 59CShown are photos of NOD / SCID / GAMMA mice in an IVIS instrument that measures photon emission from tumor cells after the mice were untreated, treated with PBS, or treated with huMNC2-CAR44 T cells. The mice were subcutaneously injected with either T47D wild-type breast cancer cells or T47D+moreMUC1*, a mixed cell population in which 95% of the cells were T47D cells stably transfected with even moreMUC1*. Both the T47D wild-type and T47D plus moreMUC1* cells were luciferase positive. Ten (10) minutes before the IVIS photos were taken, the luciferase substrate luciferin was injected into the intraperitoneal (ip) space of the mice. Figure 59A Shown are tumor-bearing mice treated with phosphate-buffered saline (PBS) alone. Figure 59B Shown are T47D wild-type tumor-bearing mice treated with two (2) doses of huMNC2-CAR44 T cells. Figure T90.1C shows T47D-MUCl* tumor-bearing mice treated with two (2) doses of huMNC2-CAR44 T cells.

[0318] Figures 60A to 60C Shown are photos of NOD / SCID / GAMMA mice in an IVIS instrument that measures photon emission from tumor cells after the mice were untreated, treated with PBS, untransduced T cells, or huMNC2-CAR44 T cells. The mice were subcutaneously injected with a mixed population of 70% T47D wild-type breast cancer cells and 30% T47D cells transfected with even more MUC1*. Both cell types were luciferase positive. Ten (10) minutes before the IVIS photos were taken, the luciferase substrate luciferin was injected into the intraperitoneal (ip) space of the mice. Figure 60A Shown are tumor-bearing mice treated with phosphate-buffered saline (PBS) alone. Figure 60B Shown are tumor-bearing mice treated with untransduced T cells alone. Figure 60C Shown are tumor-bearing mice treated with two (2) doses of huMNC2-CAR44 T cells.

[0319] Figure 61 A to Figure 61J shows a fluorescent photo of a mouse taken on an IVIS instrument. On day 0, 500K human BT-20 cells, a MUC1*-positive triple-negative breast cancer cell line, were subcutaneously injected into the flank of NSG (NOD / SCID / GAMMA) immunocompromised mice. The cancer cells were stably transfected with luciferase. The tumors were transplanted. On the 6th day after IVIS measurement, 10 million human T cells or untransduced T cells transduced with huMNC2-scFv-CAR44 were injected into the animals at one time. 5 million T cells were injected into the tumor, and 5 million were injected into the tail vein. 10 minutes before IVIS took the photo, luciferin was injected into the mouse IP, which emitted fluorescence after being cut by luciferase, thereby causing the tumor cells to emit fluorescence. Figure 61 A. Figure 61 D. Figure 61 G shows a photograph of mice treated with huMNC2-scFv-CAR44 T cells that had been pre-stimulated by co-culture with 4 μm beads attached with synthetic MUC1*PSMGFR peptide for 24 hours prior to administration: Protocol 1. Figure 61 B. Figure 61 E. Figure 61 H shows a photograph of mice treated with huMNC2-scFv-CAR44 T cells that had been pre-stimulated twice by co-culturing with MUC1*-positive cancer cells for 24 hours 24 hours prior to administration: Protocol 2. Figure 61 C. Figure 61 F. Figure 61 I shows photographs of mice treated with non-transduced human T cells. Figure 61 J is a color scale that relates fluorescence in photons / second to color.

[0320] Figure 62 A to Figure 62 M shows a fluorescent photograph of a mouse taken on an IVIS instrument. On day 0, 500K human SKOV-3 cells, a MUC1*-positive ovarian cell line, were injected intraperitoneally (IP) into the peritoneal cavity of NSG (NOD / SCID / GAMMA) immunocompromised mice. The cancer cells had been stably transfected with luciferase. Tumors were implanted. On day 4, 10M human T cells transduced with huMNC2-scFv-CAR44, untransduced T cells, or PBS were injected into the intraperitoneal space of the animals. On day 11, the animals were injected again, except that half of the cells were injected into the tail vein and the other half were injected IP. The animals were imaged by IVIS on days 3, 7, 10, and 15. 10 minutes before IVIS photography, the mice were injected IP with luciferin, which emits fluorescence after being cleaved by luciferase, causing the tumor cells to fluoresce. Figure 62 A. Figure 62 D. Figure 62 G and Figure 62 J shows a photograph of mice treated with huMNC2-scFv-CAR44 T cells that had been pre-stimulated by co-culture with 1 μm beads attached with synthetic MUC1*PSMGFR peptide for 24 hours prior to administration. Figure 62 B. Figure 62 E. Figure 62 H and Figure 62 K shows a photograph of mice treated with non-transduced human T cells. Figure 62 C. Figure 62 F. Figure 62 I and Figure 62 L shows photographs of mice treated with PBS. Figure 62 A. Figure 62 B and Figure 62 C is an IVIS image taken on day 3 before CAR T, T cells, or PBS administration. Figure 62 D. Figure 62 E and Figure 62 F shows IVIS images of animals on day 7 (only four (4) days after treatment). Figure 62 G. Figure 62 H and Figure 62 I shows IVIS images of day 10 animals. Figure 62 J. Figure 62 K and Figure 62 L shows IVIS images of day 15 animals. Figure 62 M is the IVIS color scale that relates fluorescence in photons / second to color.

[0321] Figures 63A to 63B Graphs show ELISA binding assays in which various monoclonal antibodies were tested for their ability to bind to PSMGFR peptides, N-10, C-10, N+20 / C-27, or N+9 / C-9, at concentrations of 10 μg / mL or 1 μg / mL. Note that cancer-specific anti-MUC1* monoclonal antibodies C2 and E6 have been shown to bind to the PSMGFR peptide, even if the 10 N-terminal amino acids are deleted, but not if the 10 or 9 C-terminal amino acids are deleted.

[0322] Figures 64A to 64B Figure 2 shows an ELISA binding assay. The antibodies tested were derived from animals immunized with PSMGFR peptide. The first selection criterion was confirmation that the antibody bound to the immune PSMGFR peptide. Figure 64AFigures are shown for ELISA of selected antibodies that were further tested to determine their ability to bind to 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 peptide but not the N-10 peptide, meaning its cognate epitope is located within the GTINVHDVET sequence. Except for 20A10 and C2, all showed a certain degree of binding to both the C-10 and N+9 / C-9 peptides, indicating that both 20A10 and C2 require 10 proximal membrane amino acids for binding. C2, which requires 10 proximal membrane amino acids for binding, has been shown to be cancer specific. Figure 64B The sequences of the various peptides are shown. The color of the bars for each antibody in the ELISA plots is color coded to match the putative homologous sequence of that antibody or a portion thereof.

[0323] Figures 65A to 65B Figure 1 shows an ELISA binding assay in which various monoclonal antibodies were tested for their ability to bind to 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 first selection criterion was confirmation that the antibody bound to the immune N+20 / C-27 peptide. Figure 65A Figures are shown of ELISA binding assays testing each antibody's ability to bind to various peptides. Although these antibodies were raised against N+20 / C-27, all peptides except 45C11 still bound to the PSMGFR peptide. 45C11 bound weakly, but deductive reasoning suggests that the homologous epitope must be located 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 be located within the QFNQYKTE sequence. Figure 65B The sequences of the various peptides are shown. The color of the bars for each antibody in the ELISA plots is color coded to match the putative homologous sequence of that antibody or a portion thereof.

[0324] Figures 66A to 66B Figure 2 shows an ELISA binding assay in which various monoclonal antibodies were tested for their ability to bind to 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 confirmation that the antibody bound to the immune N+9 / C-9 peptide. Figure 66AFigures of ELISA assays are shown. Except for 39H5, all were only able to bind to the immunizing peptide N+9 / C-9. 39H5 showed very weak binding to PSMGFR and the N-10 peptide, consistent with the idea that at least a portion of its cognate epitope must be located within the QFNQYKTE sequence. Figure 66B The sequences of the various peptides are shown. The color of the bars for each antibody in the ELISA plots is color coded to match the putative homologous sequence of that antibody or a portion thereof.

[0325] Figures 67A to 67D The results of ELISA assays further defining the antibody epitopes within the extracellular domain of MUC1 or MUC1* are shown. The antibodies shown in this figure were all generated by immunizing animals with PSMGFR peptides. Binding assays tested the ability of the antibodies to bind to peptides N-19, N-26, N-30, N-10 / C-5, N-19 / C-5, PSMGFR, N-10, and C-10, all of which are subsets of the PSMGFR peptides, and the numbering refers back to the PSMGFR peptides. Figure 67A Binding of various antibodies to various peptides is shown. Figure 67B The sequence of the PSMGFR peptide that has been extended by 20 amino acids at the N-terminus is shown. Figure 67C The sequences of a subset of PSMGFR-derived peptides are shown. Figure 67D Sequences encompassing all or part of the epitope necessary for antibody recognition are shown.

[0326] Figures 68A to 68D The results of ELISA assays further defining the antibody epitopes within the extracellular domain of MUC1 or MUC1* are shown. The antibodies shown in this figure were all generated by immunizing animals with the N+20 / C-27 peptide. Binding assays tested the ability of the antibodies to bind to peptides N-19, N-26, N-30, N-10 / C-5, N-19 / C-5, PSMGFR, N-10, and C-10, all of which are subsets of the PSMGFR peptides, and the numbering refers back to the PSMGFR peptide. Figure 68A Binding of various antibodies to various peptides is shown. Figure 68B The sequence of the PSMGFR peptide that has been extended by 20 amino acids at the N-terminus is shown. Figure 68C The sequences of a subset of PSMGFR-derived peptides are shown. Figure 68D Sequences encompassing all or part of the epitope necessary for antibody recognition are shown.

[0327] Figures 69A to 69DResults of ELISA assays further defining the antibody epitopes within the extracellular domain of MUC1 or MUC1* are shown. The antibodies shown in this figure were all generated by immunizing animals with the N+9 / C-9 peptide. Binding assays tested the ability of the antibodies to bind to peptides N-19, N-26, N-30, N-10 / C-5, N-19 / C-5, PSMGFR, N-10, and C-10, all of which are subsets of the PSMGFR peptides, and the numbering refers back to the PSMGFR peptide. Figure 69A Binding of various antibodies to various peptides is shown. Figure 69B The sequence of the PSMGFR peptide that has been extended by 20 amino acids at the N-terminus is shown. Figure 69C The sequences of a subset of PSMGFR-derived peptides are shown. Figure 69D Sequences encompassing all or part of the epitope necessary for antibody recognition are shown.

[0328] Figures 70A to 70B Figure 1 shows an ELISA assay. In this experiment, multiwell plates were coated with PSMGFR peptide. AB Bind to surface-immobilized PSMGFR peptide. Add various antibodies, followed by washing steps. Detection of NME7 AB The label was used to measure the NME7 that remained attached to the PSMGFR-coated plates after antibody competition. AB As a control, anti-NME7 AB Antibodies replace NME7 from PSMGFR AB ability.

[0329] Figures 71A to 71H Shown are photographs of Western blots in which antibodies were tested for their ability to bind to linear epitopes in full-length MUCl or MUCl*. Figures 71A to 71D Shown are tests of the ability of antibodies to bind to the MUCl negative cell line HCT-116 or the engineered cell lines HCT-MUCl-18, a cleavage-resistant clone expressing full-length MUCl, or HCT-MUCl*, which was engineered to express the PSMGFR sequence only in its extracellular domain. Figures 71E to 71H Shown are tests of the ability of antibodies to bind to the breast cancer cell lines T47D or 1500 (also known as ZR-75-1). Figure 71A and Figure 71E Shown is the monoclonal antibody MNC2, raised against the PSMGFR peptide, which binds to the N-10 variant, but not the C-10 variant, of the PSMGFR peptide. Figure 71B and Figure 71F Shown is the monoclonal antibody MNE6, raised against the PSMGFR peptide, which binds to the N-10 variant of the PSMGFR peptide but not to the C-10 variant. Figure 71C and Figure 71G The polyclonal antibody SDIX was raised against the PSMGFR peptide and binds to the PSMGFR peptide. Figure 71D and Figure 71H Shown is the commercially available monoclonal antibody VU4H5, which binds to the tandem repeats of the full-length MUCl. As can be seen, neither MNC2 nor MNE6 bind to the linear epitope of the MUCl species.

[0330] Figure 72 A to Figure 72 P shows a photograph of a Western blot in which the antibodies were tested for their ability to bind to a linear epitope within full-length MUCl or MUCl*. All of these antibodies were raised against and bound to the PSMGFR peptide. Figure 72 A to Figure 72 H shows testing of the ability of the antibodies to bind to the MUCl-negative cell line HCT-116 or the engineered cell line HCT-MUCl-18, a cleavage-resistant clone expressing full-length MUCl, or HCT-MUCl*, which was engineered to express the PSMGFR sequence only in its extracellular domain. Figure 72 I arrive Figure 72 P shows testing of the ability of the antibodies to bind to the breast cancer cell lines T47D or 1500 (also known as ZR-75-1). Figure 72 A and Figure 72 I shows 20A10. Figure 72 B and Figure 72 J shows 25E6. Figure 72 C and Figure 72 K shows 18B4. Figure 72 D and Figure 72 L shows 18G12. Figure 72 E and Figure 72 M shows 28F9. Figure 72 F and Figure 72 N shows 3C2B1. Figure 72 G and Figure 72 O shows 5C6F3. Figure 72 H and Figure 72 P shows 5C6F3 where the blot has been exposed for a longer period of time to make the MUCl*-specific band more visible. As can be seen, antibodies 25E6, 18B4 and 5C6F3 (to some extent) recognize the linear epitope, but 20A10, 3C2B1, 18G12 and 28F9 do not.

[0331] Figures 73A to 73JShown are photographs of Western blots in which the antibodies were tested for their ability to bind to linear epitopes within full-length MUC1 or MUC1*. All of these antibodies were raised against the N+20 / C-27 variant of the PSMGFR peptide and bound to the N+20 / C-27 peptide. Figures 73A to 73E Shown are tests of the ability of antibodies to bind to the MUCl negative cell line HCT-116 or the engineered cell lines HCT-MUCl-18, a cleavage-resistant clone expressing full-length MUCl, or HCT-MUCl*, which was engineered to express the PSMGFR sequence only in its extracellular domain. Figures 73F to 73J Shown are tests of the ability of antibodies to bind to the breast cancer cell lines T47D or 1500 (also known as ZR-75-1). Figure 73A and Figure 73F 1E4 is shown. Figure 73B and Figure 73G 45C11 is shown. Figure 73C and Figure 73H 31A1 is shown. Figure 73D and Figure 73I 32C1 is shown. Figure 73E and Figure 73J 29H1 is shown. As can be seen, antibodies 31A1 and 32C1 recognize linear epitopes.

[0332] Figures 74A to 74H Shown are photographs of Western blots in which the antibodies were tested for their ability to bind to linear epitopes within full-length MUC1 or MUC1*. All of these antibodies were raised against the N+9 / C-9 variant of the PSMGFR peptide and bound to the N+9 / C-9 peptide. Figures 74A to 74D Shown are tests of the ability of antibodies to bind to the MUCl negative cell line HCT-116 or the engineered cell lines HCT-MUCl-18, a cleavage-resistant clone expressing full-length MUCl, or HCT-MUCl*, which was engineered to express the PSMGFR sequence only in its extracellular domain. Figures 74E to 74H Shown are tests of the ability of antibodies to bind to the breast cancer cell lines T47D or 1500 (also known as ZR-75-1). Figure 74A and Figure 74E 8A9 is shown. Figure 74B and Figure 74F 17H6 is shown. Figure 74C and Figure 74G 3C5 is shown. Figure 74D and Figure 74H 39H5 is shown.

[0333] Figures 75A to 75PGraphs of FACS analysis are shown. 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 bind to the MMP9 or MMP2 cleaved forms of MUC1. Note that the first bar in each graph shows that in the absence of cleavage, no antibody binds to full-length MUC1. Each bar is labeled with the name of the antibody used in the 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 Antibody 1E4 is shown. Figure 75B Antibody 28F9 is shown. Figure 75C Antibody 18G12 is shown. Figure 75D Antibody 25E6 is shown. Figure 75E Antibody 20A10 is shown. Figure 75F Antibody 3C5 is shown. Figure 75G Antibody 29H1 is shown. Figure 75H Antibody 32C1 is shown. Figure 75I Antibody 31A1 is shown. Figure 75J Antibody 18B4 is shown. Figure 75K Antibody 45C11 is shown. Figure 75L Antibody 8A9 is shown. Figure 75M Antibody 17H6 is shown. Figure 75N Antibody 39H5 is shown. Figure 75O Antibody 3C2B1 is shown. Figure 75P Antibody 5C6F3 is shown.

[0334] Figures 76A to 76J Figures showing the binding of reference antibodies MNC2 "C2" and VU4H5 to 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 FACS analysis of MNC2 binding to 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 the majority of the extracellular domain, including the tandem repeat domain, has been cleaved and released. VU4H5 binds to hundreds of repeated epitopes within the tandem repeat domain. Figures 76A to 76E The percentage binding is shown, and Figures 76F to 76J Mean fluorescence intensity or MFI is shown.

[0335] Figures 77A to 77NA FACS analysis of the reference antibody MNC2 "C2" binding to a panel of cancer cell lines is shown, all of which are MUC1*-positive except for MDA-MB-231, which expresses very low levels of MUC1 and MUC1*, making it frequently used as a negative control. MNC2 binds to an ectopic binding site in the extracellular domain of MUC1* within the membrane-juxtaposed portion of the PSMGFR sequence. The MNC2 binding site is only accessible after the majority of the extracellular domain, including the tandem repeat domain, has been cleaved and released. Figures 77A to 77G The percentage binding is shown, and Figures 77H to 77N Mean fluorescence intensity or MFI is shown. Figure 77A and Figure 77H Antibody binding to the lung cancer cell line NCI-H292 is shown. Figure 77B and Figure 77I Antibody binding to the lung cancer cell line NCI-H1975 is shown. Figure 77C and Figure 77J Antibody binding to the ovarian cancer cell line SKOV-3 is shown. Figure 77D and Figure 77K Antibody binding to the pancreatic cancer cell line HPAF-II is shown. Figure 77E and Figure 77L Antibody binding to the pancreatic cancer cell line Capan-1 is shown. Figure 77F and Figure 77M Antibody binding to the prostate cancer cell line DU145 is shown. Figure 77G and Figure 77N Antibody binding to the breast cancer cell line MDA-MB-231, which is nearly MUCl and MUCl* negative, is shown.

[0336] Figure 78 A to Figure 78 C shows a color-coded schematic representation of the basic PSMGFR sequence with extensions or deletions at both the N and C termini. The antibodies of the invention were tested against this subset of peptides to further refine the epitope to which each antibody binds or the key amino acids within the epitope to which each antibody binds. Figure 78 A is a schematic diagram of the alignment of various peptide subsets. Figure 78 B lists the antibodies that bind to each of the color-coded sequences. Figure 78 C lists the cancer cell lines recognized by each antibody.

[0337] Figures 79A to 79I Shown are color-coded plots resulting from FACS analysis of each antibody binding to T47D breast cancer cells and its corresponding homologous sequence within the N-terminally extended PSMGFR sequence. Figures 79A to 79D are FACS graphs showing the percentage of cells recognized by each antibody. Figures 79E to 79HIt is a FACS graph showing the mean fluorescence intensity (MFI) of each antibody. Figure 79A and Figure 79E Shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 79B and Figure 79F Shown are FACS plots of antibodies produced by immunization with the N+20 / C-27 peptide. Figure 79C and Figure 79G Shown are FACS plots of antibodies produced by immunization with the N+9 / C-9 peptide. Figure 79D and Figure 79H Also shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 79I The PSMGFR sequence is shown with a 20 amino acid extension at the N-terminus.

[0338] Figures 80A to 80I Shown are color-coded plots resulting from FACS analysis of each antibody binding to 1500 (also known as ZR-75-1) breast cancer cells and its corresponding homologous sequence within the N-terminally extended PSMGFR sequence. Figures 80A to 80C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 80D to 80F It is a FACS graph showing the mean fluorescence intensity (MFI) of each antibody. Figure 80A 、 Figure 80E 、 Figure 80D and Figure 80H Shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 80B and Figure 80F Shown are FACS plots of antibodies produced by immunization with the N+20 / C-27 peptide. Figure 80C and Figure 80G Shown are FACS plots of antibodies produced by immunization with the N+9 / C-9 peptide. Figure 80I The PSMGFR sequence is shown with a 20 amino acid extension at the N-terminus.

[0339] Figures 81A to 81G Shown are color-coded plots resulting from FACS analysis of each antibody binding to NCI-H292 lung cancer cells and its corresponding homologous sequence within the N-terminally extended PSMGFR sequence. Figures 81A to 81C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 81D to 81F It is a FACS graph showing the mean fluorescence intensity (MFI) of each antibody. Figure 81A and Figure 81D Shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 81B and Figure 81EShown are FACS plots of antibodies produced by immunization with the N+20 / C-27 peptide. Figure 81C and Figure 81F Shown are FACS plots of antibodies produced by immunization with the N+9 / C-9 peptide. Figure 81G The PSMGFR sequence is shown with a 20 amino acid extension at the N-terminus.

[0340] Figures 82A to 82G Shown are color-coded plots resulting from FACS analysis of each antibody binding to NCI-H1975 lung cancer cells and its corresponding homologous sequence within the N-terminally extended PSMGFR sequence. Figures 82A to 82C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 82D to 82F It is a FACS graph showing the mean fluorescence intensity (MFI) of each antibody. Figure 82A and Figure 82D Shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 82B and Figure 82E Shown are FACS plots of antibodies produced by immunization with the N+20 / C-27 peptide. Figure 82C and Figure 82F Shown are FACS plots of antibodies produced by immunization with the N+9 / C-9 peptide. Figure 82G The PSMGFR sequence is shown with a 20 amino acid extension at the N-terminus.

[0341] Figures 83A to 83G Shown are color-coded plots resulting from FACS analysis of each antibody binding to SKOV-3 ovarian cancer cells and its corresponding homologous sequence within the N-terminally extended PSMGFR sequence. Figures 83A to 83C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 83D to 83F It is a FACS graph showing the mean fluorescence intensity (MFI) of each antibody. Figure 83A and Figure 83D Shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 83B and Figure 83E Shown are FACS plots of antibodies produced by immunization with the N+20 / C-27 peptide. Figure 83C and Figure 83F Shown are FACS plots of antibodies produced by immunization with the N+9 / C-9 peptide. Figure 83G The PSMGFR sequence is shown with a 20 amino acid extension at the N-terminus.

[0342] Figures 84A to 84HShown are color-coded plots resulting from FACS analysis of each antibody binding to DU145 prostate cancer cells and its corresponding homologous sequence within the N-terminally extended PSMGFR sequence. Figures 84A to 84C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 84D to 84F It is a FACS graph showing the mean fluorescence intensity (MFI) of each antibody. Figure 84A and Figure 84D Shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 84B and Figure 84E Shown are FACS plots of antibodies produced by immunization with the N+20 / C-27 peptide. Figure 84C and Figure 84F Shown are FACS plots of antibodies produced by immunization with the N+9 / C-9 peptide. Figure 84I The PSMGFR sequence is shown with a 20 amino acid extension at the N-terminus.

[0343] Figures 85A to 85G Shown are color-coded plots resulting from FACS analysis of each antibody binding to HPAF-II pancreatic cancer cells and its corresponding homologous sequence within the N-terminally extended PSMGFR sequence. Figures 85A to 85C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 85D to 85F It is a FACS graph showing the mean fluorescence intensity (MFI) of each antibody. Figure 85A and Figure 85D Shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 85B and Figure 85E Shown are FACS plots of antibodies produced by immunization with the N+20 / C-27 peptide. Figure 85C and Figure 85F Shown are FACS plots of antibodies produced by immunization with the N+9 / C-9 peptide. Figure 85G The PSMGFR sequence is shown with a 20 amino acid extension at the N-terminus.

[0344] Figures 86A to 86G Shown are color-coded plots resulting from FACS analysis of each antibody binding to Capan-1 pancreatic cancer cells and its corresponding homologous sequence within the N-terminally extended PSMGFR sequence. Figures 86A to 86C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 86D to 86F It is a FACS graph showing the mean fluorescence intensity (MFI) of each antibody. Figure 86A and Figure 86D Shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 86B and Figure 86EShown are FACS plots of antibodies produced by immunization with the N+20 / C-27 peptide. Figure 86C and Figure 86F Shown are FACS plots of antibodies produced by immunization with the N+9 / C-9 peptide. Figure 86G The PSMGFR sequence is shown with a 20 amino acid extension at the N-terminus.

[0345] Figures 87A to 87G Shown are color-coded plots resulting from FACS analysis of each antibody binding to MDA-MB-231 breast cancer cells (which are nearly MUCl negative) and their corresponding homologous sequence within the N-terminally extended PSMGFR sequence. Figures 87A to 87C are FACS graphs showing the percentage of cells recognized by each antibody. Figures 87D to 87F It is a FACS graph showing the mean fluorescence intensity (MFI) of each antibody. Figure 87A and Figure 87D Shown are FACS plots of antibodies produced by immunization with PSMGFR peptide. Figure 87B and Figure 87E Shown are FACS plots of antibodies produced by immunization with the N+20 / C-27 peptide. Figure 87C and Figure 87F Shown are FACS plots of antibodies produced by immunization with the N+9 / C-9 peptide. Figure 87G The PSMGFR sequence is shown with a 20 amino acid extension at the N-terminus.

[0346] Figures 88A to 88L Shown are photographs of normal liver tissue samples, each sample from the same donor but stained with a different antibody of the invention. Figures 88A to 88F The entire tissue core is shown. Figures 88G to 88L A 40X magnification of a specific region of tissue is shown. The tissue is arranged from right to left, with antibodies that bind to the most membrane-proximal (i.e., C-terminal-most) portion of the PSMGFR peptide on the right and antibodies that bind to the most N-terminal portion of the MUC1 extracellular domain (even beyond the PSMGFR region) on the left. As can be seen from the figure, the most cancer-specific antibodies are those that bind to the more membrane-proximal portion of the PSMGFR sequence, while antibodies that bind to the most distal N-terminal portion lose cancer specificity, with antibodies that bind to epitopes outside of PSMGFR having lost all cancer specificity.

[0347] Figures 89A to 89H Shown are photographs of normal heart tissue samples stained with different antibodies of the present invention. Figures 89A to 89D The entire tissue core is shown. Figures 89E to 89H A 40X magnification of a specific area of tissue is shown. Figure 89A and Figure 89EStaining with MNC2-scFv is shown. Figure 89B and Figure 89F Staining with MNE6 is shown. Figure 89C and Figure 89G Staining with 20A10 is shown. Figure 89D and Figure 89H Staining with 3C2B1 is shown. These antibodies bind to an epitope containing all or part of the sequence FPFS or PFPFSAQSGA. All of these antibodies were able to bind to the PSMGFR peptide, to the N-10 peptide, but not to the C-10 peptide. In addition, these antibodies disrupt NME7 AB Binding to the extracellular domain of MUC1*, as exemplified by the PSMGFR peptide. In addition, these antibodies recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen from the figure, these antibodies show no binding to normal cardiac tissue.

[0348] Figures 90A to 90D Shown are photographs of normal heart tissue samples stained with different antibodies of the present invention. Figures 90A to 90B The entire tissue core is shown. Figures 90C to 90D A 40X magnification of a specific area of tissue is shown. Figure 90A and Figure 90C Staining with MNC3 is shown. Figure 90B and Figure 90D Staining with 25E6 is shown. These antibodies bind to an epitope comprising all or part of the sequence ASRYNLT. All of these antibodies are able to bind to the PSMGFR peptide, the N-10 peptide, and the C-10 peptide.

[0349] Figures 91A to 91B Shown are photographs of normal heart tissue samples stained with the antibody 1E4 of the present invention. Figure 91A The entire tissue core is shown. Figure 91B A 40X magnification of a specific region of tissue is shown. Antibody 1E4 binds to an epitope comprising all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to both the N-10 peptide and the C-10 peptide. As can be seen in the figure, 1E4 binds to normal cardiac tissue.

[0350] Figures 92A to 92H Shown are photographs of normal heart tissue samples stained with different antibodies of the present invention. Figures 92A to 92D The entire tissue core is shown. Figures 92E to 92H A 40X magnification of a specific area of tissue is shown. Figure 92A and Figure 92E Staining with 18B4 is shown. Figure 92B and Figure 92F Staining with 31A1 is shown. Figure 92C and Figure 92G Staining with 32C1 is shown. Figure 92D and Figure 92H Staining with 29H1 is shown. These antibodies bind to an epitope comprising all or part of the sequence GTINVHDVET, which is the N-terminal portion of the PSMGFR peptide. None of these antibodies bind to the N-10 peptide. As can be seen in the figure, all of these antibodies, except 18B4, show binding to normal cardiac tissue.

[0351] Figures 93A to 93D Shown are photographs of normal heart tissue samples stained with the antibodies of the present invention. Figures 93A to 93B The entire tissue core is shown. Figures 93C to 93D A 40X magnification of a specific area of tissue is shown. Figure 93A and Figure 93C Staining with antibody 8A9 is shown. Figure 93B and Figure 93D Shown is staining with antibody 17H6. Both antibodies bind to an epitope outside the PSMGFR region and comprising all or part of the sequence VQLTLAFRE. As can be seen from the figure, both antibodies show strong binding to normal heart tissue.

[0352] Figures 94A to 94B Shown are photographs of normal heart tissue samples stained with the antibody 45C11 of the present invention. Figure 94A The entire tissue core is shown. Figure 94B A 40X magnification of a specific region of tissue is shown. Antibody 45C11 binds to an epitope outside the PSMGFR region and encompasses all or part of the sequence SNIKFRPGSVV. Antibody 45C11 does not bind to the N-10 peptide. As can be seen in the figure, 45C11 binds strongly to normal cardiac tissue.

[0353] Figures 95A to 95H Shown are photographs of normal liver tissue samples stained with different antibodies of the present invention. Figures 95A to 95D The entire tissue core is shown. Figures 95E to 95H A 40X magnification of a specific area of tissue is shown. Figure 95A and Figure 95E Staining with MNC2-scFv is shown. Figure 95B and Figure 95F Staining with MNE6 is shown. Figure 95C and Figure 95G Staining with 20A10 is shown. Figure 95D and Figure 95HStaining with 3C2B1 is shown. These antibodies bind to an epitope containing all or part of the sequence FPFS or PFPFSAQSGA. All of these antibodies were able to bind to the PSMGFR peptide, to the N-10 peptide, but not to the C-10 peptide. In addition, these antibodies disrupt NME7 AB Binding to the extracellular domain of MUC1*, as exemplified by the PSMGFR peptide. In addition, these antibodies recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen from the figure, these antibodies show no binding to normal liver tissue.

[0354] Figure 96 A to Figure 96 D shows photographs of normal liver tissue samples stained with different antibodies of the present invention. Figure 96 A to Figure 96 B shows the entire tissue core. Figure 96 C to Figure 96 D shows a 40X magnification of a specific area of tissue. Figure 96 A and Figure 96 C shows staining with MNC3. Figure 96 B and Figure 96 D shows staining with 25E6. These antibodies bind to an epitope containing all or part of the sequence ASRYNLT. All of these antibodies can bind to the PSMGFR peptide, the N-10 peptide, and the C-10 peptide.

[0355] Figures 97A to 97B Shown are photographs of normal liver tissue samples stained with the antibody 1E4 of the present invention. Figure 97A The entire tissue core is shown. Figure 97B A 40X magnification of a specific region of tissue is shown. Antibody 1E4 binds to an epitope comprising all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to both the N-10 peptide and the C-10 peptide. As can be seen in the figure, 1E4 binds to normal liver tissue.

[0356] Figures 98A to 98H Shown are photographs of normal liver tissue samples stained with different antibodies of the present invention. Figures 98A to 98D The entire tissue core is shown. Figures 98E to 98H A 40X magnification of a specific area of tissue is shown. Figure 98A and Figure 98E Staining with 18B4 is shown. Figure 98B and Figure 98F Staining with 31A1 is shown. Figure 98C and Figure 98G Staining with 32C1 is shown. Figure 98D and Figure 98HStaining with 29H1 is shown. These antibodies bind to an epitope containing all or part of the sequence GTINVHDVET, which is the N-terminal portion of the PSMGFR peptide. None of these antibodies bind to the N-10 peptide. As can be seen in the figure, 32C1 shows some binding to normal liver, while 29H1 shows very strong binding to normal liver tissue.

[0357] Figures 99A to 99D Shown are photographs of normal liver tissue samples stained with the antibodies of the present invention. Figures 99A to 99B The entire tissue core is shown. Figures 99C to 99D A 40X magnification of a specific area of tissue is shown. Figure 99A and Figure 99C Staining with antibody 8A9 is shown. Figure 99B and Figure 99D Staining with antibody 17H6 is shown. Both antibodies bind to an epitope outside the PSMGFR region and containing all or part of the sequence VQLTLAFRE. As can be seen from the figure, 8A9 shows strong binding to normal liver tissue. 17H6 is a weak antibody, and it is possible that the concentration used in this study was not high enough.

[0358] Figures 100A to 100B Shown are photographs of normal liver tissue samples stained with the antibody 45C11 of the present invention. Figure 100A The entire tissue core is shown. Figure 100B A 40X magnification of a specific region of tissue is shown. Antibody 45C11 binds to an epitope outside the PSMGFR region and encompassing all or part of the sequence SNIKFRPGSVV. Antibody 45C11 does not bind to the N-10 peptide. As can be seen in the figure, 45C11 binds strongly to normal liver tissue.

[0359] Figures 101A to 101H Shown are photographs of normal lung tissue samples stained with different antibodies of the present invention. Figures 101A to 101D The entire tissue core is shown. Figures 101E to 101H A 40X magnification of a specific area of tissue is shown. Figure 101A and Figure 101E Staining with MNC2-scFv is shown. Figure 101B and Figure 101F Staining with MNE6 is shown. Figure 101C and Figure 101G Staining with 20A10 is shown. Figure 101D and Figure 101HStaining with 3C2B1 is shown. These antibodies bind to an epitope containing all or part of the sequence FPFS or PFPFSAQSGA. All of these antibodies were able to bind to the PSMGFR peptide, to the N-10 peptide, but not to the C-10 peptide. In addition, these antibodies disrupt NME7 AB Binding to the extracellular domain of MUC1*, as exemplified by the PSMGFR peptide. In addition, these antibodies recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen from the figure, these antibodies show no binding to normal lung tissue.

[0360] Figures 102A to 102D Shown are photographs of normal lung tissue samples stained with different antibodies of the present invention. Figures 102A to 102B The entire tissue core is shown. Figures 102C to 102D A 40X magnification of a specific area of tissue is shown. Figure 102A and Figure 102C Staining with MNC3 is shown. Figure 102B and Figure 102D Staining with 25E6 is shown. These antibodies bind to an epitope comprising all or part of the sequence ASRYNLT. All of these antibodies are able to bind to the PSMGFR peptide, the N-10 peptide, and the C-10 peptide.

[0361] Figures 103A to 103B Shown are photographs of normal lung tissue samples stained with the antibody 1E4 of the present invention. Figure 103A The entire tissue core is shown. Figure 103B A 40X magnification of a specific area of tissue is shown. Antibody 1E4 binds to an epitope comprising all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to both the N-10 peptide and the C-10 peptide.

[0362] Figures 104A to 104H Shown are photographs of normal lung tissue samples stained with different antibodies of the present invention. Figures 104A to 104D The entire tissue core is shown. Figures 104E to 104H A 40X magnification of a specific area of tissue is shown. Figure 104A and Figure 104E Staining with 18B4 is shown. Figure 104B and Figure 104F Staining with 31A1 is shown. Figure 104C and Figure 104G Staining with 32C1 is shown. Figure 104D and Figure 104HStaining with 29H1 is shown. These antibodies bind to an epitope comprising all or part of the sequence GTINVHDVET, which is the N-terminal portion of the PSMGFR peptide. None of these antibodies bind to the N-10 peptide. As can be seen in the figure, all of these antibodies show strong binding to normal lung tissue.

[0363] Figures 105A to 105D Shown are photographs of normal lung tissue samples stained with the antibodies of the present invention. Figures 105A to 105B The entire tissue core is shown. Figures 105C to 105D A 40X magnification of a specific area of tissue is shown. Figure 105A and Figure 105C Staining with antibody 8A9 is shown. Figure 105B and Figure 105D Staining with antibody 17H6 is shown. Both antibodies bind to an epitope outside the PSMGFR region and encompassing all or part of the sequence VQLTLAFRE. As can be seen in the figure, 8A9 shows strong binding to normal lung tissue. 17H6 is a weak antibody, and it is possible that the concentration used in this study was not high enough.

[0364] Figures 106A to 106B Shown are photographs of normal lung tissue samples stained with the antibody 45C11 of the present invention. Figure 106A The entire tissue core is shown. Figure 106B A 40X magnification of a specific region of tissue is shown. Antibody 45C11 binds to an epitope outside the PSMGFR region and encompasses all or part of the sequence SNIKFRPGSVV. Antibody 45C11 does not bind to the N-10 peptide. As can be seen in the figure, 45C11 binds to normal lung tissue.

[0365] Figures 107A to 107H Shown are photographs of normal bone marrow tissue samples stained with different antibodies of the present invention. Figures 107A to 107D The entire tissue core is shown. Figures 107E to 107H A 40X magnification of a specific area of tissue is shown. Figure 107A and Figure 107E Staining with MNC2-scFv is shown. Figure 107B and Figure 107F Staining with MNE6 is shown. Figure 107C and Figure 107G Staining with 20A10 is shown. Figure 107D and Figure 107H Staining with 3C2B1 is shown. These antibodies bind to an epitope containing all or part of the sequence FPFS or PFPFSAQSGA. All of these antibodies were able to bind to the PSMGFR peptide, to the N-10 peptide, but not to the C-10 peptide. In addition, these antibodies disrupt NME7 ABBinding to the extracellular domain of MUC1*, as exemplified by the PSMGFR peptide. In addition, these antibodies recognize MUC1 cleavage products when the cleaving enzyme is MMP9. As can be seen from the figure, these antibodies show no binding to normal bone marrow tissue.

[0366] Figures 108A to 108D Shown are photographs of normal bone marrow tissue samples stained with different antibodies of the present invention. Figures 108A to 108B The entire tissue core is shown. Figures 108C to 108D A 40X magnification of a specific area of tissue is shown. Figure 108A and Figure 108C Staining with MNC3 is shown. Figure 108B and Figure 108D Staining with 25E6 is shown. These antibodies bind to an epitope comprising all or part of the sequence ASRYNLT. All of these antibodies are able to bind to the PSMGFR peptide, the N-10 peptide, and the C-10 peptide.

[0367] Figures 109A to 109B Shown are photographs of normal bone marrow tissue samples stained with the antibody 1E4 of the present invention. Figure 109A The entire tissue core is shown. Figure 109B A 40X magnification of a specific region of tissue is shown. Antibody 1E4 binds to an epitope comprising all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to both the N-10 peptide and the C-10 peptide. 1E4 binds to normal bone marrow.

[0368] Figures 110A to 110H Shown are photographs of normal bone marrow tissue samples stained with different antibodies of the present invention. Figures 110A to 110D The entire tissue core is shown. Figures 110E to 110H A 40X magnification of a specific area of tissue is shown. Figure 110A and Figure 110E Staining with 18B4 is shown. Figure 110B and Figure 110F Staining with 31A1 is shown. Figure 110C and Figure 110G Staining with 32C1 is shown. Figure 110D and Figure 110H Staining with 29H1 is shown. These antibodies bind to an epitope comprising all or part of the sequence GTINVHDVET, which is the N-terminal portion of the PSMGFR peptide. None of these antibodies bind to the N-10 peptide. As can be seen in the figure, all of these antibodies show strong binding to normal bone marrow tissue.

[0369] Figures 111A to 111D Shown are photographs of normal bone marrow tissue samples stained with the antibodies of the present invention. Figures 111A to 111B The entire tissue core is shown. Figures 111C to 111D A 40x magnification of a specific area of tissue is shown. Figure 111A and Figure 111C Staining with antibody 8A9 is shown. Figure 111B and Figure 111D Staining with antibody 17H6 is shown. Both antibodies bind to an epitope outside the PSMGFR region and encompassing all or part of the sequence VQLTLAFRE. As can be seen in the figure, 8A9 shows strong binding to normal bone marrow tissue. 17H6 is a weak antibody, and it is possible that the concentration used in this study was not high enough.

[0370] Figures 112A to 112B Shown are photographs of normal bone marrow tissue samples stained with the antibody 45C11 of the present invention. Figure 112A The entire tissue core is shown. Figure 112B A 40X magnification of a specific region of tissue is shown. Antibody 45C11 binds to an epitope outside the PSMGFR region and encompasses all or part of the sequence SNIKFRPGSVV. Antibody 45C11 does not bind to the N-10 peptide. As can be seen in the figure, 45C11 binds to normal bone marrow tissue.

[0371] Figures 113A to 113C Shown are a photograph, array map, and description of FDA normal tissue array 1021 stained with 0.25 ug / mL of anti-PSMGFR antibody 20A10. Figure 113A Photographs of tissue microarrays are shown. Figure 113B Array diagrams with abbreviated tissue descriptors are shown. Figure 113C Detailed description of tissue microarrays with deidentified donor data.

[0372] Figures 114A to 114X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 0.25 ug / mL of anti-PSMGFR antibody 20A10 at 6X and 20X magnification. Figure 114A and Figure 114E It's the adrenal glands. Figure 114B and Figure 114F It's the mammary gland. Figure 114C and Figure 114G It's the fallopian tube. Figure 114D and Figure 114H It's the kidneys. Figure 114I and Figure 114M It's the myocardium. Figure 114J and Figure 114N It's the liver. Figure 114K and Figure 114O It's the lungs. Figure 114L and Figure 114P It's the ureter. Figure 114Q and Figure 114U It's the eyes. Figure 114R and Figure 114V It is the cerebral cortex. Figure 114S and Figure 114W It's bone marrow. Figure 114T and Figure 114X It's skeletal muscle.

[0373] Figures 115A to 115C Shown are photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 0.25 ug / mL of anti-PSMGFR antibody 20A10. Figure 115A Photographs of tissue microarrays are shown. Figure 115B Array diagrams with abbreviated tissue descriptors are shown. Figure 115C Detailed description of tissue microarrays with deidentified donor data.

[0374] Figures 116A to 116F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 0.25 ug / mL of anti-PSMGFR antibody 20A10, magnified at 6X and 20X. Figure 116A and Figure 116D This is a photo of grade 2 invasive ductal carcinoma. Figure 116B and Figure 116E This is a photo of grade 2 invasive ductal carcinoma. Figure 116C and Figure 116F This is a photo of grade 2 invasive ductal carcinoma.

[0375] Figures 117A to 117C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA805c stained with 0.25 ug / mL of anti-PSMGFR antibody 20A10. Figure 117A Photographs of tissue microarrays are shown. Figure 117B Array diagrams with abbreviated tissue descriptors are shown. Figure 117C Detailed description of tissue microarrays with deidentified donor data.

[0376] Figures 118A to 118F Shown are photographs of specific tissue from pancreatic cancer tissue array PA805c stained with 0.25 ug / mL of anti-PSMGFR antibody 20A10, magnified at 6X and 20X. Figure 118A and Figure 118D This is a photo of a grade 2 papillary adenocarcinoma. Figure 118B and Figure 118E This is a photo of grade 2-3 ductal carcinoma. Figure 118C and Figure 118F This is a photo of a grade 3 invasive adenocarcinoma.

[0377] Figures 119A to 119C Shown are photographs, array maps, and descriptions of esophageal cancer tissue array BC001113 stained with 0.25 ug / mL of the anti-PSMGFR antibody 20A10. Figure 119A Photographs of tissue microarrays are shown. Figure 119B Array diagrams with abbreviated tissue descriptors are shown. Figure 119C Detailed description of tissue microarrays with deidentified donor data.

[0378] Figures 120A to 120F Shown are photographs of specific tissue from esophageal cancer tissue array BC001113 stained with 0.25 ug / mL of anti-PSMGFR antibody 20A10, magnified at 6X and 20X. Figure 120A and Figure 120D is a photograph of the sample at position A1. Figure 120B and Figure 120E is a photograph of the sample at position A7. Figure 120C and Figure 120F is a photograph of the sample at position A8.

[0379] Figures 121A to 121C Shown are a photograph, array map, and description of FDA normal tissue array 1021 stained with 20 ug / mL of anti-PSMGFR antibody 3C2B1. Figure 121A Photographs of tissue microarrays are shown. Figure 121B Array diagrams with abbreviated tissue descriptors are shown. Figure 121C Detailed description of tissue microarrays with deidentified donor data.

[0380] Figures 122A to 122X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 20 ug / mL of anti-PSMGFR antibody 3C2B1 at 6X and 20X magnification. Figure 122A and Figure 122E It's the adrenal glands. Figure 122B and Figure 122F It's the mammary gland. Figure 122C and Figure 122G It's the fallopian tube. Figure 122D and Figure 122H It's the kidneys. Figure 122I and Figure 122M It's the myocardium. Figure 122J and Figure 122N It's the liver. Figure 122K and Figure 122O It's the lungs. Figure 122L and Figure 122P It's the ureter. Figure 122Q and Figure 122U It's the eyes. Figure 122R and Figure 122V It is the cerebral cortex. Figure 122S and Figure 122W It's bone marrow. Figure 122T and Figure 122X It's skeletal muscle.

[0381] Figures 123A to 123C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 20 ug / mL of the anti-PSMGFR antibody 3C2B1. Figure 123A Photographs of tissue microarrays are shown. Figure 123B Array diagrams with abbreviated tissue descriptors are shown. Figure 123C Detailed description of tissue microarrays with deidentified donor data.

[0382] Figures 124A to 124F Shown are photographs of specific tissue from pancreatic cancer tissue array PA1003 stained with 20 ug / mL of anti-PSMGFR antibody 3C2B1 at 6X and 20X magnification. Figure 124A and Figure 124D This is a photo of a grade 2 adenocarcinoma. Figure 124B and Figure 124E This is a photo of a grade 2 adenocarcinoma. Figure 124C and Figure 124F This is a photo of a grade 2 adenocarcinoma.

[0383] Figures 125A to 125C Shown are photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 20 ug / mL of the anti-PSMGFR antibody 3C2B1. Figure 125A Photographs of tissue microarrays are shown. Figure 125B Array diagrams with abbreviated tissue descriptors are shown. Figure 125C Detailed description of tissue microarrays with deidentified donor data.

[0384] Figures 126A to 126F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 20 ug / mL of anti-PSMGFR antibody 3C2B1 at 6X and 20X magnification. Figure 126A and Figure 126D This is a photo of grade 2 invasive ductal carcinoma. Figure 126B and Figure 126E This is a photo of grade 2 invasive ductal carcinoma. Figure 126C and Figure 126F This is a photo of a grade 2 invasive carcinoma.

[0385] Figures 127A to 127C Shown are a photograph, array map, and description of the FDA normal tissue array 1021 stained with 1 ug / mL of the anti-PSMGFR antibody 5C6F3. Figure 127A Photographs of tissue microarrays are shown. Figure 127B Array diagrams with abbreviated tissue descriptors are shown. Figure 127C Detailed description of tissue microarrays with deidentified donor data.

[0386] Figures 128A to 128XShown are photographs of specific tissue from FDA normal tissue array 1021 stained with 1 ug / mL of anti-PSMGFR antibody 5C6F3 at 6X and 20X magnification. Figure 128A and Figure 128E It's the adrenal glands. Figure 128B and Figure 128F It's the mammary gland. Figure 128C and Figure 128G It's the fallopian tube. Figure 128D and Figure 128H It's the kidneys. Figure 128I and Figure 128M It's the myocardium. Figure 128J and Figure 128N It's the liver. Figure 128K and Figure 128O It's the lungs. Figure 128L and Figure 128P It's the ureter. Figure 128Q and Figure 128U It's the eyes. Figure 128R and Figure 128V It is the cerebral cortex. Figure 128S and Figure 128W It's bone marrow. Figure 128T and Figure 128X It's skeletal muscle.

[0387] Figures 129A to 129C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 1-20 ug / mL of anti-PSMGFR antibody 5C6F3. Figure 129A Photographs of tissue microarrays are shown. Figure 129B Array diagrams with abbreviated tissue descriptors are shown. Figure 129C Detailed description of tissue microarrays with deidentified donor data.

[0388] Figures 130A to 130F Shown are photographs of specific tissue from pancreatic cancer tissue array PA1003 stained with 1 ug / mL of anti-PSMGFR antibody 5C6F3, magnified at 6X and 20X. Figure 130A and Figure 130D This is a photo of a grade 2 adenocarcinoma. Figure 130B and Figure 130E This is a photo of a grade 2 adenocarcinoma. Figure 130C and Figure 130F This is a photo of a grade 2 adenocarcinoma.

[0389] Figures 131A to 131C Shown are photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 1 ug / mL of anti-PSMGFR antibody 5C6F3. Figure 131A Photographs of tissue microarrays are shown. Figure 131B Array diagrams with abbreviated tissue descriptors are shown. Figure 131CDetailed description of tissue microarrays with deidentified donor data.

[0390] Figures 132A to 132F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 1 ug / mL of anti-PSMGFR antibody 5C6F3 at 6X and 20X magnification. Figure 132A and Figure 132D This is a photo of grade 2 invasive ductal carcinoma. Figure 132B and Figure 132E This is a photo of grade 2 invasive ductal carcinoma. Figure 132C and Figure 132F This is a photo of a grade 2 invasive carcinoma.

[0391] Figures 133A to 133C Shown are a photograph, array map, and description of an FDA normal tissue array 1021 stained with 10 ug / mL of the anti-PSMGFR antibody 18B4. Figure 133A Photographs of tissue microarrays are shown. Figure 133B Array diagrams with abbreviated tissue descriptors are shown. Figure 133C Detailed description of tissue microarrays with deidentified donor data.

[0392] Figures 134A to 134X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 10 ug / mL of anti-PSMGFR antibody 18B4 at 6X and 20X magnification. Figure 134A and Figure 134E It's the adrenal glands. Figure 134B and Figure 134F It's the mammary gland. Figure 134C and Figure 134G It's the fallopian tube. Figure 134D and Figure 134H It's the kidneys. Figure 134I and Figure 134M It's the myocardium. Figure 134J and Figure 134N It's the liver. Figure 134K and Figure 134O It's the lungs. Figure 134L and Figure 134P It's the ureter. Figure 134Q and Figure 134U It's the eyes. Figure 134R and Figure 134V It is the cerebral cortex. Figure 134S and Figure 134W It's bone marrow. Figure 134T and Figure 134X It's skeletal muscle.

[0393] Figures 135A to 135C Shown are photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 10 ug / mL of anti-PSMGFR antibody 18B4. Figure 135APhotographs of tissue microarrays are shown. Figure 135B Array diagrams with abbreviated tissue descriptors are shown. Figure 135C Detailed description of tissue microarrays with deidentified donor data.

[0394] Figures 136A to 136F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 10 ug / mL of anti-PSMGFR antibody 18B4 at 6X and 20X magnification. Figure 136A and Figure 136D This is a photo of grade 2 invasive ductal carcinoma. Figure 136B and Figure 136E This is a photo of grade 2 invasive ductal carcinoma. Figure 136C and Figure 136F This is a photo of grade 2 invasive ductal carcinoma.

[0395] Figures 137A to 137C Shown are photographs, array maps, and descriptions of esophageal cancer tissue array BC001113 stained with 10 ug / mL of anti-PSMGFR antibody 18B4. Figure 137A Photographs of tissue microarrays are shown. Figure 137B Array diagrams with abbreviated tissue descriptors are shown. Figure 137C Detailed description of tissue microarrays with deidentified donor data.

[0396] Figures 138A to 138F Shown are photographs of specific tissue from esophageal cancer tissue array BC001113 stained with 10 ug / mL of anti-PSMGFR antibody 18B4, magnified at 6X and 20X. Figure 138A and Figure 138D is a photograph of the sample at position A1. Figure 138B and Figure 138E is a photograph of the sample at position A7. Figure 138C and Figure 138F is a photograph of the sample at position A8.

[0397] Figures 139A to 139C Shown are a photograph, array map, and description of an FDA normal tissue array 1021 stained with 10 ug / mL of the anti-PSMGFR antibody 18G12. Figure 139A Photographs of tissue microarrays are shown. Figure 139B Array diagrams with abbreviated tissue descriptors are shown. Figure 139C Detailed description of tissue microarrays with deidentified donor data.

[0398] Figures 140A to 140X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 10 ug / mL of anti-PSMGFR antibody 18G12 at 6X and 20X magnification. Figure 140Aand Figure 140E It's the adrenal glands. Figure 140B and Figure 140F It's the mammary gland. Figure 140C and Figure 140G It's the fallopian tube. Figure 140D and Figure 140H It's the kidneys. Figure 140I and Figure 140M It's the myocardium. Figure 140J and Figure 140N It's the liver. Figure 140K and Figure 140O It's the lungs. Figure 140L and Figure 140P It's the ureter. Figure 140Q and Figure 140U It's the eyes. Figure 140R and Figure 140V It is the cerebral cortex. Figure 140S and Figure 140W It's bone marrow. Figure 140T and Figure 140X It's skeletal muscle.

[0399] Figures 141A to 141C Shown are photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 15 ug / mL of anti-PSMGFR antibody 18G12. Figure 141A Photographs of tissue microarrays are shown. Figure 141B Array diagrams with abbreviated tissue descriptors are shown. Figure 141C Detailed description of tissue microarrays with deidentified donor data.

[0400] Figures 142A to 142F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 15 ug / mL of anti-PSMGFR antibody 18G12 at 6X and 20X magnification. Figure 142A and Figure 142D This is a photo of grade 2 invasive ductal carcinoma. Figure 142B and Figure 142E This is a photo of grade 2 invasive ductal carcinoma. Figure 142C and Figure 142F This is a photo of grade 2 invasive ductal carcinoma.

[0401] Figures 143A to 143C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 15 ug / mL of anti-PSMGFR antibody 18G12. Figure 143A Photographs of tissue microarrays are shown. Figure 143B Array diagrams with abbreviated tissue descriptors are shown. Figure 143C Detailed description of tissue microarrays with deidentified donor data.

[0402] Figures 144A to 144FShown are photographs of specific tissue from pancreatic cancer tissue array PA1003 stained with 15 ug / mL of anti-PSMGFR antibody 18G12 at 6X and 20X magnification. Figure 144A and Figure 144D This is a photo of a grade 2 adenocarcinoma. Figure 144B and Figure 144E This is a photo of a grade 2 adenocarcinoma. Figure 144C and Figure 144F This is a photo of a grade 2-3 adenocarcinoma with lymph node involvement.

[0403] Figures 145A to 145C Shown are photographs, array images, and descriptions of esophageal cancer tissue array BC001113 stained with 30 ug / mL of anti-PSMGFR antibody 18G12. Figure 145A Photographs of tissue microarrays are shown. Figure 145B Array diagrams with abbreviated tissue descriptors are shown. Figure 145C Detailed description of tissue microarrays with deidentified donor data.

[0404] Figures 146A to 146F Shown are photographs of specific tissue from esophageal cancer tissue array BC001113 stained with 30 ug / mL of anti-PSMGFR antibody 18G12, magnified at 6X and 20X. Figure 146A and Figure 146D is a photograph of the sample at position A1. Figure 146B and Figure 146E is a photograph of the sample at position A7. Figure 146C and Figure 146F is a photograph of the sample at position A8.

[0405] Figures 147A to 147C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 5.0 ug / mL of anti-PSMGFR antibody 25E6. Figure 147A Photographs of tissue microarrays are shown. Figure 147B Array diagrams with abbreviated tissue descriptors are shown. Figure 147C Detailed description of tissue microarrays with deidentified donor data.

[0406] Figures 148A to 148X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 5.0 ug / mL of anti-PSMGFR antibody 25E6 at 6X and 20X magnification. Figure 148A and Figure 148E It's the adrenal glands. Figure 148B and Figure 148F It's the mammary gland. Figure 148C and Figure 148G It's the fallopian tube. Figure 148D and Figure 148HIt's the kidneys. Figure 148I and Figure 148M It's the myocardium. Figure 148J and Figure 148N It's the liver. Figure 148K and Figure 148O It's the lungs. Figure 148L and Figure 148P It's the ureter. Figure 148Q and Figure 148U It's the eyes. Figure 148R and Figure 148V It is the cerebral cortex. Figure 148S and Figure 148W It's bone marrow. Figure 148T and Figure 148X It's skeletal muscle.

[0407] Figures 149A to 149C Shown are photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 5.0 ug / mL of anti-PSMGFR antibody 25E6. Figure 149A Photographs of tissue microarrays are shown. Figure 149B Array diagrams with abbreviated tissue descriptors are shown. Figure 149C Detailed description of tissue microarrays with deidentified donor data.

[0408] Figures 150A to 150F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 5.0 ug / mL of anti-PSMGFR antibody 25E6 at 6X and 20X magnification. Figure 150A and Figure 150D This is a photo of grade 2 invasive ductal carcinoma. Figure 150B and Figure 150E This is a photo of grade 2 invasive ductal carcinoma. Figure 150C and Figure 150F This is a photo of grade 2 invasive ductal carcinoma.

[0409] Figures 151A to 151C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 5.0 ug / mL of anti-PSMGFR antibody 25E6. Figure 151A Photographs of tissue microarrays are shown. Figure 151B Array diagrams with abbreviated tissue descriptors are shown. Figure 151C Detailed description of tissue microarrays with deidentified donor data.

[0410] Figures 152A to 152F Shown are photographs of specific tissue from pancreatic cancer tissue array PA1003 stained with 5.0 ug / mL of anti-PSMGFR antibody 25E6 at 6X and 20X magnification. Figure 152A and Figure 152D This is a photo of a grade 2 adenocarcinoma. Figure 152B and Figure 152EThis is a photo of a grade 1 adenocarcinoma. Figure 152C and Figure 152F This is a photo of a grade 1 adenocarcinoma.

[0411] Figures 153A to 153C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 15.0 ug / mL of anti-PSMGFR antibody 28F9. Figure 153A Photographs of tissue microarrays are shown. Figure 153B Array diagrams with abbreviated tissue descriptors are shown. Figure 153C Detailed description of tissue microarrays with deidentified donor data.

[0412] Figures 154A to 154X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 15.0 ug / mL of anti-PSMGFR antibody 28F9 at 6X and 20X magnification. Figure 154A and Figure 154E It's the adrenal glands. Figure 154B and Figure 154F It's the mammary gland. Figure 154C and Figure 154G It's the fallopian tube. Figure 154D and Figure 154H It's the kidneys. Figure 154I and Figure 154M It's the myocardium. Figure 154J and Figure 154N It's the liver. Figure 154K and Figure 154O It's the lungs. Figure 154L and Figure 154P It's the ureter. Figure 154Q and Figure 154U It's the eyes. Figure 154R and Figure 154V It is the cerebral cortex. Figure 154S and Figure 154W It's bone marrow. Figure 154T and Figure 154X It's skeletal muscle.

[0413] Figures 155A to 155C Shown are photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 15.0 ug / mL of anti-PSMGFR antibody 28F9. Figure 155A Photographs of tissue microarrays are shown. Figure 155B Array diagrams with abbreviated tissue descriptors are shown. Figure 155C Detailed description of tissue microarrays with deidentified donor data.

[0414] Figures 156A to 156F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 15.0 ug / mL of anti-PSMGFR antibody 28F9 at 6X and 20X magnification. Figure 156Aand Figure 156D This is a photo of grade 2 invasive ductal carcinoma. Figure 156B and Figure 156E This is a photo of grade 2 invasive ductal carcinoma. Figure 156C and Figure 156F This is a photo of grade 2 invasive ductal carcinoma.

[0415] Figures 157A to 157C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 7.5 ug / mL of N+20 / C-27 antibody 1E4. Figure 157A Photographs of tissue microarrays are shown. Figure 157B Array diagrams with abbreviated tissue descriptors are shown. Figure 157C Detailed description of tissue microarrays with deidentified donor data.

[0416] Figures 158A to 158X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 7.5 ug / mL of N+20 / C-27 antibody 1E4 at 6X and 2OX magnification. Figure 158A and Figure 158E It's the adrenal glands. Figure 158B and Figure 158F It's the mammary gland. Figure 158C and Figure 158G It's the fallopian tube. Figure 158D and Figure 158H It's the kidneys. Figure 158I and Figure 158M It's the myocardium. Figure 158J and Figure 158N It's the liver. Figure 158K and Figure 158O It's the lungs. Figure 158L and Figure 158P It's the ureter. Figure 158Q and Figure 158U It's the eyes. Figure 158R and Figure 158V It is the cerebral cortex. Figure 158S and Figure 158W It's bone marrow. Figure 158T and Figure 158X It's skeletal muscle.

[0417] Figures 159A to 159C Shown are photographs, array maps, and descriptions of breast cancer tissue array BR1007 stained with 10.0 ug / mL of N+20 / C-27 antibody 1E4. Figure 159A Photographs of tissue microarrays are shown. Figure 159B Array diagrams with abbreviated tissue descriptors are shown. Figure 159C Detailed description of tissue microarrays with deidentified donor data.

[0418] Figures 160A to 160FShown are photographs of specific tissue from breast cancer tissue array BR1007 stained with 10.0 ug / mL of N+20 / C-27 antibody 1E4 at 6X and 20X magnification. Figure 160A and Figure 160D This is a photo of a node-positive grade 2 invasive ductal carcinoma. Figure 160B and Figure 160E This is a photo of grade 2 invasive ductal carcinoma. Figure 160C and Figure 160F This is a photo of grade 2 invasive ductal carcinoma.

[0419] Figures 161A to 161C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 0.5 ug / mL of N+20 / C-27 antibody 29H1. Figure 161A Photographs of tissue microarrays are shown. Figure 161B Array diagrams with abbreviated tissue descriptors are shown. Figure 161C Detailed description of tissue microarrays with deidentified donor data.

[0420] Figures 162A to 162X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 0.5 ug / mL of N+20 / C-27 antibody 29H1 at 6X and 20X magnification. Figure 162A and Figure 162E It's the adrenal glands. Figure 162B and Figure 162F It's the mammary gland. Figure 162C and Figure 162G It's the fallopian tube. Figure 162D and Figure 162H It's the kidneys. Figure 162I and Figure 162M It's the myocardium. Figure 162J and Figure 162N It's the liver. Figure 162K and Figure 162O It's the lungs. Figure 162L and Figure 162P It's the ureter. Figure 162Q and Figure 162U It's the eyes. Figure 162R and Figure 162V It is the cerebral cortex. Figure 162S and Figure 162W It's bone marrow. Figure 162T and Figure 162X It's skeletal muscle.

[0421] Figures 163A to 163C Shown are photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 0.5 ug / mL of N+20 / C-27 antibody 29H1. Figure 163A Photographs of tissue microarrays are shown. Figure 163BArray diagrams with abbreviated tissue descriptors are shown. Figure 163C Detailed description of tissue microarrays with deidentified donor data.

[0422] Figures 164A to 164F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 0.5 ug / mL of N+20 / C-27 antibody 29H1 at 6X and 20X magnification. Figure 164A and Figure 164D This is a photo of grade 2 invasive ductal carcinoma. Figure 164B and Figure 164E This is a photo of grade 2 invasive ductal carcinoma. Figure 164C and Figure 164F This is a photo of grade 2 invasive ductal carcinoma.

[0423] Figures 165A to 165C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 0.5 ug / mL of N+20 / C-27 antibody 29H1. Figure 165A Photographs of tissue microarrays are shown. Figure 165B Array diagrams with abbreviated tissue descriptors are shown. Figure 165C Detailed description of tissue microarrays with deidentified donor data.

[0424] Figures 166A to 166F Shown are photographs of specific tissue from pancreatic cancer tissue array PA1003 stained with 0.5 ug / mL of N+20 / C-27 antibody 29H1 at 6X and 20X magnification. Figure 166A and Figure 166D This is a photo of a grade 2 adenocarcinoma. Figure 166B and Figure 166E This is a photo of a grade 2 adenocarcinoma. Figure 166C and Figure 166F This is a photo of a grade 3 adenocarcinoma.

[0425] Figures 167A to 167C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 0.5 ug / mL of N+20 / C-27 antibody 31A1. Figure 167A Photographs of tissue microarrays are shown. Figure 167B Array diagrams with abbreviated tissue descriptors are shown. Figure 167C Detailed description of tissue microarrays with deidentified donor data.

[0426] Figures 168A to 168X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 0.5 ug / mL of N+20 / C-27 antibody 31A1 at 6X and 20X magnification. Figure 168A and Figure 168E It's the adrenal glands. Figure 168Band Figure 168F It's the mammary gland. Figure 168C and Figure 168G It's the fallopian tube. Figure 168D and Figure 168H It's the kidneys. Figure 168I and Figure 168M It's the myocardium. Figure 168J and Figure 168N It's the liver. Figure 168K and Figure 168O It's the lungs. Figure 168L and Figure 168P It's the ureter. Figure 168Q and Figure 168U It's the eyes. Figure 168R and Figure 168V It is the cerebral cortex. Figure 168S and Figure 168W It's bone marrow. Figure 168T and Figure 168X It's skeletal muscle.

[0427] Figures 169A to 169C Shown are photographs, array images, and descriptions of breast cancer tissue array 1141 stained with 0.5 ug / mL of N+20 / C-27 antibody 31A1. Figure 169A Photographs of tissue microarrays are shown. Figure 169B Array diagrams with abbreviated tissue descriptors are shown. Figure 169C Detailed description of tissue microarrays with deidentified donor data.

[0428] Figures 170A to 170F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 0.5 ug / mL of N+20 / C-27 antibody 31A1 at 6X and 20X magnification. Figure 170A and Figure 170D This is a photo of grade 2 invasive ductal carcinoma. Figure 170B and Figure 170E This is a photo of grade 2 invasive ductal carcinoma. Figure 170C and Figure 170F This is a photo of grade 2 invasive ductal carcinoma.

[0429] Figures 171A to 171C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 0.5 ug / mL of N+20 / C-27 antibody 31A1. Figure 171A Photographs of tissue microarrays are shown. Figure 171B Array diagrams with abbreviated tissue descriptors are shown. Figure 171C Detailed description of tissue microarrays with deidentified donor data.

[0430] Figures 172A to 172FShown are photographs of specific tissue from pancreatic cancer tissue array PA1003 stained with 0.5 ug / mL of N+20 / C-27 antibody 31A1 at 6X and 20X magnification. Figure 172A and Figure 172D This is a photo of a grade 1 adenocarcinoma. Figure 172B and Figure 172E This is a photo of a grade 2 adenocarcinoma. Figure 172C and Figure 172F This is a photo of a grade 3 adenocarcinoma.

[0431] Figures 173A to 173C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 0.25 ug / mL of N+20 / C-27 antibody 32C1. Figure 173A Photographs of tissue microarrays are shown. Figure 173B Array diagrams with abbreviated tissue descriptors are shown. Figure 173C Detailed description of tissue microarrays with deidentified donor data.

[0432] Figures 174A to 174X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 0.25 ug / mL of N+20 / C-27 antibody 32C1 at 6X and 20X magnification. Figure 174A and Figure 174E It's the adrenal glands. Figure 174B and Figure 174F It's the mammary gland. Figure 174C and Figure 174G It's the fallopian tube. Figure 174D and Figure 174H It's the kidneys. Figure 174I and Figure 174M It's the myocardium. Figure 174J and Figure 174N It's the liver. Figure 174K and Figure 174O It's the lungs. Figure 174L and Figure 174P It's the ureter. Figure 174Q and Figure 174U It's the eyes. Figure 174R and Figure 174V It is the cerebral cortex. Figure 174S and Figure 174W It's bone marrow. Figure 174T and Figure 174X It's skeletal muscle.

[0433] Figures 175A to 175C Shown are photographs, array maps, and descriptions of breast cancer tissue array 1141 stained with 5.0 ug / mL of N+20 / C-27 antibody 32C1. Figure 175A Photographs of tissue microarrays are shown. Figure 175B Array diagrams with abbreviated tissue descriptors are shown. Figure 175CDetailed description of tissue microarrays with deidentified donor data.

[0434] Figures 176A to 176F Shown are photographs of specific tissue from breast cancer tissue array 1141 stained with 5.0 ug / mL of N+20 / C-27 antibody 32C1 at 6X and 20X magnification. Figure 176A and Figure 176D This is a photo of grade 2 invasive ductal carcinoma. Figure 176B and Figure 176E This is a photo of grade 2 invasive ductal carcinoma. Figure 176C and Figure 176F This is a photo of grade 2 invasive ductal carcinoma.

[0435] Figures 177A to 177C Shown are photographs, array images, and descriptions of esophageal cancer tissue array ES1001 stained with 1.0 ug / mL of N+20 / C-27 antibody 32C1. Figure 177A Photographs of tissue microarrays are shown. Figure 177B Array diagrams with abbreviated tissue descriptors are shown. Figure 177C Detailed description of tissue microarrays with deidentified donor data.

[0436] Figures 178A to 178F Shown are photographs of specific tissue from esophageal cancer tissue array BC001113 stained with 1.0 ug / mL of N+20 / C-27 antibody 32C1 at 6X and 20X magnification. Figure 178A and Figure 178D This is a picture of squamous cell carcinoma. Figure 178B and Figure 178E This is a picture of adenocarcinoma. Figure 178C and Figure 178F This is a picture of squamous cell carcinoma.

[0437] Figures 179A to 179C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 12.5 ug / mL of N+20 / C-27 antibody 45C11. Figure 179A Photographs of tissue microarrays are shown. Figure 179B Array diagrams with abbreviated tissue descriptors are shown. Figure 179C Detailed description of tissue microarrays with deidentified donor data.

[0438] Figures 180A to 180X Shown are photographs of specific tissue from FDA Normal Tissue Array 1021 stained with 12.5 ug / mL of N+20 / C-27 antibody 45C11 at 6X and 2OX magnifications. Figure 180A and Figure 180E It's the adrenal glands. Figure 180B and Figure 180F It's the mammary gland. Figure 180C and Figure 180G It's the fallopian tube. Figure 180D and Figure 180H It's the kidneys. Figure 180I and Figure 180M It's the myocardium. Figure 180J and Figure 180N It's the liver. Figure 180K and Figure 180O It's the lungs. Figure 180L and Figure 180P It's the ureter. Figure 180Q and Figure 180U It's the eyes. Figure 180R and Figure 180V It is the cerebral cortex. Figure 180S and Figure 180W It's bone marrow. Figure 180T and Figure 180X It's skeletal muscle.

[0439] Figures 181A to 181C Shown are photographs, array maps, and descriptions of breast cancer tissue array BR1007 stained with 10.0 ug / mL of N+20 / C-27 antibody 45C11. Figure 181A Photographs of tissue microarrays are shown. Figure 181B Array diagrams with abbreviated tissue descriptors are shown. Figure 181C Detailed description of tissue microarrays with deidentified donor data.

[0440] Figures 182A to 182F Shown are photographs of specific tissue from breast cancer tissue array BR1007 stained with 10.0 ug / mL of N+20 / C-27 antibody 45C11 at 6X and 20X magnification. Figure 182A and Figure 182D This is a photo of a node-positive grade 2 invasive ductal carcinoma. Figure 182B and Figure 182E This is a photo of grade 2 invasive ductal carcinoma. Figure 182C and Figure 182F This is a photo of grade 2 invasive ductal carcinoma.

[0441] Figures 183A to 183C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA805c stained with 12.5 ug / mL of N+20 / C-27 antibody 45C11. Figure 183A Photographs of tissue microarrays are shown. Figure 183B Array diagrams with abbreviated tissue descriptors are shown. Figure 183C Detailed description of tissue microarrays with deidentified donor data.

[0442] Figures 184A to 184FShown are photographs of specific tissue from pancreatic cancer tissue array PA805c stained with 12.5 ug / mL of N+20 / C-27 antibody 45C11 at 6X and 20X magnification. Figure 184A and Figure 184D This is a photo of a grade 2 papillary adenocarcinoma. Figure 184B and Figure 184E This is a photo of grade 2-3 ductal carcinoma. Figure 184C and Figure 184F This is a photo of a grade 3 invasive adenocarcinoma.

[0443] Figures 185A to 185C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 10.0 ug / mL of N+9 / C-9 antibody 3C5. Figure 185A Photographs of tissue microarrays are shown. Figure 185B Array diagrams with abbreviated tissue descriptors are shown. Figure 185C Detailed description of tissue microarrays with deidentified donor data.

[0444] Figures 186A to 186X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 10.0 ug / mL of N+9 / C-9 antibody 3C5 at 6X and 2OX magnifications. Figure 186A and Figure 186E It's the adrenal glands. Figure 186B and Figure 186F It's the mammary gland. Figure 186C and Figure 186G It's the fallopian tube. Figure 186D and Figure 186H It's the kidneys. Figure 186I and Figure 186M It's the myocardium. Figure 186J and Figure 186N It's the liver. Figure 186K and Figure 186O It's the lungs. Figure 186L and Figure 186P It's the ureter. Figure 186Q and Figure 186U It's the eyes. Figure 186R and Figure 186V It is the cerebral cortex. Figure 186S and Figure 186W It's bone marrow. Figure 186T and Figure 186X It's skeletal muscle.

[0445] Figures 187A to 187C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 10.0 ug / mL of N+9 / C-9 antibody 3C5. Figure 187A Photographs of tissue microarrays are shown. Figure 187B Array diagrams with abbreviated tissue descriptors are shown. Figure 187C Detailed description of tissue microarrays with deidentified donor data.

[0446] Figures 188A to 188F Shown are photographs of specific tissue from pancreatic cancer tissue array PA1003 stained with 10.0 ug / mL of N+9 / C-9 antibody 3C5 at 6X and 20X magnification. Figure 188A and Figure 188D This is a photo of a grade 2 adenocarcinoma. Figure 188B and Figure 188E This is a photo of a grade 2 adenocarcinoma. Figure 188C and Figure 188F This is a photo of a grade 2-3 adenocarcinoma with lymph node involvement.

[0447] Figures 189A to 189C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 15.0 ug / mL of N+9 / C-9 antibody 8A9. Figure 189A Photographs of tissue microarrays are shown. Figure 189B Array diagrams with abbreviated tissue descriptors are shown. Figure 189C Detailed description of tissue microarrays with deidentified donor data.

[0448] Figures 190A to 190X Shown are photographs of specific tissue from FDA normal tissue array 1021 stained with 15.0 ug / mL of N+9 / C-9 antibody 8A9 at 6X and 2OX magnification. Figure 190A and Figure 190E It's the adrenal glands. Figure 190B and Figure 190F It's the mammary gland. Figure 190C and Figure 190G It's the fallopian tube. Figure 190D and Figure 190H It's the kidneys. Figure 190I and Figure 190M It's the myocardium. Figure 190J and Figure 190N It's the liver. Figure 190K and Figure 190O It's the lungs. Figure 190L and Figure 190P It's the ureter. Figure 190Q and Figure 190U It's the eyes. Figure 190R and Figure 190V It is the cerebral cortex. Figure 190S and Figure 190W It's bone marrow. Figure 190T and Figure 190X It's skeletal muscle.

[0449] Figures 191A to 191CShown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 15.0 ug / mL of N+9 / C-9 antibody 8A9. Figure 191A Photographs of tissue microarrays are shown. Figure 191B Array diagrams with abbreviated tissue descriptors are shown. Figure 191C Detailed description of tissue microarrays with deidentified donor data.

[0450] Figures 192A to 192F Shown are photographs of specific tissue from pancreatic cancer tissue array PA1003 stained with 15.0 ug / mL of N+9 / C-9 antibody 8A9 at 6X and 20X magnification. Figure 192A and Figure 192D This is a photo of a grade 2 adenocarcinoma. Figure 192B and Figure 192E This is a photo of a grade 2 adenocarcinoma. Figure 192C and Figure 192F This is a photo of a grade 2 adenocarcinoma.

[0451] Figures 193A to 193C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 30.0 ug / mL of N+9 / C-9 antibody 17H6. Figure 193A Photographs of tissue microarrays are shown. Figure 193B Array diagrams with abbreviated tissue descriptors are shown. Figure 193C Detailed description of tissue microarrays with deidentified donor data.

[0452] Figures 194A to 194X Shown are photographs of specific tissue from FDA Normal Tissue Array 1021 stained with 30.0 ug / mL of N+9 / C-9 antibody 17H6 at 6X and 2OX magnifications. Figure 194A and Figure 194E It's the adrenal glands. Figure 194B and Figure 194F It's the mammary gland. Figure 194C and Figure 194G It's the fallopian tube. Figure 194D and Figure 194H It's the kidneys. Figure 194I and Figure 194M It's the myocardium. Figure 194J and Figure 194N It's the liver. Figure 194K and Figure 194O It's the lungs. Figure 194L and Figure 194P It's the ureter. Figure 194Q and Figure 194U It's the eyes. Figure 194R and Figure 194V It is the cerebral cortex. Figure 194S and Figure 194W It's bone marrow. Figure 194T and Figure 194X It's skeletal muscle.

[0453] Figures 195A to 195C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA805c stained with 30.0 ug / mL of N+9 / C-9 antibody 17H6. Figure 195A Photographs of tissue microarrays are shown. Figure 195B Array diagrams with abbreviated tissue descriptors are shown. Figure 195C Detailed description of tissue microarrays with deidentified donor data.

[0454] Figures 196A to 196F Shown are photographs of specific tissue from pancreatic cancer tissue array PA805c stained with 30.0 ug / mL of N+9 / C-9 antibody 17H6 at 6X and 20X magnification. Figure 196A and Figure 196D This is a photo of a grade 2 papillary adenocarcinoma. Figure 196B and Figure 196E This is a photo of a grade 2-3 ductal carcinoma with lymph node involvement. Figure 196C and Figure 196F This is a photo of a grade 3 invasive adenocarcinoma.

[0455] Figures 197A to 197C Shown are photographs, array maps, and descriptions of FDA normal tissue array 1021 stained with 5.0 ug / mL of N+9 / C-9 antibody 39H5. Figure 197A Photographs of tissue microarrays are shown. Figure 197B Array diagrams with abbreviated tissue descriptors are shown. Figure 197C Detailed description of tissue microarrays with deidentified donor data.

[0456] Figures 198A to 198X Shown are photographs of specific tissue from FDA Normal Tissue Array 1021 stained with 5.0 ug / mL of N+9 / C-9 antibody 39H5 at 6X and 2OX magnification. Figure 198A and Figure 198E It's the adrenal glands. Figure 198B and Figure 198F It's the mammary gland. Figure 198C and Figure 198G It's the fallopian tube. Figure 198D and Figure 198H It's the kidneys. Figure 198I and Figure 198M It's the myocardium. Figure 198J and Figure 198N It's the liver. Figure 198K and Figure 198O It's the lungs. Figure 198L and Figure 198P It's the ureter. Figure 198Q and Figure 198U It's the eyes. Figure 198R and Figure 198V It is the cerebral cortex. Figure 198S and Figure 198W It's bone marrow. Figure 198T and Figure 198X It's skeletal muscle.

[0457] Figures 199A to 199C Shown are photographs, array maps, and descriptions of pancreatic cancer tissue array PA1003 stained with 5.0 ug / mL of N+9 / C-9 antibody 39H5. Figure 199A Photographs of tissue microarrays are shown. Figure 199B Array diagrams with abbreviated tissue descriptors are shown. Figure 199C Detailed description of tissue microarrays with deidentified donor data.

[0458] Figure 200 A to Figure 200 F shows photographs of specific tissue from pancreatic cancer tissue array PA1003 stained with 5.0 ug / mL of N+9 / C-9 antibody 39H5, magnified at 6X and 20X. Figure 200 A and Figure 200 D is a photograph of a grade 2 adenocarcinoma. Figure 200 B and Figure 200 E is a photograph of a grade 2 adenocarcinoma. Figure 200 C and Figure 200 F is a photograph of a grade 2 adenocarcinoma.

[0459] Figures 201A to 201C Shown is a graph of an ELISA assay measuring the binding of another panel of antibodies generated by immunizing animals with PSMGFR peptide. Figure 201A Binding to the PSMGFR peptide is shown. Figure 201B Binding to the N-10 peptide is shown. Figure 201C Binding to the C-10 peptide is shown. As can be seen, no antibody 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.

[0460] Figures 202A to 202C The images show 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 at the N-terminal end of the PSMGFR peptide, while the 1E4 antibody binds to the QFNQYKTEA epitope immediately adjacent to the 18B4 epitope and at its C-terminus.

[0461] Figure 203 A to Figure 203 F shows a magnified image of a tissue sample at position A2 of the pancreatic cancer array PA1003.Figure 203 A and Figure 203 B shows a sample stained with antibody 1E4. Figure 203 C and Figure 203 D shows a sample stained with antibody 18B4. Figure 203 E and Figure 203 F shows a sample stained with polyclonal antibody SDIX.

[0462] Figure 204 A to Figure 204 D shows a magnified image of a tissue sample at position D4 of the pancreatic array PA1003. Figure 204 A and Figure 204 B shows a sample stained with antibody 18B4. Figure 204 C and Figure 204 D shows a sample stained with polyclonal antibody SDIX.

[0463] Figure 205 A to Figure 205 D shows a magnified image of a tissue sample at position E1 of the pancreatic cancer array PA1003. Figure 205 A and Figure 205 B shows a sample stained with antibody 18B4. Figure 205 C and Figure 205 D shows a sample stained with polyclonal antibody SDIX.

[0464] Figure 206 A to Figure 206 D shows a magnified image of a tissue sample at position C3 of the pancreatic cancer array PA1003. Figure 206 A and Figure 206 B shows a sample stained with antibody 1E4. Figure 206 C and Figure 206 D shows a sample stained with polyclonal antibody SDIX.

[0465] Figure 207 A to Figure 207 D shows a magnified image of a tissue sample at position D1 of the pancreatic cancer array PA1003. Figure 207 A and Figure 207 B shows a sample stained with antibody 1E4. Figure 207 C and Figure 207 D shows a sample stained with polyclonal antibody SDIX.

[0466] Figure 208 A to Figure 208 C shows a photograph of the pancreatic cancer array PA1003. Figure 208 A shows a sample stained with the polyclonal antibody SDIX. Figure 208 B shows a sample stained with antibody 20A10. Figure 208C shows a sample stained with antibody 29H1.

[0467] Figure 209 A to Figure 209 D shows photographs of esophageal cancer array ES1001 stained with various antibodies. Figure 209 A shows the array stained with polyclonal antibody SDIX. Figure 209 B shows the array stained with antibody 20A10. Figure 209 C shows the array stained with antibody 29H1. Figure 209 D shows the array stained with antibody 31A1.

[0468] Figure 210 A to Figure 210 C shows photographs of pancreatic cancer array PA1003 stained with various antibodies. Figure 210 A shows the array stained with polyclonal antibody SDIX. Figure 210 B shows the array stained with antibody 20A10. Figure 210 C shows the array stained with antibody 29H1.

[0469] Figures 211A to 211C Shown is a graph of an ELISA experiment 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 carry NFAT-inducible IL-18. Figure 211A A graph showing IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with untransduced human T cells. Figure 211B Shown is a graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T cells, which also carry the NFAT-inducible IL-18 gene inserted into a portion of the Foxp3 enhancer. Figure 211C Shown is a graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T cells, which also carry the NFAT-inducible IL-18 gene inserted into a portion of the IL-2 enhancer.

[0470] Figure 212 A to Figure 212 X shows a photograph of T47D breast cancer cells (red) doped with varying percentages 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, where the IL-18 gene was inserted into either the Foxp3 enhancer / promoter or the IL-2 enhancer / promoter. Figure 212 A to Figure 212C. Figure 212 I arrive Figure 212 K and Figure 212 Q to Figure 212 S shows cancer cells co-cultured with untransduced T cells. Figure 212 D to Figure 212 F. Figure 212 L to Figure 212 N and Figure 212 T to Figure 212 V shows cancer cells co-cultured with hiMNC2-CAR44 T cells harboring the NFAT-inducible IL-18 gene inserted into the Foxp3 enhancer / promoter. Figure 212 G to Figure 212 H. Figure 212 O to Figure 212 P and Figure 212 W to Figure 212 X shows cancer cells co-cultured with hiMNC2-CAR44 T cells harboring the NFAT-inducible IL-18 gene inserted into the IL-2 enhancer / promoter.

[0471] Figures 213A to 213B Shown is a graph of an ELISA experiment measuring the level of IL-18 secreted into conditioned media by huMNC1-CAR44 T cells harboring a NFAT-inducible IL-18 gene inserted into the Foxp3 enhancer or promoter co-cultured with MUC1*-positive cancer cells or MUC1-negative non-cancerous cells. Figure 213A Shown is IL-18 secretion by huMNC2-CAR44 T cells with NFAT-inducible IL-18 co-cultured with T47D breast cancer cells, where the population had been doped with 5%, 10%, or 30% T47D cells that had been transfected with even more MUC1*. Figure 213B Shown is IL-18 secretion by huMNC2-CAR44 T cells with NFAT-inducible IL-18 co-cultured with non-cancerous, MUC1-negative HEK293 cells, where the cell populations had been doped with 5%, 10%, or 30% T47D cells that had been transfected with more MUC1*.

[0472] Figure 214 A to Figure 214 X shows a photograph of T47D breast cancer cells (red) or non-cancerous HEK293 cells (also red), both of which were doped with varying percentages of T47D cells (green) engineered to express more MUC1*. These target cancer cells were co-cultured with huMNC2-CAR44 T cells expressing NFAT-inducible IL-18, in which the IL-18 gene had been inserted into the Foxp3 enhancer / promoter. Figure 214 A to Figure 214F shows T47D cells or HEK293 cells that have not been doped with T47D cells engineered to express high MUCl* density. Figure 214 G to Figure 214 L shows T47D cells or HEK293 cells doped with 5% T47D cells engineered to express high MUCl* density. Figure 214 M to Figure 214 R shows T47D cells or HEK293 cells doped with 10% T47D cells engineered to express high MUCl* density. Figure 214 S to Figure 214 X shows T47D cells or HEK293 cells doped with 30% T47D cells engineered to express high MUCl* density. Figure 214 AB, GH, MN, and ST show T47D breast cancer cells. Figure 214 CF, IL, OR, and UX show HEK293 cells. As can be seen from the figure, IL-18 secretion induction leads to killing of low MUC1* density T47D cells, but does not induce nonspecific killing of MUC1*-negative HEK293 cells.

[0473] Figures 215A to 215C The consensus sequences of the heavy chain CDRs generated by each set of antibodies binding to the same epitope in PSMGFR and N-terminally extended PSMGFR peptide are shown. Figure 215A The consensus sequence of the heavy chain CDR1 is shown. Figure 215B The consensus sequence of the heavy chain CDR2 is shown. Figure 215C The consensus sequence of the heavy chain CDR3 is shown.

[0474] Figures 216A to 216C The consensus sequence of the light chain CDRs generated by each set of antibodies binding to the same epitope in PSMGFR and N-terminally extended PSMGFR peptide is shown. Figure 216A The consensus sequence of the light chain CDR1 is shown. Figure 216B The consensus sequence of the light chain CDR2 is shown. Figure 216C The consensus sequence of the light chain CDR3 is shown. DETAILED DESCRIPTION

[0475] In this application, "a" and "an" are used to refer to both singular and plural items.

[0476] As used herein, polypeptides are sometimes referred to as being "transduced or transfected" into cells for simplicity. In these cases, it is understood that the nucleic acid encoding the polypeptide sequence is transduced or transfected into the cell, as it is not possible for the polypeptide to be transduced or transfected into the cell.

[0477] As used herein, sometimes when referring to the number of cells injected into an animal, or elsewhere in the context, "M" refers to millions and "K" refers to thousands.

[0478] As used herein, interchangeable designations of the various monoclonal antibodies are used, such as "MN-C2," which is interchangeable with "C2," "Min-C2," and "MNC2"; "MN-E6," which is interchangeable with "E6," "Min-E6," and "MNE6"; "MN-C3," which is interchangeable with "C3," "Min-C3," and "MNC3"; and "MN-C8," which is interchangeable with "C8," "Min-C8," and "MNC8." The monoclonal antibodies provided herein follow the same convention.

[0479] As used herein, "h" or "hu" placed before an antibody construct is shorthand for humanized.

[0480] As used herein, the term "antibody-like" means a molecule that can be engineered so that it contains portions of an antibody but is not a naturally occurring antibody. Examples include, but are not limited to, CAR (chimeric antigen receptor) T cell technology and CAR technology uses an antibody epitope fused to a portion of a T cell, allowing the body's immune system to be directed to attack a specific target protein or cell. The technology consists of an "antibody-like" library, which is a collection of synthetic human Fabs that are then screened for binding to peptide epitopes of target proteins. Selected Fab regions can then be engineered into a scaffold or framework that makes them resemble antibodies.

[0481] As used herein, "PSMGFR" is an abbreviation for the primary sequence of the MUCl growth factor receptor, which is identified by SEQ ID NO: 2, and is therefore not to be confused with the six amino acid sequence. "PSMGFR peptide" or "PSMGFR region" refers to a peptide or region that is incorporated into the primary sequence of the MUCl growth factor receptor (SEQ ID NO: 2).

[0482] As used herein, the "MUC1*" extracellular domain is primarily defined by the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 2). Because the exact site of cleavage of MUC1 depends on the enzyme that cleaves it, and the cleaving enzyme varies depending on the cell type, tissue type, or time of cell evolution, the exact sequence of the MUC1* extracellular domain may vary at the N-terminus.

[0483] Other cleaved amino acid sequences may include SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 620); or

[0484] SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621).

[0485] As used herein, the term "PSMGFR" is an acronym for the primary sequence of the MUCl growth factor receptor, as shown in 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 from the N-terminus of the 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 from the C-terminus of the PSMGFR. Mixtures of deletions and additions are also possible. For example, N+20 / C-27 refers to a peptide fragment of wild-type MUCl in which 20 amino acids are added to the N-terminus of PSMGFR and 27 amino acids are deleted from the C-terminus.

[0486] As used herein, "the extracellular domain of MUC1*" refers to the extracellular portion of the MUC1 protein that lacks the tandem repeat domain. In most cases, MUC1* is a cleavage product, where the MUC1* portion consists of a short extracellular domain lacking the tandem repeat sequence, the transmembrane domain, and the cytoplasmic tail. The exact location of MUC1 cleavage is unclear, likely because it appears to be cleaved by more than one enzyme. The extracellular domain of MUC1* will include most of the PSMGFR sequence, but may have an additional 10-20 N-terminal amino acids.

[0487] As used herein, "sequence identity" refers to the sequence homology between a particular polypeptide or nucleic acid and a reference nucleic acid or amino acid sequence, such that the homologous peptide functions in the same manner as the reference peptide or nucleic acid. This homology may be very close to the reference peptide, such that sometimes two sequences may be 90%, 95%, or 98% identical, yet function in the same manner in terms of binding or other biological activity.

[0488] As used herein, a "MUCl positive" cell refers to a cell that expresses the gene for MUCl, MUCl-Y, or MUCl-Z, or other MUCl variants.

[0489] As used herein, a "MUCl negative" cell refers to a cell that does not express the gene for MUCl.

[0490] As used herein, a "MUCl*-positive" cell refers to a cell that expresses a gene for MUCl, wherein the protein expressed by the gene is a transmembrane protein lacking tandem repeats, which may be the result of post-translational modification, cleavage, alternative splicing, or transfection or transduction of the cell with a MUCl protein lacking tandem repeats.

[0491] As used herein, a "MUCl* negative" cell refers to a cell that may or may not express the gene for MUCl but does not express the MUCl transmembrane protein lacking tandem repeats.

[0492] As used herein, "MUCl -positive" cancer cells refer to cancer cells that overexpress the MUCl gene, express MUCl in an aberrant pattern where its expression is not restricted to the apical margin, and / or express MUCl that lacks tandem repeats.

[0493] As used herein, "MUCl negative" cancer cells refer to cancer cells that may or may not express the gene for MUCl but do not overexpress MUCl or overexpress the MUCl transmembrane protein lacking tandem repeats.

[0494] As used herein, a "MUCl*-positive" cancer cell refers to a cancer cell that overexpresses the MUCl transmembrane protein lacking tandem repeats.

[0495] As used herein, a "MUCl* negative" cancer cell refers to a cancer cell that may or may not express the gene for MUCl but does not overexpress the MUCl transmembrane protein that lacks tandem repeats.

[0496] As used herein, "conformational epitope" refers to a peptide sequence that requires the antibody to be bound to exist in a specific three-dimensional structure or conformation. However, when the peptide sequence is in a three-dimensional structure or conformation, the antibody binds, but when the peptide sequence is linear, it does not bind. A common technique for determining whether an antibody binds to a linear fragment or conformational epitope is to use an antibody to probe a denatured protein blot. Proteins and peptides are linearized by passing them through a denaturing gel. Antibodies that do not work in denatured protein blots but recognize natural targets (e.g., targets expressed on intact cells) are determined to recognize conformational epitopes. As used herein, an antibody may or may not actually bind to a "conformational epitope," but the presence of a "conformational epitope" sequence is required to present a three-dimensional structure so that the MUC1* region on cancer cells can be bound by antibodies specific for cancer treatment. Therefore, a conformational epitope is an amino acid sequence that induces an antibody to bind to the MUC1* region on cancer cells. Thus, the term "conformation-inducing peptide sequence" may be used, which refers to a peptide sequence that exists within a larger peptide not as a binding site but rather induces antibody binding to the larger peptide by forming a three-dimensional structure that promotes antibody binding to the larger peptide.

[0497] MUC1* Antibodies (Anti-PSMGFR) for the Treatment or Prevention of Cancer

[0498] We discovered that a cleaved form of the MUC1 (SEQ ID NO: 1) transmembrane protein is a growth factor receptor that drives the growth of more than 75% of all human solid tumor cancers. The cleaved form of MUC1, which we call MUC1* (pronounced muk1 asterisk), is a potent growth factor receptor. Enzymatic cleavage releases most of the MUC1 extracellular domain. It is the remaining portion, comprising the truncated extracellular domain, the transmembrane domain, and the cytoplasmic tail, that is termed MUC1*. Cleavage and release of most of the extracellular domain of MUC1 exposes the activating ligands that dimerize NME1, NME6, NME8, and NME7. AB , NME7-X1 or NME7 binding sites. Cell growth assays showed that ligand-induced dimerization of the MUC1* extracellular domain promoted growth (Figures 1A to 1D). MUC1*-positive cells were treated with bivalent 'bv' anti-MUC1* antibodies, monovalent 'mv' or Fab, NM23-H1 dimer or NME7-AB. Bivalent anti-MUC1* antibodies stimulated the growth of cancer cells, while monovalent Fab inhibited growth. The classic bell-shaped curve indicates that ligand-induced dimerization stimulates growth. Dimeric NM23-H1, also known as NME1, stimulated the growth of MUC1*-positive cancer cells, but siRNA that inhibited MUC1 expression abolished its effect (Figure 1C). NME7-AB also stimulated the growth of MUC1*-positive cells (Figure 1D).

[0499] MUC1* is a perfect target for cancer drugs because it is aberrantly expressed in more than 75% of all cancers and may be overexpressed in an even higher proportion of metastatic cancers. After MUC1 cleavage, 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, and in particular competitively inhibit activating ligands (such as NME proteins, including NME1, NME6, NME8, NME7, etc.), are also known to be effective. AB , NME7-X1 and NME7), are ideal therapeutic agents and can be used to treat or prevent MUC1-positive or MUC1*-positive cancers as stand-alone antibodies, antibody fragments or variable region fragments thereof incorporated into bispecific antibodies, or chimeric antigen receptors (also known as CARs), at which time they are transfected or transduced into immune cells and then administered to patients.

[0500] Therapeutic anti-MUC1* antibodies can be monoclonal, polyclonal, antibody mimetics, engineered antibody-like molecules, whole antibodies, or antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, scFv, and scFv-Fc. Human or humanized antibodies are preferably used to treat or prevent cancer. In any of these antibody-like molecules, mutations can be introduced to prevent or minimize dimer formation. Monovalent or bispecific anti-MUC1* antibodies are preferred because MUC1* function is activated by ligand-induced dimerization. Typical binding assays show that NME1 and NME7 AB Binds to the PSMGFR peptide portion of MUC1* ( Figure 2 A. Figure 2 D). Furthermore, they showed that these activating growth factors bind to the membrane-proximal portion of MUC1*, since they did not bind to the PSMGFR peptide if the 10 C-terminal amino acids were missing. Similarly, the anti-MUC1* antibodies MN-C2 and MN-E6 bound to the PSMGFR peptide when, and only when, the 10 C-terminal amino acids were present ( Figure 2 B. Figure 2 C). Antibodies MN-C3 and MN-C8 bind to epitopes distinct from those of MN-C2 and MN-E6, as they are independent of the presence of the 10 C-terminal amino acids of the PSMGFR peptide ( Figure 2 E. Figure 2 F). Antibodies MN-C2, MN-E6, or fragments thereof can be administered to patients as independent antibodies transduced into immune cells or incorporated into bispecific antibodies, BiTEs, or chimeric antigen receptors (also known as CARs) for the treatment or prevention of cancer. MNC2 and MNE6, as well as other competitive inhibitors of NME1 and NME7 ABThe conjugated anti-MUCl* antibodies are preferably used as stand-alone antibody therapeutics.

[0501] Therapeutic anti-MUC1* antibodies for use as stand-alone antibody therapeutics or for integration into BiTEs or CARs can be selected based on specific criteria. Parent antibodies can be generated using typical methods for generating monoclonal antibodies in animals. Alternatively, they can be selected by screening antibody and antibody fragment libraries for their ability to bind to a MUC1* peptide, which can be:

[0502] (i) PSMGFR region of MUC1;

[0503] (ii) PSMGFR peptide;

[0504] (iii) a peptide having the amino acid sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10)

[0505] (iv) having an amino acid sequence

[0506] ASRYNLTISDVSVSDVPFPFSAQSGA(N-19) peptide

[0507] (v) having an amino acid sequence

[0508] NLTISDVSVSDVPFPFSAQSGA(N-23) peptide

[0509] (vi) having an amino acid sequence

[0510] ISDVSVSDVPFPFSAQSGA(N-26) peptide

[0511] (vii) having an amino acid sequence

[0512] SVSDVPFPFSAQSGA(N-30) peptide

[0513] (viii) having an amino acid sequence

[0514] Peptide of QFNQYKTEAASRYNLTISDVSVSDVPFPFS(N-10 / C-5)

[0515] (ix) having an amino acid sequence

[0516] ASRYNLTISDVSVSDVPFPFS(N-19 / C-5) peptide or

[0517] (x) has an amino acid sequence

[0518] Peptide of FPFSAQSGA(N-36).

[0519] The resulting antibodies or antibody fragments generated or selected in this manner can then be further selected through additional screening. For example, antibodies or antibody fragments can be more preferred based on their ability to bind to MUC1*-positive cancer cells or tissues but not to MUC1-negative cancer cells or normal tissues. In addition, anti-MUC1* antibodies or antibody fragments can be deselected as anti-cancer therapeutics if they bind to stem or progenitor cells. Anti-MUC1* antibodies or antibody fragments can be more preferred if they have the ability to competitively inhibit the binding of an activating ligand to MUC1*. Figures 3A to 3C It was shown that MN-E6 and MN-C2 competitively inhibited the binding of the activating ligands NME1 and NME7 to MUC1*.

[0520] Methods for selecting an anti-MUC1* antibody for treating a patient diagnosed with, at risk for developing, or suspected of having a MUC1-positive cancer include one or more of the following steps: selecting an antibody or antibody fragment that 1) binds to a PSMGFR peptide; 2) binds to an N-10 PSMGFR peptide; 3) binds to cancer cells; 4) does not bind to stem or progenitor cells; and 5) competitively inhibits binding of dimeric NME1 or NME7-AB to the PSMGFR peptide. For example, Figures 3A to 3C It was shown that monoclonal MN-E6 and MN-C2 met all five criteria, whereas monoclonal MN-C3 and MN-C8 did not competitively inhibit the binding of the activating ligands NME1 and NME7 ( Figure 3C ). Recall that the MUC1* growth factor receptor is activated by ligand-induced dimerization of its extracellular domain. Therefore, an ideal antibody therapeutic should not dimerize the MUC1* extracellular domain. Preferably, suitable antibodies in this regard include monovalent antibodies (such as those produced in alpacas and camels), Fabs, scFvs, single domain antibodies (sdAbs), and scFv-Fcs (as long as the Fc portion is constructed so that it cannot homodimerize).

[0521] FACS scans showed that the anti-MUC1* antibodies MN-C2 and MN-E6 specifically bound to MUC1*-positive solid tumor cancer cells and MUC1*-transfected cells but not to MUC1*-negative or MUC1-negative cells. In one example, humanized MN-C2 scFv was shown to bind to ZR-75-1 (also known as 1500) MUC1*-positive breast cancer cells ( Figure 4 A to Figure 4 C). MN-E6 was shown to bind only to MUC1 negative HCT-116 colon cancer cells transfected with MUC1*. MN-E6 also binds to MUC1* positive cancer cells, such as ZR-75-1 (also known as 1500) MUC1* positive breast cancer cells ( Figure 4 D to Figure 4 F) Binding assays such as ELISA, immunofluorescence, etc. all confirmed that MN-C2 and MN-E6 bound to PSMGFR peptide and live MUC1-positive cancer cells. Humanized anti-MUCl* antibodies were selected based on their ability to also bind to PSMGFR peptide or MUC1-positive cancer cells. Figure 5 The humanized MN-C2 scFv was shown to bind to the MUC1* peptide PSMGFR with high affinity, with an EC-50 of approximately 333 nM. The humanized MN-C2 scFv was as effective as the Fab in inhibiting the growth of MUC1* positive cancer cells, as shown in FIG. Figure 6A 、 6B As shown in an example in . As with the parent antibody, the humanized scFv showed the same binding pattern. huMNE6-scFv bound to PSMGFR peptide, bound to N-10 peptide, but not to C-10 peptide (SEQ ID NO: 825) ( Figure 8 ). Murine or humanized MNC3-scFv bound to PSMGFR peptide, bound to N-10 peptide, and bound to C-10 peptide ( FIG. 9 ).

[0522] Fabs of MN-E6 and MN-C2, or comparable single-chain variable regions derived therefrom, effectively inhibited the growth of MUC1*-positive cancers in vitro and in vivo. In several examples, Fabs of anti-MUC1* antibodies inhibited the growth of human MUC1*-positive cancers in vivo. In one instance, immunocompromised mice were implanted with human breast tumors and then treated with MN-E6 Fab after tumor engraftment. Figure 7A MN-E6 Fab was shown to effectively inhibit the growth of MUC1* positive breast cancer. Six million T47D human breast cancer cells mixed with Matrigel 50 / 50 were implanted into female nu / nu mice that had been implanted with estrogen pellets for 90 days. 3 Mice with three consecutive increases in tumor volume were used for treatment. Animals were injected subcutaneously with 80 mg / kg MN-E6 Fab twice a week, and the same number of mice meeting the same selection criteria were injected with vehicle alone ( Figure 7A ).

[0523] On the other hand, MN-E6 was shown to prevent the growth of prostate cancer. Figure 7BMN-E6 Fab was shown to effectively inhibit the growth of MUC1*-positive prostate cancer. Male NOD / SCID mice were implanted with 6 million DU-145 human prostate cancer cells mixed with Matrigel 50 / 50. Mice bearing tumors of at least 150 mm^3 and with three consecutive increases in tumor volume were selected for treatment. Animals were injected subcutaneously with 160 mg / kg MN-E6 Fab every 48 hours, and an equal number of mice meeting the same selection criteria were injected with vehicle alone ( Figure 7B Tumors were independently measured and recorded twice weekly by two researchers. Statistics were calculated blindly by an independent statistician, yielding a P value of 0.0001 for each comparison. Anti-MUC1* Fab inhibited breast cancer growth and prostate cancer growth. Treatment had no effect on body weight or bone marrow cell type or number. MN-E6 Fab effectively inhibited tumor growth, while tumors in the control group continued to grow until sacrifice. No adverse effects of treatment were observed or detected.

[0524] Recombinant forms of MN-E6 and MNC2 were constructed to be monomeric, like the Fab. In this case, MN-E6 is humanized, and MN-C2 is humanized. Numerous methods for humanizing antibodies are known to those skilled in the art. In addition to humanization, human antibody libraries can also be screened to identify other fully human antibodies that bind to PSMGFR.

[0525] A single chain of the humanized MN-E6 variable region, called scF, was genetically engineered so that it could be attached to the Fc portion of an antibody (SEQ ID NOs: 256 and 257). The Fc region confers certain benefits to antibody fragments for use as therapeutic agents. The Fc portion of an antibody recruits complement, which generally means it can recruit other aspects of the immune system and thereby amplify the anti-tumor response, rather than just inhibiting the target. The addition of the Fc portion also increases the half-life of the antibody fragment (Czajkowsky DM, Hu J, Shao Z, and Pleass RJ. (2012) Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med. 4(10): 1015-1028). However, the Fc portion of an antibody homodimerizes, which is not optimal in the case of therapeutics based on anti-MUC1* antibodies because ligand-induced dimerization of the MUC1* receptor stimulates growth. Therefore, for anti-MUC1* anticancer therapeutics, Fc region mutations that resist dimer formation are preferred. Deletions of the hinge region and other mutations in the Fc region are made that render Fc mutants resistant to dimerization and are useful as therapeutic agents.

[0526] Human or humanized MN-E6 antibody or antibody fragment, Fab, MN-E6 scFv or hu MN-E6 scFv-Fc mut is an effective anti-cancer agent that can be administered to a person diagnosed with, suspected of having, or at risk of developing a MUC1 or MUC1*-positive cancer.

[0527] Humanization

[0528] Humanized antibodies or antibody fragments or fully human antibodies that bind to the extracellular domain of -MUC1* are preferably used for therapeutic purposes. The techniques described herein for humanizing antibodies are only a few of the many methods known to those skilled in the art. The present invention is not intended to be limited by the technique used to humanize antibodies.

[0529] Humanization is the process of replacing the non-human regions of a therapeutic antibody (usually a mouse monoclonal antibody) with human regions without changing its binding specificity and affinity. The main goal of humanization is to reduce the immunogenicity of therapeutic monoclonal antibodies when administered to humans. Three different types of humanization are possible. First, chimeric antibodies are prepared by replacing the non-human constant regions of an antibody with human constant regions. Such antibodies will contain mouse Fab regions and will contain approximately 80%-90% human sequences. Second, humanized antibodies are prepared by transplanting mouse CDR regions (responsible for binding specificity) onto the variable regions of a human antibody, replacing human CDRs (CDR grafting). Such antibodies will contain approximately 90%-95% human sequences. Third and finally, fully human antibodies (100% human sequences) can be produced by phage display (wherein human antibody libraries are screened to select antigen-specific human antibodies) or by immunizing transgenic mice expressing human antibodies.

[0530] The general technique for humanizing antibodies is roughly as follows. Monoclonal antibodies are produced in a host animal, usually a mouse. The monoclonal antibodies are then screened for affinity and specificity in binding to the target. Once a monoclonal antibody with the desired effect and desired characteristics is identified, it is sequenced. The sequence of the antibody produced by the animal is then compared with the sequences of many human antibodies to find a human antibody with the most homologous sequence to the animal antibody. Biochemical techniques are used to paste the human and animal antibody sequences together. Typically, the non-human CDRs are transplanted into the human antibody with the highest homology to the non-human antibody. This process can generate many candidate humanized antibodies that need to be tested to identify which one or more antibodies have the desired affinity and specificity.

[0531] Once a human antibody or humanized antibody has been produced, it can be further modified to be used as a Fab fragment, a complete antibody or an antibody-like entity, such as a single-chain molecule comprising a variable region, such as scFv or scFv-Fc. In some cases, it is necessary to mutate the Fc region of the antibody or antibody-like molecule so that it does not dimerize.

[0532] In addition to methods that incorporate human sequences into antibodies produced by non-human species, fully human antibodies can also be obtained by screening human antibody libraries with peptide fragments of the antigen. Fully human antibodies with functions similar to MN-E6 or MN-C2 can be generated by screening human antibody libraries with peptides having the sequence of the PSMGFR N-10 peptide. Humanized anti-MUC1* antibodies have been generated based on the sequences of the mouse monoclonal antibodies MN-E6 and MN-C2. In one aspect of the invention, patients diagnosed with MUC1*-positive cancers are treated with an effective amount of murine or camelid MNC2, MNE6, 20A10 (SEQ ID NOs: 1574-1581), 3C2B1 (SEQ ID NOs: 1572-1573), 5C6F3, 25E6 (SEQ ID NOs: 1598-1601), 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. In another aspect of the invention, a patient diagnosed with a MUC1*-positive cancer is treated with an effective amount of humanized MN-E6 or MN-C2. In a preferred embodiment, a patient diagnosed with a MUC1*-positive cancer is treated with an effective amount of humanized MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. In another aspect of the invention, a patient diagnosed with a MUC1*-positive cancer is treated with an effective amount of a humanized monovalent MNC2, MNE6, 20A10 (SEQ ID NOs: 1574-1581), 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, wherein monovalent refers to the corresponding Fab fragment, the corresponding scFv, or the corresponding scFv-Fc fusion. In a preferred embodiment, a patient diagnosed with a MUC1*-positive cancer is treated with an effective amount of a humanized scFv or monomeric humanized scFv-Fc of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. Because the MUCl* growth factor receptor is activated by ligand-induced dimerization of its extracellular domains, and because the Fc portion of antibodies homodimerizes, constructs comprising the Fc portion preferably use a mutated Fc region to prevent or minimize dimerization.

[0533] Antibodies that bind to the PSMGFR (SEQ ID NO: 2) peptide, and more specifically the N-10 peptide, of the extracellular domain of the MUC1* receptor are potent anticancer therapeutics that are effective in treating or preventing MUC1*-positive cancers. They have been shown to inhibit the activation of ligand dimerization of NME1 (SEQ ID NO: 1781) and NME7. AB (SEQ ID NO:827) binds to the extracellular domain of MUC1*. Anti-MUC1* antibodies that bind to the PSMGFR sequence inhibit the growth of MUC1*-positive cancer cells, particularly when they inhibit ligand-induced receptor dimerization. Fabs of anti-MUC1* antibodies have been shown to block tumor growth in animals. Therefore, antibodies or antibody fragments that bind to the extracellular domain of MUC1* would be beneficial in treating cancers where cancerous tissue expresses MUC1*.

[0534] Antibodies that bind to the PSMGFR region of MUCl* or to synthetic PSMGFR peptides are preferred.We have identified several monoclonal antibodies that bind to the extracellular domain of MUCl*. This group includes mouse monoclonal antibodies MNC2 (SEQ ID NOs: 118-131, 144-158, 163-164, 168-181, 194-209), MNE6 (SEQ ID NOs: 12-25, 39-59, 65-78, 93-114), 20A10 (SEQ ID NOs: 988-1019, 1574-1597, 1659-1666), 3C2B1 (SEQ ID NOs: 1386-1413, 1572-1573), 5C6F3 (SEQ ID NOs: 1356-1385), 25E6 (SEQ ID NOs: 1020-1051, 1598-1617, 1667-1674), 18G12 (SEQ ID NOs: 956-987), 28F9 (SEQ ID NOs: 1 The variable regions of the mouse antibodies were sequenced and given as MN-E6 (SEQ ID NOs: 12-13 and 65-66) and MN-C2 (SEQ ID NOs: 118-119 and 168-169). The CDRs of these antibodies constitute the recognition unit of the antibody and are the most important part of the mouse antibody that should be retained when transplanted into a human antibody. The CDR sequences for each mouse monoclonal antibody are as follows, with the heavy chain sequence followed by the light chain sequence: MN-E6 CDR1 (SEQ ID NOs: 16-17 and 69-70), CDR2 (SEQ ID NOs: 20-21 and 73-74), CDR3 (SEQ ID NOs: 24-25 and 77-78), MN-C2 CDR1 (SEQ ID NOs: 122-123 and 172-173), CDR2 (SEQ ID NOs: 126-127 and 176-177), CDR3 (SEQ ID NOs: 130-131 and 180-181). In some cases, portions of the framework regions considered important for the three-dimensional structure of the CDRs by modeling were also imported from the mouse sequence.

[0535] Monoclonal antibodies MN-E6 and MN-C2 have higher affinity for MUC1* when it is present on cancer cells, while monoclonal antibodies MN-C3 and MN-C8 have higher affinity for MUC1* when it is present on stem cells.

[0536] All four antibodies have been humanized, and the process has resulted in several humanized versions of each antibody. The CDRs from the variable regions of the mouse antibodies were biochemically grafted into the homologous human antibody variable region sequences. The humanized variable regions of MN-E6 (SEQ ID NOs: 38-39 and 93-94), MN-C2 (SEQ ID NOs: 144-145 and 194-195), MN-C3 (SEQ ID NOs: 439-440 and 486-487), and MN-C8 (SEQ ID NOs: 525-526 and 543-544) were generated by grafting the mouse CDRs into the variable regions of the homologous human antibodies. The humanized heavy chain variable constructs were then fused to the constant region of either the human IgG1 heavy chain constant region (SEQ ID NOs: 58-59) or the human IgG2 heavy chain constant region (SEQ ID NOs: 54-55), which were then paired with humanized light chain variable constructs fused to the constant region of either the human kappa chain (SEQ ID NOs: 109-110) or the human lambda chain (SEQ ID NOs: 113-114). Other IgG isotypes can be used as constant regions, including IgG3 or IgG4.

[0537] Examples of humanized MN-E6 variable regions fused to IgG2 heavy chains (SEQ ID NOs: 52-53) and IgG1 heavy chains (SEQ ID NOs: 56-57), humanized MN-C2 variable regions fused to IgG1 heavy chains (SEQ ID NOs: 157-158) or IgG2 heavy chains (SEQ ID NOs: 163-164), wherein the heavy chains are paired with lambda light chains (SEQ ID NOs: 111-112 and 216-219) or kappa chains (SEQ ID NOs: 107-108 and 210-213), as well as humanized MN-C3 (SEQ ID NOs: 455-456, 453-454 and 500-501, 502-503) and MN-C8 (SEQ ID NOs: 541-542, 539-540 and 579-580, 581-582) antibodies were generated. Which IgG constant region is fused to the humanized variable region depends on the desired effect, as each isotype has its own characteristic activity. The isotype of the human constant region is selected based on considerations such as whether antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) is desired, but can also depend on the yield of the antibody produced in a cell-based protein expression system. In a preferred embodiment, the humanized anti-MUC1* antibody or antibody fragment is administered to a human diagnosed with or at risk of developing a MUC1-positive cancer.

[0538] One method for testing and selecting humanized anti-MUC1* antibodies that would be most useful for treating humans who have cancer or are at risk of developing cancer is to test their ability to inhibit the binding of activating ligands to the extracellular domain of MUC1*. Dimeric NME1 can bind to the extracellular domain of MUC1* and dimerize it, thereby stimulating cancer cell growth. Therefore, antibodies and antibody fragments that compete with NME1 for binding to the extracellular domain of MUC1* are anticancer agents. NME7 AB is another activating ligand of MUC1*. In some cases, it is preferred to identify NME7 that blocks NME7 or NME7-X1. ABAntibodies that bind to the extracellular domain of MUC1* by competing with truncated or cleaved products. Antibodies and antibody fragments that compete with NME7 and NME7 variants for binding to the extracellular domain of MUC1* are effective as anticancer therapeutics. These antibodies include, but are not limited to, MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, as well as single-chain versions (such as scFv) of these antibodies and their humanized versions. Other NME proteins also bind to MUC1 or MUC1*, including NME6 and NME8. Antibodies that compete with these proteins for binding to MUC1* can also be used as therapeutic agents. In a preferred embodiment, a murine, camelid, human, or humanized anti-MUC1* antibody or antibody fragment is administered to a person diagnosed with a MUC1-positive cancer or at risk of developing a MUC1-positive cancer. In a more preferred embodiment, a single-chain antibody fragment or monomeric scFv-Fc fusion derived from the humanized sequence of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 or H11 is administered to a human diagnosed with or at risk of developing a MUC1-positive cancer.

[0539] Single-chain variable fragments (scFvs) or other formats for producing monovalent antibodies or antibody-like proteins are also available. In some cases, it is desirable to prevent dimerization of the MUC1* extracellular domain. Single-chain variable fragments, Fabs, and other monovalent antibody-like proteins have been shown to effectively bind to the MUC1* extracellular domain and block MUC1* dimerization. These single-chain variable fragments, Fabs, and other monovalent antibody-like molecules effectively blocked cancer growth in vitro and in animals xenografted with human MUC1-positive cancer cells. Therefore, humanized single-chain variable fragments or monovalent anti-MUC1* antibodies or antibody-like molecules would be highly effective as anti-cancer therapeutics. Therefore, such humanized single-chain antibodies, Fabs, and other monovalent antibody-like molecules that bind to the MUC1* extracellular domain or PSMGFR peptide can be used as anti-cancer therapeutics. Anti-MUC1* single-chain variable fragments are generated by grafting the non-human CDRs of an antibody that binds to the MUC1* extracellular domain or PSMGFR peptide into the framework of a cognate variable region human antibody. The resulting humanized heavy and light chain variable regions are then linked to each other via a suitable linker, wherein the linker should be flexible and of a length that allows the heavy chain to bind to the light chain but prevents the heavy chain of one molecule from binding to the light chain of another molecule. For example, a linker of about 10-15 residues. Preferably, the linker comprises [(glycine)4(serine)1]3 (SEQ ID NOs: 401-402), but is not limited to this sequence, as other sequences are also possible.

[0540] In one aspect, humanized variable regions from MN-E6 (SEQ ID NOs: 38-39 and 93-94), MN-C2 (SEQ ID NOs: 144-145 and 194-195), or other antibodies of the invention were biochemically grafted into a construct in which the heavy and light chains were connected by a linker. Examples of humanized single-chain anti-MUCl* antibodies were generated that comprised humanized sequences of the variable regions from MN-E6 and MN-C2. Several humanized MN-E6 single-chain proteins were generated (SEQ ID NOs: 232-237). Several humanized MN-C2 single-chain proteins were generated (SEQ ID NOs: 238-243). In a preferred embodiment, humanized anti-MUC1* antibody fragments, including variable fragments, scFv antibody fragments MN-E6 scFv, MN-C2 scFv, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 or H11 scFv, are administered to a human diagnosed with or at risk of developing a MUC1-positive cancer.

[0541] One aspect of the invention is a method for treating a patient diagnosed with, suspected of having, or at risk of developing a MUC1-positive or MUC1*-positive cancer, wherein an effective amount of monomeric MN-E6 scFv, MN-C2 scFv or MN-E6 scFv-Fc, MN-C2 scFv-Fc, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 is administered to the patient, wherein the antibody variable fragment portion is human or has been humanized, and wherein the Fc portion of the antibody-like protein has been mutated to render it resistant to dimer formation.

[0542] CAR T and cancer immunotherapy technologies

[0543] In another aspect of the invention, some or all of the single-chain portions of the anti-MUC1* antibody fragment are biochemically fused to immune system molecules using several different chimeric antigen receptors, a 'CAR' strategy. The idea is to fuse the recognition portion of the antibody (usually as a single-chain variable fragment) with an immune system molecule having a transmembrane domain and a cytoplasmic tail capable of transmitting a signal that activates the immune system. The recognition unit can be an antibody fragment, a single-chain variable fragment, a scFv or a peptide. In one aspect, the recognition portion of the extracellular domain of CAR is composed of sequences from the humanized variable regions of MN-E6 (SEQ ID NO: 38-39 and 93-94), MN-C2 (SEQ ID NO: 144-145 and 194-195), 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 or H11. Examples of murine or humanized antibodies or single-chain fragments scFv of the present invention that can be incorporated into CAR, BiTE or ADC are given as follows: 3C2B1 (SEQ ID NO: 1572-1573), 20A10 (SEQ ID NO: 1574-1581), 25E6 (SEQ ID NO: 1598-1601). On the other hand, it consists of sequences from single-chain variable fragments. Examples of single-chain constructs are given. Several humanized MN-E6 single-chain protein scFvs (SEQ ID NO: 232-237) were generated. Several humanized MN-C2 single-chain protein scFvs (SEQ ID NO: 238-243) were generated. The transmembrane region of CAR can be derived from CD8, CD4, antibody domains or other transmembrane regions, including the transmembrane region of the proximal cytoplasmic co-stimulatory domain, such as CD28, 4-1BB, etc. The cytoplasmic tail of CAR can be composed of one or more motifs that send immune system activation signals. This group of cytoplasmic signaling motifs, sometimes referred to as costimulatory cytoplasmic domains, include but are not limited to CD3-ζ, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7, and Fc receptor gamma domains. Minimal CAR may have CD3-ζ or Fc receptor gamma domains, and then one or two of the above domains are connected in series in the cytoplasmic tail. In one aspect, the cytoplasmic tail includes CD3-ζ, CD28, 4-1BB, and / or OX40.

[0544] The extracellular domain recognition unit of the CAR targeting MUC1* can include the variable region of any non-human, humanized or human antibody, which can bind to at least 12 consecutive amino acids of PSMGFR peptide (SEQ ID NO: 2) or N-10 peptide. In one aspect, the MUC1* targeting portion of CAR includes a variable region from non-human, humanized or human MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 or H11. Examples of some antibodies of the present invention incorporated into CAR in mouse or humanized form are given as 20A10 (SEQ ID NO: 1582-1597) and 25E6 (SEQ ID NO: 1602-1617). During the humanization process, the antibody CDRs can be inserted into a variety of different framework regions; as a demonstration, we have produced three versions of humanized 20A10 that differ only in the framework region. These have been incorporated into CARs (SEQ ID NOs: 1675, 1678, 1685), which, when transduced into human T cells, are able to recognize and kill cells expressing the target MUC1*. In one aspect, the extracellular domain recognition unit of the CAR consists essentially of humanized MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 or H11 single-chain variable fragment scFv. The transmembrane region of CAR can be derived from CD8 (SEQ ID NO: 363-364), or can be the transmembrane domain of CD3-ζ, CD28, 41bb, OX40 or other transmembrane region (SEQ ID NO: 361-372), and the cytoplasmic domain of CAR with the antibody fragment targeting MUC1* extracellular domain can be composed of one or more selected from the group comprising immune system costimulatory cytoplasmic domains. The group of immune system costimulatory domains includes but is not limited to CD3-ζ, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7 and Fc receptor gamma domain (SEQ ID NO: 373-382).

[0545] The CAR can be transfected or transduced into cells of the immune system. In a preferred embodiment, the CAR targeting MUC1* is transfected or transduced into T cells. In one aspect, the T cell is a CD3+ / CD28+T cell. In another case, it is a dendritic cell. In another case, it is a B cell. In another case, it is a mast cell. In yet another case, it is a natural killer NK cell. The recipient cell can be from the patient or the donor. If it comes from a donor, it can be engineered to remove molecules that trigger rejection. The cells transfected or transduced with the CAR of the present invention can be expanded ex vivo or in vitro and then administered to the patient. The route of administration is selected from a group including but not limited to bone marrow transplantation, intravenous injection, in situ injection or transplantation. In a preferred embodiment, the CAR targeting MUC1* is administered to a person diagnosed with MUC1-positive cancer or at risk of developing MUC1-positive cancer.

[0546] There are many possible anti-MUC1* CAR constructs that can be transduced into T cells or other immune cells for the treatment or prevention of MUC1*-positive cancers. CARs are composed of modules, and the identity of some modules is relatively unimportant, while the identity of others is crucial.

[0547] We and others have shown that intracellular signaling modules, such as CD3-ζ (SEQ ID NOs: 373-376), CD28 (SEQ ID NOs: 377-378), and 41BB (SEQ ID NOs: 379-380), alone or in combination, stimulate immune cell expansion, cytokine secretion, and immune cell-mediated killing of targeted tumor cells (Pulè MA, Straathof KC, Dotti G, Heslop HE, Rooney CM, and Brenner MK (2005) A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells. Mol Ther. 12(5):933-941; Hombach AA, Heiders J, Foppe M, Chmielewski M, and Abken H. (2012) OX40 costimulation by a chimeric antigen receptor abrogates CD28 and IL-2induced IL-10 secretion by redirected CD4(+)Tcells.Oncoimmunology.1(4):458-466; Kowolik CM, Topp MS, Gonzalez S, Pfeiffer T, Olivares S, Gonzalez N, Smith DD, Forman SJ, Jensen MC and Cooper LJ. (2006) CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res. 66(22):10995-11004; Loskog A, Giandomenico V, Rossig C, PuleM, Dotti G and Brenner MK.(2006) Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells. Leukemia. 20(10):1819 - 1828; Milone MC, Fish JD, Carpenito C, Carroll RG, Binder GK, Teachey D, Samanta M, Lakhal M, Gloss B, Danet-Desnoyers G, Campana D, Riley JL, Grupp SA and June CH. (2009) Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther. 17(8):1453 - 1464; Song DG, Ye Q, Carpenito C, Poussin M, Wang LP, Ji C, Figini M, June CH, Coukos G, Powell DJ Jr. (2011) In vivo persistence, tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137(4-1BB). Cancer Res.71(13):4617-4627). Antibodies of the present invention, including but not limited to fragments of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 or H11, may also be incorporated into CARs having mutated cytoplasmic tails (such as mutated tyrosine or ITAM). In any of the CARs described above, the cytoplasmic tail may comprise a mutation that weakens signal transduction. Such mutations include but are not limited to mutations to tyrosine that inhibit phosphorylation and signal transduction (Salter et al., 2018). In any of the CARs described above, the ITAM of CD3-ζ may mutate to inhibit or weaken signal transduction (Feucht et al. 2019). In any of the CARs described above, the CD3 of the cytoplasmic tail may comprise mutations in ITAM, including those mutations known as 1XX. Examples of antibodies of the present invention incorporated into CARs with 1XX mutations in the ITAMs of CD3-ζ are given in the following sequences: MNC2 (SEQ ID NO: 1618-1625), MNE6 (SEQ ID NO: 1626-1633), 20A10 (SEQ ID NO: 1590-1595), 25E6 (SEQ ID NO: 1610-1617). We note that the CDRs of an antibody can be inserted into the context of many different framework regions. For example, the 20A10 CDRs were inserted into three different sets of framework regions (SEQ ID NO: 1692, 1699, and 1706), and all were able to function when transduced into T cells. In any of the CARs described above, T cells can be engineered to overexpress c-Jun as a method to inhibit T cell exhaustion (Lynn et al. 2019). A variety of promoters can be used upstream of genes used for CAR and other compositions of the present invention, including insertion into naturally occurring promoters in cells, such as the TRAC locus, using CRISPR, Sleeping Beauty, or similar technologies for site-directed gene insertion. Commonly used promoters include the CMV promoter, or mini-CMV (SEQ ID NO: 1634), the minimal IL-2 promoter (SEQ ID NO: 1635), or the minimal promoter minip (SEQ ID NO: 1636).

[0548] A single-chain antibody fragment containing the variable domains of a monoclonal anti-MUC1* antibody, known as MN-E6 or MN-C2, can be engineered into a panel of CARs. The MUC1*-targeting CARs are then transduced into immune cells, either alone or in combination. When challenged with surfaces presenting MUC1* peptides, antigen-presenting cells transfected with MUC1*, or MUC1*-positive cancer cells, immune cells transduced with MUC1*-targeting CARs elicit an immune response, including cytokine release, targeted cell killing, and immune cell expansion.

[0549] For example, the gene encoding CAR described herein and activated T cell induction gene can be virally transduced into immune cells using a virus, or inserted into the downstream region of one of the cell promoters or enhancers (such as TRAC (T cell receptor α chain) locus). Viral delivery systems and viral vectors can be used, including but not limited to retroviruses including gamma-retrovirus, slow virus, adenovirus, adeno-associated virus, baculovirus, poxvirus, herpes simplex virus, oncolytic virus, HF10, T-Vec, etc. In addition to viral transduction, methods such as CRISPR technology, CRISPR-Cas9 and CRISPR-CPF1, TALEN, Sleeping Beauty transposon system and SB100X can be used to directly splice CAR described herein and activated T cell induction genes into the genome of the recipient cell.

[0550] Similarly, the identity of the molecules that make up the non-targeting portion of the CAR (such as the extracellular domain, the transmembrane domain, and the membrane-proximal portion of the cytoplasmic domain) is not essential for the function of the CAR targeting MUC1*. For example, the extracellular domain, the transmembrane domain, and the membrane-proximal portion of the cytoplasmic domain can be composed of portions of CD8, CD4, CD28, or universal antibody domains such as Fc, CH2CH3, or CH3. In addition, the non-targeting portion of the CAR can be a complex of portions of one or more of these molecules or other family members.

[0551] One aspect of the present invention is a method for treating a patient diagnosed with MUC1-positive or MUC1*-positive cancer, suspected of having MUC1-positive or MUC1*-positive cancer, or at risk of developing MUC1-positive or MUC1*-positive cancer, wherein an effective amount of immune cells transduced with a CAR targeting MUC1* are administered to the patient. In another aspect of the present invention, the immune cells are T cells separated from the patient, which are then transduced with a CAR, wherein the targeting head of the CAR binds to MUC1*, and after the transduced T cells are expanded, an effective amount of CART cells are administered to the patient. In another aspect of the present invention, the immune cells are T cells separated from the patient, which are then transduced with a CAR, wherein the targeting head of the CAR comprises a portion of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 or H11, and after the optional transduced T cells are expanded, an effective amount of CAR T cells are administered to the patient.

[0552] Specificity of anti-MUC1* targeting antibodies

[0553] As these experiments demonstrate, the key part of the CAR is the antibody fragment that directs the immune cells to the tumor cells. As we will show in the next section, MN-E6 and MN-C2 are specific for the form of MUC1* expressed on tumor cells. The next most important part of the CAR is the cytoplasmic tail that carries the immune system's co-stimulatory domains. The identity of these domains modulates the extent of the immune response but does not affect specificity. As shown, the identity of the transmembrane portion of the CAR is the least important. It seems that as long as the transmembrane portion has some flexibility and is long enough to allow the antibody fragment to reach its cognate receptor on the tumor cell, it is sufficient. CARs that contain the MN-E6 targeting antibody fragment and the intracellular co-stimulatory domains 41BB and CD3-ζ but with a variety of different extracellular, transmembrane, and short cytoplasmic tails all work because they specifically kill the targeted cells while stimulating host T cells to expand.

[0554] The most accurate way to demonstrate antibody specificity is to test the antibody on normal human tissue samples compared to cancerous tissue samples. MN-C2 and MN-E6 were shown to bind specifically to MUC1 or MUC1* positive cancer cells. Several breast tumor arrays were assayed using several anti-MUC1 or MUC1* antibodies. Essentially, a study involving serial sections of breast cancer tissue samples from over 1,200 different breast cancer patients showed that very little full-length MUC1 was retained on breast cancer tissue. The vast majority of MUC1 expressed was MUC1* and stained with MN-C2. Analysis was performed by Clarient Diagnostics and tissue staining was scored using the Allred method. For example, Figure 10 Shown are serial sections of breast cancer tissue arrays stained with VU4H5 (a commercially available anti-MUCl antibody that binds to tandem repeats) or MN-C2 that binds to MUCl*. Figure 10 and Figure 11 Figure 2 is a photograph of breast cancer tissue arrays stained with either VU4H5, which recognizes MUC1-FL (full length), or MN-C2, which recognizes cancerous MUC1*. Tissue staining was scored using the Allred scoring method, which combines an intensity score and a distribution score. Below the photograph of the tissue array is a color-coded graph showing the results. As can be seen, despite published reports that MUC1 is aberrantly expressed in over 96% of all breast cancers as demonstrated by nucleic acid-based diagnostics, the arrays stained with VU4H5 were very pale, and many tissues did not stain at all. In contrast, the arrays stained with MN-C2 were very dark (red versus yellow or white in the figure). In addition, many tissues did not stain at all with anti-full-length MUC1, but stained very darkly with MN-C2 (see green box in the figure). Similarly, we stained normal or cancerous breast tissue with humanized MN-E6 scFv-Fc. The antibody fragment is biotinylated so it can be visualized with a streptavidin-based secondary antibody. As seen from Figure 12 As can be seen in Figure 3, hMN-E6 scFv-Fc does not stain normal breast tissue but stains cancerous breast tissue. In addition, the intensity and uniformity of staining increase with the patient's tumor grade and / or metastasis grade ( Figures 12 to 13 Similarly, hMN-E6 scFv-Fc did not stain normal lung tissue but stained lung cancer tissue ( Figures 14 to 18 ), and the intensity and distribution of staining increased with increasing tumor grade or metastasis grade. Figure 19 Photographs of normal and cancerous small intestinal tissue stained with 5 μg / mL humanized MN-E6-scFv-Fc biotinylated anti-MUC1* antibody followed by streptavidin-HRP secondary staining are shown. A) Normal small intestinal tissue. B) Small intestinal cancer from the patient shown. C and D are photographs of corresponding serial sections stained with the respective secondary antibodies. Figure 20 Shown are photographs of normal small intestinal tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are normal small intestinal tissue. EH are photographs of corresponding series of sections stained with the secondary antibody alone. Figure 21 Shown are photographs of cancerous small intestinal tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are cancerous small intestinal tissue from the patients shown in the figure. EH are photographs of corresponding serial sections stained with the secondary antibodies alone. Figure 22Shown are photographs of cancerous small intestinal tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are cancerous small intestinal tissue from the patients shown in the figure. EH are photographs of corresponding serial sections stained with the secondary antibodies alone. Figure 23 Shown are photographs of normal colon tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are normal colon. EH are photographs of corresponding series of sections stained with the secondary antibody alone. Figure 24 Shown are photographs of colon cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are colon cancer tissues from the metastatic patients shown in the figure. EH are photographs of corresponding serial sections stained with the secondary antibodies alone. Figure 25 Shown are photographs of colon cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are colon cancer tissues from the grade 2 patients shown in the figure. EH are photographs of corresponding serial sections stained with the secondary antibodies alone. Figure 26 Shown are photographs of colon cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are colon cancer tissues from the metastatic patients shown in the figure. EH are photographs of corresponding serial sections stained with the secondary antibodies alone. Figure 27 Shown are photographs of prostate cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are prostate cancer tissues from the patients shown in the figure. EH are photographs of corresponding serial sections stained with the secondary antibodies alone. Figure 28 Shown are photographs of prostate cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are prostate cancer tissues from the patients shown in the figure. EH are photographs of corresponding serial sections stained with the secondary antibodies alone. Figure 29 Shown are photographs of prostate cancer tissue stained with 50 μg / mL humanized MN-E6-scFv-Fc anti-MUC1* antibody and then stained with goat anti-human HRP secondary antibody. AD are prostate cancer tissues from the patients shown in the figure. EH are photographs of corresponding serial sections stained with the secondary antibodies alone.

[0555] One aspect of the invention is a method for treating a patient diagnosed with, suspected of having, or at risk of developing a MUC1-positive or MUC1*-positive cancer, wherein a sample is obtained from the patient's cancer and tested for reactivity with an antibody that binds to PSMGFR SEQ ID NO: 2, or more specifically, the N-10 peptide. The patient is then treated with an scFv, scFv-Fc, or CAR T comprising an antibody variable fragment from an antibody that reacted with their cancer sample or which can be selected from MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. Another aspect of the invention is a method for treating a patient diagnosed with, suspected of having, or at risk of developing a MUC1-positive or MUC1*-positive cancer, wherein a sample is obtained from the patient's cancer and tested for reactivity with MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11; and the patient is then treated with an antibody, antibody fragment, scFv, scFv-Fc-mut, BiTE, or CAR T comprising a portion of an antibody that reacts with their cancer sample.

[0556] As we have previously reported, it is MUC1*, the transmembrane cleavage product, rather than full-length MUC1, that is the growth factor receptor that drives tumor growth. Growth factors that activate MUC1* bind to ectopic sites that are exposed only after cleavage and release of the tandem repeat portion of MUC1. The antibodies of the present invention, like the activating growth factors, cannot bind to full-length MUC1. FACS analysis clearly showed that the anti-MUC1* antibody MNC2 could not bind to HCT-116 MUC1-negative cells ( Figure 35 A), robust binding to these cells if transfected with MUC1* ( Figure 35 B), but not to HCT cells transfected with full-length MUC1 ( Figure 35 C). The commercially available anti-tandem repeat antibody VU4H5 clearly recognizes full-length MUC1 ( Figure 35 D).

[0557] We found that MUC1 can be cleaved into MUC1* by more than one cleavage enzyme, and that the cleavage site affects its folding and therefore which monoclonal antibody can recognize this form of MUC1*. Different cancer cells or cancerous tissues express different cleavage enzymes. We tested various cleavage enzyme inhibitors on different cancer cell lines and found that inhibitors that inhibit MUC1 cleavage in one cancer cell line did not inhibit MUC1 cleavage in another cancer cell line. Similarly, PCR experiments showed that the cleavage enzymes are expressed at different levels in different cells or cell lines. For example, hematopoietic stem cells of the bone marrow express MUC1* that is recognized by the monoclonal antibody MNC3 but not by MNE6 or MNC2 (Figure 39). The growth of DU145 prostate cancer cells and T47D breast cancer cells was inhibited by Fabs of MNC2 and MNE6, but not by Fabs of MNC3 or MNC8, indicating that the cancer cell lines express MUC1* that is recognized by MNE6 and MNC2 but not by MNC3 or MNC8 ( Figure 42 PCR experiments showed that CD34-positive cells in the bone marrow expressed approximately 2,500 times more MMP2 and approximately 350 times more ADAM28 than T47D breast cancer cells, while DU145 prostate cancer cells expressed approximately 2,000 times more ADAM TS16, approximately 400 times more MMP14, and approximately 100 times more MMP1 than T47D breast cancer cells ( Figure 43 and Figure 44 In contrast, T47D breast cancer cells express about 80 times more MMP9 than myeloid cells and about twice as much as DU145 prostate cancer cells. Various cleavage enzyme inhibitors were tested for their ability to inhibit cleavage in different types of cancer cells.

[0558] General strategy using antibodies, antibody fragments, and CARs targeting the extracellular domain of MUC1*

[0559] In one aspect of the present invention, a second factor (which can be a cutting enzyme, an antibody, a cytokine or the second CAR) and CAR are transduced into the same T cell. In another aspect of the present invention, the second factor is on an inducible promoter so that its expression is activated when CAR engages with the targeted cancer cells. In some cases, the expression of the second factor is controlled by an inducible promoter. In one aspect of the present invention, when immune cells are activated, for example, when it recognizes or engages its target, the expression of the second factor is induced. In one example, T cells are transfected or transduced with the second factor, and its expression is induced when T cells recognize target cancer cells. One way is to induce the expression of the second factor when or shortly after NFAT protein expression or translocation to the nucleus. For example, the sequence derived from the NFAT promoter region is placed upstream of the second factor gene. In this way, when the transcription factor bound to the promoter of NFAT protein exists with a concentration sufficient to bind to the NFAT protein and induce the transcription of NFAT protein, they will also be combined with the same promoter of the engineering in front of the sequence for the second factor transcription. 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 aspect of the invention, the NFAT is NFATc1, NFATc3 or NFATc2. In one aspect of the invention, the NFAT is NFAT2, also known as NFATc1. SEQ ID NO: 646 shows the nucleic acid sequence of the upstream transcriptional regulatory region of NFAT2. The promoter sequence of the NFAT gene can include, for example, the nucleic acid sequences SEQ ID NO: 781-783 or SEQ ID NO: 815, but it can be seen that the optimal sequence or minimal sequence for expression of the second factor can be obtained by preparing fragments, extensions or mutations of the promoter and testing the strength of the promoter relative to the expression of the second factor. In one aspect of the invention, the transcriptional regulatory region of NFAT2 is engineered upstream of the gene encoding the second factor, the second factor being for the cleavage enzyme MMP9 (SEQ ID NO: 647) or the catalytic subunit of MMP9 (SEQ ID NO: 648). In one aspect of the invention, the NFAT is NFATc3 and the promoter sequence of NFATc3 includes the nucleic acid sequence from SEQ ID NO: 816. In one aspect of the invention, the transcriptional regulatory region of NFATc3 is engineered upstream of the gene encoding the second factor, here using MMP9 as an example. In another aspect of the invention, the NFAT is NFATc2. SEQ ID NOs: 817-818 show the nucleic acid sequence of the upstream transcriptional regulatory region of NFATc2.In one aspect of the present invention, the transcriptional regulatory region of NFATc2 is engineered upstream of the gene encoding the second factor, which may be the cleavage enzyme MMP9 (SEQ ID NO: 647) or the catalytic subunit of MMP9 (SEQ ID NO: 648).

[0560] Another method for making the expression of the second factor be induced when T cells or CAR T cells are activated is to place the gene of the second factor on an inducible promoter, on which the NFAT protein itself binds to the second factor and induces its transcription. In this case, the NFAT response element (NFAT RE) can be located upstream of the gene of the second factor or a fragment of the second factor. NFAT can bind to its response element upstream of the second factor alone or as a part of a complex. The NFAT protein can be NFATc1, NFATc2, NFATc3, NFATc4 or NFAT5. In a preferred embodiment, the NFAT protein is NFAT2, also known as NFATc1, also known as NFATc. The gene of the second factor or its fragment is cloned into the downstream of the NFAT response element (SEQ ID NO: 649) (which can be a repeat of the response element (SEQ ID NO: 650)) and the CMV minimal promoter (mCMV) (SEQ ID NO: 651) to induce the expression of the second factor by the NFAT protein. The NFAT response element can include a nucleic acid sequence NFAT consensus sequence (SEQ ID NO: 804). The NFAT response element may comprise, for example, a nucleic acid sequence SEQ ID NO: 805-814, but it can be seen that the optimal sequence or minimal sequence for expression of the second factor can be obtained by preparing a fragment, extension, or mutation of the response element nucleic acid and testing the response element relative to the intensity of expression of the second factor. The enhancer region of Foxp3 further comprises a NFAT response element (SEQ ID NO: 821) within 120 bp from 2079 to 2098. The NFAT response element may comprise a nucleic acid NFAT consensus sequence (5'-cattttttccat-3') (SEQ ID NO: 819) or (5'-tttttcca-3') (SEQ ID NO: 820) or a repetition thereof, to which NFATc1 specifically binds (Xu et al., Closely related T-memorystem cells correlate with in vivo expansion of CAR.CD19-T cells and arepreserved by IL-7and IL-15, Blood2014 123:3750-3759). NFAT response elements may also be separated by nucleic acid spacer sequences. Other NFAT response elements may exist and may be further discovered, and those skilled in the art, when directed to identify NFAT response elements, can perform molecular biological assays to obtain them based on the guidance of at least the response elements shown in SEQ ID NOS:804-814 (although only as examples). In one aspect of the invention, the cleavage enzyme located downstream of the NFAT response element and the CMV minimal promoter is MMP9 (SEQ ID NO:652). In another aspect of the invention, the cleavage enzyme is the catalytic subunit of MMP9 (SEQ ID NO:653).

[0561] Since NFAT 1-4 is regulated by the calcineurin pathway, the potential toxicity that may occur in patients can be prevented by treatment with immunosuppressants (such as FK506, cyclosporine, cyclosporine A or tacrolimus) that block calcineurin activity and inhibit NFAT translocation to the nucleus. T cells transduced or transfected with a cleavage enzyme on an inducible promoter can also be transfected or transduced with a CAR that recognizes a protein or molecule on a cancer cell. In a specific example, the cleavage enzyme is an enzyme that can cut the full length of MUC1, and the CAR carries an antibody fragment that directs it to the MUC1* on the surface of the cancer cell.

[0562] To determine which enzymes cleave MUC1 in cancer cells, we tested a series of inhibitors of MMPs and ADAM enzymes. These experiments implicated MMP9 as a key cleavage enzyme in cancer cells. To confirm that MMP9 cleaves MUC1 in cancer cells, we transfected HCT-116 MUC1-negative colon cancer cells with a mimic of full-length MUC1 containing 41 tandem repeats: HCT-MUC1-41TR. Through single-cell cloning, we were able to establish this cell line in which MUC1 is minimally cleaved to MUC1*. Figures 36A to 36D Western blot and FACS analysis are shown, which show that HCT-MUC1-41TR is 95% positive for full-length MUC1, but only 5%-10% positive for the cleaved form MUC1*. HCT-MUC1-41TR cells were incubated with different concentrations of MMP9 and then assayed by immunofluorescence to measure the binding of MNC2 monoclonal antibodies to the resulting cells. Figures 37A to 37C As can be seen in Figure 2, MNC2 binding increases with increasing concentrations of MMP9 added to cells. These experiments show that MMP9 cleaves MUC1 into a form that can be recognized by MNC2. Figure 30 A to Figure 30 F. Figure 31 A to Figure 31 F. Figure 32 A to Figure 32 F. Figure 33 A to Figure 33 F) Demonstrates that MNC2 recognizes a cleaved form of MUC1 that is present on cancerous tissue but not on healthy cells or tissues ( Figure 34 A to Figure 34 I). Importantly, MNC2 does not recognize the cleaved form of MUC1 expressed on healthy hematopoietic stem cells in the bone marrow (Figures 39 to Figure 41 ).

[0563] In one aspect of the invention, immune cells are transduced with CAR and a cleavage enzyme that targets immune cells to tumors. CAR and the cleavage enzyme can be encoded on the same plasmid or two different plasmids. In one aspect, the cleavage enzyme is on an inducible promoter. On the other hand, the expression of the cleavage enzyme is induced by a protein expressed when the immune cell is activated. In one case, the expression of the cleavage enzyme is induced by NFAT protein. On the other hand, the expression of the cleavage enzyme is induced by NFATc1. On the other hand, when one of the NFAT proteins is bound to an NFAT response element upstream of a gene into which a cleavage enzyme or a catalytically active fragment thereof is inserted, the expression of the cleavage enzyme is induced. In one aspect, the cleavage enzyme is a catalytically active fragment of MMP9 or MMP9.

[0564] In one aspect of the invention, the cleavage enzyme is MMP9 (SEQ ID NO: 643). Some cleavage enzymes are naturally expressed as zymogens that need to be activated. This can be achieved by biochemical means, by expressing a coenzyme that activates the cleavage enzyme, or by engineering the enzyme into an activated form. The present invention contemplates overcoming this problem by co-expressing the cleavage enzyme with its activator. In one aspect of the invention, the cleavage enzyme is MMP9 and the coactivator is MMP3. In another aspect of the invention, the cleavage enzyme is expressed in an already active form, for example, by expressing a cleavage enzyme fragment that still has catalytic function. In one case, the cleavage enzyme is a catalytically active MMP9 fragment. An example of an MMP9 catalytic fragment is given as SEQ ID NO: 645.

[0565] MMP9, which must be activated by MMP3, is overexpressed in a large proportion of solid tumors. Furthermore, the MNC2 anti-MUC1* monoclonal antibody is known to recognize MUC1 after cleavage by MMP9. Figures 30 to 33 The various breast, ovarian, pancreatic, and lung cancer tissue arrays shown in Figure 3 were probed with MNC2-scFv, further demonstrating that MUC1 in these cancers is cleaved by MMP9. To investigate whether MMP9 cleavage of tumors would increase T cell access to tumors, we used the cell line HCT-MUC1-41TR expressing full-length MUC1, a breast cancer cell line that highly expresses full-length MUC1 and MUC1*, and a cell line we used with MUC1*.45 A series of experiments were conducted using a transfected MUC1-negative cell line. We transfected the cells with MMP9 and MMP3, which activates MMP9. We removed the supernatant from these cells, which contains activated MMP9, and added it to various cells, which were then co-cultured with T cells transduced with an anti-MUC1* CAR (huMNC2-CAR44). The result was a significant increase in CAR T cell killing of the targeted MUC1 / MUC1*-positive cancer cells compared to control cells not incubated with the MUC1-cleaving enzyme.

[0566] APMA is a biochemical that activates MMPs. We used APMA and conditioned medium from cells that we transfected with MMP9 or ADAM17 to see if either of these cleaving enzymes would cleave MUC1 on the HCT-MUC1-41TR cell line, which only expresses full-length MUC1. As a control, we also tested the enzyme on HCT-MUC1* cells. Cells expressing MUC1 and MUC1* were stained with the red dye CMTMR. Human T cells transduced with anti-MUC1* CAR, CAR44, or CAR50 were co-cultured with cancer cells. Untransduced T cells were used as a control ( Figure 45 A to Figure 45 P). Figure 45 B. Figure 45 C and Figure 45 As can be seen in Figure D, anti-MUC1* CAR T cells effectively recognized and aggregated HCT-MUC1* cancer cells, a hallmark of T cell activation and killing. However, no CAR T cell-induced aggregation was observed in wells containing HCT-MUC1-41TR (cells expressing full-length MUC1). Figure 45 F. Figure 45 G and Figure 45 H). However, cells incubated with activated MMP9 showed a significant increase in CAR T cell-induced aggregation ( Figure 45 J. Figure 45 K and Figure 45 L), indicating that MMP9 cleaves full-length MUC1 into the MUC1* form, which is recognized by the MNC2 monoclonal antibody and, more specifically, by the huMNC2-scFv. ADAM17 has no apparent effect. ADAM17 either does not cleave MUC1 or, more likely, cleaves at a site not recognized by MNC2 ( Figure 45 N to Figure 45 P).

[0567] We performed the same experiment, this time using T47D breast cancer cells, which are refractory to killing using anti-MUC1* CAR T cells, presumably because they express high levels of full-length MUC1 and MUC1* ( Figure 46 A to Figure 46 T). Figure 46 B. Figure 46 C and Figure 46 As can be seen in D, anti-MUC1*CAR44 and CAR50 had little effect on T47D cancer cells. Figure 46 In D, which is the highest CAR expression level in T cells, CAR44, we see a small amount of CAR T cell-induced aggregation. However, activated MMP2 ( Figure 46 J. Figure 46 K. Figure 46 L) or activated MMP9 ( Figure 46 R. Figure 46 S. Figure 46 The presence of APMA showed a significant increase in CAR T cell recognition, aggregation, and killing, indicating that cleavage of full-length MUC1 increases T cell access to cancer cells. To ensure that the addition of APMA did not induce cleavage or anti-MUC1*CAR T recognition through some other mechanism, we prepared a catalytically active form of MMP9 and added it to T47D cells, which were then co-cultured with MNC2-CAR44 T cells ( Figure 47 A to Figure 47 I). As can be seen from the figure, MNC2-CAR T cells recognize and accumulate cells transfected with MUC1* ( Figure 47 B to Figure 47 C), T47D breast cancer cells expressing both full-length MUCl and MUCl* aggregate poorly ( Figure 47 E to Figure 47 F), but after addition of catalytically active MMP9, it robustly binds to and aggregates T47D cells ( Figure 47 H to Figure 47 I). This result supports the claim that MNC2 does not recognize full-length MUC1 but rather the growth factor receptor MUC1*. Note that full-length MUC1 expressed on this cell line may sterically hinder CAR T cell binding near the cell membrane.

[0568] In another example, T47D MUC1-positive tumor cells were incubated with 100 ng / mL or 500 ng / mL of the recombinant catalytic domain of MMP9 (Enzo Life Sciences, Inc., Farmingdale, NY). Western blot analysis showed that MUC1 / MUC1*-positive cancer cells underwent extensive cleavage of MUC1 into MUC1*. In another example, T47D breast cancer cells were pre-incubated with human recombinant MMP9 catalytic domain protein and then co-cultured with anti-MUC1* CAR44 T cells. The specific killing of T47D cells by CAR44 T cells was monitored in real time on an xCelligence instrument, which measured changes in impedance over time. This analysis uses an electrode array on which cancer cells are plated. Adherent cancer cells insulate the electrodes and cause an increase in impedance as they grow. In contrast, T cells do not adhere and remain suspended, so there is no increase or decrease in impedance. However, if T cells or CAR T cells kill cancer cells on the electrode plate, the cancer cells will sphericalize and float as they die, resulting in a decrease in impedance. The addition of the MMP9 catalytic domain significantly increased the killing of T47D cancer cells. Figure 48 The xCelligence graph shows T47D breast cancer cells co-cultured with untransduced T cells (as a control) or huMNC2-CAR44 T cells for a period of 45 hours. After 18 hours of cancer cell growth, the catalytic subunit MMP9 was added to some cells. At 25 hours, T cells were added. As can be seen, when T47D cells were pre-incubated with the cleavage enzyme MMP9, the killing of huMNC2-CAR44 T cells was greatly improved. In the xCelligence system, adherent target cancer cells are plated on the electrode array plate. Adherent cells insulate the electrodes and increase impedance. The number of adherent cancer cells is proportional to the impedance. T cells do not adhere and do not generate impedance. Therefore, increasing impedance reflects the growth of cancer cells, and decreasing impedance reflects the killing of cancer cells. The prostate cancer cell line DU145 expresses both MUC1 and MUC1*, but at much lower levels than T47D cells. In the presence or absence of the cleavage enzyme, DU145 cells are effectively killed by anti-MUC1*CAR T cells.

[0569] Figure 49The xCelligence graph shows DU145 prostate cancer cells co-cultured with untransduced T cells (as a control) or huMNC2-CAR44 T cells for a period of 45 hours. After the cancer cells grew for 18 hours, the catalytic subunit MMP9 was added to some cells. At 25 hours, T cells were added. As can be seen, the killing of low-density MUC1 / MUC1*-positive cancer cells by huMNC2-CAR44 T cells was not affected by pre-incubation with the cleavage enzyme MMP9. DU145 cancer cells express significantly lower amounts of MUC1, which includes both full-length forms and MUC1*. The lower density of full-length MUC1 does not spatially hinder T cells from accessing the membrane-proximal MUC1*. DU145 cells represent early-stage cancers, which express both full-length and cleaved MUC1, but at lower levels so that T cells are not spatially hindered from approaching. T47D cells represent mid-stage cancers, which express high levels of both MUC1 and MUC1*, where the density of full-length MUC1 spatially hinders T cells from accessing the tumor. HCT-MUC1* cells are a MUC1-negative cell line that has been treated with MUC1* 45 Stable transfection, and they represent advanced cancer cells. Importantly, MUC1 cleaved into MUC1* by MMP9 is recognized by the anti-MUC1* antibody MNC2, which is the targeting head of the CAR. The presence of bulky extracellular domain proteins or other obstructive factors (also known as the tumor microenvironment) may spatially hinder immune cells from approaching tumor antigens on the surface of cancer cells. The foregoing serves as an example of an approach that can be expanded to improve the efficacy of CAR T therapy targeting other tumor antigens. In one aspect of the invention, immune cells are transfected or transduced with a CAR comprising an antibody fragment targeting a tumor antigen and a cleavage enzyme. In another aspect of the invention, immune cells are transfected or transduced with both a CAR comprising an antibody fragment targeting a tumor antigen and a cleavage enzyme that cuts the tumor antigen into a form recognized by the antibody fragment of the CAR. In one aspect, immune cells are transfected or transduced with both a CAR comprising an antibody fragment targeting a tumor antigen and a cleavage enzyme that cuts the tumor antigen into a form recognized by the antibody fragment of the CAR, wherein the antibody fragment of the CAR recognizes the MUC1* extracellular domain and the cleavage enzyme cuts MUC1 into MUC1*. In one aspect, an immune cell (which can be a T cell or a NK cell) is transfected or transduced with a CAR and a cleavage enzyme, wherein the CAR comprises an antibody fragment derived from MNC2, MNE6, MNC3 or MNC8, and the cleavage enzyme is selected from the group comprising MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP11, MMP12, MMP13, MMP14, MMP16, ADAM9, ADAM10, ADAM17, ADAM 19, ADAMTS16, ADAM28 or a catalytically active fragment thereof.

[0570] In one aspect of the invention, a sufficient amount of immune cells transduced with both CAR and a cleavage enzyme is administered to a person diagnosed with cancer or at risk of developing cancer. In another aspect of the invention, a sufficient amount of immune cells transduced with both CAR and a cleavage enzyme is administered to a person diagnosed with cancer or at risk of developing cancer, wherein the cleavage enzyme is on an inducible promoter activated by a protein expressed when the immune cells are activated. In another aspect of the invention, a sufficient amount of immune cells transduced with both CAR and a cleavage enzyme is administered to a person diagnosed with cancer or at risk of developing cancer, wherein the cleavage enzyme is on an inducible promoter activated by one or more NFATs. In one case, NFAT is NFATc1. In another aspect, NFAT is NFATc3. In another aspect, NFAT is NFATc2. In any of the above cases, the extracellular domain of CAR comprises a fragment of an anti-MUC1* antibody. In one aspect, the anti-MUC1* antibody is a humanized form of MNC2scFv or MNC2scFv. In another aspect, the anti-MUC1* antibody is a humanized form of MNE6scFv or MNE6scFv. In any of the above cases, the immune cell may be a T cell, a NK cell, a mast cell or a dendritic cell.

[0571] The present invention is not intended to be limited to one or two ad hoc methods for expressing the cleavage enzyme induced by activated T cells. We have demonstrated that the cleavage enzyme is specifically expressed only when T cells are activated by constructing a plasmid with a cleavage enzyme gene downstream of the downstream of the NFAT promoter sequence or one or more repetitions of the NFAT response element. In another aspect of the invention, by constructing a plasmid, the plasmid is then inserted into an immune cell to induce the expression of the cleavage enzyme, in which the cleavage enzyme gene is inserted downstream of the IL-2 promoter sequence or downstream of the IL-2 response element. In another aspect of the invention, by constructing a plasmid, the plasmid is then inserted into an immune cell and then administered to a patient to induce the expression of the cleavage enzyme for the treatment or prevention of cancer, in which the cleavage enzyme gene is inserted downstream of the calcineurin promoter sequence or downstream of the calcineurin response element. There are also drug-inducible plasmids, which can be used to induce the expression of the cleavage enzyme or to prevent the expression of the elements induced by activated T cells. These drug-inducible systems may include tetracycline-inducible systems, Tet-on, Tet-off, tetracycline-responsive elements, doxycycline, tamoxifen-inducible systems, ecdysone-inducible systems, and the like.

[0572] The present invention is not intended to be limited to one or two specific promoters used in the plasmid encoding CAR or inducible cleavage enzyme. As known to those skilled in the art, many promoters can be interchangeable, including SV40, PGK1, Ubc, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1 and U6. Another method for solving the steric hindrance problem of CAR T cells approaching caused by bulky cell surface proteins such as MUC1-FL is to increase the length of the joint region of the CAR expressed by T cells. In the standard design CAR, the length of the extracellular joint region between the transmembrane portion and the antibody fragment is about 45-50 amino acids in length. We prepared long-arm CAR in which the length of the extracellular joint extends from about 50 amino acids to 217-290 amino acids. Co-culture assays show that CAR with a longer extracellular joint can improve access to tumor-associated antigens on target cancer cells.

[0573] BiTE

[0574] Bivalent (or bivalent) single chain variable fragments (di-scFv, bi-scFv) can be engineered by linking two scFvs. This can be achieved by generating a single chain variable fragment with two V H and two V L The single peptide chain of the variable region is formed to obtain a series scFv. Another possibility is to form an scFv with a linker peptide that is too short (about five amino acids) to fold the two variable regions together, thereby forcing the scFv to dimerize. This type is called a diabody. It has been shown that the dissociation constant of diabodies is up to 40 times lower than that of the corresponding scFv, which means that their affinity for their targets is much higher. Therefore, the dosage of diabody drugs can be much lower than other therapeutic antibodies and can target tumors with high specificity in vivo. Shorter linkers (one or two amino acids) will lead to the formation of trimers, so-called tribodies or tribodies. Tetrabodies have also been produced. Compared with diabodies, they show even higher affinity for their targets.

[0575] All of these formats can be composed of variable fragments specific for two different antigens, in which case they are bispecific antibody types. The most developed of these fragments is the bispecific tandem di-scFv, known as bispecific T cell engagers (BiTE antibody constructs). BiTEs are fusion proteins consisting of two scFvs of different antibodies, which are located on a single peptide chain of approximately 55 kilodaltons. One of the scFvs can bind to T cells, such as through the CD3 receptor, while the other can bind to tumor cells through a tumor-specific molecule, such as abnormally expressed MUC1*.

[0576] Another aspect of the invention is a method for treating a patient diagnosed with, suspected of having, or at risk of developing a MUC1-positive or MUC1*-positive cancer, wherein an effective amount of a BiTE is administered to the patient, wherein one variable fragment of the BiTE binds to a T cell surface antigen and another variable fragment of the BiTE binds to PSMGFR (SEQ ID NO: 2), or more specifically, to an N-10 peptide. In one embodiment, the variable fragment of the BiTE that binds to MUC1* comprises a portion of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11.

[0577] In another aspect of the invention, MUC1* peptides, including PSMGFR (SEQ ID NO: 2) or most or all of the N-10 peptide, are used in adoptive T cell transfer. In this approach, the patient's T cells are exposed to the MUC1* peptide and, after multiple rounds of maturation, the T cells develop MUC1*-specific receptors. The adapted T cells are then expanded and administered to a donor patient diagnosed with, suspected of having, or at risk of developing a MUC1*-positive cancer.

[0578] A series of CARs with MNC2 and humanized MNC2 as the extracellular targeting head of CAR were also prepared. The constructs of these CARs were inserted into plasmids, and then the plasmids were inserted into lentiviral vectors. Human T cells were then transduced with lentiviral vectors carrying MNC2 CAR and huMNC2CAR. Mouse sequence or humanized MNC2-scFv-CAR was produced. In one aspect of the present invention, CAR comprises huMNC2-scFv-short hinge region-transmembrane domain derived from CD8-short intracellular fragment-4-1BB-3ζ. On the other hand, the transmembrane domain is derived from the CD4 transmembrane sequence. On the other hand, the intracellular costimulatory domain is CD28-3ζ. On the other hand, the intracellular costimulatory domain is CD28-4-1BB-3ζ.

[0579] There are several methods for assessing whether T cells recognize target cells and are in the process of initiating an immune response. T cells aggregate when they recognize target or foreign cells. This can be easily seen with the naked eye or under low magnification. The aggregation of CAR T cells when co-cultured with target cancer cells is a measure of: a) whether they recognize the cells as target cells; and b) whether they are activated to attack the targeted cells, which in this case are cancer cells. Figures 45 to 47Shown are photos of MUC1*-positive T47D breast cancer cells stably transfected with mCherry or stained with CMTMR (thus red), co-cultured with human T cells without CAR or transduced with huMNC2-scFv-CAR44 or huMNC2-scFv-CAR50. CAR T cells are transparent. As can be seen, when T cells do not carry CAR, there is no T cell-induced cancer cell aggregation. However, in the case of T cells carrying MUC1*-targeting CAR, there is a significant aggregation of MUC1*-positive cancer cells.

[0580] After T cells recognize and recruit target cells, they overexpress perforin and granzyme B. Together, these two molecules activate cell death pathways in the targeted cells. Perforin is thought to create a hole in the target cell, into which the T cell injects granzyme B, which then activates apoptotic proteases, leading to the lysis of the target cell. Figure 55 and Figure 56 Shown are huMNC2-scFV-CAR44 T cells bound to target MUC1*-positive prostate and pancreatic cancer cells and infused with granzyme B.

[0581] Another measure of whether a T cell has recognized a target cell and has been activated to kill the cell is the upregulation and secretion of cytokines, interferon gamma (IFN-g) and interleukin-2 (IL-2) by the T cell. CAR T cell activation can be easily measured in vitro, as demonstrated by IFN-g and IL-2 secretion. CAR T cells are co-cultured with target cells, and after the incubation period, the conditioned medium is assayed by ELISA to detect secreted IFN-g and IL-2. To determine the cancer specificity of CAR T cells, in which the targeting head of the CAR is huMNC2 or huMNE6, these experiments were performed with huMNC2-CAR44 T cells and huMNE6-CAR44 T cells co-cultured with MUC1*-positive cancer cells and normal cells. Table 1 details the MUC1-positive normal or primary cells tested.

[0582] Table 1: Normal cell lines and primary cells

[0583]

[0584]

[0585]

[0586] Figure 5051 and 52). In addition to testing the IFN-g and IL-2 secretion of CAR T cells, normal cells were also assayed for signs of cell death, which may be induced by CAR T cells if the antibody targeting head is not very cancer-specific. After co-culture with huMNC2-CAR44 T cells, the cells were incubated with cell death markers and then assayed by FACS. huMNC2-CAR44 T cells do not induce cell death in normal cells ( Figures 53A to 53J ).

[0587] In addition to FACS analysis, many researchers now use xCELLigence instruments to measure cancer cell killing by CAR T cells. FACS is not the optimal method for tracking T cell-induced cell killing because T cells lyse target cells. Dead cells are difficult to measure with FACS because they are excluded as cellular debris, so a certain amount of cell killing must be inferred and various methods must be used to determine whether the missing cells are T cells or cancer cells.

[0588] The xCELLigence instrument uses an electrode array onto which cancer cells are plated. Adherent cancer cells insulate the electrodes, causing impedance to increase as they grow. In contrast, T cells do not adhere and remain suspended, thus not contributing to the electrode insulation that increases impedance. However, if T cells or CAR T cells kill cancer cells on the electrode plate, the cancer cells pellet and float out as they die, resulting in a decrease in impedance. The xCELLigence instrument measures changes in impedance over time, which correlates with cancer cell killing. The electrode plate also has an observation window. When CAR T cells effectively kill adsorbed target cancer cells, the impedance decreases, but no cancer cells remain on the plate surface.

[0589] Figure 55 A to Figure 55 H shows the cytotoxic effect of huMNC2-CAR44T cells on MUC1*-positive DU145 prostate cancer cells as measured by various assays. Figure 55 A is a fluorescent photograph of untransduced T cells co-cultured with prostate cancer cells, in which granzyme B was stained with a red fluorophore. Figure 55C is a fluorescent photograph of huMNC2-CAR44T cells co-cultured with prostate cancer cells, in which granzyme B was stained with a red fluorophore. Figure 55 D is the merge of DAPI and granzyme B. Figure 55 E is a FACS scan of fluorescently labeled granzyme B in non-transduced T cells incubated with cancer cells. Figure 55 F is a FACS scan showing the positive increase of fluorescently labeled granzyme B in hu MNC2-CAR44 T cells incubated with cancer cells. Figure 55 G is a graph of mean fluorescence intensity. Figure 55 H is an xCEL Ligence scan tracking real-time killing of DU145 cancer cells by huMNC2-CAR44 T cells (blue trace) but not untransduced T cells (green). Figure 56 A to Figure 56 H shows the cytotoxic effect of huMNC2-CAR44 T cells on MUC1*-positive CAPAN-2 pancreatic cancer cells as measured by various assays. Figure 56 A is a fluorescent photograph of untransduced T cells co-cultured with pancreatic cancer cells, in which granzyme B was stained with a red fluorophore. Figure 56 B is the merge of DAPI and granzyme B. Figure 56 C is a fluorescent photograph of huMNC2-CAR44T cells co-cultured with pancreatic cancer cells, in which granzyme B was stained with a red fluorophore. Figure 56 D is the merge of DAPI and granzyme B. Figure 56 E is a FACS scan of fluorescently labeled granzyme B in non-transduced T cells incubated with cancer cells. Figure 56 F is a FACS scan showing the positive increase of fluorescently labeled granzyme B in huMNC2-CAR44 T cells incubated with cancer cells. Figure 56 G is a graph of mean fluorescence intensity. Figure 56 H is an xCELLigence scan tracking real-time killing of CAPAN-2 cancer cells by huMNC2-CAR44 T cells (blue trace), but not untransduced T cells (green). Figures 57A to 57C Shown are xCELLigence scans tracking real-time killing of MUC1*-positive cancer cells, but not MUC1*-negative cells, by huMNC2-CAR44 T cells. Figure 57A huMNC2-CAR44 T cells were shown to effectively kill HCT colon cancer cells that had been stably transfected with MUC1*. Figure 57BThe results showed that huMNC2-CAR44 T cells had little effect on HCT-MUC1-41TR, a MUC1-negative cancer cell line that had been stably transfected with full-length MUC1. In this cell line, only about 10% of cells had MUC1 cleaved into MUC1*. Figure 57C It was shown that huMNC2-CAR44 T cells had no effect on HCT-116 cells, a MUC1 negative colon cancer cell line.

[0590] These data demonstrate that T cells transduced with a CAR in which the antibody fragment targeting head is MNC2 can effectively kill MUC1*-positive cancer cells. These data clearly indicate that huMNC2-scFV-CAR44 transduced into human T cells can effectively kill MUC1*-positive cancer cells. Because we and others have now demonstrated that the most important aspect of CAR T function is the targeting antibody fragment, immune cells or T cells transduced with any CAR containing the antibody fragment MNC2-scFV or huMNC2-scFV will have similar efficacy against MUC1 or MUC1*-positive tumors. For example, the hinge region connecting the scFv to the transmembrane portion can be any flexible linker. The intracellular costimulatory domain can be CD28-3ζ, CD28-4-1BB-3ζ, or any combination of immune cell costimulatory domains.

[0591] Figure 61 An experiment is shown in which the ability of human T cells transduced with huMNC2-scFv-CAR44 that were stimulated with beads (Scheme 1) or cancer cells (Scheme 2) to inhibit tumor growth in animals was tested. Human cancer cells stably transfected with luciferase were injected into female NOD / SCID / GAMMA (NSG) mice aged 11 to 15 weeks. 500,000 BT-20 breast cancer cells were injected subcutaneously into the flank. Tumor transplantation was verified by injecting luciferin into the animals and then imaging the fluorescent cancer cells using an IVIS instrument. IVIS images taken on the 5th day after implantation showed the presence of tumor cells. On the 6th day after IVIS measurement, the animals were injected with 10 million human T cells transduced with huMNC2-scFv-CAR44 or untransduced T cells. 5 million T cells were injected into the tumor and 5 million were injected into the tail vein. Ten minutes before IVIS imaging, mice were IP injected with luciferin, which fluoresces after being cleaved by luciferase, thereby making tumor cells fluorescent. Figure 61 A. Figure 61 D. Figure 61G shows a photograph of mice treated with huMNC2-scFv-CAR44 T cells that had been pre-stimulated by co-culture with 4 μm beads attached with synthetic MUC1*PSMGFR peptide for 24 hours prior to administration, "Scheme 1." Figure 61 B. Figure 61 E. Figure 61 H shows a photograph of mice treated with huMNC2-scFv-CAR44 T cells that had been pre-stimulated by co-culturing with MUC1*-positive cancer cells for 24 hours twice before administration, "Scheme 2". Figure 61 As can be seen in the figure, peptide-bead-stimulated huMNC2-CAR44 T cells inhibited tumor growth better than cells pre-stimulated by incubation with live cancer cells, which could contaminate target cells and increase tumor volume.

[0592] The ability of huMNC2-scFv-CAR44 transduced human T cells stimulated by beads (Scheme 1) or cancer cells (Scheme 2) to inhibit tumor growth in animals was also tested. Human cancer cells stably transfected with luciferase were injected into female NOD / SCID / GAMMA (NSG) mice aged 11 to 15 weeks. In another experiment, 500,000 BT-20MUC1* positive triple-negative breast cancer cells were subcutaneously injected into the flank. Tumor transplantation was verified by injecting luciferin into the animals and then imaging the fluorescent cancer cells using an IVIS instrument. IVIS images taken on the 6th day after implantation showed the presence of tumor cells. On the 6th day, after IVIS imaging, 10M huMNC2-scFv-CAR44 T cells were administered to the animals. 5M CAR T cells were administered by intratumoral injection, and another 5M were administered by tail vein injection. The control group was injected with the same number of untransduced T cells by the same route of administration. Tumor burden was measured by IVIS on days 6, 8, and 12. As from Figure 61 A to Figure 61 As can be seen in Figure 5J, both groups of mice treated with huMNC2-CAR44 T cells showed reduced tumor burden compared to the control group.

[0593] The ability of bead-stimulated (Scheme 1) huMNC2-scFv-CAR44 transduced human T cells to inhibit ovarian cancer growth in animals was also tested. Human SKOV-3 MUC1*-positive ovarian cancer cells stably transfected with luciferase were injected into 11- to 15-week-old female NOD / SCID / GAMMA (NSG) mice. In one experiment, 500,000 SKOV-3 cancer cells were injected into the peritoneal cavity to simulate human metastatic ovarian cancer. Tumor engraftment was verified by injecting the animals with luciferin and then imaging the fluorescent cancer cells using an IVIS instrument. IVIS images taken on day 3 after implantation showed the presence of tumor cells. On days 4 and 11, after tumor implantation, 10M huMNC2-scFv-CAR44 T cells were administered IP to the animals. On day 4, CAR T cells were injected IP. On day 11, half of the CAR T cells were injected into the peritoneal cavity, while the other half were injected into the tail vein. The control group was injected with the same number of non-transduced T cells or the same volume of PBS by the same administration route. Subsequent IVIS measurements of tumor burden were performed on days 7, 10, and 15. Figure 62 A to Figure 62 As can be seen in Figure 5, tumors in control mice grew much faster than in mice treated with huMNC2-CAR44 T cells. Figure 62 M shows the IVIS color bar relating photons / second to color.

[0594] One aspect of the invention is a method for treating a patient diagnosed with, suspected of having, or at risk of developing a MUC1-positive or MUC1*-positive cancer, wherein the patient is administered an effective amount of immune cells transduced with a CAR targeting MUC1*, wherein the CAR is selected from the group consisting of: MN-E6-CD8-CD28-3z (SEQ ID NOs: 297-298); MN-E6-CD4-CD28-3z (SEQ ID NOs: 748-749); MN-E6-CD8-41BB-3z (SEQ ID NOs: 300-301); MN-E6-CD4-41BB-3z (SEQ ID NOs: 750-751); MN-E6-CD8-CD28-41BB-3z (SEQ ID NOs: 751-751); NO:303-304); MN-E6-CD4-CD28-41BB-3z (SEQ ID NO:754-755); MN-E6scFv-Fc-8-41BB-CD3z (SEQ ID NO:310-311); MN-E6scFv-IgD-Fc-8-41BB-CD3z (SEQ ID NO:770-771); MN-E6scFv-FcH-8-41BB-CD3z (SEQ ID NO:315-316); MN-E6scFv-IgD-FcH-8-41BB-CD3z (SEQ ID NO:772-773); MN-E6scFv-Fc-4-41BB-CD3z (SEQID NO:318-319); MN-E6scFv-FcH-4-41BB-CD3z (SEQ ID NO:321-322); MN-E6scFv-IgD-8-41BB-CD3z (SEQ ID NO:323-324); MN-E6scFv-IgD-4-41BB-CD3z (SEQ ID NO:327-328); MN-E6scFv-X4-8-41BB-CD3z (SEQ ID NO:330-331); MN-E6scFv-X4-4-41BB-CD3z (SEQ ID NO:333-334); MN-E6scFv-8-4-41BB-CD3z (SEQ ID NO:336-337) or any of the above CARs, wherein MN-E6 is replaced by a fragment derived from MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3 or H11.Another aspect of the present invention is a method for treating a patient diagnosed with, suspected of having, or at risk of developing cancer, wherein an effective amount of immune cells transduced with one of the above-described CARs is administered to the patient, wherein MN-E6 is replaced with a peptide comprising an antibody variable domain fragment specific for a cancer antigen. In any of the above methods, the immune cells may be T cells and may be further isolated from the patient to be treated.

[0595] Other MUC1 cleavage sites

[0596] It is known that on some healthy cells, besides cancer cells, MUC1 is cleaved into its growth factor receptor form, MUC1*. For example, on healthy stem cells and progenitor cells, MUC1 is cleaved into MUC1*. Most bone marrow cells are MUC1*-positive. Parts of the intestine are MUC1*-positive.

[0597] The inventors have discovered that MUC1 can be cleaved at different locations relatively close to each other, but the location of the cleavage alters the folding of the remaining extracellular domain. As a result, monoclonal antibodies can be identified that bind to MUC1* cleaved at the first location but not to MUC1* cleaved at the second location. This discovery is disclosed in WO2014 / 028668, filed on August 14, 2013, the contents of which are incorporated herein by reference in their entirety. We identified a group of anti-MUC1* monoclonal antibodies that bind to MUC1* present on cancer cells but not to MUC1* present on stem and progenitor cells. Conversely, we identified a second group of monoclonal antibodies that bind to stem and progenitor cells but not to cancer cells. A method for identifying stem cell-specific antibodies is as follows: Supernatants from monoclonal hybridomas are adsorbed onto two multiwell plates. Stem cells, which are non-adherent cells, are placed in one plate, and adherent cancer cells are placed in the same plate. After the incubation period, the plates are rinsed and inverted. If non-adherent stem cells adhere to the plate, the monoclonal antibodies in a specific well will recognize the stem cells but not the cancer cells. Antibodies that do not capture stem cells or those that capture cancer cells are identified as cancer-specific antibodies. FACS analysis has confirmed the effectiveness of this method.

[0598] Antibodies MN-E6 and MN-C2 are examples of cancer-specific antibodies. Antibodies MN-C3 and MN-C8 are examples of stem cell-specific antibodies. Although both sets of antibodies can bind to peptides with PSMGFR sequences, FACS analysis showed that anti-MUC1* polyclonal antibodies and MN-C3 bound to MUC1*-positive bone marrow cells, but MN-E6 did not. The MUC1* polyclonal antibody was generated by immunizing rabbits with PSMGFR peptides. Similarly, MN-C3 bound to stem cells in intestinal crypts, but MN-E6 did not. Conversely, the MN-E6 antibody bound to cancerous tissue, while the stem cell-specific MN-C3 did not. Competition ELISA experiments demonstrated that the C-terminal 10 amino acids of the PSMGFR peptide are required for binding of MN-E6 and MN-C2, but not MN-C3 and MN-C8. Therefore, another method for identifying cancer-specific antibodies is to immunize with a peptide having the sequence of the PSMGFR peptide minus the N-terminal 10 amino acids, or to use such peptides to screen for cancer-specific antibodies or antibody fragments. An antibody that binds to a peptide having the sequence of the PSMGFR peptide minus the N-terminal 10 amino acids (referred to herein as the N-10 peptide) but does not bind to a peptide having the sequence of the PSMGFR peptide minus the C-terminal 10 amino acids (C-10 peptide) is a cancer-specific antibody for treating or preventing cancer.

[0599] The extracellular domain of MUC1 is also cleaved on stem cells and some progenitor cells, where activation of the cleaved MUC1 by the dimeric ligands NME1 or NME7 promotes growth and pluripotency and inhibits differentiation. The transmembrane portion of MUC1 that remains after cleavage is called MUC1*, and the extracellular domain consists essentially of the primary sequence of the MUC1 growth factor receptor (PSMGFR) sequence. However, the exact site of cleavage may vary depending on the cell type, tissue type, or the cleavage enzyme expressed or overexpressed by a particular person. In addition to the cleavage site we previously identified that leaves behind the transmembrane portion of MUC1* containing most or all of PSMGFR (SEQ ID NO:2), other cleavage sites may result in a sequence consisting of most or all of SNIKFRPGSVVVQTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:620); or

[0600] An extended MUCl* consisting of SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 621).

[0601] To test this hypothesis and determine whether antibodies directed against N-terminally extended PSMGFR would yield antibodies that are more cancer-specific than antibodies that bind to PSMGFR alone, we generated monoclonal antibodies by immunization with the following peptides:

[0602] (PSMGFR)GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 2),

[0603] (N+20 / C-27)SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTE(SEQ ID NO:822), or

[0604] (N+9 / C-9)VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVP(SEQ ID NO:824)

[0605] Monoclonal antibodies produced by immunization with the same peptide also show different reactivity to the same cancerous tissue samples. These results indicate that monoclonal antibodies recognize different conformations of the truncated MUC1 extracellular domain produced by immunization with peptides of different lengths, mimicking different cleavage sites, or cleavage at different sites in the host animal. Antibodies that recognize different cleavage site conformations may be cancer subtype specific or patient specific, depending on which cleavage enzyme their tumor expresses. In one aspect of the invention, a patient diagnosed with a certain type of cancer is treated with an antibody of the invention that recognizes cleaved MUC1, wherein the antibody is specific for cleavage with a specific enzyme known to be commonly expressed by the cancer subtype. On the other hand, the patient's tumor is analyzed to determine which enzyme his or her tumor expresses, and th...

Claims

1. An antibody or a fragment thereof for use in diagnosing, treating or preventing cancer, wherein the antibody specifically binds to PSMGFR peptide (SEQ ID NO: 2) or a fragment of the peptide.

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

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

4. The antibody or fragment thereof of claim 1 , which interacts with a peptide comprising the conformational epitopes ASRYNLT (SEQ ID NO: 1745), SVSDV (SEQ ID NO: 1751), and FPSA (SEQ ID NO: 1747) within the N-19 sequence ASRYNLT ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4), wherein mutation or deletion of ASRYNLT (SEQ ID NO: 1745) disrupts binding of the antibody or fragment thereof to the N-26 peptide. The antibody or fragment thereof according to claim 1 , which does not bind to the C-10 peptide (SEQ ID NO: 825). 6 . The antibody or fragment thereof according to claim 5 , which binds to the N-10 peptide (SEQ ID NO: 3) but does not bind to the C-10 peptide (SEQ ID NO: 825).

7. An antibody or fragment thereof for use in diagnosing, treating or preventing cancer, wherein the binding-inducing peptide sequence is located within the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6), wherein a mutation or deletion within FPFS (SEQ ID NO: 1747) disrupts the binding of the antibody or fragment thereof to PSMGFR.

8. An antibody or fragment thereof for use in diagnosing, treating or preventing cancer, which requires the presence of the antibody-binding conformation-inducing peptide ASRYNLT (SEQ ID NO: 1745) of PSMGFR (SEQ ID NO: 2).

9. An antibody or fragment thereof for use in diagnosing, treating or preventing cancer, which requires the presence of the antibody-binding conformation-inducing peptide SVSDV (SEQ ID NO: 1761) of PSMGFR (SEQ ID NO: 2).

10. An antibody or fragment thereof that specifically binds to PSMGFR (SEQ ID NO: 2) and N-10 (SEQ ID NO: 3); and (i) does not bind to full-length MUC1; (ii) does not bind to C-10 (SEQ ID NO: 825); (iii) Competitive inhibition of NME1 or NME7 AB Binding to the MUC1* extracellular domain or PSMGFR peptide; (iv) recognizes MUC1* produced by cleavage by the cleavage enzyme; (v) recognizes conformational epitopes and does not recognize linear epitopes; or (vi) Cancer selectivity by immunohistochemistry of tissues.

Images