Diagnostic method using anti-MUCI antibody

Abstract

To provide a method of determining the suitability of treating a patient suffering from cancer or metastasis of cancer characterized by aberrant expression of MUC1, with a MUC1* targeting therapeutic.SOLUTION: The present invention provides a method comprising contacting cells or tissue of a patient diagnosed with or suspected of having cancer, with an antibody that binds to a form of MUC1 that is devoid of the tandem repeat domain, wherein the presence of specific binding of the antibody to the cleaved or truncated form of a MUC1* targeting therapeutic indicates that the MUC1* targeting therapeutic is suitable to be used to treat the patient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for diagnosing cancer and determining the suitability of treating patients suffering from cancer or cancer metastasis characterized by abnormal expression of MUC1 with a MUC1 * targeted therapeutic agent, the method comprising contacting cells or tissues of a patient diagnosed with cancer or suspected of having cancer with an antibody that binds to a form of MUC1 lacking a tandem repeat domain, wherein the MUC1 * presence of specific binding of the antibody to a cleaved or cleaved form of the targeted therapeutic agent indicates that the * targeted therapeutic agent is suitable for use in treating the patient.

Background Art

[0002] Here, MUC1 * is defined as a transmembrane cleavage product of MUC1 that functions as a growth factor receptor and lacks a tandem repeat sequence. However, MUC1 can be cleaved by different enzymes that cleave at different sites. Which cleavage enzyme clips MUC1 can be tissue-specific or patient-specific. The * conformation of the extracellular domain of MUC1 can vary depending on which cleavage enzyme cleaves it. Anti-MUC1 * antibodies may bind to the extracellular domain of the transmembrane receptor remaining after cleavage.

[0003] In one aspect, the antibody can bind to peptides of the primary sequences of MUC1 proliferating factor (PSMGFR), PSMGFR N-10, PSMGFR N-10, PSMGFR C-10, or can bind to PSMGFR N-10 but cannot bind to PSMGFR N-10, or cannot bind to PSMGFR N-10, or can bind to PSMGFR N+20 peptides such as N+20 / C-22, N+20 / C-41, or N+20 / C-27 peptides, or N+9 / C-9 peptides. The antibody can bind to peptides having sequences that are extended terminally beyond the PSMGFR sequence. The antibody can bind to peptides of the sequence N+20-PSMGFR or N+9-PSMGFR. In one embodiment of the invention, anti-MUCl * Diagnostic assays using the antibody or fragments thereof are used to screen patients to determine their potential benefit from MUC1 * targeted therapeutics. In one embodiment of the invention, the antibody used in the diagnosis, and the antibody or fragments thereof incorporated into the therapeutic agent, are derived from the same antibody. The species of the diagnostic antibody and the therapeutic antibody need not be the same.

[0004] In one example, (i) a suspicious cell or tissue sample, which may be a biopsy, from a patient diagnosed with or suspected of developing cancer is contacted with an anti-MUC1 * antibody; (ii) a normal cell or tissue sample from the patient or a healthy donor is contacted with the same anti-MUCl * antibody, which may be a stored reference sample; (iii) antibody binding is detected; (iv) the degree and pattern of antibody binding to the suspicious sample are compared to those of the normal sample; (v) the determination that the suspicious sample overexpresses MUC1 * or expresses MUC1 * in a uniform pattern as opposed to apical border-limited expression indicates that the patient has a MUC1 * positive cancer; (vi) a therapeutic agent incorporating the anti-MUCl * antibody or fragments thereof is administered to the patient.

[0005] In another aspect of the present invention, the anti-MUC1 * antibody can be conjugated to a contrast agent for use in a patient as a whole body diagnostic to determine whether the patient has a MUC1 * positive tumor or, depending on the specific antibody used, whether the patient will benefit from a therapeutic agent comprising all or a fragment of the antibody conjugated to the contrast agent. The types of diagnostic and therapeutic antibodies need not be the same. Antibodies produced in camelid species are particularly useful in in vivo diagnostic assays because camelids produce small monovalent antibodies that have a short half-life in humans.

[0006] In another aspect of the present invention, the anti-MUC1 * antibody that can be conjugated to a contrast agent is used during surgery to detect or mark cancerous tissue so that it can be excised during surgery.

[0007] In another aspect of the present invention, an anti-MUC1 * antibody or fragment thereof that binds to a peptide having some or all of the sequence of the PSMGFR peptide is used for the diagnosis and / or treatment of breast cancer.

[0008] In another aspect of the present invention, an anti-MUC1 * antibody or fragment thereof that binds to a peptide having some or all of the sequence of the PSMGFR peptide extended at the N-terminus by 20 amino acids is used for the diagnosis and / or treatment of pancreatic cancer.

[0009] In another aspect of the present invention, an anti-MUC1 * antibody or fragment thereof that binds to a peptide having a part or all of the sequence of the PSMGFR peptide and extended at the N-terminus by about 20 amino acids is used for the diagnosis and / or treatment of esophageal cancer.

[0010] In another aspect of the present invention, an anti-MUC1* An antibody or fragment thereof is used for the diagnosis and / or treatment of prostate cancer.

[0011] In one aspect, the treatment agent targets MUC1 * which can be cancer immunotherapy. MUC1 * The targeted therapeutic agent can be a CAR T, BiTE, ADC (antibody-drug conjugate), bispecific antibody or antibody mimetic.

[0012] MUC1 * The targeted therapeutic agent may be an antibody that binds to the cleaved form of MUC1, and the cleaved form is the extracellular domain of the transmembrane receptor remaining after cleavage. The antibody can bind to a peptide known as the primary sequence of the MUC1 growth factor (PSMGFR), or a peptide with an N-terminal extension of up to 20 amino acids beyond the PSMGFR sequence. The antibody used in the treatment may be derived from the antibody used in the diagnostic assay, but does not have to be produced in the same species of animal.

[0013] The method of the present invention can be an in vitro assay. This assay can be performed on tissue specimens, body fluid samples, or blood samples.

[0014] In another aspect, the assay can be an in vivo assay. A contrast agent can be attached to the antibody.

[0015] In another aspect, the present invention may include a second antibody, and the step may include determining the ratio of the amount of the first antibody to the second antibody. The first antibody can bind to the extracellular domain of the transmembrane receptor remaining after cleavage, and the second antibody can bind to a part of the extracellular domain of MUC1 that is the N-terminus of the cleavage site such as a tandem repeat sequence.

[0016] In another aspect, for all of the above methods, a non-human, human or humanized anti-MUC1 * antibody or antibody fragment or antibody-like protein can specifically bind to the following

[0017] (i) The PSMGFR region of MUC1;

[0018] (ii) The PSMGFR peptide described in SEQ ID NO:4;

[0019] (iii) A peptide having the following amino acid sequence of PSMGFR N+20 / C-22 SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:5);

[0020] (iv) A peptide having the amino acid sequence of PSMGFR N+12 / C-22 SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:6);

[0021] (v) A peptide having the amino acid sequence of PSMGFR N+9 / C-30 VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO:7);

[0022] (vi) A peptide having the following amino acid sequence of PSMGFR N+20 / C-41 SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO:8)

[0023] (vii) A peptide having the following amino acid sequence of PSMGFR N+20 / C-27 SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTE (SEQ ID NO:9)

[0024] (viii) A peptide having the amino acid sequence of PSMGFR N+9 / C-9 VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVP (SEQ ID NO:10).

[0025] An antibody that binds to the extracellular domain of the transmembrane receptor remaining after cleavage, The SDIX SRY polyclonal antibody, MNC2 monoclonal antibody, MNE6 monoclonal antibody, or monoclonal antibodies 1E4, 29H1, 31A1, 32C1, 45C11 that are reactive with PSMGFR N+20 / C-27; 17H6, 39H5, 3C5, 8A9 that are reactive with PSMGFR N+9 / C-9; 18G12, 20A10, 25E6, 28F9, 18B4 that are reactive with PSMGFR, and it may be MNC2 and MNE6 that are reactive with PSMGFR. These antibodies can be of human, humanized, mouse, camel, llama, alpaca, camel, rabbit, goat, hamster or other non-human species.

[0026] These and other objects of the present invention will be more fully understood from the following description of the invention, the accompanying drawings and the appended claims.

Brief Description of the Drawings

[0027] The present invention will be more fully understood from the following detailed description of the invention herein and the accompanying drawings, which are given by way of illustration only and thus do not limit the present invention;

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[0083] Figure 55C shows the MNC2 monoclonal anti-MUC1 *An array stained with an antibody, where the antibody binds to the PSMGFR peptide. Figure 55D shows an array stained with the 45C11 monoclonal anti-MUC1 * An array stained with an antibody, where the antibody binds to the PSMGFR N+20 / C-27 peptide. These results are consistent with the idea that in most esophageal cancers, MUC1 is cleaved by an enzyme that exposes a potential epitope at the N-terminus of the PSMGFR sequence.

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[0102] In addition to the monoclonal antibodies MNC2, MNE6, MNC3, MNC8, and 18B4, 18G12, 20A10, 25E6, 1E4, 29H1, 31A1, 32C1, 4501, 3C5, 8A9, 17H6, and 39H5 disclosed in the present application, other monoclonal antibody sequences made from inoculation with PSMGFR peptides are listed in SEQ ID NOs: 237 - 349.

Mode for Carrying Out the Invention

[0103] In the present application, "a" and "an" are used to refer to both single objects and plural objects.

[0104] As used herein, sometimes briefly stated, a polypeptide is shown as "transduced or transfected" into a cell. In these occurrences, it is understood that the nucleic acid encoding the polypeptide sequence is transduced or transfected into the cell, since it is not possible for the polypeptide to be transduced or transfected into the cell itself.

[0105] As used herein, when referring to the number of cells injected into an animal, or in other contexts where the number of cells is referenced, "M" refers to millions and "K" refers to thousands.

[0106] As used herein, interchangeable names for various monoclonal antibodies are used, such as "MN-C2" which is interchangeable with "C2", "Min-C2" and "MNC2"; "MNE6" 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".

[0107] As used herein, "h" or "hu" placed before an antibody construct is an abbreviation for human or humanized.

[0108] As used herein, "antibody-like" means a molecule that includes a portion of an antibody but is engineered to not be a naturally occurring antibody. Examples include, but are not limited to, CAR (chimeric antigen receptor) T cell technology and Ylanthia <登録商標> technology. CAR technology uses antibody epitopes fused to a portion of a T cell so that the body's immune system attacks specific target proteins or cells. Ylanthia <登録商標> technology consists of a library of "antibody-like" synthetic human Fabs, which are then screened for binding to peptide epitopes from a target protein. The selected Fab regions can then be engineered into scaffolds or frameworks so that they resemble antibodies.

[0109] As used herein, "PSMGFR" is an abbreviation for the primary sequence of the MUC1 growth factor receptor identified by SEQ ID NO: 4 and should not be confused with the 6 amino acid sequence. "PSMGFR peptide" or "PSMGFR region" refers to a peptide or region that incorporates the primary sequence of the MUC1 growth factor receptor (SEQ ID NO: 4).

[0110] As used herein, the term "PSMGFR" is an acronym for the primary sequence of the MUC1 growth factor receptor described below; GTINVHDVETQFNQYKTEAASRYNFTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4). In this regard, "N-10 PSMGFR", "N-15 PSMGFR" or "N-20 PSMGFR" refer to the number of amino acid residues deleted at the N-terminus of PSMGFR, and "N+10 PSMGFR", "N+15 PSMGFR" refer to the number of amino acid residues added at the N-terminus of PSMGFR. Fibrous "C-numbers" such as "C-10 PSMGFR", "C-15 PSMGFR", or "C-20 PSMGFR" mean the number of amino acid residues deleted at the C-terminus of PSMGFR, and "C+10 PSMGFR", "C+15 PSMGFR", or "C+20 PSMGFR" mean the number of amino acid residues added at the C-terminus of PSMGFR. Also, possible combinations such as "N+20 / C-27 PSMGFR", "PSMGFR N+20 / C-27" or "N+20 / C-27" mean a peptide in which 20 amino acids of the MUC1 peptide are added to the N-terminus of PSMGFR and 27 amino acids are deleted from the C-terminus of PSMGFR.

[0111] As used herein, when it is desired to refer to the genus of PSMGFR peptides, they are referred to as "PSMGFR groups". For example, an "N+20 PSMGFR group" refers to a peptide having an additional 20 amino acids at the N-terminus, regardless of how the C-terminus is modified, whether amino acids are deleted or added, etc.

[0112] As used herein, "MUC1 * extracellular domain" refers to the extracellular portion of the MUC1 protein lacking the tandem repeat domain. In most cases, MUC1 * is MUC1 *The cleavage product consists of a short extracellular domain without tandem repeats, a transmembrane domain, and a cytoplasmic tail. The exact position of MUC1 cleavage is not known, presumably because it can be cleaved by two or more enzymes. MUC1 * The extracellular domain of MUC1 contains most of the PSMGFR sequence, but may have an additional 10-20 N-terminal amino acids.

[0113] As used herein, the "MUC1 * " extracellular domain mainly consists of the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNFTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4)). The exact site of MUC1 cleavage depends on the enzyme that cleaves it, and since the cleavage enzyme varies depending on cell type, tissue type, or time in the evolution of the cell, the exact sequence of the MUC1 * extracellular domain can vary at the N-terminus.

[0114] Other clipped amino acid sequences may include SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 5); or SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 6).

[0115] As used herein, "sequence identity" means homology to a reference sequence of nucleic acid or amino acid in the sequence of a particular polypeptide or nucleic acid, such that the function of the homologous peptide can be the same as that of the reference peptide or nucleic acid. Such homology is very close to the reference peptide, such that the two sequences can sometimes be 90%, 95% or 98% identical, but retain the same function in binding or other biological activities.

[0116] As used herein, "MUC1 positive" cells refer to cells that express the gene for MUC1, MUC1-Y or MUC1-Z or other MUC1 variants.

[0117] As used herein, a "MUC1-negative" cell refers to a cell that does not express the gene for MUC1.

[0118] As used herein, "MUC1" * "positive" cells refer to cells that express the MUC1 gene, where the protein expressed by that gene is a transmembrane protein that does not contain tandem repeats, which can be the result of post-translational modification, cleavage, alternative splicing, or transfection or transduction of cells with a MUC1 protein that does not contain tandem repeats.

[0119] As used herein, "MUC1" * "negative" cells refer to cells that may or may not express the gene for MUC1, but do not express a MUC1 transmembrane protein lacking tandem repeats.

[0120] As used herein, "MUC1-positive" cancer cells refer to cancer cells that overexpress the MUC1 gene, express MUC1 in an abnormal pattern, where the expression is not limited to the apical border, and / or express a MUC1 lacking tandem repeats.

[0121] As used herein, "MUC1-negative" cancer cells refer to cancer cells that may or may not express the MUC1 gene, but do not overexpress MUC1 or do not overexpress a MUC1 transmembrane protein lacking tandem repeats.

[0122] As used herein, "MUC1" * "positive" cancer cells refer to cancer cells that overexpress a MUC1 transmembrane protein lacking tandem repeats.

[0123] As used herein, "MUC1" * "negative" cancer cells refer to cancer cells that may or may not express the MUC1 gene, but do not overexpress a MUC1 transmembrane protein lacking tandem repeats.

[0124] The present invention generally includes cancer-related diagnostic assays characterized by abnormal expression of a class of cell surface receptors characterized by increased cleavage of the interchain binding region or extracellular domain in cancerous tissue. Such a set of cancers are cancers characterized by abnormal expression of mucin family proteins such as MUC1, MUC2, MUC3, MUC4 up to MUC16. Much of the description of the present invention herein is directed to cells and tissues that abnormally express MUC1 as an example of a larger class of proteins involved in cancers that have extracellular domains that are increasingly cleaved in cancer and / or have an interchain binding region (IBR). In these cases, this description should be considered illustrative, and it should be understood that the principles of the present invention apply to other transmembrane proteins that function by similar mechanisms. Using the disclosure herein, one of ordinary skill in the art can readily identify other transmembrane proteins that function by this or similar mechanisms and apply the present invention to cancers characterized by abnormal expression of the receptor. The present invention is based on a novel mechanism involving transmembrane proteins that self-aggregate and / or have regions of their extracellular domains that are progressively cleaved, which is exemplified by MUC1 elucidated by the inventors.

[0125] MUC1 contains several regions that are referred to herein as follows. From the C-terminal inside the cell to the N-terminal outside the cell, the MUC1 protein is composed of 1) a cytoplasmic tail; 2) a transmembrane portion; 3) an MGFR; 4) an IBR (interchain binding region); 5) a UR (unique region); and 6) a tandem repeat domain.

[0126] One aspect of the inventors' previous invention was characterized by the discovery that a specific region of the MUC1 receptor (i.e., the IBR) strongly binds to the same region of other MUC1 molecules. That is, the MUC1 receptor has the ability to aggregate (i.e., self-aggregate) with other MUC1 receptors via the IBR of each receptor. MUC1 or its cleavage product MUC1 *A gold nanoparticle experiment was conducted to show that the IBR that can block the binding of ligands thereto aggregates with itself. The boundary between IBR and MGFR changes depending on where MUC1 is cleaved, which is determined by which cleavage enzyme cleaves it.

[0127] This self-aggregation may contribute to the formation of MUC1 receptor clusters observed in healthy cells. The finding that the IBR portion of the MUC1 receptor self-aggregates is consistent with the following mechanistic model in which the inventors present supporting evidence. (1) The aggregated IBR portions act as functional receptors to block the access of ligands such as growth factors and modifying enzymes to the adjacent extracellular portion of the MUC1 receptor, so receptor cluster formation is associated with a healthy state; cluster formation also blocks the access of the intracellular tail to intracellular modifying enzymes and signaling ligands; (2) When the MUC1 receptor is cleaved at the position where it releases some or all of the self-assembled portion, the critical force that keeps the receptor clustered is lost, and the receptor moves freely within the cell membrane or interacts with activating ligands or growth factors or modifying enzymes such as other cell surface receptors, secreted ligands. These interactions involve new induced multimerization states such as dimerization that trigger the cell growth signaling cascade.

[0128] Cleavage of MUC1 releases most of the extracellular domain containing the tandem repeat domain, leaving a transmembrane protein with a cleaved extracellular domain containing at least the PSMGFR region. Cleavage and release of most of the tandem repeat domain exposes the binding site for ligands that bind to the cleaved extracellular domain and dimerize, leading to activation of the growth and survival pathways. We refer to the MUC1 cleavage product as "MUC1 * ".

[0129] MUC1 *is a growth factor receptor activated by ligand-induced dimerization of its cleaved extracellular domain. A bivalent antibody that binds to the PSMGFR peptide, a 45-amino acid sequence in the membrane-proximal portion of MUC1, dimerizes MUC1 * and stimulates proliferation. Anti-PSMGFR antibodies stimulated the proliferation of T47D MUC1-positive cancer cells in a concentration-dependent manner. In a similar experiment, the concentration of the anti-PSMGFR antibody that was identified to maximize cancer cell proliferation was added to the first group of T47D tumor cells grown as described above. The same amount of anti-PSMGFR antibody was added to a set of control cells, K293 cells. Addition of the anti-PSMGFR antibody to MUC1 tumor cells (T47D) enhanced proliferation up to 180% by 24 hours, but had no effect on the control cells.

[0130] MUC1 * ligands that digest the extra-cellular region of MUC1 induce cell growth and survival. The MUC1 * ligands identified by the inventors are NME1, NME2, NME6, NME7-AB and the alternative splice variant NME7-X1.

[0131] MUC1 * is a growth factor receptor that promotes the proliferation of cancer cells, but full-length MUC1 is not. Thus, detecting an amount of MUC1 * above normal levels serves as an indicator of cancer, and the greater the amount of MUC1 * , the worse the cancer. Cleavage of MUC1 can occur at multiple sites depending on which cleavage enzymes the tumor expresses. Cleavage of MUC1 releases a portion of the extracellular domain that contains tandem repeats and, depending on the cleavage site, may include a portion of the unique region of the IBR or a portion of the IBR. The amount of cleaved MUC1 can be inferred by measuring the amount of full-length MUC1 remaining on the cell or tissue. This can be accomplished by contacting the cell or tissue with an antibody that binds to the tandem repeats, or the unique region or IBR. Antibodies that bind to the tandem repeat domain have the sequence PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 235) The antibody can bind to a peptide having the following structure: Commonly used antibodies that bind to tandem repeat domains include, but are not limited to, VU4H5 (SantaCruz Biotechnology, Dallas Texas Cat. No. SC-7313), HMPV,5E5 (Sorensen et al., Glycobiology, Vol. 16, no. 2, pp. 96-107,2006), PR81, and LDQ10. In these cases, MUC1 expressed on the same cells or tissues * It is most useful to measure the amount of full-length MUC1 relative to the amount of MUC1. * The ratio of MUC1 to full-length is an indicator of cancer and cancer aggressiveness, * The more MUC1 is present, the more aggressive the cancer is. * or MUC1 to determine the suitability of MUC1-targeted cancer therapy. * or MUC1 relative to full-length MUC1 * Similarly, the efficacy of such treatments can be assessed by measuring the ratio of MUC1 * amount of MUC1 * The ratio of expressed MUC1 to full-length MUC1 can be assessed. * Decreased amount of MUC1 * A shift in the ratio to full length MUC1 is indicative of efficacy.

[0132] There may be alternative splice isoforms of MUC1 that do not contain IBR or tandem repeats. For example, MUC1-Y or MUC1-X. These alternative splice isoforms still have the extracellular domain composed of the sequence of the PSMGFR peptide, since this is the part that interacts with growth factors to promote cancer and survival. Thus, the MUC1 expressed by a cell or tissue may be expressed by the cytoplasmic domain of the PSMGFR peptide. * Detection of the amount of cancer remains a valid indicator of cancer and cancer aggressiveness.

[0133] The predominant MUC1 species in breast cancer tissues is the transmembrane cleavage product MUC1 *and is not full-length MUC1. Breast tumor microarrays were probed with either VET4H5 or MNC2. VU4H5 is a monoclonal antibody that binds only to full-length MUC1 because it recognizes an epitope (PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 235)) within the tandem repeat domain of full-length MUC1. This epitope is repeated hundreds of times within the tandem repeat domain of full-length MUC1. Therefore, antibody VU4H5 should give a stronger signal than an antibody that binds to a single epitope on the molecule. MNC2 is a monoclonal antibody produced by immunizing animals with the PSMGFR peptide (SEQ ID NO: 4). Transfection experiments show that MNC2 does not bind to full-length MUC1. MNC2 binds to MUC1 when MUC1 is cleaved into a form that includes at least the first 35 membrane-proximal amino acids of the extracellular domain * of MUC1 * and binds to a potential epitope that is exposed after cleavage, indicating that its cognate epitope is at least partially contained within the 10 membrane-proximal amino acids of the extracellular domain of MUC1 * . Importantly, MNC2 competitively inhibits the binding of the activation growth factors NME1 and NME7-AB to MUC1 * .

[0134] Figures 1A - 1D show photographs of adjacent consecutive sections of a breast cancer tissue array and graphical representations of the pathological scores according to the Allred scoring system. The pathological score ranges from 0 to 3, where 0 indicates no staining and 3 indicates maximum staining. The graph is also color-coded, with a pathological score of zero being black, 1 being yellow, 2 being orange, and 3 being red; tissues that scored zero when probed with an antibody that recognizes full-length MUC1 but were scored positive when probed with an antibody that recognizes MUC1 * are green; missing or uninterpretable tissues were scored -1. Figure 1A shows a photograph of a breast cancer tissue array after staining with VU4H5, an antibody that binds to the tandem repeat domain of full-length MUC1. Figure 1B shows a graph of the pathological scores for the tissues depicted in Figure 1A. Figure 1C shows a photograph of a breast cancer tissue array after staining with MNC2, an antibody that binds to an epitope within the PSMGFR region of MUC1 * Figure 1D shows a graph of the pathological scores for the tissues depicted in Figure 1C. Figure 1D shows a graph of the pathological scores for the tissues depicted in Figure 1C. Figures 2A - 2B show pie charts of the pathological scores of the arrays shown in Figures 1A and 1C. Figure 2A shows that antibodies that bind to the tandem repeats of full-length MUC1 miss 30% of breast cancers. Figure 2B shows that the anti-MUCl * antibody MNC2 recognizes 95% of breast cancers. Anti-MUCl-full length binds strongly only to 10% of breast tumors, while the anti-MUCl * antibody MNC2 binds strongly to approximately 50% of breast tumors. Taken together, these data demonstrate that MUC1 * , rather than full-length MUC1, is the predominant MUC1 species on cancer tissues. The anti-MUCl * antibody would detect or diagnose almost all breast cancers, while the antibody that binds to full-length MUC1 would not detect approximately 30% of breast cancers. Furthermore, since MUC1 * is a growth factor receptor that promotes cancer growth, the degree of anti-MUCl * staining of tissue or cell specimens would likely be proportional to the degree or stage of cancer, while the expression of full-length MUC1 appears to be inversely proportional to the stage of cancer.

[0135] A wide range of cancer cells and tumor specimens were probed with the anti-MUCl * antibody MNC2. Using MNC2, MUC1 was assayed in a wide range of assays including fluorescence-activated cell sorting (FACS), immunofluorescence (IF), and immunohistochemistry (IHC) *Positive cancer was detected. FACS and IF are generally used to study cell lines, which are single immortalized cells that have been grown in the laboratory for decades. After growth in unnatural growth solutions for decades, these cell lines show little resemblance even to single cells within the patient's original tumor and do not represent the tumors of recently diagnosed patients seeking treatment. For these reasons, the inventors analyzed thousands of tumor microarrays. Here, each dot within the array is a tumor specimen from a single patient's biopsy. In most cases, the biopsy is from a recently diagnosed patient, and the attached anonymized patient data indicates the patient's age, cancer subtype, and cancer stage or malignancy. In some cases, tissue microarrays in which all breast cancers were either all HER2+ or all ER+ / PR+ were analyzed. In other cases, we analyzed tumor microarrays comparing the first biopsy specimen to later metastases. In these studies, tumor recognition by MNC2 was also compared to staining using the anti-full length MUC1 antibody VU4H5 or the novel antibody 5E5 that binds to captured O-linked glycans within the tandem repeat domain of full length MUC1. MNC2 and other anti-MUCl * antibodies consistently recognized tumor tissue better than VU4H5 or 5E5. Normal tissues and normal tissue microarrays were also extensively studied to determine the binding of MNC2 or its humanized single chain form huMNC2-scFv or huMNC2-scFv-Fc to normal tissues. In normal tissues, MNC2-reactive MUC1 * expression was limited to the ducts and apical margins of glands in only a few tissues. In all cases, MNC2-reactive MUC1 * was expressed at much higher levels in cancer tissues than in normal tissues, with expression exceeding 50 - 100% in cancer tissues compared to 0.2% - 5% expression in normal tissues expressing MNC2-reactive MUC1 * .

[0136] Figures 3 - 19 show that the monoclonal anti-MUCl * antibody MNC2 binds to a high percentage of breast, ovarian, pancreatic, lung, and esophageal cancers, while binding to normal tissues is minimal if present. Figures 3A-3B show a pie chart of the pathological score and a photograph of breast cancer array BR1141 after staining with the anti-MUC1 * antibody huMNC2-scFv-Fc. Figures 4A-4C show photographs of individual breast cancer specimens from breast cancer array BR1141 at two different magnifications after staining with the anti-MUC1 * antibody huMNC2-scFv-Fc. The position within the array, cancer subtype, tumor malignancy, TNM (Tumor stage, Node involvement, and Metastasis), and pathological score were graphed. Standard immunohistochemical methods were used. Antibody titers were measured using the highest concentration at which the antibody showed the expected staining of normal tissue without staining the stroma. To avoid false positives by host antibodies and anti-human secondary antibodies that stain B cell follicles, the antibody was conjugated to biotin through its Fc region. Figure 4A shows the specimen at position A7 that was negative for huMNC2-reactive cells. Figure 4B shows the specimen at position A9, a grade 2 cancer with lymph node metastasis, scored +1 for humMNC2 reactivity. Figure 4C shows the specimen at position B10, a larger grade 2 tumor with lymph node metastasis, scored +2 for humMNC2 reactivity. Figures 5A-5B show photographs of individual breast cancer specimens from breast cancer array BR1141 at two different magnifications after staining with the anti-MUC1 * antibody huMNC2-scFv-Fc. Figure 5A shows the specimen at position D7, a grade 2 cancer without lymph node metastasis, scored +3 for humMNC2 reactivity. Figure 5B shows the specimen at position F6, a grade 2 tumor with lymph node metastasis, scored +4 for humMNC2 reactivity. Figures 6A-6B show a pie chart of the pathological score and a photograph of ovarian cancer array BCl l 5a after staining with the anti-MUC1 * antibody huMNC2-scFv-Fc. Figures 7A-7C show the anti-MUC1 *Shows magnified photographs of different cancer subtypes after staining with the antibody huMNC2-scFv-Fc. Figure 7A shows a photograph of a grade 2 breast tumor with a pathology score of +4. Figure 7B shows a photograph of a grade 2 ovarian tumor with a pathology score of +3. Figure 7C shows a photograph of a grade 3 pancreatic tumor with a pathology score of +3. In an IHC study involving over 1,000 tumor specimens, it was shown that huMNC2-scFv recognized 95% of breast cancers (90% triple-negative), 83% of ovarian cancers, 78% of pancreatic cancers, and 71% of lung cancers. Figures 8A - 8D show anti-MUCl * Shows magnified photographs of different cancer subtypes after staining with the antibody huMNC2-scFv-Fc. Figure 8A shows a photograph of a grade 2 breast tumor with a pathology score of +2. Figure 8B shows a photograph of a grade 3 ovarian tumor with a pathology score of +3. Figure 8C is a photograph of a grade 3 pancreatic tumor with lymph node metastasis and a pathology score of +3. Figure 8D shows a photograph of a lung cancer with a pathology score of +3. Figures 9A - 9I show anti-MUCl * Shows magnified photographs of various normal tissues after staining with the antibody huMNC2-scFv-Fc. The conditions and concentrations used were the same as those used for studying cancer tissues. Figure 9A shows normal adrenal tissue. Figure 9B shows normal brain tissue. Figure 9C shows normal breast tissue. Figure 9D shows normal gastric tissue. Figure 9E shows normal heart tissue. Figure 9F shows normal kidney tissue. Figure 9G shows normal testicular tissue. Figure 9H shows normal intestinal tissue. Figure 9I shows normal liver tissue. Figures 10A - 10F show anti-MUCl* Figure showing a photograph of normal kidney tissue after staining with the antibody huMNC2-scFv-Fc. The conditions and concentrations used were the same as those used for studying cancer tissues. Figure 10A shows normal kidney tissue with huMNC2 reactivity limited to the apical edge of normal expression. Figure 10B shows the same tissue at a higher magnification. Figure 10C shows another example of normal kidney tissue with undetectable huMNC2 reactivity. Figure 10D shows the same tissue at a higher magnification. Figure 10E shows another example of normal kidney tissue with huMNC2 reactivity limited to the apical boundary of normal expression. Figure 10F shows the same tissue at a higher magnification. Further studies showed that less than 10% of normal kidney tissue showed huMNC2 reactivity in the distal collecting tubules, and such reactivity was strictly limited to the apical boundary of the normal expression pattern. Figures 11A-11B are pie charts of the pathological scores and anti-MUCl * Figure showing a photograph of esophageal cancer array BC001113 after staining with the antibody huMNC2-scFv-Fc. Figures 12A-12F are anti-MUCl * Figures showing photographs of individual esophageal cancer specimens from esophageal cancer array BC001113 at two different magnifications after staining with the antibody huMNC2-scFv-Fc. The positions within the array, cancer subtypes, tumor malignancy, and pathological scores are shown in the figures. Figure 12A shows the specimen at position A4 that was negative for huMNC2-reactive cells. Figure 12B shows the same specimen at a higher magnification. Figure 12C shows the specimen at position D2 that was scored as having minimal reactivity to huMNC2 by pathology. Figure 12D shows the same specimen at a higher magnification. Figure 12E shows the specimen at position B8 that was scored as having a +1 reactivity to huMNC2 by pathology. Figure 12F shows the same specimen at a higher magnification. Figures 13A-13D are anti-MUCl* Photographs of individual esophageal cancer specimens from esophageal cancer array BC001113 at two different magnifications after staining with the antibody huMNC2-scFv-Fc are shown. The position within the array, cancer subtype, tumor malignancy, and pathological score are shown in the figure. Figure 13A shows the specimen at position D6, which is a grade 4 tumor with a pathological score of +2. Figure 13B shows the same specimen at a higher magnification. Figure 13C shows the specimen at position D5, which is a grade 3 tumor with a pathological score of +3. Figure 12D shows the same specimen at a higher magnification. Figures 14A - 14B are a pie chart of the pathological scores and anti-MUCl * A photograph of pancreatic cancer array PA805b after staining with the antibody huMNC2-scFv-Fc is shown. Figures 15A - 15D are anti-MUCl * Photographs of individual pancreatic cancer specimens from pancreatic cancer array PA805b at two different magnifications after staining with the antibody huMNC2-scFv-Fc are shown. The position within the array, cancer subtype, tumor malignancy, and pathological score are shown in the figure. Figure 15A shows the specimen at position F3, which is a grade 3 tumor with a pathological score of +3. Figure 15B shows the same specimen at a higher magnification. Figure 15C shows the specimen at position B1, which is a grade 1 tumor with a pathological score of +2. Figure 15D shows the same specimen at a higher magnification. Figures 16A - 16D are anti-MUCl * Photographs of individual pancreatic cancer specimens from pancreatic cancer array PA805b at two different magnifications after staining with the antibody huMNC2-scFv-Fc are shown. The position within the array, cancer subtype, tumor malignancy, and pathological score are shown in the figure. Figure 16A shows the specimen at position A2, which is a grade 1 tumor with a pathological score of +2. Figure 16B shows the same specimen at a higher magnification. Figure 16C shows the specimen at position C3, which is a grade 2 tumor with a pathological score of +2. Figure 16D shows the same specimen at a higher magnification. Figures 17A-17D show * photographs at two different magnifications of individual pancreatic cancer specimens from pancreatic cancer array PA805b after staining with the anti-MUC1 antibody huMNC2-scFv-Fc. The position within the array, cancer subtype, tumor malignancy, and pathology score are shown graphically. Figure 17A shows a specimen at position C6 that is a grade 2 tumor scored as pathology +2. Figure 17B shows the same specimen at a higher magnification. Figure 17C shows a specimen at position D1 that is a higher grade 3 tumor with lymph node metastasis scored as pathology +3. Figure 17D shows the same specimen at a higher magnification. * Figures 18A-18D show photographs at two different magnifications of individual pancreatic cancer specimens from pancreatic cancer array PA805b after staining with the anti-MUC1 antibody huMNC2-scFv-Fc. The position within the array, cancer subtype, tumor malignancy, and pathology score are shown graphically. Figure 18A shows a specimen at position E2 that is a grade 1 tumor scored as pathology +2. Figure 18B shows the same specimen at a higher magnification. Figure 18C shows a specimen at position E10 that is a smaller grade 3 tumor with lymph node metastasis scored as pathology +3.

[0137] MNC2 recognized approximately 95% of breast cancers across all breast cancer subtypes, but it was confirmed that some cancer subtypes did not express MNC2-reactive MUC1 * as strongly as breast cancer. In particular, in pancreatic, esophageal, and prostate cancers, the expression level of MNC2-reactive MUC1 * was low. In the pancreatic cancer array, 78% of the tumors were MNC2-reactive, but the intensity of staining proportional to the expression level of the tumor was relatively weak. The pie chart in FIG. 14A shows that 65% of pancreatic tumors had a score of +1 or +2, only 5% had a score of +3, and none had a score of +4. The pie chart in FIG. 3A shows that more than half of breast tumors had scores of +2 to +3, 6% had a score of +4, and only 4% were negative for MNC2 MUC1 * reactivity. Both arrays were stained with the same MNC2 anti-MUCl * antibody and scored by the same board-certified pathologist. The difference in MUC1 * MNC2 staining between breast and pancreatic cancers was inferred to be due to differences in the cleavage enzymes that cleave MUC1 at different positions that induce conformational or linear changes in the extracellular domain of MUC1 * to MUC1 * . To investigate, we stained the same pancreatic cancer array with the anti-MUCl * polyclonal antibody SDIX. Both MNC2 and SDIX were generated by immunizing animals with the PSMGFR peptide, but they showed different binding properties to tumor tissue. In general, SDIX recognized more pancreatic tissue and stained more strongly than MNC2, but there were cases where MNC2 recognized tumors that SDIX did not.

[0138] On cancer tissue, MUC1 * is expressed in most tissues, is characteristic of cancer, and all anatomical barriers are disrupted in cancer tissue. In contrast, in normal tissue, MUC1 * expression is limited to the ductal and apical margins of glands. The expression of MNC2-reactive MUC1 * is even more restricted. For example, FIG. 6B shows a photograph of an ovarian cancer microarray. However, row J consists of normal ovarian tissue. As can be confirmed, the expression of MNC2-reactive MUC1 * is not observed. Normal kidney expresses some MNC2-reactive MUC1 * . As seen in FIGS. 10A - 10F, normal MUC1 * expression is weak and limited to the apical margins of approximately 10% of the distal convoluted tubules of normal kidney. Normal pancreas expresses MUC1 *is expressed, and this is also strictly limited to the apical edge of the acinar cells (Figure 20). Those skilled in the art can easily identify cancerous tissues and distinguish the MUC1 * expression on normal tissues and cancerous tissues. Generally, MUC1 * is macroscopically overexpressed on cancer tissues, and its expression is not limited to the apical pattern of expression.

[0139] In Figures 20 - 34, we showed that a series of pancreatic tumors did not show staining with the monoclonal antibody MNC2 or showed minimal staining, while staining of the same tissues with the SDIX polyclonal antibody resulted in strong staining. Both MNC2 and SDIX were produced by immunizing animals with the same peptide: PSMGFR. However, MNC2 recognizes only a subset recognized by SDIX. These results strongly suggest that MNC2 recognizes epitopes generated only in a subset of tumors. The data suggest that the MNC2-reactive subset of MUC1 * may be cancer subtype-specific or patient-specific and may be due to cleavage by different proteases.

[0140] The hypothesis that anti-MUCl * antibody specificity depends on a protease that cleaves MUC1 into MUC1 * is supported by the data shown in Figures 35 - 37. MNC2, MNC3, and SDIX were all produced by immunizing animals with the PSMGFR peptide. However, the monoclonal antibody MNC3 recognizes almost 100% of hematopoietic stem cells, similar to the polyclonal antibody SDIX, while the monoclonal antibody MNC2 does not. Conversely, MNC2 binds to almost 95% of breast tumors, while MNC3 does not. Importantly, MNC2 binds to MUC1 after it has been cleaved by the protease MMP9, which is overexpressed in most breast cancers but not in hematopoietic stem cells. *has been shown to recognize. The expression of MMP9 is a predictor of poor prognosis in most solid tumor cancers (Yousef et al., BMC Cancer 2014, 14:609; Mehner et al., Oncotarget, Vol. 5, No. 9, pp 2736 - 2749, 2014; Radisky et al., Front Biosci (Landmark Ed); 20:1144 - 1163, 2015; Gong et al., Journal of Surgical Oncology 2000; 73:95 - 99; Latinovic et al., Arch Oncol 2013; 21(3 - 4):109 - 14; Sillanpa et al., Gynecologic Oncology 104(2007)296 - 303).

[0141] A wide range of MUC1 that can better address cancer subtype - specific, patient - specific, or tumor heterogeneity * A new anti - MUC1 that can recognize * To generate monoclonal antibodies, the inventors immunized animals with one of the following peptides derived from the sequence of the extracellular domain of MUC1: * One of the following peptides derived from the sequence of the extracellular domain of MUC1:

[0142] (i) PSMGFR peptide GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 4);

[0143] (ii) PSMGFR N + 20 / C - 27 SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTE (SEQ ID NO: 9)

[0144] (iii) PSMGFR N + 9 / C - 9 VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVP (SEQ ID NO: 10).

[0145] Antibody clones were isolated, and a subset from each immunization was first selected based on their ability to bind to the immunizing peptide and then, next, selected based on their ability to recognize cancer tissue over normal tissue. FIGS. 38A - 38C show a table of selected antibodies organized according to the immunizing peptide. In the table, a designation of -1 or -2 indicates that these are sister clones, and after sequencing, it was shown that these are actually the same antibody. Throughout the remainder of the present disclosure, antibodies are referred to without the -1 or -2 designation.

[0146] FIGS. 39 - 44 show the binding characteristics of the new anti-MUC1 * antibodies. All antibodies were first selected based on the fact that they bind to the immunizing peptide. For comparison with MNC2 and MNC3, the new antibodies were tested for their ability to bind to PSMGFR, the N-10 peptide, and the C-10 peptide. The new anti-MUC1 * antibodies were also tested by FACS to determine their ability to bind to the T47D breast cancer cell line. For the analysis of antibody binding to single cell lines created from patients decades ago, we expanded the analysis of the new antibodies to hundreds of tumor tissues across multiple cancer subtypes. The number of patients represented in each array varied. Normal tissues were also probed with the antibodies.

[0147] FIGS. 45 - 52 compare the binding of the new anti-MUC1 * antibodies to SDIX polyclonal to examine antibodies that bind to the region that is the N-terminus of the PSMGFR sequence. Since in previous studies MNC2 recognized approximately 78% of pancreatic cancers but the binding was not very strong and it was shown that very poor tumors were not recognized at all by MNC2 or the SDIX polyclonal, the pancreatic cancer array was started.

[0148] Some anti-PSMGFR antibodies (e.g., 18B4) appear to recognize the same pancreatic tumor tissue as the polyclonal anti-PSMGFR antibody SDIX (Figures 45A-45BC). In this small pancreatic cancer array, the anti-PSMGFR N+20 / C-27 antibody 1E4 appears to recognize the same tumors as SDIX and 18B4, although magnified views of these tumor specimens show that antibody 1E4 recognizes a cancer cell population within the tumor that is different from the anti-PSMGFR antibodies (Figures 46A-46F), and a portion of the tumor was not well recognized by SDIX but was recognized by the monoclonal antibody 18B4 (Figures 47A-48D). Other pancreatic tumors were better recognized by the anti-PSMGFR N+20 / C-27 antibody 1E4 (Figures 49A-49D). Similarly, the anti-PSMGFR N+20 / C-27 antibody 29H1 recognizes some pancreatic tumors that were missed by the anti-PSMGFR antibodies SDIX and 20A10 (Figures 51A-51C).

[0149] These studies generally show that antibodies that bind to the extracellular domain of MUC1 that extends beyond PSMGFR at the N-terminus recognize pancreatic cancer better than SDIX polyclonal. However, antibody specificity for pancreatic tumors also appears to be patient-specific. Some patient specimens stained much better with the SDIX anti-PSMGFR antibody than with the new antibodies that bind to PSMGFR N+20 / C-27 or PSMGFR N+9 / C-9. This supports the idea that patient tumors must be probed with a panel of MUC1 * antibodies to determine which treatment is most suitable for the elimination of those tumors. In one aspect of the invention, a therapeutic agent incorporates some or all of an antibody that is a diagnostic agent, or some or all of an antibody derived from an antibody that is a diagnostic antibody. *

[0150] Figure 53 shows that these new antibodies extended at the N-terminus recognize more pancreatic tumors than the antibody that binds to full-length MUC1. This figure compares the binding of the standard antibody VU4H5 that binds to the tandem repeat sequence of full-length MUC1, the new antibody 5E5 that binds to the captured O-linked glycans present in some cancer cells, and 29H1.​

[0151] Next, esophageal and prostate tumors were examined. These studies were motivated by the authors' previous findings that both the monoclonal antibody MNC2 and the polyclonal antibody SDIX, which binds to both the PSMGFR peptide, showed poor recognition of esophageal and prostate tumors. In fact, these tumors that showed MNC2 reactivity in the well-differentiated parts of the tumor specimens lost that reactivity in the poorly-differentiated parts of the same specimens. These results advocated that cleavage enzymes other than MMP9 are dominant in most esophageal and prostate cancers. These studies support that idea.

[0152] New anti-MUC1 antibodies that bind to the peptide PSMGFR N+20 / C-27 and / or PSMGFR N+9 / C-9 * showed significantly better recognition of esophageal and prostate tumors when compared to MNC2, SDIX, and the full-length MUC1 antibodies 5E5 and VU4H5.

[0153] Figures 54A - 54C show photographs of adjacent serial sections from an esophageal cancer array, which were stained by standard IHC methods using various anti-MUC1 * antibodies. Figure 54A shows the array stained with the SDIX polyclonal anti-MUC1 * antibody, where the immunogen for the antibody was the PSMGFR peptide. Figure 54B shows the array stained with the 20A10 monoclonal anti-MUC1 * antibody, and the immunogen for the antibody was the PSMGFR peptide. Figure 54C shows the array stained with the 29H1 monoclonal anti-MUC1 * antibody, where the immunogen for the antibody was the PSMGFR N+20 / C-27 peptide. Figure 54D shows the array stained with the 31A1 monoclonal anti-MUC1 * antibody, where the immunogen for the antibody was the PSMGFR N+20 / C-27 peptide. This figure shows that the antibodies SDIX and 20A10, both of which bind to the PSMGFR peptide, recognize the same tumor tissue specimens to different extents, while the antibody that binds to the PSMGFRN+20 / C27 peptide binds to more esophageal tumor specimens, as well as to most of those recognized by the anti-PSMGFR antibody. These results indicate that antibodies that bind to the PSMGFR N+20 / C-27 peptide are generally more specific for esophageal cancer than antibodies that bind to the PSMGFR peptide, although certain patients may have esophageal cancers that are better recognized by anti-MUCl * antibodies that bind to the PSMGFR peptide.

[0154] Figures 55A - 55C show photographs of adjacent consecutive sections from an esophageal cancer array, which were stained by standard IHC methods using various anti-MUCl * antibodies. Figure 55A shows the array stained with the SDIX polyclonal anti-MUCl * antibody, where the immunogen for the antibody was the PSMGFR peptide. Figure 55B shows the array stained with the 17H6 monoclonal anti-MUCl * antibody, where the antibody binds to the PSMGFR N+9 / C-9 peptide. Figure 55C shows the array stained with the MNC2 monoclonal anti-MUCl * antibody, where the antibody binds to the PSMGFR peptide. Figure 55D shows the array stained with the 45C11 monoclonal anti-MUCl * antibody, where the antibody binds to the PSMGFR N+20 / C-27 peptide. These results are consistent with the idea that in most esophageal cancers, MUC1 is cleaved by an enzyme that exposes a potential epitope at the N-terminus of the PSMGFR sequence.

[0155] Figures 56A - 56F show photographs and graphical representations of the pathologist staining scores of adjacent consecutive sections from an esophageal cancer array, which were stained with antibodies that recognize full-length MUC1 or MUC1 *Stained by standard IHC method with any of the antibodies that recognize only Figure 56A shows an esophageal cancer array stained with antibody 5E5, an antibody that binds to captured O-linked glycans in the tandem repeat domain of full-length MUC1. Figure 56B shows the pathologist's score for each specimen in the array. Figure 56C shows MUC1 * An esophageal cancer array stained with anti-MUCl * antibody 29H1, which is an antibody that binds to the PSMGFR N+20 / C-27 peptide of Figure 56D shows the pathologist's score for each specimen in the array. Figure 56E shows an esophageal cancer array stained with antibody VU4H5, an antibody that binds to an epitope in the tandem repeat domain of full-length MUC1. Figure 56F shows the pathologist's score for each specimen in the array. As can be seen from the figure, antibody 5E5 recognizes some specimens that are not recognized by VU4H5, but anti-MUCl * antibody 29H1 recognizes specimens recognized by both antibodies that recognize full-length MUC1 and other specimens not recognized by other anti-MUCl antibodies. These findings indicate that anti-MUCl * antibodies that bind to peptides containing amino acids extended N-terminally beyond the PSMGFR sequence do not recognize full-length MUC1, and that antibodies that bind to the PSMGFR N+20 / C-27 peptide recognize epitopes prevalent in esophageal cancer.

[0156] Figures 57A-57G show photographs of prostate cancer arrays stained with either antibody 5E5 or VU4H5, both of which recognize only * MUC1 and recognize full-length MUC1 or 29H1 that binds to the PSMGFR N+20 / C-27 peptide. Figure 57A shows an esophageal cancer array stained with antibody 5E5. Figure 57B shows an esophageal cancer array stained with antibody 29H1. Figure 57B shows an esophageal cancer array stained with antibody 29H1. Figure 57C shows an esophageal cancer array stained with the antibody VU4H5. Figure 57D shows, as a control, an esophageal cancer array stained with the secondary antibody only. Figure 57E shows the tissue marked by the red box in Figure 57A at a higher magnification, and the staining was performed with 5E5. Figure 57F shows the tissue marked by the red box in Figure 57B at a higher magnification, where the staining was performed with 29H1. Figure 57G shows the tissue marked by the red box in Figure 57C at a higher magnification, where the staining was performed using VU4H5. The dashed red box is negatively stained for antibodies that recognize full-length MUC1, but is highly positive when probed with anti-MUCl * antibodies, particularly antibodies that bind to the PSMGFR N+20 / C-27 peptide, showing just one patient specimen out of many esophageal tumor specimens.

[0157] Figures 58A - 58C show photographs of adjacent consecutive sections from a prostate cancer array stained by standard IHC methods using various anti-MUCl * antibodies. Figure 58A shows the array stained with the SDIX polyclonal anti-MUCl * antibody, where the immunogen for the antibody was the PSMGFR peptide. Figure 58B shows the array stained with the 18B4 monoclonal anti-MUCl * antibody, where the antibody binds to the PSMGFR peptide. Figure 58C shows the array stained with the 1E4 monoclonal anti-MUCl * antibody, where the antibody binds to the PSMGFR N+20 / C-27 peptide.

[0158] Figures 59A - 59E show photographs of adjacent consecutive sections from a prostate cancer array stained by standard IHC methods using various anti-MUCl * antibodies. Figure 59A shows an array stained with the MNC2 monoclonal antibody that binds to the PSMGFR peptide but not to the C-10 peptide. Figure 59B shows an array stained with the 18B4 antibody that binds to the PSMGFR peptide. Figure 59C shows an array stained with the 32C1 antibody that binds to the PSMGFR N+20 / C-27 peptide. Figure 59D shows an array stained with the SDIX polyclonal anti-MUC1 * antibody, where the immunogen for the antibody was the PSMGFR peptide. Figure 59E shows an array stained with the 31A1 monoclonal anti-MUC1 * antibody that binds to the PSMGFR N+20 / C-27 peptide.

[0159] Figures 60A - 60F show photomicrographs and graphical representations of the pathological staining scores of adjacent consecutive sections from a prostate cancer array stained by standard IHC methods with either an antibody that recognizes full-length MUC1 or an antibody that recognizes only MUC1. * Figure 60A shows a prostate cancer array stained with antibody 5E5, which is an antibody that binds to the captured O-linked glycan in the tandem repeat domain of full-length MUC1. Figure 60B shows the pathological scores for each specimen within the array. Figure 60C shows a prostate cancer array stained with the anti-MUC1 * antibody 29H1, which is an antibody that binds to the PSMGFR N+20 / C-27 peptide of MUC1. * Figure 60D shows the pathological scores for each specimen within the array. Figure 60E shows a prostate cancer array stained with antibody VU4H5, which is an antibody that binds to an epitope in the tandem repeat domain of full-length MUC1. Figure 60F shows the pathological scores for each specimen within the array. As can be seen from the figures, antibody 5E5 recognizes some specimens that VU4H5 does not recognize, but the anti-MUC1 *Antibody 29H1 recognizes specimens recognized by both antibodies that recognize full-length MUC1 and other specimens not recognized by other anti-MUC1 antibodies. These findings indicate that anti-MUC1 antibodies that bind to peptides containing amino acids extended N-terminally beyond the PSMGFR sequence do not recognize full-length MUC1, and that antibodies that bind to the PSMGFR N+20 / C-27 peptide recognize epitopes prevalent in prostate cancer. * Antibodies that bind to the PSMGFR N+20 / C-27 peptide recognize epitopes prevalent in prostate cancer.

[0160] Figures 61A-61G show photographs of prostate cancer arrays stained with either antibody 5E5 or VU4H5, both of which recognize only full-length MUC1 that recognizes MUC1 only and binds to the PSMGFR N+20 / C-27 peptide. * Figures 61A-61G show photographs of prostate cancer arrays stained with either antibody 5E5 or VU4H5, both of which recognize only full-length MUC1 that recognizes MUC1 only and binds to the PSMGFR N+20 / C-27 peptide. Figure 61A shows a prostate cancer array stained with antibody 5E5. Figure 61B shows a prostate cancer array stained with antibody 29H1. Figure 61B shows a prostate cancer array stained with antibody 29H1. Figure 61C shows a prostate cancer array stained with antibody VU4H5. Figure 61D shows, as a control, a prostate cancer array stained with secondary antibody only. Figure 61E shows the tissue marked by the red box in Figure 61A at a higher magnification, and the staining was performed with 5E5. Figure 61F shows the tissue marked by the red box in Figure 61B at a higher magnification, where the staining was performed with 29H1. Figure 61G shows the tissue marked by the red box in Figure 61C at a higher magnification, where the staining was performed with VU4H5. The dashed red boxes are negatively stained for antibodies that recognize full-length MUC1, but are very positive and show only one patient specimen out of many prostate tumor specimens when probed with anti-MUC1 antibodies, particularly antibodies that bind to the PSMGFR N+20 / C-27 peptide. * The dashed red boxes are negatively stained for antibodies that recognize full-length MUC1, but are very positive and show only one patient specimen out of many prostate tumor specimens when probed with anti-MUC1 antibodies, particularly antibodies that bind to the PSMGFR N+20 / C-27 peptide.

[0161] MNC2 recognizes MUC1, which is present in the majority of breast cancers. * However, due to tumor heterogeneity and the fact that MUC1, a growth factor receptor, * is cleaved by different proteases, thereby increasing the possibility of tumor escape by * propagating cell populations recognized by different anti-MUCl * antibodies, it is suggested that treatment with more than one anti-MUCl * antibody is beneficial. For this purpose, the inventors compared the recognition of new anti-MUCl * antibodies to MNC2 more closely (Figs. 62 - 73).

[0162] The breast cancer array BR1141 was stained with either MNC2 or 20A10, both of which bind to the PSMGFR peptide and the N - 10 peptide but not to the C - 10 peptide. In a first approximation, the two antibodies recognize the same or very close epitopes of MUC1 * expressed in breast cancer (Figs. 62A - 62B). Figs. 63A - 65B show the same breast cancer array compared to 25E6, 18B4, and 18G12, but with MNC2. It should be recalled that, unlike MNC2, this new set of anti - PSMGFR antibodies can bind to the C - 10 peptide (Fig. 41). As can be seen from the figure, there are differences between the binding of MNC2 and these new anti - PSMGFR antibodies. When comparing MNC2 with the anti - PSMGFR N + 9 / C - 9 antibody 8A9 (Figs. 66A - 66B) and the anti - PSMGFR antibody 28F9 (Figs. 67A - 67B), the differences in the recognition of breast cancer populations among patients are more prominent, as well as within the same tumor. Referring to Fig. 41, the antibody 28F9 showed the highest degree of binding to the C - 10 peptide, while MNC2 did not bind to the C - 10 peptide, and it was argued that these antibodies bind to very different epitopes on the cleaved extracellular domain of MUC1 * . The difference between the binding of the anti - PSMGFR N + 9 / C - 9 antibody 3C5 and MNC2 is clearly seen in Figs. 69A - 69B. Differences in breast cancer recognition between anti-PSMGFR antibodies 20A10 and 18B4 and other antibodies that bind to the peptide PSMGFR N+20 / C-27 (e.g., 29H1, 45C11, and 32C1, 31A1) or antibodies that bind to the PSMGFR N+9 / C-9 peptide (e.g., 17H6) are shown in FIGS. 70A-70G.

[0163] A smaller breast cancer array, BR1007, was probed with the anti-MUC1 * antibody 29H1 and compared to the recognition of the same array when probed with anti-full length MUC1 antibodies 5E5 and VU4H5 (FIGS. 71A-71F). As can be seen from the figure, antibody 5E5 recognizes some specimens that are not recognized by VU4H5, while the anti-MUC1 * antibody 29H1 recognizes specimens that are recognized by both antibodies that recognize full length MUC1 and other specimens that are not recognized by other anti-MUC1 antibodies. These findings indicate that anti-MUC1 * antibodies that bind to peptides containing amino acids extended N-terminally beyond the PSMGFR sequence do not recognize full length MUC1.

[0164] In FIGS. 72A-72F, the binding of MNC2 to breast cancer array BR1141 was compared to a panel of anti-PSMGFR antibodies. All of these antibodies bind to the PSMGFR peptide and generally produce the same staining pattern on this breast cancer array. However, there are some differences in how these antibodies recognize individual specimens within the array, which may represent MUC1-MUC1 * cleavage by different enzymes. Referring to FIG. 39, MNC2 and 20A10 bind to the N-10 peptide but not to the C-10 peptide, indicating that the 10 membrane-proximal amino acids are important for their binding. Antibodies 18B4, 18G12, and 25E6 show some binding to the C-10 peptide, and 28F9 shows even more binding to the C-10 peptide. In particular, 18B4 does not bind to the N-10 peptide, indicating that it binds to an epitope that is more N-terminal within PSMGFR than the others. Despite the aforementioned exceptions, the recognition of tumors within this array by anti-PSMGFR antibodies was highly similar.

[0165] In contrast, antibodies that bind to the PSMGFR N+9 / C-9 peptide strongly recognized a subset of tumors that were not recognized by MNC2 or were weakly recognized by MNC2 and other anti-PSMGFR antibodies (Figures 73A-73F). The photographs shown are adjacent serial sections of breast cancer tissue array BR1141 stained with various anti-MUC1 * monoclonal antibodies, where antibodies that bind to the PSMGFR N+9 / C-9 peptide were compared to MNC2 and its humanized single form huMNC2-scFv-Fc, both of which bind to PSMGFR N-10 but not to the C-10 peptide. Figure 73A shows a breast cancer specimen stained with MNC2. Figure 73B shows a breast cancer specimen stained with 8A9. Figure 73C shows a breast cancer specimen stained with 17H6. Figure 73D shows a breast cancer specimen stained with huMNC2-scFv-Fc. Figure 73E shows a breast cancer specimen stained with 3C5. Figure 73F shows a breast cancer specimen stained with 39H5. Referring to the patient specimens marked with red circles here, it is clear that antibodies that bind to the PSMGFR N+9 / C-9 peptide recognize populations of breast cancer cells that are missed or weakly bound by the MNC2 anti-PSMGFR antibody. Anti-MUC1 * antibodies 8A9, 17H6, 3C5, and 39H5 recognize unique subsets of cancer cells that are not recognized or are recognized to a lesser extent by anti-PSMGFR antibodies such as MNC2, 20A10, 25E6, 28F9, 18G12, or 18B4.

[0166] In summary, these data show that (i) the diagnosis of MUC1-positive cancer, even within cancer subtypes such as breast cancer, is not by antibodies that bind to full-length MUC1 but by anti-MUC1 *more accurate when probed with an antibody; (ii) the diagnosis of MUC1-positive cancer is more accurate even within a cancer subtype such as breast cancer when the tumor is probed with one or more anti-MUC1 * antibodies; (iii) the diagnosis of MUC1-positive cancer is more accurate even within a cancer subtype such as breast cancer when the tumor is probed with one or more anti-MUC1 * antibodies, where at least two different antibodies are selected from two different groups, the groups being antibodies that bind to the PSMGFR peptide, PSMGFR N+20 / C-27, and antibodies that bind to the PSMGFR N+9 / C-9 peptide.

[0167] The anti-MUC1 antibodies of the present invention that can be used for use in the diagnosis of cancer * bind to the PSMGFR peptide, the PSMGFR N+20 / C-27 peptide, the PSMGFR N+9 / C-9 peptide, or more specifically, a peptide having at least 15 consecutive amino acids of the following sequences, and include antibodies having up to 4 amino acid substitutions;

[0168] (i) the PSMGFR region of MUC1;

[0169] (ii) the PSMGFR peptide set forth in SEQ ID NO:4;

[0170] (iii) PSMGFR N+20 / C-22; a peptide having the amino acid sequence SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:5).

[0171] (iv) PSMGFR N+12 / C-22; a peptide having the amino acid sequence SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:6).

[0172] (v) PSMGFR N+9 / C-30; A peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO: 7)

[0173] (vi) PSMGFR N+20 / C-41; A peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO: 8).

[0174] vii) PSMGFR N+20 / C-27; A peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTE (SEQ ID NO: 9).

[0175] viii) PSMGFR N+9 / C-9; A peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVP (SEQ ID NO: 10). Specifically, the anti-PSMGFR antibodies MNC2, MNE6, 18B4, 18G12, 20A10, 25E6, the anti-PSMGFR N+20 / C-27 antibodies 1E4, 29H1, 31A1, 32C1, 4501, and the anti-PSMGFR N+9 / C-9 antibodies 3C5, 8A9, 17H6, and 39H5 are antibodies that can be used for diagnosing cancer. These antibodies can be human, humanized or non-human. They can be intact antibodies or antibody fragments. The antibodies can be produced by immunizing an animal with the peptides of the above sequences (i)-(viii). For producing antibodies, MUC1 * The animals immunized with the extracellular domain peptide can be human, rabbit, mouse, goat, donkey, camelid, llama, alpaca or other non-human species.

[0176] The antibodies of the present invention can be derivatized with imaging agents, dyes, fluorescent entities, chromogenic reagents, or any other element that makes the antibody optically, visually, electrically or radioactively detectable, or can be used in diagnostic assays attached to imaging agents. The antibodies of the present invention can be used in various diagnostic formats.

[0177] In another example, the anti-MUC1 * antibody of the present invention can be conjugated to a contrast agent for use in living patients as a whole body diagnostic to determine whether the patient has a MUC1 * positive tumor, or to determine whether the patient can benefit from a therapeutic agent that includes all or a fragment of an anti-MUC1 * antibody that may be derived from or have similar binding characteristics to the antibody used for diagnosis. The types of diagnostic and therapeutic antibodies need not be the same. Antibodies produced in camelid species are particularly useful in in vivo diagnostic assays because camelids produce small monovalent antibodies that have a short half-life in humans.

[0178] In yet another example, the anti-MUC1 * antibody of the present invention can be conjugated to a contrast agent and used during surgery to detect or mark cancerous tissue so that the cancerous tissue can be completely resected during surgery.

[0179] In one aspect of the present invention, a body fluid or tissue sample from a patient diagnosed with cancer or suspected of being at risk of cancer is contacted with one or more anti-MUC1 * antibodies of the present invention; analysis of the binding of the antibody to the cells of the sample indicates a level or pattern of binding indicative of cancer. A therapeutic agent for the treatment of cancer is then administered to the patient. In one aspect of the present invention, the therapeutic agent includes all or a fragment of an anti-MUC1 * antibody.

[0180] In one example, a diagnostic assay using an anti-MUC1 * antibody or a fragment thereof is used to screen patients to determine their potential benefit from MUC1 * targeted therapeutic agents. The anti-MUC1 *Antibodies and antibodies or fragments thereof incorporated into therapeutic agents may be derived from the same antibody. The types of diagnostic antibodies and therapeutic antibodies need not be the same. Diagnostic assays may include the use of one or more anti-MUC1 * antibodies. A patient specimen reactive with one or more anti-MUC1 * antibodies indicates that the patient may benefit from administration of a therapeutic agent comprising one or more reactive antibodies or fragments thereof.

[0181] One example is (i) contacting a suspect cell or tissue specimen from a patient diagnosed with or suspected of developing cancer with an anti-MUC1 * antibody; (ii) contacting with a normal cell or tissue specimen, which may be a stored reference specimen; (iii) detecting antibody binding; (iv) the measurement that the suspect specimen overexpresses MUC1 * or expresses MUC1 * in a uniform pattern as opposed to apical border-limited expression indicates that the patient has a MUC1 * positive cancer; (vi) then a therapeutic agent for the treatment of cancer is administered to the patient, which can incorporate an anti-MUC1 * antibody or fragment thereof.

[0182] In one aspect of the invention, a body fluid or tissue specimen from a patient diagnosed with or suspected of having cancer is contacted with an anti-MUC1 * antibody of the invention, and a higher than normal level of MUC1 * is detected, or an abnormal pattern of MUC1 * is detected, which indicates that the patient has a MUC1 * positive cancer, and then a therapeutic agent is administered to the patient, which is anti-MUC1 *It shows the incorporation of an antibody or antibody fragment. In one case, the therapeutic agent into which the antibody or antibody fragment is incorporated is an immuno-oncology agent such as CAR T cells, engineered NK cells or dendritic cells. In another case, the therapeutic agent into which the antibody or antibody fragment is incorporated is huMNC2-CAR44 T cells. In yet another aspect of the present invention, the therapeutic agent into which the antibody or antibody fragment is incorporated is a bispecific antibody. In yet another aspect of the present invention, the therapeutic agent into which the antibody or antibody fragment is incorporated is an antibody-drug conjugate (ADC). In yet another aspect of the present invention, the therapeutic agent into which the antibody or antibody fragment is incorporated is a bispecific T cell engager (BiTE).

[0183] In another embodiment, the diagnostic assay may comprise an anti-MUCl * antibody and a second antibody, and the step may comprise determining the ratio of the amount of the first antibody to the second antibody. The first antibody can bind to the extracellular domain of MUC1 * and the second antibody can bind to a portion of the MUC1 extracellular domain that is the N-terminus of a cleavage site such as a tandem repeat. When contacting a tissue specimen, the higher the ratio of the full-length MUC1 * to MUC1, the more advanced the cancer and the more likely the patient is to benefit from treatment targeting MUC1 * .

[0184] The present invention includes antibodies, as well as antibody-like proteins including but not limited to polyclonal, monoclonal, chimeric, humanized, single-chain, antibody fragments, etc. Further, the present invention includes the use of protein scaffolds for generating antibody mimetics for obtaining proteins that can be characterized by the binding assays described herein, and the present invention further includes using the methods described herein to identify antibodies that recognize specific epitopes within the extracellular domain of MUC1 * that are differentially expressed on cancer cells.

[0185] In one aspect, the present invention relates to a human or humanized anti-MUC1 antibody, antibody fragment or antibody-like protein that binds to a cleavage product lacking a region or tandem repeat domain on the extracellular domain of the MUC1 isoform. The human or humanized anti-MUC1 antibody, antibody fragment or antibody-like protein can specifically bind to * (i) the PSMGFR region of MUC1; * (ii) the PSMGFR peptide set forth in SEQ ID NO:4;

[0186] (iii) PSMGFR N+20 / C-22;

[0187] (iv) PSMGFR N+12 / C-22;

[0188] (v) PSMGFR N+9 / C-30; a peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:5).

[0189] (vi) PSMGFR N+20 / C-41; a peptide having the amino acid sequence of SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:6).

[0190] (vii) PSMGFR N+20 / C-27; a peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO:7).

[0191] (viii) PSMGFR N+20 / C-41; a peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO:8).

[0192] (ix) PSMGFR N+20 / C-27; a peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTE (SEQ ID NO:9).

[0193] viii) PSMGFR N+9 / C-9; It is a peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVP (SEQ ID NO: 10).

[0194] The human or humanized antibody can be IgG1, IgG2, IgG3, IgG4 or IgM. The human or humanized antibody fragment or antibody-like protein can be scFv or scFv-Fc.

[0195] The human or humanized antibody, antibody fragment or antibody-like protein as described above is derived from the mouse monoclonal MN-E6 antibody and may contain a heavy chain variable region and a light chain variable region having at least 80%, 90%, 95% or 98% sequence identity with the mouse monoclonal MN-E6 antibody.

[0196] The human or humanized antibody, antibody fragment or antibody-like protein as described above may contain complementarity-determining regions (CDRs) in the heavy chain variable region and the light chain variable region having at least 90%, 95% or 98% sequence identity with the CDR1, CDR2 or CDR3 regions of the antibodies 1E4, 29H1, 31A1, 32C1, and 45C11 that are reactive with PSMGFR N+20 / C-27; 17H6, 39H5, 3C5, 8A9 that are reactive with PSMGFR N+9 / C-9; 18G12, 20A10, 25E6, 28F9, 18B4, MNC2, and MNE6 that are reactive with PSMGFR.

[0197] In another aspect, the present invention relates to the above-mentioned human or humanized anti-MUC1 * antibody or antibody fragment or antibody-like protein that inhibits the binding of the NME protein to MUC1. The NME may be NME1, NME6, NME7AB, NME7 or NME8. *

[0198] ​In yet another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising a single-chain variable fragment (scFv) or a humanized variable region that binds to the extracellular domain of MUC1 lacking tandem repeats, linker molecules, transmembrane domains, and cytoplasmic domains. The single-chain antibody fragment can bind to

[0199] (i) the PSMGFR region of MUC1;

[0200] (ii) the PSMGFR peptide set forth in SEQ ID NO:4;

[0201] (iii) PSMGFR N+20 / C-22; a peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:5).

[0202] (iv) PSMGFR N+12 / C-22; a peptide having the amino acid sequence of SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:6).

[0203] (v) PSMGFR N+9 / C-30; a peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO:7)

[0204] (vi) PSMGFR N+20 / C-41; a peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO:8).

[0205] vii) PSMGFR N+20 / C-27; a peptide having the amino acid sequence of SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTE (SEQ ID NO:9).

[0206] viii) PSMGFR N+9 / C-9; It is a peptide having the amino acid sequence of VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVP (SEQ ID NO: 10).

[0207] In this regard, a preferred embodiment is huMNC2-CAR44 described in SEQ ID NO: 236

[0208] In one aspect, the present invention involves administering to a human an effective amount of a cancer-specific antibody (e.g., MNC2 or MNE6, or a fragment thereof) (where the antibody can be human, humanized, or of a non-human species) for the treatment of a human diagnosed with, having, or at risk of developing MUC1 or MUC1 * positive cancer. In a particular aspect of the present invention, the MUC1 * targeted therapeutic agent is an immune cell transduced with a chimeric antigen receptor also known as CAR T, where the antibody fragment of the CAR is derived from an antibody specific for cancer cells of MUC1 * cancer cells. In one aspect, it is derived from MNC2. In another case, it is derived from MNE6.

[0209] In another aspect, the present invention relates to a diagnostic assay for identifying humans who may benefit from the treatment of MUC1 or MUC1 * positive cancer using a therapeutic agent comprising an antibody or a fragment thereof selected from the group consisting of the 1E4, 29H1, 31A1, 32C1, 4501, 17H6, 39H5, 3C5, 8A9, 18G12, 20A10, 25E6, 28F9, 18B4, MNC2, and MNE6 antibodies. In one aspect of the present invention, the anti-MUCl * antibody or a fragment thereof may comprise all or part of the therapeutic agent and may be derived from the antibody or a fragment thereof used for diagnosis, where the therapeutic agent and the diagnostic agent need not be of the same species. In another example, the anti-MUCl * antibody or a fragment thereof comprising all or part of the therapeutic agent is not derived from the antibody or a fragment thereof used for diagnosis, where the therapeutic agent and the diagnostic agent need not be of the same species.

[0210] In one aspect of the present invention, the therapeutic agent targets MUC1 * In another aspect of the present invention, the therapeutic agent comprising some or all of the anti-MUCl * antibodies is a cancer immunotherapy composition, CAR T, BiTE, antibody or antibody-drug conjugate, ADC.

[0211] In one aspect of the present invention, the diagnosis is a companion diagnosis for determining the eligibility of treatment by treatment. In another aspect of the present invention, the diagnosis is used to evaluate the efficacy of a therapeutic treatment. In yet another aspect of the present invention, the diagnosis is analyzed together with the results of a clinical trial of a treatment, such that the results of the diagnosis can be used to predict which patients will benefit from the treatment. In another aspect of the present invention, the cancer cell antibody or a fragment thereof is derivatized with a contrast agent and then this composition is administered to a patient to enable visualization of reactive tumors within the patient. In this way, the antibody + contrast agent can be used to diagnose cancer, to evaluate the response to a therapeutic treatment, or to evaluate the response to a therapeutic treatment, where the treatment targets MUC1 * and may include some or all of the cancer cell antibodies used in the diagnosis. In one aspect of the present invention, the antibody attached to the imaging agent is a camelid antibody, including but not limited to camels, alpacas, and llamas.

[0212] The diagnostic assays described herein can be used on specimens that can be tissues, biopsy specimens, cells, or body fluids taken from a subject, patient, or a normal person as a control. The diagnostic assays can be performed in vitro or in vivo. The diagnostic assays can be used during surgery (e.g., the tissue at the surgical site can be studied without removing the tissue from the subject). In this way, the diagnostic assay can detect all detectable MUC1 regardless of whether the tissue appears to be part of a tumor *Guide the surgeon to remove the positive tissue. In any of these studies, the main indicator of tumor formation or the potential for tumor formation is MUC1 on the cell or tissue surface accessible to the anti-PSMGFR antibody or cancer cell antibody * is the amount of. When extended, the exposed cancer cell antibody-binding epitope is MUC1 * The PSMGFR region of is also accessible to growth factors that bind to and activate the growth and survival functions mediated by MUC1 * by growth factor receptors. In another technique, antibodies against the MUC1 * region and tandem repeats, IBR or UR can be exposed to the sample, and the ratio of binding to the full-length MUC1 of MUC1 * can be measured. Healthy samples show little or no antibody binding to the MUC1 * region. Samples showing tumor formation show a non-zero ratio of anti-MUCl * antibody against tandem repeat antibody or anti-IBR antibody, where the ratio of MUC1 * to MUC1-containing tandem repeats, IBR or UR increases as the stage / malignancy of the cancer increases.

[0213] In addition to detecting the amount of MUC1 * or tandem repeats containing MUC1 on cells and tissues, the portion of MUC1 containing tandem repeats excreted from the tissue can be detected in body fluids such as blood, breast milk or secretions, urine, lung effluent, etc. In these cases, the level of MUC1 cleavage relative to transmembrane MUC1 * is inferred by measuring the amount of shed MUC1 using antibodies that bind to tandem repeats, IBR or antibodies including but not limited to the unique region that is N-terminal to IBR itself.

[0214] MUC1 on cells or tissues *Measuring or inferring the amount that is greater than that in a previous sample from normal tissue or a patient is an indicator of the potential for tumor formation, the presence of a tumor, or tumor progression, and thereby can serve as a diagnostician and / or evaluator of the effectiveness of treatment for cancer in a patient. In one embodiment, MUC1 * The amount of is determined by contacting a tissue specimen with an anti-MUCl * antibody and determining that the amount of MUC1 * is greater than the amount expressed in normal tissue or in a healthy human.

[0215] All references cited herein are incorporated by reference in their entirety. Images available in the "original document"

[0216] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. (Sequence determination list free text) In the following antibody sequences, the underlined sequences refer to CDR sequences and the double-underlined regions refer to framework regions. Full-length MUC1 receptor (mucin 1 precursor, GenBank accession number: P15941) MTPGTQSPFF LLLLLTVLTV VTGSGHASST PGGEKETSAT QRSSVPSSTE KNAVSMTSSV LSSHSPGSGS STTQGQDVTL APATEPASGS AATWGQDVTS VPVTRPALGS TTPPAHDVTS APDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDNRPALGS TAPPVHNVTS ASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHS SVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQI YKQGGFLGLS NIKFRPGSW VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR DTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASAND (SEQ ID NO : 1 ) A truncated MUC1 receptor isoform comprising the transmembrane and cytoplasmic sequences of the full-length MUC1 receptor that has nat-PSMGFR and PSIBR at its N-terminus and can be cleaved after translation and before expression of the receptor on the cell surface: GFLGLS NIKFRPGSW VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSAQSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR DTYHPMSEYP TYHTHGRYVPPSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL (SEQ ID NO:2) A truncated MUC1 receptor isoform that has nat-PSMGFR + PSIBR + Unique Region at its N-terminus and comprises the transmembrane and cytoplasmic regions of the full-length MUC1 receptor: ATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSTVPPLTSSNHSTSPQLSTGVSFFFLSFHIS NLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSWVQLTLAFREGTIVHDVETQ FNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKN YGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQ ID NO:3) PSMGFR GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:4 ) PSMGFR N+20 / C-22 SNIKFRPGSVWQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:5 ) PSMGFR N+12 / C-22 SWVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:6 ) PSMGFR N+9 / C-30 VQLTLAFREGTINVHDVETQFNQY (SEQ ID NO:7 ) PSMGFR N+20 / C-41 SNIKFRPGSVWQLTLAFREGTIN (SEQ ID NO:8 ) PSMGFR N+20 / C-27 SNIKFRPGSVWQLTLAFREGTINVHDVETQFNQYKTE (SEQ ID NO:9 ) PSMGFR N+9 / C-9 VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTI SDVSVSDVP (SEQ ID NO:10 )

[0215]

Table 1

[0216]

Table 2

[0217]

Table 3

[0218]

Table 4

[0219]

Table 5

[0220]

Table 6

[0221]

Table 7

[0222]

Table 8

[0223]

Table 9

[0224]

Table 10

[0225]

Table 11

[0226] [Table 12]

[0227] [Table 13]

[0228] [Table 14]

[0229] [Table 15]

[0230] [Table 16]

[0231] [Table 17]

[0232] [Table 18]

[0233] [Table 19]

[0234] [Table 20]

[0235] [Table 21]

[0236]

Table 22

[0237]

Table 23

[0238]

Table 24

[0239]

Table 25

[0240]

Table 26

[0241]

Table 27

[0242]

Table 28

[0243]

Table 29

[0244]

Table 30

[0245]

Table 31

Claims

1. An anti-mucin 1 that binds to a peptide having the following sequence ＊ (MUC1 ＊ ) antibody or antibody fragment, wherein the sequence is (i) SEQ ID NO: 4, wherein said anti-MUC1 ＊ antibody or antibody fragment comprises the following six complementarity determining regions (CDRs), SEQ ID NO: 4, 18B4 (SEQ ID NOs: 144, 146, 148, 150, 152, and 154), 18G12 (SEQ ID NOs: 80, 82, 84, 86, 88, and 90), or 28F9 (SEQ ID NOs: 128, 130, 132, 134, 136, and 138), (ii) SEQ ID NO: 9, wherein said anti-MUC1 ＊ antibody or antibody fragment comprises the following six CDRs, SEQ ID NO: 9, 1E4 (SEQ ID NOs: 160, 162, 164, 166, 168, and 170), 29H1 (SEQ ID NOs: 176, 178, 180, 182, 184, and 186), 31A1 (SEQ ID NOs: 192, 194, 196, 198, 200, and 202), or 32C1 (SEQ ID NOs: 208, 210, 212, 214, 216, and 218), or (iii) SEQ ID NO: 10, wherein said anti-MUC1 ＊ antibody or antibody fragment comprises the following six CDRs, SEQ ID NO: 10, 3C5 (SEQ ID NOs: 48, 50, 52, 54, 56, and 58), 8A9 (SEQ ID NOs: 64, 66, 68, 70, 72, and 74), 17H6 (SEQ ID NOs: 16, 18, 20, 22, 24, and 26), or 39H5 (SEQ ID NOs: 32, 34, 36, 38, 40, and 42) and having The six CDRs in each antibody are, respectively, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, anti-MUC1 ＊ Antibody or antibody fragment.

2. The anti-MUC1 according to claim 1, which binds to a peptide having the sequence of SEQ ID NO: 4 ＊ antibody or antibody fragment.

3. The anti-MUC1 according to claim 1, comprising six CDRs in 18B4 (SEQ ID NOs: 144, 146, 148, 150, 152, and 154). ＊ Antibody or antibody fragment.

4. The anti-MUC1 according to claim 1, comprising six CDRs in 18G12 (SEQ ID NOs: 80, 82, 84, 86, 88, and 90). ＊ antibody or antibody fragment.

5. The anti-MUC1 according to claim 1, which is a humanized antibody or antibody fragment ＊ antibody or antibody fragment.

6. The anti-MUC1 according to claim 1, which is a rabbit antibody or antibody fragment, a goat antibody or antibody fragment, a donkey antibody or antibody fragment, a murine antibody or antibody fragment, a rodent antibody or antibody fragment, a camelid antibody or antibody fragment, a llama antibody or antibody fragment, or an alpaca antibody or antibody fragment. ＊ An antibody or antibody fragment.

7. The anti-MUC1 according to any one of claims 1 to 6, to which an imaging agent, a dye, a fluorescent substance, a chromogenic reagent, or any other substance that enables optical, visual, electrical, or radiolabeled detection of the anti-MUC1* antibody or antibody fragment is attached. ＊ A conjugate comprising an antibody or antibody fragment.

8. MUC1 ＊ A composition comprising the conjugate according to claim 7, which is used for the diagnosis of positive cancer.

9. The diagnosis involves contacting a biological sample in vitro with the anti-MUC1 ＊ antibody or antibody fragment, the composition according to claim 8.

10. The composition according to claim 9, wherein the biological specimen comprises a single cell, a plurality of cells or a tissue.

11. By determining that the amount of the antibody or antibody fragment that binds to the biological sample is greater than the amount of the antibody or antibody fragment that binds to a normal sample, it is indicated that the patient has MUC1 ＊ The composition according to claim 9, which indicates that the patient is suffering from a MUC1 ＊ positive cancer.

12. The anti-MUC1 ＊ antibody or antibody fragment pattern that binds to the biological specimen is not limited to the apical edge, by determining this, it indicates that the patient has MUC1 ＊ positive cancer, the composition according to claim 9.

13. The composition according to claim 8, which is administered to a subject suffering from cancer.

14. A composition comprising the conjugate according to claim 7, which is used for determining the suitability of treating a patient suffering from cancer or cancer metastasis with a MUC1 ＊ targeted therapeutic agent.

15. said anti-MUC1 ＊ The specific binding of the antibody or antibody fragment to the cleaved form of MUC1 indicates that the ＊ composition according to claim 14, wherein the targeted therapeutic agent is suitable for the treatment of a patient.

16. the aforementioned MUC1 ＊ The composition according to claim 14, wherein the targeted therapeutic agent is for cancer immunotherapy.

17. the aforementioned MUC1 ＊ The composition according to claim 14, wherein the targeted therapeutic agent is a CAR T cell.

18. the aforementioned MUC1 ＊ The composition according to claim 14, wherein the targeted therapeutic agent is a bispecific T cell engager (BiTE).

19. said MUC1 ＊ The composition according to claim 14, wherein the targeted therapeutic agent is an antibody-drug conjugate.

20. said MUC1 ＊ The targeted therapeutic agent is the anti-MUC1 ＊ The composition according to claim 14, comprising six CDRs of the antibody or antibody fragment.

21. The anti-MUC1 according to any one of claims 1 to 6 ＊ antibody or antibody fragment, for MUC1 ＊ targeted therapeutic agent.

22. The MUC1 according to claim 21, which is a CAR T cell ＊ Targeted therapeutic agent.

23. The MUC1 according to claim 21, which is a bispecific T cell engager (BiTE). ＊ A targeted therapeutic agent.

24. The MUC1 according to claim 21, which is an antibody-drug conjugate ＊ Targeted therapeutic agent.

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