WO2022027039A1 - Anti-variable muc1* antibodies and uses thereof - Google Patents

Anti-variable muc1* antibodies and uses thereof Download PDF

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Publication number
WO2022027039A1
WO2022027039A1 PCT/US2021/071017 US2021071017W WO2022027039A1 WO 2022027039 A1 WO2022027039 A1 WO 2022027039A1 US 2021071017 W US2021071017 W US 2021071017W WO 2022027039 A1 WO2022027039 A1 WO 2022027039A1
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antibody
cells
consensus sequence
seq
peptide
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PCT/US2021/071017
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English (en)
French (fr)
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Cynthia Bamdad
Benoit Smagghe
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Minerva Biotechnologies Corporation
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Priority to AU2021316042A priority Critical patent/AU2021316042A1/en
Priority to EP21850106.2A priority patent/EP4188959A1/en
Priority to IL300252A priority patent/IL300252A/he
Priority to JP2023506494A priority patent/JP2023537326A/ja
Priority to CA3187555A priority patent/CA3187555A1/en
Priority to KR1020237006591A priority patent/KR20230059789A/ko
Priority to CN202180067049.7A priority patent/CN116234828A/zh
Publication of WO2022027039A1 publication Critical patent/WO2022027039A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0634Cells from the blood or the immune system
    • C12N5/0636T lymphocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/7051T-cell receptor (TcR)-CD3 complex
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/60Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622Single chain antibody (scFv)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/01Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/33Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00Genetically modified cells

Definitions

  • the CAR T cells can just eliminate all of the patient’s B cells without causing serious harm to the patient.
  • Most tumor associated antigens are also expressed on normal tissues; they are just expressed at a higher level in cancerous tissues.
  • the challenge is to develop an antibody that recognizes an epitope on a tumor associated antigen that is somehow different in the context of the tumor compared to normal tissue.
  • the antibody should recognize and bind to cancerous tissues at least two-times more than normal tissues. Antibodies that are not so cancer selective may be used therapeutically if they are inducibly expressed at the tumor site.
  • the invention is directed to an antibody that is “like” MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 in that they have the same or very similar pattern of binding to subsets of peptides derived from the PSMGFR peptide, also do not recognize a linear epitope, competitively inhibit the binding of NME1 or NME7AB to MUC1*, recognize a MUC1 transmembrane cleavage product produced by cleavage by MMP9 or contain CDR sequences that are at least 80% homologous to the MNE6, MNC2, MN18G12, MN20A10, MN25E6, MN28F9, MN5C6F3, MN3C2B1, and MN1E4 CDR consensus sequences.
  • the antibody may be a fragment, including a single chain fragment, scFv, of one of the antibodies.
  • the antibody or antibody fragment may be administered directly to the patient or incorporated into a multi-specific antibody-like molecule, a bispecific antibody, a bispecific T cell engager , BiTE, or an antibody drug conjugate, ADC.
  • the antibody or antibody fragment may be incorporated into a T cell receptor, TCR.
  • the sequence of the antibody or antibody fragment may be incorporated into a chimeric antigen receptor, a “CAR”, or other similar entity, then introduced into an immune cell, ex vivo, then administered to a patient diagnosed with or at risk of developing a cancer.
  • the invention is directed to a chimeric antigen receptor (CAR) comprising a scFv or a humanized variable region that binds to the extracellular domain of a MUC1 that is devoid of tandem repeats, a linker molecule, a transmembrane domain and a cytoplasmic domain.
  • CAR chimeric antigen receptor
  • the CD3 of the cytoplasmic tail may comprise deletions or mutations in or of the ITAMs including those referred to as 1XX (Feucht et al 2019; SEQ ID NO:1796- 1797).
  • the T cell may be engineered to overexpress c- Jun as a method to inhibit T cell exhaustion (Lynn et al 2019).
  • the CAR constructs described above may be expressed in a T cell, an NK cell, a dendritic cell or other immune cell, which may be autologous or allogeneic. Allogeneic cells may be derived from human stem cells.
  • the invention is directed to a composition that includes at least two CARs with different extracellular domain units transfected into the same cell, which may be an immune cell, which may be derived from the patient requiring treatment for a cancer.
  • the expression of the second CAR may be inducible and driven by the recognition of a target by the first CAR.
  • the nucleic acid encoding the second CAR may be linked to an inducible promoter.
  • the expression of the second CAR may be induced by an event that occurs specifically when the immune cell mounts an immune response to a target tumor cell.
  • the antibody fragments of one or both of the CARs may direct the cell to a MUC1* positive tumor.
  • the antibody fragments of the first and second CARs may bind to a MUC1* that is produced when MUC1 is cleaved by two different cleavage enzymes.
  • Expression of the second CAR by the inducible promoter may be induced when the antibody fragment of the first CAR engages or binds to a MUC1 or MUC1* on the tumor.
  • One way to do this is to induce expression of the second CAR when, or shortly after, an NFAT protein is expressed or translocated to the nucleus.
  • a sequence derived from an NFAT promoter region is put upstream of the gene for the second CAR.
  • the NFAT protein may be NFAT1 also known as NFATc2, NFAT2 also known as NFATc or NFATc1, NFAT3 also known as NFATc4, NFAT4 also known as NFATc3, or NFAT5.
  • the NFAT is NFATc1, NFATc3 or NFATc2.
  • the NFAT is NFAT2 also known as NFATc1.
  • one of the extracellular domain recognition units may be an antibody fragment and the other is a peptide, which may be devoid of transmembrane and signaling motifs; the peptide may be a single chain antibody fragment or antibody.
  • one of the recognition units may bind PD-1 or PDL-1.
  • one extra cellular domain recognition unit is an anti-MUC1* antibody, antibody fragment or scFv chosen from the group consisting of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11.
  • the cell may be cancer cell.
  • the invention is directed to a method for testing a subject’s cancer for suitability of treatment with a composition comprising antibodies of the invention, which may be murine, camelid, human or humanized, or fragments thereof, or portions of the variable regions of antibodies MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, comprising the steps of contacting a bodily specimen from the patient, in vitro, ex-vivo, or in vivo, with the antibody and determining that the patient exhibits aberrant expression of MUC1* compared to normal tissue or specimen and concluding that the patient’s cancer will beneficially respond to treatment with an agent comprising the antibody or a fragment thereof.
  • the invention is directed to a method of treating a subject suffering from a disease comprising, exposing immune cells, which may be T cells or NK cells from the subject, or from a donor, to MUC1* peptides wherein through various rounds of maturation, the T cells or NK cells develop MUC1* specific receptors, creating adapted T cells or NK cells, and expanding and administering the adapted cells to the donor patient who is diagnosed with, suspected of having, or is at risk of developing a MUC1* positive cancer.
  • immune cells which may be T cells or NK cells from the subject, or from a donor
  • MUC1* peptides wherein through various rounds of maturation, the T cells or NK cells develop MUC1* specific receptors, creating adapted T cells or NK cells, and expanding and administering the adapted cells to the donor patient who is diagnosed with, suspected of having, or is at risk of developing a MUC1* positive cancer.
  • the MUC1* peptide is chosen from among the group: [0093] (i) PSMGFR region of MUC1; [0094] (ii) PSMGFR peptide; [0095] (iii) a peptide having amino acid sequence of QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) [0096] (iv) a peptide having amino acid sequence of [0097] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19) [0098] (v) a peptide having amino acid sequence of [0099] NLTISDVSVSDVPFPFSAQSGA (N-23) [00100] (vi) a peptide having amino acid sequence of [00101] ISDVSVSDVPFPFSAQSGA (N-26) [00102] (vii) a peptide having amino acid sequence of [00103] SVSDVPFPFSAQSGA (N-30) [00104] (viii) a peptide having amino acid sequence
  • the antibody can be administered alone, as a monovalent antibody, as an scFv, or a fragment of the antibody can be incorporated into a CAR, a BiTE or an ADC.
  • the antibody that is administered to a patient for the treatment or prevention of a MUC1 or MUC1* positive cancer is selected for its ability to bind to the QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) peptide, wherein the presence of the FPFSAQSGA (N-36) sequence is required for binding.
  • the invention is directed to a method of treating cancer in a patient comprising administering to the patient the antibody, antibody fragment, BiTE, ADC or CAR expressed in an immune cell of any of the above, in combination with a checkpoint inhibitor.
  • any of the antibodies, or variable regions thereof, set forth in the following may be used: MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11.
  • the extracellular domain may be comprised of humanized single chain antibody fragments of an MN-E6 scFv set forth as SEQ ID NOS: 233, 235, or 237), MN-C2 scFv (SEQ ID NOS:239, 241, or 243).
  • the cytoplasmic tail may be comprised of one or more of signaling sequence motifs and co- stimulatory domains, including but not limited to CD3-zeta-1XX, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7 and CD3-zeta or variants 1XX, X2X, XX3, 12X, or 23X.
  • the sequence of the intracellular signaling domain may contain mutations that dampen the signal to improve persistence or target cell killing.
  • the method above may include two CARs with different extracellular domain units transfected into the same cell.
  • One of the extracellular domain recognition units may bind to MUC1* extracellular domain.
  • One of the extracellular domain recognition units may bind to PD-1.
  • One of the extracellular domain recognition units may be an antibody fragment and the other may be a peptide or an anti-MUC1* antibody fragment.
  • the method may include an immune cell transfected or transduced with a plasmid encoding a CAR and a plasmid encoding a non-CAR species that is expressed from an inducible promoter.
  • the non-CAR species may be expressed from an inducible promoter that is activated by elements of an activated immune cell.
  • the non-CAR species may be expressed from an NFAT inducible promoter.
  • the NFAT may be NFATc1, NFATc3 or NFATc2.
  • the cleavage enzyme may be MMP2, MMP3, MMP9, MMP13, MMP14, MMP16, ADAM10, ADAM17, or ADAM28, or a catalytically active fragment thereof.
  • the non-CAR species may be a cytokine.
  • the cytokine may be IL-7, IL-12, IL-15 or IL-18.
  • the antibody interacts with a peptide comprising conformational epitope SVSDV (SEQ ID NO:1751) and FPFSA (SEQ ID NO:1747) within N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO:6), wherein mutation or deletion of FPFS (SEQ ID NO:1747) destroys binding of the antibody or fragment thereof to the N-26 peptide.
  • the antibody inhibits interaction between NME7AB and MUC1*.
  • the antibody inhibits interaction between NME7 AB and PSMGFR peptide (SEQ ID NO:2).
  • the antibody inhibits interaction between NME7AB and N-10 peptide (SEQ ID NO:3), N- 19 peptide (SEQ ID NO:4), N-23 peptide (SEQ ID NO:5), N-26 peptide (SEQ ID NO:6), N-30 peptide (SEQ ID NO:7), N-10/C-5 peptide (SEQ ID NO:8), N-19/C-5 peptide (SEQ ID NO:9), or C-5 peptide (SEQ ID NO:825).
  • the antibody does not bind to a linear form of the binding conformationally inducing peptide sequence wherein the linear form of the peptide is a denatured form.
  • the binding conformationally inducing peptide sequence is in the N-26 peptide sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO:6), wherein mutation or deletion of FPFS (SEQ ID NO:1747) destroys binding of the antibody or fragment thereof to the N-26 peptide.
  • An antibody binding to a conformational epitope within a peptide having the N-26 sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO:6), wherein mutation or deletion within FPFS (SEQ ID NO:1747), SVSDV (SEQ ID NO:1751), or ASRYNLT (SEQ ID NO:1745) destroys binding of the antibody or fragment thereof to PSMGFR.
  • heavy chain CDR1 comprises consensus sequence at least 90% identical to sequence: F or I at position 1, T or A at position 2, F at position 3, S at position 4, T, G, or R at position 5, Y or F at position 6, A, G or T at position 7, M at position 8 and S at position 9;
  • heavy Chain CDR2 comprises consensus sequence at least 90% identical to sequence: T or A at position 1, I or S at position 2, I or S at position 3, N, S, T or G at position 4, G or R at position 5, G or A at position 6, G, T, or D at position 7, Y, K, H or S at position 8, T or I at position 9, Y or F at position 10, Y at position 11, P or S at position12 and D at position 13, S or T at position 14, V or L at position 15 and KG for positions 16-17;
  • heavy chain CDR3 comprises consensus sequence at least 90% identical to sequence: G, L, or N at position 2, G, T, or or N at position 2, G, T, or or R at position 5, Y or F at position 6, A, G or T at
  • heavy chain CDR1 comprises consensus sequence FTFSGYAMS; [00160] heavy Chain CDR2 comprises consensus sequence TISSGGTYIYYPDSVKG; [00161] heavy chain CDR3 comprises consensus sequence -LGGDNYYEYFDV--; [00162] light chain CDR1 comprises consensus sequence RASKS--VSTSGYSYMH; [00163] light Chain CDR2 comprises consensus sequence LASNLES; and [00164] light chain CDR3 comprises consensus sequence QHSRELPFT.
  • heavy chain CDR1 comprises consensus sequence FTFSRYGMS
  • heavy Chain CDR2 comprises consensus sequence TISGGGTYIYYPDSVKG
  • heavy chain CDR3 comprises consensus sequence DNYGRNYDYGMDY--
  • light chain CDR1 comprises consensus sequence -------SATSSVSYIH
  • light Chain CDR2 comprises consensus sequence STSNLAS
  • light chain CDR3 comprises consensus sequence QQRSSSPFT.
  • heavy chain CDR1 comprises consensus sequence FAFSTFAMS; [00174] heavy Chain CDR2 comprises consensus sequence AISNGGGYTYYPDTLKG; [00175] heavy chain CDR3 comprises consensus sequence ----RYYDLYFDL--; [00176] light chain CDR1 comprises consensus sequence RSSQNIV-HSNGNTYLE; [00177] light Chain CDR2 comprises consensus sequence KVSNRFS; and [00178] light chain CDR3 comprises consensus sequence FQDSHVPLT.
  • heavy chain CDR1 comprises consensus sequence FTFSTYAMS; [00195] heavy Chain CDR2 comprises consensus sequence AISNGGGYTYYPDSLKG; [00196] heavy chain CDR3 comprises consensus sequence ----RYYDHYFDY--; [00197] light chain CDR1 comprises consensus sequence --RASESVATYGNNFMQ; [00198] light Chain CDR2 comprises consensus sequence LASTLDS; and [00199] light chain CDR3 comprises consensus sequence QQNNEDPPT.
  • heavy chain CDR1 comprises consensus sequence FAFSTFAMS; [00202] heavy Chain CDR2 comprises consensus sequence AISNGGGYTYYPDTLKG; [00203] heavy chain CDR3 comprises consensus sequence ----RYYDLYFDL--; [00204] light chain CDR1 comprises consensus sequence RSSQNIV-HSNGNTYLE; [00205] light Chain CDR2 comprises consensus sequence KVSNRFS; and [00206] light chain CDR3 comprises consensus sequence FQDSHVPLT.
  • heavy chain CDR1 comprises consensus sequence SYGVH; [00209] heavy Chain CDR2 comprises consensus sequence VIWPGGSTNYNSTLMSRM; [00210] heavy chain CDR3 comprises consensus sequence DRTPRVGAWFAY; and [00211] light chain CDR1 comprises consensus sequence RASESVATYGNNFMQ; [00212] light Chain CDR2 comprises consensus sequence LASTLDS; and [00213] light chain CDR3 comprises consensus sequence QQNNEDPPT.
  • heavy chain CDR1 comprises consensus sequence FTFSTYAMS; [00216] heavy Chain CDR2 comprises consensus sequence -SIGRAGSTYYSDSVKG; [00217] heavy chain CDR3 comprises consensus sequence ---GPIYNDYDEFAY; [00218] light chain CDR1 comprises consensus sequence KSSQSVLYSSNQKNYLA; [00219] light Chain CDR2 comprises consensus sequence WASTRES; and [00220] light chain CDR3 comprises consensus sequence HQYLSSLT.
  • 3C2B1 having [00222] heavy chain CDR1 comprises consensus sequence ITFSTYTMS; [00223] heavy Chain CDR2 comprises consensus sequence TISTGGDKTYYSDSVKG; [00224] heavy chain CDR3 comprises consensus sequence -GTTAMYYYAMDY; [00225] light chain CDR1 comprises consensus sequence RASKS---ISTSDYNYIH ; [00226] light Chain CDR2 comprises consensus sequence LASNLES; and [00227] light chain CDR3 comprises consensus sequence QHSRELPLT.
  • the invention is directed to an antibody, or fragment thereof, for the diagnosis, treatment or prevention of cancers that requires presence of antibody binding conformationally inducing peptide ASRYNLT (SEQ ID NO:1745) of PSMGFR (SEQ ID NO:2).
  • the antibody may be 25E6, having [00229] heavy chain CDR1 comprises consensus sequence FTFSSYGMS; [00230] heavy Chain CDR2 comprises consensus sequence TISNGGRHTFYPDSVKG; [00231] heavy chain CDR3 comprises consensus sequence QTGTEGWFAY; [00232] light chain CDR1 comprises consensus sequence KSSQSLLDSDGKTYLN; [00233] light Chain CDR2 comprises consensus sequence LVSKLDS _; and [00234] light chain CDR3 comprises consensus sequence WQGTHFPQT.
  • the invention is directed to an antibody, or fragment thereof, for the diagnosis, treatment or prevention of cancers that requires presence of antibody binding conformationally inducing peptide SVSDV (SEQ ID NO:1761) of PSMGFR (SEQ ID NO:2).
  • the antibody may be 5C6F3, having [00236] heavy chain CDR1 comprises consensus sequence FTFSTYAMS ; [00237] heavy Chain CDR2 comprises consensus sequence AISNGGGYTYYPDSLKG; [00238] heavy chain CDR3 comprises consensus sequence RYYDHYFDY; [00239] light chain CDR1 comprises consensus sequence RSSQTIVHSNGNTYLE; [00240] light Chain CDR2 comprises consensus sequence KVSNRFS; and [00241] light chain CDR3 comprises consensus sequence FQDSHVPLT. [00242] The antibody or fragment thereof according all of the above may be murine, camelid, human or humanized.
  • the antibody fragment may be scFv or scFv-Fc, which variable regions thereof may be murine, camelid, human or humanized.
  • the invention is directed to a chimeric antigen receptor (CAR) comprising the antibody fragments of above, and may further comprise mutations in the co- stimulatory domain or mutations or deletions of one or two of the ITAMs of the CD3-zeta signaling domain.
  • Tyrosines may be mutated in CD28 or 4-1BB.
  • CD3-zeta may contain a single ITAM such as only ITAM1 also known as 1XX, ITAM2 also known as X2X, or ITAM3 also known as XX3.
  • the immune cell is derived from a stem cell that has been directed to differentiate to that immune cell type in vitro.
  • a CAR containing sequences of the antibody are expressed in a stem cell, which then may be differentiated into an immune cell.
  • the invention is directed to a cell composition expressed in a cell comprising a CARs of above, and second entity having a biological recognition unit that has a specificity that is different from that of the CAR.
  • the second entity may bind PD-1, PDL-1, or other checkpoint inhibitor, or NME7, or a cytokine such as IL-12 or IL-18, or c- Jun.
  • the nucleic acids encoding the second entity may be inserted into a Foxp3 promoter or enhancer region, wherein the cytokine is IL-18.
  • the cytokine may be expressed from an NFAT inducible promoter.
  • the invention is directed to a BiTE construct comprising the antibody fragment of above.
  • the invention is directed to an antibody drug conjugate (ADC) comprising the antibody or antibody fragment of above.
  • ADC antibody drug conjugate
  • the cancer may be breast cancer, pancreatic cancer, ovarian cancer, lung cancer, colon cancer, gastric cancer or esophageal cancer.
  • the present invention is also directed to a method of diagnosing, treating or preventing cancer by administering the antibodies and fragments disclosed herein to a cancer patient in need thereof that has been identified as expressing MUC1 aberrantly and expressing truncated MUC1, such as MUC1*.
  • FIGS 1A-1D show cell growth assay graphs of MUC1* positive cells treated with either bivalent ‘bv’ anti-MUC1* antibody, monovalent ‘mv’ or Fab, NM23-H1 dimers or NME7-AB.
  • Bivalent anti-MUC1* antibodies stimulate growth of cancer cells whereas the monovalent Fab inhibits growth (Fig. 1A-1B).
  • Classic bell-shaped curve indicates ligand induced dimerization stimulates growth.
  • FIG. 1C Dimeric NM23-H1, aka NME1, stimulates growth of MUC1* positive cancer cells but siRNA to suppress MUC1 expression eliminate its effect (Fig.1C). NME7-AB also stimulates the growth of MUC1* positive cells (Fig.1D).
  • Figures 2A-2I show results of ELISA assays. MUC1* peptides PSMGFR, PSMGFR minus 10 amino acids from the N-terminus aka N-10, or PSMGFR minus 10 amino acids from the C-terminus, aka C-10 are immobilized on the plate and the following are assayed for binding: NME7-AB (Fig. 2A), MNC2 monoclonal antibody (Fig. 2B), MNE6 monoclonal antibody (Fig.
  • MUC1* peptides PSMGFR minus 10 amino acids from the N-terminus aka N-10, or PSMGFR minus 10 amino acids from the C-terminus, aka C-10, are immobilized on the plate and the following are assayed for binding: MNC3 (Fig. 2E) and MNC8 (Fig. 2F).
  • Fig. 2G shows the amino acid sequence of the PSMGFR peptide.
  • Fig. 2H shows the amino acid sequence of the N-10 peptide.
  • FIG.2I shows the amino acid sequence of the C-10 peptide.
  • Figures 3A-3C show results of competitive ELISA assays.
  • the PSMGFR MUC1* peptide is immobilized on the plate and dimeric NM23-H1, aka NME1, is added either alone or after the MNE6 antibody has been added (Fig.3A).
  • NM23-H7, NME7-AB is added alone or after MNE6 has been added
  • Results show that MNE6 competitively inhibits the binding of MUC1* activating ligands NME1 and NME7.
  • Fig. 3A shows that MNE6 competitively inhibits the binding of MUC1* activating ligands NME1 and NME7.
  • ZR-75-1 aka 1500, MUC1* positive breast cancer cells were stained with 1:2 or 1:10 dilutions of the 1.5 ug/ml humanized MNC2. After two washes, cells were stained with secondary antibody, Anti-Penta-His antibody conjugated to Alexa 488 (Qiagen) dilutions of 1:200 (Fig. 4A), 1:50 (Fig. 4B), or 1:10 (Fig. 4C) to detect the 6x His tag on the huMNC2 scFv.
  • Fig. 4A shows huMNC2 binding to ZR-75-1 breast cancer cells where secondary antibody is added at a 1:200 dilution.
  • FIG. 4B shows huMNC2 binding to ZR-75-1 breast cancer cells where secondary antibody is added at a 1:50 dilution.
  • Fig. 4C shows huMNC2 binding to ZR-75-1 breast cancer cells where secondary antibody is added at a 1:10 dilution.
  • Flow cytometric analysis revealed a concentration-dependent shift of a subset of cells, indicating specific binding, which is unseen in the absence of the MNC2 scFv (Fig.4A- 4C).
  • Fig. 4D shows anti-MUC1* antibody MNE6 staining of MUC1 negative HCT-116 colon cancer cells transfected with the empty vector, single cell clone #8.
  • FIG.4E shows anti- MUC1* antibody MNE6 staining of HCT-116 colon cancer cells transfected with MUC1* single cell clone #10.
  • Fig. 4F shows anti-MUC1* antibody MNE6 staining of ZR-75-1, aka 1500, MUC1* positive breast cancer cells. As the FACS scans show, both MNC2 and MNE6 only stain MUC1* positive cells and not MUC1 or MUC1* negative cells.
  • Figure 5 shows a graph of an ELISA in which surface is coated with either the MUC1* PSMGFR peptide or a control peptide. Humanized MNC2 scFv is then incubated with the surface, washed and detected according to standard methods.
  • Figures 7A-7B show graphs of tumor growth in immune compromised mice that have been implanted with human tumors then treated with anti-MUC1* antibody MNE6 Fab or mock treatment.
  • Female nu/nu mice implanted with 90-day estrogen pellets were implanted with 6 million T47D human breast cancer cells that had been mixed 50/50 with Matrigel. Mice bearing tumors that were at least 150 mm 3 and had three successive increases in tumor volume were selected for treatment. Animals were injected sub cutaneously twice per week with 80 mg/kg MNE6 Fab and an equal number of mice fitting the same selection criteria were injected with vehicle alone (Fig. 7A).
  • Figures 9A-9B show graphs of ELISAs wherein the assay plate surface was immobilized with either PSMGFR peptide, PSMGFR minus 10 amino acids from the N- terminus or minus 10 amino acids from the C-terminus. The MNC3 antibody variants were then assayed for binding to the various MUC1* peptides.
  • Fig. 9A shows purified mouse monoclonal MNC3 antibody; and
  • Figure 9B shows the humanized MNC3 scFv-Fc.
  • ELISAs show binding to the PSMGFR peptide as well as to certain deletion peptides.
  • Figures 10A-10J Figs. 10A10B are photographs of breast cancer tissue arrays. Fig.
  • FIG. 10A was stained with VU4H5 which recognizes MUC1-FL (full length); Fig. 10B was stained with mouse monoclonal antibody MNC2 which recognizes cancerous MUC1*. Following automated staining (Clarient Diagnostics), the tissue staining was scored using Allred scoring method which combines an intensity score and a distribution score.
  • Figs. 10C10F are color coded graphs showing the score calculated for MUC1 full-length staining for each patient’s tissue.
  • Figs. 10G10J are color coded graphs showing the score calculated for MUC1* staining for each patient’s tissue.
  • Figures 11A-11J. Figs. 11A11B are photographs of breast cancer tissue arrays. Fig.
  • Figs. 11A was stained with VU4H5 which recognizes MUC1-FL (full length); Fig. 11B was stained with mouse monoclonal antibody MNC2 which recognizes cancerous MUC1*. Following automated staining (Clarient Diagnostics), the tissue staining was scored using Allred scoring method which combines an intensity score and a distribution score.
  • Figs.11C- 11F are color coded graphs showing the score calculated for MUC1 full-length staining for each patient’s tissue.
  • Figs. 11G-11J are color coded graphs showing the score calculated for MUC1* staining for each patient’s tissue.
  • Figures 12A-12H show photographs of normal breast and breast cancer tissues stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 2.5 ug/mL, then stained with a secondary streptavidin HRP antibody.
  • Fig. 12A is a normal breast tissue.
  • Figs.12B-12D are breast cancer tissues from patients as denoted in the figure.
  • Figs.12E-12H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 13A-13F show photographs of normal breast and breast cancer tissues stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 2.5 ug/mL, then stained with a secondary streptavidin HRP antibody.
  • Fig. 13A is a normal breast tissue.
  • Figs.13B-13C are breast cancer tissues from patients as denoted in the figure.
  • Figs.13D-13F are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 14A-14H show photographs of breast cancer tissues stained with MNE6 anti-MUC1* antibody at 10 ug/mL, then stained with a rabbit anti mouse secondary HRP antibody.
  • FIGS. 14A-14D are breast cancer tissues from patient #300.
  • Figs. 14E-14H are breast cancer tissues from metastatic patient #291.
  • Figures 15A-15F show photographs of normal lung and lung cancer tissues stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 2.5 ug/mL, then stained with a secondary streptavidin HRP antibody.
  • Fig. 15A is a normal lung tissue.
  • Figs. 15B15C are lung cancer tissues from patients as denoted in the figure.
  • Figs. 15D-15F are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 16A-16F show photographs of normal lung and lung cancer tissues stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 2.5 ug/mL, then stained with a secondary streptavidin HRP antibody.
  • Fig. 16A is a normal lung tissue.
  • Figs. 16B16C are lung cancer tissues from patients as denoted in the figure.
  • Figs. 16D-16F are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 17A-17F show photographs of normal lung and lung cancer tissues stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 25 ug/mL, then stained with a secondary streptavidin HRP antibody.
  • Fig. 17A is a normal lung tissue.
  • Figs. 17B-17C are lung cancer tissues from patients as denoted in the figure.
  • Figs. 17D-17F are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 18A-18F show photographs of normal lung and lung cancer tissues stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 25 ug/mL, then stained with a secondary streptavidin HRP antibody.
  • Fig. 18A is a normal lung tissue.
  • Figs. 18B-18C are lung cancer tissues from patients as denoted in the figure.
  • Figs. 18D-18F are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 19A-19D show photographs of normal small intestine and cancerous small intestine tissues stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 5 ug/mL, then stained with a secondary streptavidin HRP antibody.
  • Fig. 19A is a normal small intestine tissue.
  • Fig.19B is small intestine cancer from patient as denoted in the figure.
  • Figs. 19C-19D are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 20A-20H show photographs of normal small intestine tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • Figs. 20A-20D are normal small intestine tissue.
  • Figs. 20E- 20H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 21A-21H show photographs of cancerous small intestine tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • FIGS. 21A-21D are cancerous small intestine tissue from a patient as denoted in figure.
  • Figs. 21E-21H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 22A-22H show photographs of cancerous small intestine tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • Figs. 22A-22D are cancerous small intestine tissue from a patient as denoted in figure.
  • Figs. 22E-22H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 23A-23H show photographs of normal colon tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • Figs. 23A-23D are normal colon.
  • Figs. 23E-23H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 24A-24H show photographs of colon cancer tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • Figs. 24A-24D are colon cancer tissue from a metastatic patient as denoted in figure.
  • FIGs. 24E-24H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 25A-25H show photographs of colon cancer tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • Figs. 25A-25D are colon cancer tissue from a Grade 2 patient as denoted in figure.
  • Figs. 25E-25H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 26A-26H show photographs of colon cancer tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • Figs. 26A-26D are colon cancer tissue from a metastatic patient as denoted in figure.
  • Figs. 26E-26H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 27A-27H show photographs of prostate cancer tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • FIGS. 27A-27D are prostate cancer tissue from a patient as denoted in figure.
  • FIGs. 27E-27H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 28A-28H show photographs of prostate cancer tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • Figs. 28A-28D are prostate cancer tissue from a patient as denoted in figure.
  • Figs. 28E-28H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 29A-29H show photographs of prostate cancer tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • Figs. 29A-29D are prostate cancer tissue from a patient as denoted in figure.
  • Figs. 29E-29H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Figures 30A-30F show photographs of a triple negative breast cancer array stained with anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score and the second is the tumor grade.
  • FIG. 30A shows the pie chart of score of anti- MUC1* antibody staining.
  • Fig. 30B shows a photograph of the array stained with the antibody.
  • Figs. 30C-30D show magnified photographs of two of the breast cancer specimens from the array.
  • Figs. 30E-30F show more magnified photographs of the portion of the specimen that is marked by a box.
  • Figures 31A-31F show photographs of an ovarian cancer array stained with anti- MUC1* antibody huMNC2scFv. The first score shown is the Allred score and the second is the tumor grade.
  • FIG. 31A shows the pie chart of score of anti-MUC1* antibody staining.
  • Fig. 31B shows a photograph of the array stained with the antibody.
  • Figs. 31C-31D show magnified photographs of two of the breast cancer specimens from the array.
  • Figs.31E- 31F show more magnified photographs of the portion of the specimen that is marked by a box.
  • Figures 32A-32F show photographs of a pancreatic cancer array stained with anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score and the second is the tumor grade.
  • FIG. 32A shows the pie chart of score of anti-MUC1* antibody staining.
  • Fig. 32B shows a photograph of the array stained with the antibody.
  • Figs. 32C-32D show magnified photographs of two of the breast cancer specimens from the array.
  • Figs. 32E-32F show more magnified photographs of the portion of the specimen that is marked by a box.
  • Figures 33A-33F show photographs of a lung cancer array stained with anti- MUC1* antibody huMNC2scFv. The first score shown is the Allred score and the second is the tumor grade.
  • FIG. 33A shows the pie chart of score of anti-MUC1* antibody staining.
  • Fig. 33B shows a photograph of the array stained with the antibody.
  • Figs. 33C-33D show magnified photographs of two of the breast cancer specimens from the array.
  • Figs.33E- 33F show more magnified photographs of the portion of the specimen that is marked by a box.
  • Figures 34A-34I show photographs of normal tissues stained with anti-MUC1* antibody huMNC2scFv.
  • Figures 35A-35D show FACS scans of cells expressing either no MUC1, MUC1* or full-length MUC1, wherein the cells were probed with either MNC2 or VU4H5.
  • Fig. 35A shows MUC1 negative HCT-116 colon cancer cells probed with antibody MNC2.
  • Fig. 35B shows HCT cells that have been transfected with MUC1* wherein the extra cellular domain is just the sequence of the PSMGFR peptide wherein the cells are probed with antibody MNC2.
  • Fig. 35A shows MUC1 negative HCT-116 colon cancer cells probed with antibody MNC2.
  • Fig. 35B shows HCT cells that have been transfected with MUC1* wherein the extra cellular domain is just the sequence of the PSMGFR peptide wherein the cells are probed with antibody MNC2.
  • HCT-MUC1-18 cells which are a cleavage resistant single cell clone of HCT cells transfected with full-length MUC1, also referred to herein as HCT-MUC1- 41TR, and cells were probed with antibody MNC2.
  • Fig. 35D shows HCT-MUC1-18 cells probed with antibody VU4H5 which is an antibody that recognizes the hundreds of tandem repeats epitopes in full-length MUC1.
  • VU4H5 is an antibody that recognizes the hundreds of tandem repeats epitopes in full-length MUC1.
  • MNC2 recognizes an ectopic epitope that is not accessible in full-length MUC1.
  • Figures 36A-36D show Western blots and corresponding FACs analysis of HCT- 116 cells which are a MUC1 negative colon cancer cell line, that were then stably transfected with either MUC1* or MUC1 full-length.
  • the single cell clones that are shown are HCT- MUC1-41TR, and HCT-MUC1*.
  • Fig.36A shows a Western blot of the parent cell line HCT- 116, HCT-MUC1-41TR and HCT-MUC1* wherein the gel has been probed with a rabbit polyclonal antibody, SDIX, that only recognizes cleaved MUC1.
  • Fig. 36B is a Western blot that was probed with a mouse monoclonal antibody VU4H5 that recognizes the tandem repeats of full-length MUC1. As can be seen, only HCT-MUC1-41TR contains full-length MUC1.
  • FIG. 36C shows FACS scans showing that HCT-MUC1* is 95.7% positive for SDIX which only binds to MUC1* and essentially not at all for MUC1 full-length.
  • Fig. 36D shows FACS scans that show that HCT-MUC1-41TR cells are 95% positive for full-length MUC1 and only about 11% positive for the cleaved form, MUC1*.
  • Figure 37A-37C shows western blots and a bar graph of FACS analysis assessing the ability of MNC2to recognize a full-length MUC1 after it has been cleaved by MMP9. Fig.
  • FIG. 37A shows a Western blot of HCT-MUC1-18 cells, which are a cleavage resistant cell line, to which was added cleavage enzyme MMP9.
  • the cell lysate fraction was run on a gel and probed with a polyclonal anti-PSMGFR antibody.
  • the photo shows that in a dose dependent manner, MMP9 cleaved MUC1 to MUC1*, the ⁇ 25kDa species.
  • Fig.37B shows the Western blot of the conditioned media from the same experiment.
  • the photo shows that the addition of cleavage enzyme MMP9, in a dose dependent manner, increased the release of the tandem repeat domain into the conditioned media.
  • Fig. 37C shows FACS analysis of the experiment.
  • FIG. 38 shows a photograph of a Western blot in which HCT-MUC1-18 cells, labeled here as HCT-18, a cleavage resistant single cell clone of HCT cells transfected with full-length MUC1, are treated with varying amounts of a catalytically active ADAM17 or MMP14.
  • FIG. 39A-39B shows fluorescence activated cell sorting (FACS) measurements of human CD34+ hematopoietic stem cells of human bone marrow stained with anti-MUC1* monoclonal antibodies MNC3, MNC2, MNE6 or an isotype control antibody.
  • FIG. 40A-40G shows the details of FACS analysis of the hematopoietic stem cells probed with either MNC3 or MNE6.
  • Fig.40A shows the FACS scatter plot of total bone marrow cells.
  • FIG. 40B shows the FACS scatter plot of the CD34+ cells.
  • Fig. 40C shows the FACS histogram of the CD34+ cells.
  • Fig. 40D shows the FACS scatter plot of the earliest hematopoietic stem cells, which are CD34+/CD38-, stained with either MNC3 or MNE6.
  • Fig. 40E shows the histogram of the experiment.
  • Fig. 40F shows the histogram overlay of MNC3 binding to CD34+/CD38- cells versus MNE6.
  • Fig. 40G shows the bar graph of that FACS experiment.
  • Figure 41A-42H shows the details of FACS analysis of CD34+/CD38 -/lo hematopoietic stem cells probed with a polyclonal anti-PSMGFR antibody SDIX, MNE6 or MNC2.
  • Fig.41A shows the FACS scatter plot of the CD34+/CD38 -/lo population of cells.
  • Fig. 41E shows a table of the detailed analysis.
  • Fig. 41B shows the FACS scatter plot of the CD34+/CD38 -/lo population of cells probed with the anti-PSMGFR polyclonal antibody SDIX.
  • Fig. 41F shows a table of the detailed analysis.
  • Fig. 41A shows the FACS scatter plot of the CD34+/CD38 -/lo population of cells probed with the anti-PSMGFR polyclonal antibody SDIX.
  • Fig. 41F shows a table of the detailed analysis.
  • Fig. 41A shows the FACS scatter plot of the CD34+/CD38
  • Figs. 45A, 45E, 45I, 45M show photographs of cells co-cultured with untransduced human T cells.
  • Figs. 45B, 45F, 45J, 45N show photographs of cells co-cultured with human T cells transduced with anti-MUC1* CAR44 at an MOI of 10.
  • Figs. 45C, 45G, 45K, 45O show photographs of cells co-cultured with human T cells transduced with anti- MUC1* CAR50 at an MOI of 10.
  • Figs. 45A, 45E, 45I, 45M show photographs of cells co-cultured with untransduced human T cells.
  • Figs. 45B, 45F, 45J, 45N show photographs of cells co-cultured with human T cells transduced with anti-MUC1* CAR44 at an MOI of 10.
  • Figs. 45C, 45G, 45K, 45O show photographs of cells co-cultured with human T cells transduced with anti- MUC1* CAR50 at an
  • FIG. 76A-76E show percent binding and Fig.76F-76J show Mean Fluorescent Intensity or MFI.
  • Figure 77A-77N show graphs of FACS analyses of reference antibody MNC2, “C2”, binding to a panel of cancer cell lines that are MUC1* positive, with the exception of MDA-MB-231, which expresses MUC1 and MUC1* at a level that is so low that it is often used as a negative control.
  • Figure 97A-97B show photographs of normal liver tissue specimens, stained with an antibody of the invention 1E4.
  • Fig. 97A show the entire tissue core.
  • Fig. 97B show the 40X magnification of a particular area of the tissue.
  • Antibody 1E4 binds to an epitope that comprises all or part of the sequence QFNQYKTEA.
  • Antibody 1E4 can bind to the N-10 peptide but also binds to the C-10 peptide.
  • 1E4 binds to normal liver tissue.
  • Figure 98A-98H show photographs of normal liver tissue specimens, stained with different antibodies of the invention.
  • FIG. 102A-102D show photographs of normal lung tissue specimens, stained with different antibodies of the invention.
  • Fig. 102A-102B show the entire tissue core.
  • Fig. 102C-102D show the 40X magnification of a particular area of the tissue.
  • Figure 108A-108D show photographs of normal bone marrow tissue specimens, stained with different antibodies of the invention.
  • Fig. 108A-108B show the entire tissue core.
  • Fig. 108C-108D show the 40X magnification of a particular area of the tissue.
  • Fig. 108A and Fig. 108C show staining with MNC3.
  • Fig. 108B and Fig. 108D show staining with 25E6.
  • These antibodies bind to an epitope that comprises all or part of the sequence ASRYNLT.
  • Figure 109A-109B show photographs of normal bone marrow tissue specimens, stained with an antibody of the invention 1E4.
  • Fig. 109A show the entire tissue core.
  • Fig. 109B show the 40X magnification of a particular area of the tissue.
  • Antibody 1E4 binds to an epitope that comprises all or part of the sequence QFNQYKTEA.
  • Antibody 1E4 can bind to the N-10 peptide but also binds to the C-10 peptide.1E4 binds to normal bone marrow.
  • Figure 111A-111D show photographs of normal bone marrow tissue specimens, stained with antibodies of the invention.
  • Fig. 111A-111B show the entire tissue core.
  • Fig. 111C-111D show the 40X magnification of a particular area of the tissue.
  • Fig. 111A and Fig. 111C show staining with antibody 8A9.
  • Fig. 111B and Fig. 111D show staining with antibody 17H6. Both antibodies bind to an epitope that that is outside of the PSMGFR region and comprises all or part of the sequence VQLTLAFRE.
  • FIG. 113A-113C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 20A10 at 0.25ug/mL.
  • Fig. 113A shows photographs of the tissue micro array.
  • Fig. 113B shows map of the array with abbreviated tissue descriptors.
  • Fig. 113C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 114A-114X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 20A10 at 0.25ug/mL, magnified to 6X and 20X.
  • Fig. 114A and Fig. 114E are adrenal gland.
  • Fig. 114B and Fig. 114F are breast.
  • Fig. 114C and Fig. 114G are fallopian tube.
  • Fig. 114D and Fig. 114H are kidney.
  • Fig. 114I and Fig. 114M are heart muscle.
  • Fig. 114J and Fig. 114N are liver.
  • Fig. 114K and Fig. 114O are lung.
  • Fig. 114L and Fig. 114P are ureter.
  • Fig. 114Q and Fig. 114U are eye.
  • Fig. 114R and Fig. 114V are cerebral cortex.
  • Fig. 114S and Fig. 114W are bone marrow.
  • FIG.114T and Fig.114X are skeletal muscle.
  • Figure 115A-115C shows photographs, array map and description of breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 20A10 at 0.25ug/mL.
  • Fig. 115A shows photographs of the tissue micro array.
  • Fig. 115B shows map of the array with abbreviated tissue descriptors.
  • Fig. 115C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 116A-116F shows photographs of specific tissues from breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 20A10 at 0.25ug/mL, magnified to 6X and 20X.
  • FIG. 116D are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig. 116B and Fig. 116E are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig.116C and Fig.116F are photographs of a Grade 2 invasive ductal carcinoma.
  • Figure 117A-117C shows photographs, array map and description of pancreatic cancer tissue array PA805c stained with the anti-PSMGFR antibody 20A10 at 0.25ug/mL.
  • Fig. 117A shows photographs of the tissue micro array.
  • Fig. 117B shows map of the array with abbreviated tissue descriptors.
  • Fig. 117C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 124A-124F shows photographs of specific tissues from pancreatic cancer tissue array PA1003 stained with the anti-PSMGFR antibody 3C2B1 at 20ug/mL, magnified to 6X and 20X.
  • Fig. 124A and Fig.124D are photographs of a Grade 2 adenocarcinoma.
  • Fig. 124B and Fig. 124E are photographs of a Grade 2 adenocarcinoma.
  • Fig. 124C and Fig. 124F are photographs of a Grade 2 adenocarcinoma.
  • Fig. 126B and Fig. 126E are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig.126C and Fig.126F are photographs of a Grade 2 invasive carcinoma.
  • Figure 127A-127C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 5C6F3 at 1ug/mL.
  • Fig. 127A shows photographs of the tissue micro array.
  • Fig. 127B shows map of the array with abbreviated tissue descriptors.
  • Fig. 127C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 128A-128X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 5C6F3 at 1ug/mL, magnified to 6X and 20X.
  • Fig. 128A and Fig. 128E are adrenal gland.
  • Fig. 128B and Fig. 128F are breast.
  • Fig. 128C and Fig. 128G are fallopian tube.
  • Fig. 128D and Fig. 128H are kidney.
  • Fig.128I and Fig.128M are heart muscle.
  • Fig.128J and Fig.128N are liver.
  • Fig.128K and Fig. 128O are lung.
  • Fig. 128L and Fig. 128P are ureter.
  • FIG. 128Q and Fig. 128U are eye.
  • Fig.128R and Fig.128V are cerebral cortex.
  • Fig. 128S and Fig.128W are bone marrow.
  • Fig. 128T and Fig.128X are skeletal muscle.
  • Figure 129A-129C shows photographs, array map and description of pancreatic cancer tissue array PA1003 stained with the anti-PSMGFR antibody 5C6F3 at 1-20ug/mL.
  • Fig. 129A shows photographs of the tissue micro array.
  • Fig. 129B shows map of the array with abbreviated tissue descriptors.
  • Fig. 129C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 130A-130F shows photographs of specific tissues from pancreatic cancer tissue array PA1003 stained with the anti-PSMGFR antibody 5C6F3 at 1ug/mL, magnified to 6X and 20X.
  • Fig. 130A and Fig. 130D are photographs of a Grade 2 adenocarcinoma.
  • Fig. 130B and Fig. 130E are photographs of a Grade 2 adenocarcinoma.
  • Fig. 130C and Fig. 130F are photographs of a Grade 2 adenocarcinoma.
  • Figure 131A-131C shows photographs, array map and description of breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 5C6F3 at 1ug/mL.
  • Figure 133A-133C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 18B4 at 10ug/mL.
  • Fig. 133A shows photographs of the tissue micro array.
  • Fig. 133B shows map of the array with abbreviated tissue descriptors.
  • Fig. 133C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 134A-134X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 18B4 at 10ug/mL, magnified to 6X and 20X.
  • Fig. 134A and Fig. 134E are adrenal gland.
  • Fig. 134B and Fig. 134F are breast.
  • Figure 135A-135C shows photographs, array map and description of breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 18B4 at 10ug/mL.
  • Fig. 135A shows photographs of the tissue micro array.
  • Fig. 135B shows map of the array with abbreviated tissue descriptors.
  • Fig. 135C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 136A-136F shows photographs of specific tissues from breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 18B4 at 10ug/mL, magnified to 6X and 20X.
  • Fig. 136A and Fig. 136D are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig. 135A shows photographs of the tissue micro array.
  • Fig. 135B shows map of the array with abbreviated tissue descriptors.
  • Fig. 135C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 138A-138F shows photographs of specific tissues from esophageal cancer tissue array BC001113 stained with the anti-PSMGFR antibody 18B4 at 10ug/mL, magnified to 6X and 20X.
  • Fig. 138A and Fig. 138D are photographs of the specimen at position A1.
  • Fig. 138B and Fig. 138E are photographs of the specimen at position A7.
  • Fig. 138C and Fig. 138F are photographs of the specimen at position A8.
  • Figure 139A-139C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 18G12 at 10ug/mL.
  • Fig. 139A shows photographs of the tissue micro array.
  • Fig. 139A shows photographs of the tissue micro array.
  • FIG. 139B shows map of the array with abbreviated tissue descriptors.
  • FIG. 139C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 140A-140X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 18G12 at 10ug/mL, magnified to 6X and 20X.
  • Fig. 140A and Fig. 140E are adrenal gland.
  • Fig. 140B and Fig. 140F are breast.
  • Fig. 140C and Fig. 140G are fallopian tube.
  • Fig. 140D and Fig. 140H are kidney.
  • Fig. 140I and Fig. 140M are heart muscle.
  • Fig. 140J and Fig. 140N are liver.
  • FIG. 140O are lung.
  • Fig. 140L and Fig. 140P are ureter.
  • Fig. 140Q and Fig. 140U are eye.
  • Fig. 140R and Fig. 140V are cerebral cortex.
  • Fig. 140S and Fig. 140W are bone marrow.
  • Fig.140T and Fig.140X are skeletal muscle.
  • Figure 141A-141C shows photographs, array map and description of breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 18G12 at 15ug/mL.
  • Fig. 141A shows photographs of the tissue micro array.
  • Fig. 141B shows map of the array with abbreviated tissue descriptors.
  • Fig. 141C detailed description of the tissue micro array with non-identifying donor data.
  • FIG. 143A shows photographs of the tissue micro array.
  • Fig. 143B shows map of the array with abbreviated tissue descriptors.
  • Fig. 143C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 144A-144F shows photographs of specific tissues from pancreatic cancer tissue array PA1003 stained with the anti-PSMGFR antibody 18G12 at 15ug/mL, magnified to 6X and 20X.
  • Fig. 144A and Fig.144D are photographs of a Grade 2 adenocarcinoma.
  • Fig. 144B and Fig. 144E are photographs of a Grade 2 adenocarcinoma.
  • FIG. 148Q and Fig. 148U are eye.
  • Fig. 148R and Fig. 148V are cerebral cortex.
  • Fig. 148S and Fig. 148W are bone marrow.
  • Fig. 148T and Fig.148X are skeletal muscle.
  • Figure 149A-149C shows photographs, array map and description of breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 25E6 at 5.0ug/mL.
  • Fig. 149A shows photographs of the tissue micro array.
  • Fig. 149B shows map of the array with abbreviated tissue descriptors.
  • Fig. 149C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 150A-150F shows photographs of specific tissues from breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 25E6 at 5.0ug/mL, magnified to 6X and 20X.
  • Fig. 150A and Fig. 150D are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig. 150B and Fig. 150E are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig.150C and Fig.150F are photographs of a Grade 2 invasive ductal carcinoma.
  • Figure 151A-151C shows photographs, array map and description of pancreatic cancer tissue array PA1003 stained with the anti-PSMGFR antibody 25E6 at 5.0ug/mL.
  • FIG. 151A shows photographs of the tissue micro array.
  • Fig. 151B shows map of the array with abbreviated tissue descriptors.
  • Fig. 151C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 152A-152F shows photographs of specific tissues from pancreatic cancer tissue array PA1003 stained with the anti-PSMGFR antibody 25E6 at 5.0ug/mL, magnified to 6X and 20X.
  • Fig. 152A and Fig. 152D are photographs of a Grade 2 adenocarcinoma.
  • Fig. 152B and Fig. 152E are photographs of a Grade 1 adenocarcinoma.
  • FIG. 152F are photographs of a Grade 1 adenocarcinoma.
  • Figure 153A-153C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 28F9 at 15.0ug/mL.
  • Fig. 153A shows photographs of the tissue micro array.
  • Fig. 153B shows map of the array with abbreviated tissue descriptors.
  • Fig. 153C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 154A-154X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 28F9 at 15.0ug/mL, magnified to 6X and 20X.
  • Fig. 154E are adrenal gland.
  • Fig. 154B and Fig. 154F are breast.
  • Fig. 154C and Fig. 154G are fallopian tube.
  • Fig. 154D and Fig. 154H are kidney.
  • Fig. 154I and Fig. 154M are heart muscle.
  • Fig. 154J and Fig. 154N are liver.
  • Fig. 154K and Fig. 154O are lung.
  • Fig. 154L and Fig. 154P are ureter.
  • Fig. 154Q and Fig. 154U are eye.
  • Fig. 154R and Fig. 154V are cerebral cortex.
  • Fig. 154S and Fig. 154W are bone marrow.
  • FIG.154T and Fig.154X are skeletal muscle.
  • Figure 155A-155C shows photographs, array map and description of breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 28F9 at 15.0ug/mL.
  • Fig. 155A shows photographs of the tissue micro array.
  • Fig. 155B shows map of the array with abbreviated tissue descriptors.
  • Fig. 155C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 156A-156F shows photographs of specific tissues from breast cancer tissue array BR1141 stained with the anti-PSMGFR antibody 28F9 at 15.0ug/mL, magnified to 6X and 20X.
  • FIG. 156D are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig. 156B and Fig. 156E are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig.156C and Fig.156F are photographs of a Grade 2 invasive ductal carcinoma.
  • Figure 157A-157C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the N+20/C-27 antibody 1E4 at 7.5ug/mL.
  • Fig. 157A shows photographs of the tissue micro array.
  • Fig. 157B shows map of the array with abbreviated tissue descriptors.
  • Fig. 157C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 158A-158X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the N+20/C-27 antibody 1E4 at 7.5ug/mL, magnified to 6X and 20X.
  • Fig. 158A and Fig. 158E are adrenal gland.
  • Fig. 158B and Fig. 158F are breast.
  • Fig.158C and Fig. 158G are fallopian tube.
  • Fig. 158D and Fig. 158H are kidney.
  • Fig.158I and Fig. 158M are heart muscle.
  • Fig. 158J and Fig. 158N are liver.
  • Fig. 158K and Fig. 158O are lung.
  • Fig. 158L and Fig. 158P are ureter.
  • FIG. 158Q and Fig. 158U are eye.
  • Fig. 158R and Fig. 158V are cerebral cortex.
  • Fig. 158S and Fig. 158W are bone marrow.
  • Fig. 158T and Fig.158X are skeletal muscle.
  • Figure 159A-159C shows photographs, array map and description of breast cancer tissue array BR1007 stained with the N+20/C-27 antibody 1E4 at 10.0ug/mL.
  • Fig. 159A shows photographs of the tissue micro array.
  • Fig. 159B shows map of the array with abbreviated tissue descriptors.
  • Fig. 159C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 160A-160F shows photographs of specific tissues from breast cancer tissue array BR1007 stained with the N+20/C-27 antibody 1E4 at 10.0ug/mL, magnified to 6X and 20X.
  • Fig. 160A and Fig. 160D are photographs of a Grade 2 invasive ductal carcinoma with positive lymph nodes.
  • Fig. 160B and Fig. 160E are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig. 160C and Fig. 160F are photographs of a Grade 2 invasive ductal carcinoma.
  • Figure 161A-161C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the N+20/C-27 antibody 29H1 at 0.5ug/mL.
  • FIG. 161A shows photographs of the tissue micro array.
  • Fig. 161B shows map of the array with abbreviated tissue descriptors.
  • Fig. 161C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 162A-162X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the N+20/C-27 antibody 29H1 at 0.5ug/mL, magnified to 6X and 20X.
  • Fig. 162A and Fig. 162E are adrenal gland.
  • Fig. 162B and Fig. 162F are breast.
  • Fig.162C and Fig. 162G are fallopian tube.
  • Fig. 162D and Fig. 162H are kidney.
  • FIG. 162M are heart muscle.
  • Fig. 162J and Fig. 162N are liver.
  • Fig. 162K and Fig. 162O are lung.
  • Fig. 162L and Fig. 162P are ureter.
  • Fig. 162Q and Fig. 162U are eye.
  • Fig. 162R and Fig. 162V are cerebral cortex.
  • Fig. 162S and Fig. 162W are bone marrow.
  • Fig. 162T and Fig.162X are skeletal muscle.
  • Figure 163A-163C shows photographs, array map and description of breast cancer tissue array BR1141 stained with the N+20/C-27 antibody 29H1 at 0.5ug/mL.
  • Fig. 163A shows photographs of the tissue micro array.
  • FIG. 163B shows map of the array with abbreviated tissue descriptors.
  • FIG. 163C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 164A-164F shows photographs of specific tissues from breast cancer tissue array BR1141 stained with the N+20/C-27 antibody 29H1 at 0.5ug/mL, magnified to 6X and 20X.
  • Fig. 164A and Fig. 164D are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig. 164B and Fig. 164E are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig.164C and Fig.164F are photographs of a Grade 2 invasive ductal carcinoma.
  • Figure 165A-165C shows photographs, array map and description of pancreatic cancer tissue array PA1003 stained with the N+20/C-27 antibody 29H1 at 0.5ug/mL.
  • Fig. 165A shows photographs of the tissue micro array.
  • Fig. 165B shows map of the array with abbreviated tissue descriptors.
  • Fig. 165C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 166A-166F shows photographs of specific tissues from pancreatic cancer tissue array PA1003 stained with the N+20/C-27 antibody 29H1 at 0.5ug/mL, magnified to 6X and 20X.
  • FIG. 166D are photographs of a Grade 2 adenocarcinoma.
  • Fig. 166B and Fig. 166E are photographs of a Grade 2 adenocarcinoma.
  • Fig. 166C and Fig. 166F are photographs of a Grade 3 adenocarcinoma.
  • Figure 167A-167C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the N+20/C-27 antibody 31A1 at 0.5ug/mL.
  • Fig. 167A shows photographs of the tissue micro array.
  • Fig. 167B shows map of the array with abbreviated tissue descriptors.
  • Fig. 167C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 168A-168X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the N+20/C-27 antibody 31A1 at 0.5ug/mL, magnified to 6X and 20X.
  • Fig. 168A and Fig. 168E are adrenal gland.
  • Fig. 168B and Fig. 168F are breast.
  • Fig.168C and Fig. 168G are fallopian tube.
  • Fig. 168D and Fig. 168H are kidney.
  • Fig.168I and Fig. 168M are heart muscle.
  • Fig. 168J and Fig. 168N are liver.
  • Fig. 168K and Fig. 168O are lung.
  • Fig. 168L and Fig. 168P are ureter.
  • FIG. 168Q and Fig. 168U are eye.
  • Fig. 168R and Fig. 168V are cerebral cortex.
  • Fig. 168S and Fig. 168W are bone marrow.
  • Fig. 168T and Fig.168X are skeletal muscle.
  • Figure 169A-169C shows photographs, array map and description of breast cancer tissue array BR1141 stained with the N+20/C-27 antibody 31A1 at 0.5ug/mL.
  • Fig. 169A shows photographs of the tissue micro array.
  • Fig. 169B shows map of the array with abbreviated tissue descriptors.
  • Fig. 169C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 170A-170F shows photographs of specific tissues from breast cancer tissue array BR1141 stained with the N+20/C-27 antibody 31A1 at 0.5ug/mL, magnified to 6X and 20X.
  • Fig. 170A and Fig. 170D are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig. 170B and Fig. 170E are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig.170C and Fig.170F are photographs of a Grade 2 invasive ductal carcinoma.
  • Figure 171A-171C shows photographs, array map and description of pancreatic cancer tissue array PA1003 stained with the N+20/C-27 antibody 31A1 at 0.5ug/mL.
  • FIG. 173A-173C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the N+20/C-27 antibody 32C1 at 0.25ug/mL.
  • Fig. 173A shows photographs of the tissue micro array.
  • Fig. 173B shows map of the array with abbreviated tissue descriptors.
  • Fig. 173C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 174A-174X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the N+20/C-27 antibody 32C1 at 0.25ug/mL, magnified to 6X and 20X.
  • 174A and Fig. 174E are adrenal gland.
  • Fig. 174B and Fig. 174F are breast.
  • Fig. 174C and Fig. 174G are fallopian tube.
  • Fig. 174D and Fig. 174H are kidney.
  • Fig.174I and Fig.174M are heart muscle.
  • Fig.174J and Fig.174N are liver.
  • Fig.174K and Fig. 174O are lung.
  • Fig. 174L and Fig. 174P are ureter.
  • Fig. 174Q and Fig. 174U are eye.
  • Fig.174R and Fig.174V are cerebral cortex.
  • Fig. 174S and Fig.174W are bone marrow.
  • FIG. 174T and Fig.174X are skeletal muscle.
  • Figure 175A-175C shows photographs, array map and description of breast cancer tissue array BR1141 stained with the N+20/C-27 antibody 32C1 at 5.0ug/mL.
  • Fig. 175A shows photographs of the tissue micro array.
  • Fig. 175B shows map of the array with abbreviated tissue descriptors.
  • Fig. 175C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 176A-176F shows photographs of specific tissues from breast cancer tissue array BR1141 stained with the N+20/C-27 antibody 32C1 at 5.0ug/mL, magnified to 6X and 20X.
  • Figure 178A-178F shows photographs of specific tissues from esophageal cancer tissue array BC001113 stained with the N+20/C-27 antibody 32C1 at 1.0ug/mL, magnified to 6X and 20X.
  • Fig. 178A and Fig. 178D are photographs of a squamous cell carcinoma.
  • Fig. 178B and Fig. 178E are photographs of an adenocarcinoma.
  • Fig. 178C and Fig. 178F are photographs of a squamous cell carcinoma.
  • Figure 179A-179C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the N+20/C-27 antibody 45C11 at 12.5ug/mL.
  • Fig.180J and Fig.180N are liver.
  • Fig.180K and Fig. 180O are lung.
  • Fig. 180L and Fig. 180P are ureter.
  • Fig. 180Q and Fig. 180U are eye.
  • Fig.180R and Fig.180V are cerebral cortex.
  • Fig. 180S and Fig.180W are bone marrow.
  • Fig. 180T and Fig.180X are skeletal muscle.
  • Figure 181A-181C shows photographs, array map and description of breast cancer tissue array BR1007 stained with the N+20/C-27 antibody 45C11 at 10.0ug/mL.
  • Fig. 181A shows photographs of the tissue micro array.
  • Fig. 181A shows photographs of the tissue micro array.
  • FIG. 181B shows map of the array with abbreviated tissue descriptors.
  • FIG. 181C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 182A-182F shows photographs of specific tissues from breast cancer tissue array BR1007 stained with the N+20/C-27 antibody 45C11 at 10.0ug/mL, magnified to 6X and 20X.
  • Fig. 182A and Fig. 182D are photographs of a Grade 2 invasive ductal carcinoma with positive lymph nodes.
  • Fig. 182B and Fig. 182E are photographs of a Grade 2 invasive ductal carcinoma.
  • Fig. 182C and Fig. 182F are photographs of a Grade 2 invasive ductal carcinoma.
  • Figure 183A-183C shows photographs, array map and description of pancreatic cancer tissue array PA805c stained with the N+20/C-27 antibody 45C11 at 12.5ug/mL.
  • Fig. 183A shows photographs of the tissue micro array.
  • Fig. 183B shows map of the array with abbreviated tissue descriptors.
  • Fig. 183C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 184A-184F shows photographs of specific tissues from pancreatic cancer tissue array PA805c stained with the N+20/C-27 antibody 45C11 at 12.5ug/mL, magnified to 6X and 20X.
  • FIG. 184D are photographs of a Grade 2 papillary adenocarcinoma.
  • Fig. 184B and Fig. 184E are photographs of a Grade 2-3 ductal carcinoma.
  • Fig.184C and Fig.184F are photographs of a Grade 3 invasive adenocarcinoma.
  • Figure 185A-185C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the N+9/C-9 antibody 3C5 at 10.0ug/mL.
  • Fig. 185A shows photographs of the tissue micro array.
  • Fig. 185B shows map of the array with abbreviated tissue descriptors.
  • Fig. 185C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 186A-186X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the N+9/C-9 antibody 3C5 at 10.0ug/mL, magnified to 6X and 20X.
  • Fig. 186A and Fig. 186E are adrenal gland.
  • Fig. 186B and Fig. 186F are breast.
  • Fig.186C and Fig. 186G are fallopian tube.
  • Fig. 186D and Fig. 186H are kidney.
  • Fig.186I and Fig. 186M are heart muscle.
  • Fig. 186J and Fig. 186N are liver.
  • Fig. 186K and Fig. 186O are lung.
  • Fig. 186L and Fig. 186P are ureter.
  • FIG. 186Q and Fig. 186U are eye.
  • Fig. 186R and Fig. 186V are cerebral cortex.
  • Fig. 186S and Fig. 186W are bone marrow.
  • Fig. 186T and Fig.186X are skeletal muscle.
  • Figure 187A-187C shows photographs, array map and description of pancreatic cancer tissue array PA1003 stained with the N+9/C-9 antibody 3C5 at 10.0ug/mL.
  • Fig. 187A shows photographs of the tissue micro array.
  • Fig. 187B shows map of the array with abbreviated tissue descriptors.
  • Fig. 187C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 188A-188F shows photographs of specific tissues from pancreatic cancer tissue array PA1003 stained with the N+9/C-9 antibody 3C5 at 10.0ug/mL, magnified to 6X and 20X.
  • Fig. 188A and Fig.188D are photographs of a Grade 2 adenocarcinoma.
  • Fig.188B and Fig. 188E are photographs of a Grade 2 adenocarcinoma.
  • Fig. 188C and Fig. 188F are photographs of a Grade 2-3 adenocarcinoma with lymph node involvement.
  • Figure 189A-189C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the N+9/C-9 antibody 8A9 at 15.0ug/mL.
  • Fig. 189A shows photographs of the tissue micro array.
  • Fig. 189B shows map of the array with abbreviated tissue descriptors.
  • Fig. 189C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 190A-190X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the N+9/C-9 antibody 8A9 at 15.0ug/mL, magnified to 6X and 20X.
  • Fig. 190A and Fig. 190E are adrenal gland.
  • Fig.190F are breast.
  • Fig.190C and Fig. 190G are fallopian tube.
  • Fig. 190D and Fig.190H are kidney.
  • Fig.190I and Fig. 190M are heart muscle.
  • Fig. 190J and Fig. 190N are liver.
  • Fig. 190K and Fig. 190O are lung.
  • Fig. 190L and Fig. 190P are ureter.
  • Fig. 190Q and Fig. 190U are eye.
  • Fig. 190R and Fig. 190V are cerebral cortex.
  • Fig. 190S and Fig. 190W are bone marrow.
  • Fig. 190T and Fig.190X are skeletal muscle.
  • Figure 191A-191C shows photographs, array map and description of pancreatic cancer tissue array PA1003 stained with the N+9/C-9 antibody 8A9 at 15.0ug/mL.
  • Fig. 191A shows photographs of the tissue micro array.
  • Fig. 191B shows map of the array with abbreviated tissue descriptors.
  • Fig. 191C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 192A-192F shows photographs of specific tissues from pancreatic cancer tissue array PA1003 stained with the N+9/C-9 antibody 8A9 at 15.0ug/mL, magnified to 6X and 20X.
  • FIG. 193A-193C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the N+9/C-9 antibody 17H6 at 30.0ug/mL.
  • Fig. 193A shows photographs of the tissue micro array.
  • Fig. 193B shows map of the array with abbreviated tissue descriptors.
  • Fig. 193C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 194A-194X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the N+9/C-9 antibody 17H6 at 30.0ug/mL, magnified to 6X and 20X.
  • Fig. 194A and Fig. 194E are adrenal gland.
  • Fig. 194B and Fig. 194F are breast.
  • Fig.194C and Fig. 194G are fallopian tube.
  • Fig. 194D and Fig. 194H are kidney.
  • Fig.194I and Fig. 194M are heart muscle.
  • Fig. 194J and Fig. 194N are liver.
  • Fig. 194K and Fig. 194O are lung.
  • Fig. 194L and Fig. 194P are ureter.
  • FIG. 194Q and Fig. 194U are eye.
  • Fig. 194R and Fig. 194V are cerebral cortex.
  • Fig. 194S and Fig. 194W are bone marrow.
  • Fig. 194T and Fig.194X are skeletal muscle.
  • Figure 195A-195C shows photographs, array map and description of pancreatic cancer tissue array PA805c stained with the N+9/C-9 antibody 17H6 at 30.0ug/mL.
  • Fig. 195A shows photographs of the tissue micro array.
  • Fig. 195B shows map of the array with abbreviated tissue descriptors.
  • Fig. 195C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 196A-196F shows photographs of specific tissues from pancreatic cancer tissue array PA805c stained with the N+9/C-9 antibody 17H6 at 30.0ug/mL, magnified to 6X and 20X.
  • Fig. 196A and Fig. 196D are photographs of a Grade 2 papillary adenocarcinoma.
  • Fig. 196B and Fig. 196E are photographs of a Grade 2-3 ductal carcinoma with lymph node involvement.
  • Fig. 196C and Fig. 196F are photographs of a Grade 3 invasive adenocarcinoma.
  • Figure 197A-197C shows photographs, array map and description of FDA normal tissue array MNO1021 stained with the N+9/C-9 antibody 39H5 at 5.0ug/mL.
  • Fig. 197A shows photographs of the tissue micro array.
  • Fig. 197B shows map of the array with abbreviated tissue descriptors.
  • Fig. 197C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 198A-198X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the N+9/C-9 antibody 39H5 at 5.0ug/mL, magnified to 6X and 20X.
  • Fig. 198A and Fig. 198E are adrenal gland.
  • Fig.198F are breast.
  • Fig.198C and Fig. 198G are fallopian tube.
  • Fig. 198D and Fig.198H are kidney.
  • Fig.198I and Fig. 198M are heart muscle.
  • Fig. 198J and Fig. 198N are liver.
  • Fig. 198K and Fig. 198O are lung.
  • Fig. 198L and Fig. 198P are ureter.
  • Fig. 198Q and Fig. 198U are eye.
  • Fig. 198R and Fig. 198V are cerebral cortex.
  • Fig. 198S and Fig. 198W are bone marrow.
  • Fig. 198T and Fig.198X are skeletal muscle.
  • Figure 199A-199C shows photographs, array map and description of pancreatic cancer tissue array PA1003 stained with the N+9/C-9 antibody 39H5 at 5.0ug/mL.
  • Fig. 199A shows photographs of the tissue micro array.
  • Fig. 199B shows map of the array with abbreviated tissue descriptors.
  • Fig. 199C detailed description of the tissue micro array with non-identifying donor data.
  • Figure 200A-200F shows photographs of specific tissues from pancreatic cancer tissue array PA1003 stained with the N+9/C-9 antibody 39H5 at 5.0ug/mL, magnified to 6X and 20X.
  • Fig. 200A and Fig. 200D are photographs of a Grade 2 adenocarcinoma.
  • FIG. 200B and Fig. 200E are photographs of a Grade 2 adenocarcinoma.
  • Fig. 200C and Fig. 200F are photographs of a Grade 2 adenocarcinoma.
  • Figure 201A-201C show graphs of ELISA assays to determine the binding of another set of antibodies generated by immunizing animals with the PSMGFR peptide.
  • Fig. 201A shows binding to the PSMGFR peptide.
  • Fig. 201B shows binding to the N-10 peptide.
  • Fig.201C shows binding to the C-10 peptide. As can be seen, none of the antibodies bound to the C-10 peptide.
  • FIG. 202A-202C shows photographs of pancreatic cancer tissue array PA1003 that has been stained with monoclonal antibody 1E4, monoclonal antibody 18B4 or the polyclonal anti-PSMGFR antibody SDIX.
  • 18B4 binds to the GTINVHDVET epitope at the most N-terminal portion of the PSMGFR peptide, while the 1E4 antibody binds to the QFNQYKTEA epitope that is immediately adjacent and C-terminal to the 18B4 epitope.
  • Figure 203A-203F shows magnified images of the tissue specimen at position A2 of the pancreatic cancer array PA1003.
  • Fig. 203A and Fig. 203B show the specimen stained with antibody 1E4.
  • Fig. 203C and Fig. 203D show the specimen stained with antibody 18B4.
  • Fig.203E and Fig.203F show the specimen stained with polyclonal antibody SDIX.
  • Figure 204A-204D shows magnified images of the tissue specimen at position D4 of the pancreatic array PA1003.
  • Fig. 204A and Fig. 204B show the specimen stained with antibody 18B4.
  • Fig.204C and Fig. 204D show the specimen stained with polyclonal antibody SDIX.
  • Figure 205A-205D shows magnified images of the tissue specimen at position E1 of the pancreatic cancer array PA1003.
  • Fig. 205A and Fig. 205B show the specimen stained with antibody 18B4.
  • Fig. 205C and Fig. 205D show the specimen stained with polyclonal antibody SDIX.
  • Figure 206A-206D shows magnified images of the tissue specimen at position C3 of the pancreatic cancer array PA1003.
  • Fig. 206A and Fig. 206B show the specimen stained with antibody 1E4.
  • Fig. 206C and Fig. 206D show the specimen stained with polyclonal antibody SDIX.
  • Figure 207A-207D shows magnified images of the tissue specimen at position D1 of the pancreatic cancer array PA1003.
  • Fig. 207A and Fig. 207B show the specimen stained with antibody 1E4.
  • Fig. 207C and Fig. 207D show the specimen stained with polyclonal antibody SDIX.
  • Figure 208A-208C shows photographs of the pancreatic cancer array PA1003.
  • Fig. 208A shows the specimen stained with polyclonal antibody SDIX.
  • Fig. 208B shows the specimen stained with antibody 20A10.
  • Fig. 208C shows the specimen stained with antibody 29H1.
  • Figure 209A-209D shows photographs of the esophageal cancer array ES1001 stained with various antibodies.
  • Fig. 209A shows the array stained with polyclonal antibody SDIX.
  • Fig. 209B shows the array stained with antibody 20A10.
  • Fig. 209C shows the array stained with antibody 29H1.
  • Fig.209D shows the array stained with antibody 31A1.
  • Figure 210A-210C shows photographs of the pancreatic cancer array PA1003 stained with various antibodies.
  • Fig. 210A shows the array stained with polyclonal antibody SDIX.
  • Fig. 210B shows the array stained with antibody 20A10.
  • Fig. 210C shows the array stained with antibody 29H1.
  • Figure 211A- 211C show graphs of an ELISA experiment measuring the amount of IL-18 secreted into the condition media of MUC1* positive cancer cells co-cultured with huMNC2-CAR44 T cells wherein the cells also bear an NFAT inducible IL-18.
  • Fig. 211A shows the graph of IL-18 secreted into the supernatant of T47D breast cancer cells co- cultured with untransduced human T cells.
  • Fig. 211B shows the graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T cells that also bore an NFAT inducible IL-18 gene inserted into a portion of the Foxp3 enhancer.
  • FIG. 211C shows the graph of IL-18 secreted into the supernatant of T47D breast cancer cells co- cultured with huMNC2-CAR44 T cells that also bore an NFAT inducible IL-18 gene inserted into a portion of the IL-2 enhancer.
  • Figure 212A- 212X shows photographs of T47D breast cancer cells (red) doped with varying percentages of T47D cells engineered to express more MUC1* (green). The target cancer cells have been co-cultured with huMNC2-CAR44 T cells with NFAT inducible IL-18 wherein the IL-18 gene has been inserted into either the Foxp3 enhancer/promoter or the IL-2 enhancer/promoter.
  • Fig.214A-214F shows either T47D cells or HEK293 cells that have not been doped with T47D cells engineered to express high MUC1* density.
  • Fig. 214G-214L shows either T47D cells or HEK293 cells that have been doped with 5% T47D cells engineered to express high MUC1* density.
  • Fig. 214M-214R shows either T47D cells or HEK293 cells that have been doped with 10% T47D cells engineered to express high MUC1* density.
  • FIG. 216B shows consensus sequences for light chain CDR2.
  • Fig.216C shows consensus sequences for light chain CDR3.
  • Figure 217 shows alternative formats for bispecific antibodies and other bispecific immunotherapeutics subdivided into five major classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. Heavy chains are shown in dark blue, dark pink and dark green and corresponding light chains are in lighter shades of the same colors. Connecting peptide linkers are shown by thin black lines and engineered disulfide bonds by thin green lines. Approximate molecular weights are shown assuming ⁇ 12.5 kDa per immunoglobulin domain. BsAb formats that have advanced into clinical testing are highlighted (*).
  • Tumors were made heterogeneous, comprised of two different tumor cell types.
  • a first tumor cell population was T47D-wt, a breast cancer cell line that expresses both full-length MUC1 and the growth factor receptor form MUC1*, which we engineered to express mCherry fluorescence.
  • the second tumor cell population was the same T47D breast cancer cells, except that they had been stably transduced to express even more MUC1* and GFP fluorescence, referred to here as T47D-MUC1*.
  • animals were implanted with T47D-wt plus T47D-MUC1*, wherein the population of T47D- MUC1* made up 30%, 15% or 7.5% of the tumor population.
  • FIG.219A shows IVIS photographs and graphs of IVIS tumor volume measurement.
  • Fig.219A shows photographs of the mice that had been implanted with tumors in which 30% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells.
  • FIG. 219B shows a graph of the tumor volume by IVIS measurement by day.
  • animals injected with the huMNC2-CD28-1XX had much smaller tumors than the animals treated huMNC2-4-1BB-3z or huMNC2-CD28-3z, which is the same CAR T except without the 1XX mutations in the CD3-zeta domain.
  • Figure 220A-220T shows the IVIS graphs for 30% tumors treated at a CAR T to Tumor ratio of 10:1.
  • Figure 221A-221B shows IVIS photographs and graphs of IVIS tumor volume measurement.
  • Fig.221A shows photographs of the mice that had been implanted with tumors in which 30% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells.
  • the various CAR T cells were administered at a CAR T to tumor cell ratio of 1:1 wherein 250,000 tumor cells were implanted and the animals were injected 5 days later with 250,000 CAR T cells.
  • Fig. 221B shows a graph of the tumor volume by IVIS measurement by day.
  • FIG. 222A-222T shows the IVIS graphs for 30% tumors treated at a CAR T to Tumor ratio of 1:1. Here graphs are shown for each individual animal rather than the average of the treatment group.
  • Figure 223A-223B shows IVIS photographs and graphs of IVIS tumor volume measurement.
  • Fig.223A shows photographs of the mice that had been implanted with tumors in which 7.5% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells.
  • the various CAR T cells were administered at a CAR T to tumor cell ratio of 10:1 wherein 250,000 tumor cells were implanted and the animals were injected 5 days later with 2,500,000 CAR T cells.
  • Fig. 223B shows a graph of the tumor volume by IVIS measurement by day.
  • FIG. 224A-224T shows the IVIS graphs for 7.5% tumors treated at a CAR T to Tumor ratio of 10:1. Here graphs are shown for each individual animal rather than the average of the treatment group.
  • Figure 225A-225B shows IVIS photographs and graphs of IVIS tumor volume measurement.
  • Fig.225A shows photographs of the mice that had been implanted with tumors in which 7.5% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells.
  • the various CAR T cells were administered at a CAR T to tumor cell ratio of 1:1 wherein 250,000 tumor cells were implanted and the animals were injected 5 days later with 250,000 CAR T cells.
  • Fig. 225B shows a graph of the tumor volume by IVIS measurement by day.
  • FIG. 226A-226T shows the IVIS graphs for 7.5% tumors treated at a CAR T to Tumor ratio of 1:1. Here graphs are shown for each individual animal rather than the average of the treatment group.
  • Figure 227 shows the tabulation of CD3 positive human T cells that were harvested from the spleens of the test animals post sacrifice.
  • cells isolated from mice implanted with tumors comprised of 30% T47D-MUC1* and treated with CAR T cells at a 10:1 ratio.
  • the huMNC2-CD28-1XX treated mice that had smaller tumors, have the greater numbers of CAR T cells and CD8 positive killer T cells.
  • TIM3, LAG3 and PD-1 are molecular markers of T cell exhaustion.
  • FIG. 1 shows that the huMNC2-CD28-1XX CAR T cells harvested from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutations in CD3-zeta increase CAR T cell persistence in vivo.
  • Figure 228 shows the tabulation of CD3 positive human T cells that were harvested from the spleens of the test animals post sacrifice.
  • cells isolated from mice implanted with tumors comprised of 30% T47D-MUC1* and treated with CAR T cells at a 1:1 ratio.
  • the huMNC2-CD28-1XX treated mice, that had smaller tumors have the greater numbers of CAR T cells and CD8 positive killer T cells.
  • TIM3, LAG3 and PD-1 are molecular markers of T cell exhaustion.
  • the table shows that the huMNC2-CD28- 1XX CAR T cells harvested from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutations in CD3-zeta increase CAR T cell persistence in vivo.
  • Figure 229 shows the tabulation of CD3 positive human T cells that were harvested from the blood of the test animals post sacrifice. In this table, cells isolated from mice implanted with tumors comprised of 30% T47D-MUC1* and treated with CAR T cells at a 1:1 ratio.
  • Figure 230 shows the tabulation of CD3 positive human T cells that were harvested from the spleens of the test animals post sacrifice.
  • cells isolated from mice implanted with tumors comprised of 7.5% T47D-MUC1* and treated with CAR T cells at a 10:1 ratio.
  • the huMNC2-CD28-1XX treated mice that had smaller tumors, have the greater numbers of CAR T cells and CD8 positive killer T cells.
  • TIM3, LAG3 and PD-1 are molecular markers of T cell exhaustion.
  • the table shows that the huMNC2-CD28-1XX CAR T cells harvested from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutations in CD3-zeta increase CAR T cell persistence in vivo.
  • Figure 231 shows the tabulation of CD3 positive human T cells that were harvested from the blood of the test animals post sacrifice. In this table, cells isolated from mice implanted with tumors comprised of 7.5% T47D-MUC1* and treated with CAR T cells at a 10:1 ratio.
  • TIM3, LAG3 and PD-1 are molecular markers of T cell exhaustion.
  • the table shows that the huMNC2-CD28-1XX CAR T cells harvested from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutations in CD3-zeta increase CAR T cell persistence in vivo.
  • Figure 232 shows the tabulation of CD3 positive human T cells that were harvested from the spleens of the test animals post sacrifice.
  • mice implanted with tumors comprised of 7.5% T47D-MUC1* and treated with CAR T cells at a 1:1 ratio.
  • the huMNC2-CD28-1XX treated mice that had smaller tumors, have the greater numbers of CAR T cells and CD8 positive killer T cells.
  • TIM3, LAG3 and PD-1 are molecular markers of T cell exhaustion.
  • the table shows that the huMNC2-CD28- 1XX CAR T cells harvested from the animals express lower levels of exhaustion markers, consistent with the idea that the 1XX mutations in CD3-zeta increase CAR T cell persistence in vivo.
  • Figure 233 shows the tabulation of CD3 positive human T cells that were harvested from the blood of the test animals post sacrifice.
  • cells isolated from mice implanted with tumors comprised of 7.5% T47D-MUC1* and treated with CAR T cells at a 1:1 ratio.
  • the huMNC2-CD28-1XX treated mice that had smaller tumors, have the greater numbers of CAR T cells and CD8 positive killer T cells.
  • TIM3, LAG3 and PD-1 are molecular markers of T cell exhaustion.
  • 235K-235T show tumor cells excised from the animals treated with huMNC2-41BB-3z CAR T cells.
  • Fig. 235U- 235D2 show tumor cells excised from the animals treated with huMNC2-CD28-1XX CAR T cells.
  • Fig. 235E2-235N2 show tumor cells excised from the animals treated with huMNC2- CD28-3z CAR T cells.
  • Figure 236A-236U shows photographs of the tumors excised from test animals and shows their weight in grams. Tumors were excised from animals implanted with tumors made up of 30% T47D-MUC1* high antigen density cells and 70% T47D-wt low antigen density cells.
  • FIG. 237U- 237D2 show tumor cells excised from the animals treated with huMNC2-CD28-1XX CAR T cells.
  • Fig. 237E2-237N2 show tumor cells excised from the animals treated with huMNC2- CD28-3z CAR T cells.
  • Figure 238A-238T shows photographs of the tumors excised from test animals and shows their weight in grams. Tumors were excised from animals implanted with tumors made up of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector to target ratio of 10:1.
  • Figure 239A-239M2 shows magnified photographs of dissociated tumors excised from animals implanted with tumors made up of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector to target ratio of 10:1. Shown are overlays of bright field images and fluorescent images, wherein the red fluorescence, mCherry, shows the low antigen density cells and the green fluorescence, GFP, shows the low antigen density cells.
  • Fig. 239A-239J show tumor cells excised from animals mock treated with PBS. Fig.
  • FIG. 239K-239T show tumor cells excised from the animals treated with huMNC2-41BB-3z CAR T cells.
  • Fig. 239U- 239C2 show tumor cells excised from the animals treated with huMNC2-CD28-1XX CAR T cells.
  • Fig. 239D2-239M2 show tumor cells excised from the animals treated with huMNC2- CD28-3z CAR T cells.
  • Figure 240A-240O shows photographs of the tumors excised from test animals and shows their weight in grams. Tumors were excised from animals implanted with tumors made up of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low antigen density cells.
  • FIG. 241A-241D2 shows magnified photographs of dissociated tumors excised from animals implanted with tumors made up of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low antigen density cells. Animals were treated with CAR T cells at an effector to target ratio of 1:1. Shown are overlays of bright field images and fluorescent images, wherein the red fluorescence, mCherry, shows the low antigen density cells and the green fluorescence, GFP, shows the low antigen density cells.
  • Fig. 241A-241J show tumor cells excised from control animals treated only with PBS. Fig.
  • FIG. 241K-241T show tumor cells excised from the animals treated with huMNC2-41BB-3z CAR T cells.
  • Fig. 241U-241D2 show tumor cells excised from the animals treated with huMNC2-CD28-1XX CAR T cells.
  • Fig. 241E2-241N2 show tumor cells excised from the animals treated with huMNC2-CD28- 3z CAR T cells.
  • Figure 242A-242R shows photographs of live animals, where IVIS measures tumor volume, mCherry detects the low antigen cells within the tumor and GFP detects the high antigen cells within the tumor. Post sacrifice photographs are shown of the excised tumors and a graph of tumor weights.
  • FIG. 243A-243F shows photographs taken at two different timepoints.
  • IVIS photographs measure tumor volume
  • mCherry fluorescent photographs measure low antigen cells
  • GFP fluorescent photographs measure high antigen cells.
  • the animals were implanted with tumors made up of 30% high antigen density cells (GFP+) and 70% low antigen density cells (mCherry+).
  • FIG. 244 shows graphs of IVIS tumor volume measurements over time. Arrows indicate timepoints when fluorescent photographs, mCherry and GFP, of live animals were taken. In this case, the animals were implanted with tumors made up of 30% high antigen density cells (GFP+) and 70% low antigen density cells (mCherry+). Animals had been given a single dose of CAR T cells at a 10:1 or a 1:1 CAR T to tumor cell ratio.
  • GFP+ high antigen density cells
  • mCherry+ 70% low antigen density cells
  • Figure 245 shows graphs of IVIS measurements of tumor volume, mCherry measurements of the growth rate of low antigen cells and GFP measurements of the growth rate of high antigen cells, between two timepoints.
  • GFP high antigen density cells
  • FIG.246A shows photographs of the mice that had been implanted with tumors in which 15% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells.
  • the various CAR T cells were administered at a CAR T to tumor cell ratio of 10:1 wherein 250,000 tumor cells were implanted and the animals were injected 5 days later with 2,500,000 CAR T cells.
  • Fig. 246B shows a graph of the tumor volume by IVIS measurement by day.
  • animals outlined in red were implanted with 250,000 more 100% high antigen density tumor cells.
  • the animals outlined in green received an additional dose of 2,500,000 CAR T cells.
  • Figure 247A-247T shows graphs of the IVIS measured growth of the tumors in each individual animal.
  • the red arrows indicate injection of more tumor cells and the green arrows indicate injection of 2,500,000 additional CAR T cells.
  • the injection of additional tumor cells does not increase the tumor growth in animals treated with huMNC2-CD28-1XX CAR T cells.
  • Fig. 248A shows cartoon of animals implanted with tumors in which 30% of the tumors express high levels of MUC1* and those tumor cells fluoresce green.
  • Fig. 248B shows cartoon of animals implanted with tumors in which 15% of the tumors express high levels of MUC1* and those tumor cells fluoresce green.
  • Fig. 248C shows cartoon of animals implanted with tumors in which 7.5% of the tumors express high levels of MUC1* and those tumor cells fluoresce green.
  • FIG.248D lists the variables used in these experiments.
  • Figure 249A-247F shows cartoons of the experimental strategy and data.
  • Fig. 249A shows cartoons of MUC1 full-length as it appears on normal epithelial cells.
  • Fig.249B shows four tissue specimens stained with huMNC2-scFv-Fc.
  • Fig. 249C shows cartoons depicting heterogeneous tumors expressing either high (30%-left) or low (7.5%-right) percentages of high MUC1* expressing tumor cells.
  • Fig. 249D shows flow cytometry verifying the percentages of the heterogeneous tumors before their implantation.
  • FIG. 249E shows bar graphs of bioluminescence measured on an IVIS instrument for animals implanted with 30% high MUC1* cells and treated with the various CAR T cells at an effector to target ratio of 10:1 (top) or 1:1 (bottom).
  • Fig. 249F shows bar graphs of bioluminescence measured on an IVIS instrument for animals implanted with 7.5% high MUC1* cells and treated with the various CAR T cells at an effector to target ratio of 10:1 (top) or 1:1 (bottom).
  • Figure 250A-250F shows bar graphs of bioluminescence of the tumors measured on an IVIS instrument for animals implanted with 30% high MUC1* cells or 7.5% high MUC1* tumors and treated with the various CAR T cells at various effector to target ratios.
  • Fig. 250A shows IVIS graph of animals implanted with tumors in which 30% expressed high levels of MUC1* and where animals were treated with CAR T cells at an effector to target ratio of 10:1.
  • Fig. 250B shows IVIS graph where effector to target ratio was 5:1.
  • Fig. 250C shows IVIS graph where effector to target ratio was 1:1.
  • FIG. 250D shows IVIS graph of animals implanted with tumors in which 7.5% expressed high levels of MUC1* and where animals were treated with CAR T cells at an effector to target ratio of 10:1.
  • Fig. 250E shows IVIS graph where effector to target ratio was 5:1.
  • Fig.250F shows IVIS graph where effector to target ratio was 1:1.
  • Figure 251A-251D shows photographs of bioluminescence of the tumors measured on an IVIS instrument for animals implanted with 30% high MUC1* cells or 7.5% high MUC1* tumors and treated with the various CAR T cells at effector to target ratios of 10:1 or 1:1.
  • 251A shows IVIS photographs for animals implanted with tumors in which 30% expressed high levels of MUC1* and where animals were treated with of CAR T cells at an effector to target ratio of 10:1.
  • Fig.250B shows IVIS photographs where effector to target ratio was 1:1.
  • Fig.251C shows IVIS photographs for animals implanted with tumors in which 7.5% expressed high levels of MUC1* and where animals were treated with of CAR T cells at an effector to target ratio of 10:1.
  • Fig. 250D shows IVIS photographs where effector to target ratio was 1:1.
  • M refers to millions
  • K refers to thousands.
  • MNC2 which is interchangeable with “C2”, “Min-C2” and “MNC2”
  • MNE6 which is interchangeable with “E6”, “Min-E6” and “MNE6”
  • MNC3 which is interchangeable with “C3”, “Min-C3” and “MNC3”
  • MNC8 which is interchangeable with “C8”, “Min-C8” and “MNC8”.
  • antibody-like means a molecule that may be engineered such that it contains portions of antibodies but is not an antibody that would naturally occur in nature. Examples include but are not limited to CAR (chimeric antigen receptor) T cell technology and the Ylanthia ® technology.
  • CAR chimeric antigen receptor
  • Ylanthia ® Ylanthia
  • the Ylanthia ® technology consists of an “antibody- like” library that is a collection of synthetic human Fabs that are then screened for binding to peptide epitopes from target proteins. The selected Fab regions can then be engineered into a scaffold or framework so that they resemble antibodies.
  • PSMGFR is abbreviation for Primary Sequence of the MUC1 Growth Factor Receptor which is identified by SEQ ID NO:2, and thus is not to be confused with a six 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:2).
  • the “MUC1*” extra cellular domain is defined primarily by the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:2)). Because the exact site of MUC1 cleavage depends on the enzyme that clips it, and that the cleavage enzyme varies depending on cell type, tissue type or the time in the evolution of the cell, the exact sequence of the MUC1* extra cellular domain may vary at the N-terminus.
  • PSMGFR is an acronym for Primary Sequence of MUC1 Growth Factor Receptor as set forth as GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:2).
  • N-10 PSMGFR or simply “N-10”, “N-15 PSMGFR” or simply “N-15”, or “N-20 PSMGFR” or simply “N-20” refers to the number of amino acid residues that have been deleted at the N-terminal end of PSMGFR.
  • C-number as in “C-10 PSMGFR” or simply “C-10”, “C-15 PSMGFR” or simply “C-15”, or “C-20 PSMGFR” or simply “C-20” refers to the number of amino acid residues that have been deleted at the C-terminal end of PSMGFR. A mixture of deletions and additions is also possible.
  • N+20/C-27 refers to a peptide fragment of wild-type MUC1 in which 20 amino acids are added to the PSMGFR at the N-terminus and 27 amino acids are deleted from the C-terminus.
  • the “extracellular domain of MUC1*” refers to the extracellular portion of a MUC1 protein that is devoid of the tandem repeat domain. In most cases, MUC1* is a cleavage product wherein the MUC1* portion consists of a short extracellular domain devoid of tandem repeats, a transmembrane domain and a cytoplasmic tail.
  • sequence identity means homology in sequence of a particular polypeptide or nucleic acid to a reference sequence of nucleic acid or amino acid such that the function of the homologous peptide is the same as the reference peptide or nucleic acid. Such homology can be so close with the reference peptide such that at times the two sequences may be 90%, 95% or 98% identical yet possess the same function in binding or other biological activities.
  • MUC1 positive cell refers to a cell that expresses a gene for MUC1, MUC1-Y or MUC1-Z or other MUC1 variant.
  • MUC1 negative cell refers to a cell that does not express a gene for MUC1.
  • a term “conformational inducing peptide sequence” may be used, which indicates that a peptide sequence is present within a larger peptide not as a binding site but that induces binding of an antibody to the larger peptide by causing a three- dimensional structure to form that facilitates the binding of the antibody to the larger peptide.
  • MUC1* antibodies anti-PSMGFR
  • a cleaved form of the MUC1 (SEQ ID NO:1) transmembrane protein is a growth factor receptor that drives the growth of over 75% of all human solid tumor cancers.
  • MUC1* The cleaved form of MUC1, which we called MUC1* (pronounced muk 1 star), is a powerful growth factor receptor. Enzymatic cleavage releases the bulk of the MUC1 extracellular domain. It is the remaining portion comprising a truncated extracellular domain, transmembrane domain and cytoplasmic tail that is called MUC1*. Cleavage and release of the bulk of the extracellular domain of MUC1 unmasks a binding site for activating ligands dimeric NME1, NME6, NME8, NME7 AB , NME7-X1 or NME7. Cell growth assays show that it is ligand-induced dimerization of the MUC1* extracellular domain that promotes growth (Fig. 1A-1D).
  • Bivalent anti-MUC1* antibodies stimulate growth of cancer cells whereas the monovalent Fab inhibits growth.
  • Classic bell-shaped curve indicates ligand induced dimerization stimulates growth.
  • Dimeric NM23-H1, aka NME1 stimulates growth of MUC1* positive cancer cells but siRNA to suppress MUC1 expression eliminate its effect (Fig. 1C).
  • NME7-AB also stimulates the growth of MUC1* positive cells (Fig.1D).
  • MUC1* is an excellent target for cancer drugs as it is aberrantly expressed on over 75% of all cancers and is likely overexpressed on an even higher percentage of metastatic cancers. After MUC1 cleavage, most of its extracellular domain is shed from the cell surface. The remaining portion has a truncated extracellular domain that at least comprises the primary growth factor receptor sequence, PSMGFR (SEQ ID NO:2).
  • Antibodies that bind to the PSMGFR sequence and especially those that competitively inhibit the binding of activating ligands such as NME proteins, including NME1, NME6, NME8, NME7AB, NME7- X1 and NME7, are ideal therapeutics and can be used to treat or prevent MUC1 positive or MUC1* positive cancers, as stand-alone antibodies, antibody fragments or variable region fragments thereof incorporated into multi-specific antibody-like molecules, bispecific antibodies, antibody-drug conjugates or chimeric antigen receptors also called CARs, which are then transfected or transduced into immune cells, then administered to a patient.
  • these activating growth factors bind to the membrane proximal portion of MUC1*, as they do not bind to the PSMGFR peptide if the 10 C-terminal amino acids are missing.
  • anti-MUC1* antibodies MNC2 and MNE6 bind to the PSMGFR peptide if an only if the 10 C-terminal amino acids are present (Fig. 2B, 2C).
  • Antibodies MNC3 and MNC8 bind to epitopes that are different from MNC2 and MNE6, as they do not depend on the presence of the 10 C-terminal amino acids of the PSMGFR peptide (Fig. 2E, 2F).
  • the antibody or antibody fragment may be murine, human, humanized, camelid, rabbit or other non-human species.
  • BiTEs or chimeric antigen receptors also called CARs that have been transduced into immune cells. MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11 antibody and other anti-MUC1* antibodies that competitively inhibit the binding of NME1 and NME7AB are preferred.
  • the antibody or antibody fragment may be murine, human, humanized, camelid, rabbit or other non-human species.
  • Therapeutic anti-MUC1* antibodies for use as a stand-alone antibody therapeutic or for integration into a BiTE, a CAR, an ADC, or any of the multi-specific antibody-like molecules can be selected based on specific criteria.
  • the parent antibody can be generated using typical methods for generating monoclonal antibodies in animals. Alternatively, they can be selected by screening antibody or antibody fragment libraries, including but not limited to strategies described in Beckman U.S. Patent No. 9,944,719B2, which is incorporated by reference herein for description of methods of screening antibodies.
  • Antibodies suitable for therapeutic use are chosen based on their ability to bind to a MUC1* peptide, which can be: [00577] (i) PSMGFR region of MUC1; [00578] (ii) PSMGFR peptide; [00579] (iii) a peptide having amino acid sequence of QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) [00580] (iv) a peptide having amino acid sequence of [00581] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19) [00582] (v) a peptide having amino acid sequence of [00583] NLTISDVSVSDVPFPFSAQSGA (N-23) [00584] (vi) a peptide having amino acid sequence of [00585] ISDVSVSDVPFPFSAQSGA (N-26) [00586] (vii) a peptide having amino acid sequence of [00587] SV
  • Resultant antibodies or antibody fragments generated or selected in this way can then be further selected by passing additional screens. For example, antibodies or antibody fragments become more preferred based on their ability to bind to MUC1* positive cancer cells or tissues but not to MUC1 negative cancer cells or to normal tissues. Further, anti- MUC1* antibodies or antibody fragments may be de-selected as anti-cancer therapeutics if they bind to stem or progenitor cells. Anti-MUC1* antibodies or antibody fragments become more preferred if they have the ability to competitively inhibit the binding of activating ligands, such as NME7AB or NME7-X1, to MUC1*. Figs.
  • a process for selecting anti-MUC1* antibodies for use in treating a patient diagnosed with a MUC1 positive cancer, at risk of developing a MUC1 positive cancer or suspected of having a MUC1 positive cancer comprises one or more of the following steps of selecting antibodies or antibody fragments that 1) bind to the PSMGFR peptide; 2) bind to the N-10 PSMGFR peptide; 3) selectively bind to cancer cells; 4) do not bind to C-10 PSMGFR peptide; and 5) competitively inhibited the binding of dimeric NME1 or NME7- AB to the PSMGFR peptide.
  • suitable antibodies in this regard include monovalent antibodies such as those generated in lamas and camels, Fabs, scFv’s, single domain antibodies (sdAb), scFv-Fc as long as the Fc portion is constructed such that it does not homo-dimerize.
  • FACS scans show that anti-MUC1* antibodies MNC2 and MNE6 specifically bind to MUC1* positive solid tumor cancer cells and MUC1* transfected cells but not MUC1* negative or MUC1 negative cells.
  • a humanized MNC2 scFv is shown to bind to ZR-75-1, aka 1500, MUC1* positive breast cancer cells (Fig. 4A-4C).
  • MNE6 was shown to bind to MUC1 negative HCT-116 colon cancer cells if an only if they were transfected with MUC1*. MNE6 also bound to MUC1* positive cancer cells such as ZR-75-1, aka 1500, MUC1* positive breast cancer cells (Fig. 4D-4F). Binding assays such as ELISAs, immunofluorescence, and the like all confirm that MNC2 and MNE6 bind to the PSMGFR peptide and to live MUC1 positive cancer cells. Humanized anti-MUC1* antibodies are selected based on their ability to also bind to the PSMGFR peptide or to MUC1 positive cancer cells.
  • murine or humanized MNC3-scFv which is less suitable for the treatment of cancers, binds to the, PSMGFR peptide, binds to the N-10 peptide and also binds to the C-10 peptide (Fig.9), which we know is the epitope to which the activating ligand NME7AB binds.
  • the Fabs of MNE6 and MNC2 or the comparable single chain variable regions derived from them potently inhibit the growth of MUC1* positive cancers in vitro and in vivo.
  • the Fabs of Anti-MUC1* antibodies inhibited the growth of human MUC1* positive cancers in vivo.
  • MNE6 was shown to halt the growth of prostate cancer.
  • Fig.7B shows that MNE6 Fab potently inhibited the growth of MUC1* positive prostate cancers.
  • Male NOD/SCID mice were implanted with 6 million DU-145 human prostate cancer cells that had been mixed 50/50 with Matrigel. Mice bearing tumors that were at least 150 mm ⁇ 3 and had three successive increases in tumor volume were selected for treatment. Animals were injected sub-cutaneously every 48 hours with 160 mg/kg MNE6 Fab and an equal number of mice fitting the same selection criteria were injected with vehicle alone (Fig. 7B). Tumors were measured independently by two researchers twice per week and recorded.
  • scFv humanized MNE6 variable region
  • SEQ ID NO:256 and 257 A single chain of the humanized MNE6 variable region, called an scFv, was genetically engineered such that it was connected to the Fc portion of the antibody (SEQ ID NO:256 and 257). Fc regions impart certain benefits to antibody fragments for use as therapeutics.
  • the Fc portion of an antibody recruits complement, which in general means it can recruit other aspects of the immune system and thus amplify the anti-tumor response beyond just inhibiting the target.
  • the addition of the Fc portion also increases the half-life of the antibody fragment (Czajkowsky DM, Hu J, Shao Z and Pleass RJ.
  • a human or humanized MNE6 antibody or antibody fragment, Fab, MNE6 scFv or hu MNE6 scFv-Fcmut are effective anti-cancer agents that can be administered to a person diagnosed with a MUC1 or MUC1* positive cancer, suspected of having a MUC1 or MUC1* positive cancer or is at risk of developing a MUC1 or MUC1* positive cancer.
  • Humanizing [00603] Humanized antibodies or antibody fragments or fully human antibodies that bind to the extracellular domain of -MUC1* are preferred for therapeutic use. The techniques described herein for humanizing antibodies are but a few of a variety of methods known to those skilled in the art.
  • Humanization is the process of replacing the non-human regions of a therapeutic antibody (usually mouse monoclonal antibody) by human one without changing its binding specificity and affinity.
  • the main goal of humanization is to reduce immunogenicity of the therapeutic monoclonal antibody when administered to human.
  • Three distinct types of humanization are possible.
  • a chimeric antibody is made by replacing the non-human constant region of the antibody by the human constant region.
  • Such antibody will contain the mouse Fab region and will contain about 80-90% of human sequence.
  • a monoclonal antibody that has the desired effect and desired characteristics is identified, it is sequenced.
  • the sequence of the animal-generated antibody is then aligned with the sequences of many human antibodies in order to find human antibodies with sequences that are the most homologous to the animal antibody.
  • Biochemistry techniques are employed to paste together the human antibody sequences and the animal antibody sequences.
  • the non-human CDRs are grafted into the human antibodies that have the highest homology to the non- human antibody. This process can generate many candidate humanized antibodies that need to be tested to identify which antibody or antibodies has the desired affinity and specificity.
  • a human antibody or a humanized antibody can be further modified for use as an Fab fragment, as a full antibody, or as an antibody-like entity such as a single chain molecule containing the variable regions, such as scFv or an scFv-Fc. In some cases it is desirable to have Fc region of the antibody or antibody-like molecule mutated such that it does not dimerize.
  • fully human antibodies can be obtained by a variety of methods known to those skilled in the art, including screening human antibody libraries with a peptide fragment of an antigen.
  • a fully human antibody that functions like MNE6 or MNC2, 20A10 or other antibodies of the invention can be generated by screening a human antibody library or library of antibody fragments with a peptide having the sequence of the PSMGFR N-10 peptide.
  • human antibodies are generated in genetically modified mice. Humanized anti-MUC1* antibodies or antibody fragments were generated based on the sequences of the mouse monoclonal antibodies MNE6, MNC2, 20A10, 3C2B1, 5C6F3 and 25E6.
  • a patient diagnosed with a MUC1* positive cancer is treated with an effective amount of a murine or camelid antibody or antibody fragment comprising sequences from MNC2 (SEQ ID NO:118-119 and 168-169), MNE6 (SEQ ID NOS: 12-13 and 65-66), 20A10 (SEQ ID NOS:988-989 and 1004-1005), 3C2B1 (SEQ ID NOS:1820-1821 and 1822-1823), 5C6F3 (SEQ ID NO:1816-1817 and 1818-1819), 25E6 (SEQ ID NO:1020-1021 and 1036-1037 ), 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11.
  • MNC2 SEQ ID NO:118-119 and 168-169
  • MNE6 SEQ ID NOS: 12-13 and 65-66
  • 20A10 SEQ ID NOS:988-989 and 1004-1005
  • 3C2B1 SEQ ID NOS:1820
  • a patient diagnosed with a MUC1* positive cancer is treated with an effective amount of human or humanized antibody or antibody fragment comprising sequences from MNE6 (SEQ ID NOS:56-57 and 107-108, or 341-342, or 391- 392, or 393-394) or MNC2 (SEQ ID NO:144-145 and 194-195, or 654-655, or 239-249, or 5017-5020), 20A10 (SEQ ID NOS:1576-1581 or 5001-5012), 3C2B1 (SEQ ID NOS:1820-1823 or 1812-1813), 5C6F3 (SEQ ID NOS:1816-1819, or 1814-1815), 25E6 (SEQ ID NOS:1020- 1021 and 1036-1037, or 1600-1601), 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11.
  • MNE6 SEQ ID NOS:56-57 and 107-108, or 341-342, or 391-
  • a patient diagnosed with a MUC1* positive cancer is treated with an effective amount of humanized antibody or antibody fragment comprising sequences of MNC2 (SEQ ID NO:654-655), MNE6 (SEQ ID NO:341-342), 20A10 (SEQ ID NO:1580- 1581), 3C2B1 (SEQ ID NO:1812-1813), 5C6F3 (SEQ ID NO:1814-1815), 25E6 (SEQ ID NO:1600-1601).
  • a patient diagnosed with a MUC1* positive cancer is treated with an effective amount of humanized monovalent form of the antibodies such as MNC2 (SEQ ID NOS:239, 241, 243,396 or 5018-5020), MNE6 (SEQ ID NO:), 20A10 (SEQ ID NOS:1574-1581 or SEQ ID NOS:5001-5012) , 3C2B1 (SEQ ID NO:1813), 5C6F3 (SEQ ID NO:1815), 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11, wherein monovalent means the corresponding Fab fragment, the corresponding scFv or the corresponding scFv-Fc fusion.
  • MNC2 SEQ ID NOS:239, 241, 243,396 or 5018-5020
  • MNE6 SEQ ID NO:
  • 20A10 SEQ ID NOS:1574-1581 or SEQ ID NOS:5001-5012
  • a patient diagnosed with a MUC1* positive cancer is treated with an effective amount of a humanized scFv or monomeric humanized scFv-Fc of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11.
  • MUC1* growth factor receptor is activated by ligand induced dimerization of its extracellular domain, and because the Fc portion of an antibody homo-dimerizes, it is preferable that a construct that includes an Fc portion uses a mutated Fc region that prevents or minimizes dimerization.
  • Antibodies that bind to PSMGFR region of MUC1* or bind to a synthetic PSMGFR peptide are preferred. Especially preferred are antibodies that bind to the N-10 peptide but not to the C-10 peptide. Still more preferred are antibodies that bind to the N-26 peptide wherein mutation or deletion of the PFPFS sequence (SEQ ID NO:1747) destroys binding of the antibody or fragment thereof to the N-26 peptide.
  • the CDRs of these antibodies make up the recognition units of the antibodies and are the most important parts of the mouse antibody that should be retained when grafting into a human antibody.
  • the sequences of the CDRs for each mouse monoclonal are as follows, heavy chain sequence followed by light chain: MNE6 CDR1 (SEQ ID NO:16-17 and 69-70) CDR2 (SEQ ID NO:20-21 and 73-74) CDR3 (SEQ ID NO: 24-25 and 77-78), MNC2 CDR1 (SEQ ID NO:122-123 and 172-173) CDR2 (SEQ ID NO:126-127 and 176-177) CDR3 (SEQ ID NO:130-131 and 180-181), 20A10 CDR1 (SEQ ID NO:991-992 and 1008-1009) CDR2 (SEQ ID NO:996-997 and 1012-1013) CDR3 (SEQ ID NO:1000-1001 and 1016-1017), 3C2B1 CDR1 (SEQ ID NO:1388-1389 and 1402-1
  • MNE6 Humanized variable regions of MNE6 (SEQ ID NOS: 38-39 and 93-94), MNC2 (SEQ ID NOS: 144-145 and 194-195), 20A10 (SEQ ID NOS:1576-1581 and 5001-5012), 3C2B1 (SEQ ID NOS:1812-1813), 5C6F3 (SEQ ID NOS: 1814-1815), 25E6 (SEQ ID NOS:1600-1601).
  • MNC3 SEQ ID NOS: 439-440 and 486-487
  • MNC8 SEQ ID NOS: 525-526 and 543-544
  • humanized heavy chain variable constructs were then fused into constant regions of either human IgG1 heavy chain constant region (SEQ ID NOS:58-59) or human IgG2 heavy chain constant region (SEQ ID NO:54-55), which are then paired with either humanized light chain variable constructs fused to a human kappa chain (SEQ ID NO: 109-110) or human lambda chain (SEQ ID NO: 113-114) constant region.
  • Other IgG isotypes could be used as constant region including IgG3 or IgG4.
  • IgG constant region which IgG constant region is fused to the humanized variable region depends on the desired effect since each isotype has its own characteristic activity.
  • the isotype of the human constant region is selected on the basis of things such as whether antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) is desired but can also depend on the yield of antibody that is generated in cell-based protein expression systems.
  • ADCC antibody dependent cell cytotoxicity
  • CDC complement dependent cytotoxicity
  • humanized anti-MUC1* antibodies or antibody fragments are administered to a person diagnosed with or at risk of developing a MUC1-positive cancer.
  • One method for testing and selecting the humanized anti-MUC1* antibodies that would be most useful for the treatment of persons with cancer or at risk of developing cancers is to test them for their ability to inhibit the binding of activating ligands to the MUC1* extracellular domain.
  • Dimeric NME1 can bind to and dimerize the MUC1* extracellular domain and in so doing stimulates cancer cell growth.
  • Antibodies and antibody fragments that compete with NME1 for binding to the MUC1* extracellular domain are therefore anti- cancer agents.
  • NME7AB is another activating ligand of MUC1*.
  • NME proteins also bind to MUC1 or MUC1* including NME1, NME6 and NME8. Antibodies that compete with these proteins for binding to MUC1* may also be useful as therapeutics.
  • murine, camelid, human or humanized anti-MUC1* antibodies or antibody fragments are administered to a person diagnosed with or at risk of developing a MUC1-positive cancer.
  • Anti-MUC1* single chain variable fragments are generated by grafting non-human CDRs of antibodies, which bind to extracellular domain of MUC1* or bind to PSMGFR peptide, into a framework of a homologous variable region human antibody.
  • the resultant humanized heavy and light chain variable regions are then connected to each other via a suitable linker, wherein the linker should be flexible and of length that it allows heavy chain binding to light chain but discourages heavy chain of one molecule binding to the light chain of another.
  • a linker of about 10-15 residues.
  • the linker includes [(Glycine)4 (Serine)1]3 (SEQ ID NOS: 401-402), but is not limited to this sequence as other sequences are possible.
  • the recognition portion of the extracellular domain of the CAR is comprised of sequences from the human, humanized or non-human variable regions of MNE6 (SEQ ID NOS:12-13 and 65-66, 56-57 and 107-108, 38-39 and 93-94, 341-342, 391-394), MNC2 (SEQ ID NOS:118-119 and 168- 169, or 144-145 and 194-195, 654-655, 239-243, or 5017-5020), 20A10 (SEQ ID NOS:988- 989 and 1004-1005, or 1574-1581, 1677, 1687 or 5001-5012), 3C2B1 (SEQ ID NOS:1386- 1413, or 1820-1823, 1572-1573, or 1812-1813), 5C6F3 (SEQ ID NOS:1816-1819, or 1384- 1385, or 1814-1815), 25E6 (SEQ ID NOS:1020-1021, or 1036-1037, or 1598-1601), 18
  • Humanized single chain proteins were also derived from 3C2B1 (SEQ ID NOS:1812-1813), 5C6F3 (SEQ ID NOS:1814-1815) and 25E6 (SEQ ID NOS:1600-1601).
  • the extracellular hinge of the CAR can be derived from a variety of proteins, including CD8 (SEQ ID NOS:345-346), CD4 (SEQ ID NOS:347-348) or CD28 (SEQ ID NOS:349-350).
  • the transmembrane region of the CAR can also be derived from CD3-zeta (SEQ ID NOS:361-362), CD8 (SEQ ID NOS:363- 364), CD4 (SEQ ID NOS:365-366), CD28 (SEQ ID NOS:367-368), 4-1BB (SEQ ID NOS:369-370), OX40 (SEQ ID NOS:371-372), antibody domains or other transmembrane region, including the transmembrane region of the proximal cytoplasmic co-stimulatory domain, such as CD28, 4-1BB or other.
  • the cytoplasmic tail of the CAR can be comprised of one or more motifs that signal immune system activation.
  • a minimal CAR may have the CD3-zeta or an Fc receptor gamma domain then one or two of the above domains in tandem on the cytoplasmic tail.
  • the cytoplasmic tail comprises CD3-zeta, or a mutant thereof such as 1XX, plus a co-stimulatory domain such as CD28, 4-1BB and/or OX40.
  • one or two ITAMs of CD3-zeta are deleted or mutated to slow signaling which increases persistence and decreases differentiation of the immune cell.
  • MNE6 SEQ ID NOS:297-298, 300-301, 303-304, 1626-1633 and 5045-5048
  • MNC2 SEQ ID NOS:306- 307, 608-611, 718-719, 1618-1625, 5041-5044, and 1784-1785
  • 20A10 SEQ ID NOS:1582- 1597, 5021-5028, 1798-1799, 1692,1699, and 1706
  • 25E6 SEQ ID NOS:1602-1617, 5033-5040.
  • single chain antibodies derived from 3C2B1 (SEQ ID NOS:1572- 1573 or 1812-1813) or 5C6F3 (SEQ ID NOS:1384-1385 or 1814-1815) can be substituted for the single chain antibody fragment in any of the CARs listed above.
  • the antibody CDRs can be inserted into a number of different framework regions; as a demonstration we generated three versions of a humanized 20A10 which differ only in the framework regions. These have been incorporated into CARs (SEQ ID NOS:1675, 1678, 1685) that when transduced into human T cells are able to recognize target MUC1* expressing cells and kill them.
  • the transmembrane region of the CAR can be derived from a number of protein transmembrane domains, including but not limited to CD8 (SEQ ID NOS:363-364), or can be the transmembrane domain of CD3-zeta (SEQ ID NOS:361-362), CD4 (SEQ ID NOS:365-366), CD28 (SEQ ID NOS:367-368), 41BB (SEQ ID NOS:369-370), OX40 (SEQ ID NOS:371- 372) or other transmembrane region.
  • CD8 SEQ ID NOS:363-364
  • CD4 SEQ ID NOS:365-366
  • CD28 SEQ ID NOS:367-368
  • 41BB SEQ ID NOS:369-370
  • OX40 SEQ ID NOS:371- 372
  • the cytoplasmic domain of a CAR with antibody fragment targeting MUC1* extracellular domain can be comprised of one or more selected from the group comprising an immune system co-stimulatory cytoplasmic domain and a cytoplasmic signaling domain.
  • the group of immune system co-stimulatory domains includes but is not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7 and Fc receptor gamma domain (SEQ ID NOS:373-382).
  • the group of immune system signaling domains includes but is not limited to CD3-zeta (SEQ ID NOS:373-376) and CD3-zeta-1XX (SEQ ID NOS:1796-1797).
  • the CD3-zeta signaling domain may be wild type or may contain deletions or mutations of one or two of the three ITAMs.
  • the CD3-zeta domain contains only one functional ITAM.
  • ITAM is ITAM1 also known as the 1XX variation of CD3-zeta.
  • CAR constructs that can be transduced into T cells or other immune cells for the treatment or prevention of MUC1* positive cancers.
  • CARs are made up of modules and the identity of some of the modules is relatively unimportant, while the identity of other modules is critically important.
  • intracellular signaling modules such as CD3-zeta (SEQ ID NOS: 373-376), CD28 (SEQ ID NOS: 377-378) and 41BB (SEQ ID NOS: 379-380), alone or in combinations stimulate immune cell expansion, cytokine secretion and immune cell mediated killing of the targeted tumor cells (Pulè MA, Straathof KC, Dotti G, Heslop HE, Rooney CM and Brenner MK (2005) A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells. Mol Ther.12(5):933-941; Hombach AA, Heiders J, Foppe M, Chmielewski M and Abken H.
  • CD3-zeta SEQ ID NOS: 373-376
  • CD28 SEQ ID NOS: 377-3708
  • 41BB SEQ ID NOS: 379-380
  • the ITAMs of CD3-zeta may be mutated to inhibit or dampen signaling (Feucht et al 2019).
  • the CD3-zeta of the cytoplasmic tail may comprise mutations or deletions in the ITAMs including those referred to as 1XX (SEQ ID NOS:1796-1797).
  • one or two ITAMs are deleted, leaving only one or two ITAMs (Feucht et al 2019).
  • the position of the included ITAM or ITAMs is moved to a position proximal to the co-stimulatory domain.
  • Suitable ITAM configurations for increased persistence of CARs include but are not limited to 1XX, X2X, XX3, 12X and 23X, wherein the numeral 1, 2 or 3 refers to ITAM1, ITAM2, or ITAM3 and X refers to the deletion of that ITAM.
  • ITAM1 is the only functional ITAM included in the CAR construct, also known as 1XX.
  • Examples of antibodies of the invention incorporated into CARs with 1XX mutations in ITAMs of CD3- zeta are given in the following sequences: MNC2 (SEQ ID NOS: 1618-1625, 5041-5044 and 1784-1785), MNE6 (SEQ ID NOS:1626-1633 and 5045-5048 ), 20A10 (SEQ ID NOS:1590- 1597, 5021-5028, and 1798-1799), 25E6 (SEQ ID NOS:1610-1617 and 5037-5040).
  • the transmembrane and extracellular hinge region of the CAR may or may not be derived from sequences of the adjacent co-stimulatory domain.
  • a CAR comprising the 4-1BB co-stimulatory domain may have a transmembrane and hinge region derived from CD8 or CD28.
  • a CAR comprising the CD28 co-stimulatory domain may have a transmembrane and hinge region derived from CD28.
  • Examples of antibodies of the invention incorporated into CARs with 1XX mutations in ITAMs of CD3-zeta, which have a CD28 co-stimulatory domain as well as transmembrane and hinge region derived from CD28, are given in the following sequences: MNC2 (SEQ ID NOS:5041-5044 and 1784-1785) MNE6 (SEQ ID NOS:5045-5048), 20A10 (SEQ ID NOS:5025-5028, 1798-1799, 1692,1699, and 1706); 25E6 (SEQ ID NOS:5037-5040).
  • the cytoplasmic region may be comprised of one or more of signaling sequence motifs and co- stimulatory domains, including but not limited to CD3-zeta, CD3-zeta-1XX, CD27, CD28, 4- 1BB, OX40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7.
  • the sequence of the intracellular signaling domain may contain mutations, such as CD3-zeta-1XX (SEQ ID NOS:1796-1797) that dampen the signal to improve persistence or target cell killing.
  • Signaling domain CD3-zeta may be wild type or may contain mutations or deletions of one or two ITAMs.
  • ITAMs 2 and 3 are deleted or inactivated, leaving a single ITAM, which is ITAM1 also known as the 1XX construct.
  • the hinge and transmembrane regions of CAR are derived from CD8 (SEQ ID NO: 301, 719, 1675 or 1605).
  • the hinge and transmembrane regions of CAR are derived from CD28 (SEQ ID NO:5048, 5044, 5024 or 5036).
  • the co-stimulatory domain is CD28 (SEQ ID NO: 298, 609, 1589, 1609).
  • the antibody fragment that is the targeting head of the CAR binds to N-10 (SEQ ID NO:3) but does not bind to C-10 (SEQ ID NO:825).
  • the antibody fragment that is the targeting head of the CAR binds to N-10 (SEQ ID NO:3), does not bind to C-10 (SEQ ID NO:825) and either does not bind to a linear epitope, that is to say doesn’t work in a standard Western blot, or competes with NME7 AB for binding to the N-10 peptide (SEQ ID NO:3).
  • the extracellular domain may include a murine, camelid, human, non- human or humanized single chain antibody fragment with framework region IV of the light chain having variable lengths as set forth as MNE6 scFv (SEQ ID NOS:5014 or 5016), MNC2 scFv (SEQ ID NOS:5018 or 5020) or 20A10 scFv (SEQ ID NOS 5002, 5004, 5006, 5008, 5010 or 5012), 25E6 scFv (SEQ ID NOS: 5030 or 5032).
  • the Framework region IV of the light chain of the single chain antibody fragment may have the terminal amino acids R and T deleted or just T deleted.
  • the CDRs of antibodies can be inserted into a background of a number of different framework regions.
  • 20A10 CDRs were inserted into three different sets of framework regions (SEQ ID NOS:1692, 1699 and 1706) and all were able to function when transduced into T cells.
  • the T cell may be engineered to overexpress c-Jun as a method to inhibit T cell exhaustion (Lynn et al 2019).
  • a variety of promoters can be used upstream of the genes for CARs and other compositions of the invention, including insertion into a naturally occurring promoter in the cell, such as the TRAC locus, using CRISPR, Sleeping Beauty or similar technology for site directed insertion of a gene.
  • promoters commonly used are the CMV promoter, or a mini CMV (SEQ ID NO: 1634), a minimal IL-2 promoter (SEQ ID NO: 1635), or Minimal Promoter minip (SEQ ID NO: 1636).
  • the MUC1* targeting CARs were then transduced, separately or in combinations, into immune cells.
  • immune cells When challenged with surfaces presenting a MUC1* peptide, an antigen presenting cell transfected with MUC1*, or MUC1* positive cancer cells, the immune cells that were transduced with MUC1* targeting CARs elicited immune responses, including cytokine release, killing of the targeted cells and expansion of the immune cells.
  • the gene encoding the CARs and activated T cell induced genes described herein can be virally transduced into an immune cell using viruses, or inserted into a region downstream of one of the cell’s promoters or enhancers, such as the TRAC (T cell receptor alpha chain) locus.
  • Virus delivery systems and viral vectors including but not limited to retroviruses, including gamma-retroviruses, lentivirus, adenoviruses, adeno-associated viruses, baculoviruses, poxvirus, herpes simplex viruses, oncolytic viruses, HF10, T-Vec and the like can be used.
  • retroviruses including gamma-retroviruses, lentivirus, adenoviruses, adeno-associated viruses, baculoviruses, poxvirus, herpes simplex viruses, oncolytic viruses, HF10, T-Vec and the like
  • CARs and activated T cell induced genes described herein can be directly spliced into the genome of the recipient cell using methods such as CRISPR technology, CRISPR-Cas9 and -CPF1, TALEN, Sleeping Beauty transposon system, and SB 100X.
  • the identity of molecules that make up the non-targeting portions of the CAR are not essential to the function of a MUC1*-targeting CAR.
  • the extracellular domain, transmembrane domain and membrane proximal portion of the cytoplasmic domain can be comprised of portions of CD8, CD4, CD28, or generic antibody domains such as Fc, CH2CH3, or CH3.
  • the non-targeting portions of a CAR can be a composite of portions of one or more of these molecules or other family members.
  • CD3-zeta is critical, as mutations, such as those referred to as 1XX or CD3-zeta-1XX, greatly affect in vivo persistence of CAR T cells.
  • CAR T cells that express CARs whose cytoplasmic tail includes CD3-zeta-1XX have prolonged activity in vivo because they do not get exhausted as quickly as cells containing wild-type CD3-zeta.
  • CARs with the 1XX signaling domain are more effective against cells characterized by low antigen density. Cancer cells with low antigen density may comprise a sub-population of a heterogeneous tumor. Cancer cells with low antigen density may be characteristic of early cancer cells that can lead to cancer recurrence.
  • tumors at the time of treatment may be comprised of cancer cells that express low levels of a particular cancer antigen.
  • patients diagnosed with a cancer or at risk of developing a cancer or a cancer recurrence are treated with immune cells that express a CAR comprising a 1XX signaling domain.
  • the patient is diagnosed with or at risk of developing a MUC1* cancer.
  • the recognition unit of the CAR comprises an antibody fragment that binds to the N-10 peptide (SEQ ID NO:3) but does not bind to the C-10 peptide (SEQ ID NO:825).
  • the antibody fragment is derived from MNC2, MNE6, 20A10, 3C2B1, 5C6F3, or 25E6.
  • a patient diagnosed with a cancer comprised of tumor cells that express low levels of a targeted antigen, or diagnosed with an early cancer, or a patient who has been treated but still has residual tumor cells and is at risk of a cancer recurrence is treated with immune cells that express a CAR comprising a 1XX signaling domain, which enables the CAR T cells to kill both high and low antigen density cancer cells.
  • the patient is diagnosed with or at risk of developing a MUC1* cancer.
  • the recognition unit of the CAR comprises an antibody fragment that binds to the N-10 peptide (SEQ ID NO:3) but does not bind to the C-10 peptide (SEQ ID NO:825).
  • the antibody fragment is derived from MNC2, MNE6, 20A10, 3C2B1, 5C6F3, or 25E6.
  • the tumor is attacked by an immune cell expressing a CAR with full CD3-zeta signaling that efficiently kills off the high antigen expressing cells, but which are prematurely exhausted, while the cells expressing a CAR with a mutated CD3-zeta, such as CD3-zeta-1XX, persist longer in the patient and kill of the low antigen expressing cells that likely give rise to tumor recurrence.
  • the patient is diagnosed with or at risk of developing a MUC1* cancer.
  • the recognition unit of the CAR comprises an antibody fragment that binds to the N-10 peptide (SEQ ID NO:3) but does not bind to the C-10 peptide (SEQ ID NO:825).
  • a patient is treated with a CAR T cell in which the CAR has a CD3-zeta-1XX signaling domain, wherein the CAR is chosen from among the group comprising MNC2 CARs (SEQ ID NO:1618-1625, 5041-5044, 1784-1785), MNE6 CARs (SEQ ID NO:1626-1633, 5045-5048), 20A10 CARs (SEQ ID NO:1590-1597, 5025-5028, 1798-1799), 25E6 CARs (SEQ ID NOS:1610-1617, 5037-5040), a CAR comprising an antibody fragment derived from 3C2B1 wherein the signaling domain is CD3-zeta-1XX, and a CAR comprising an antibody fragment derived from 5C6F3 wherein the signaling domain is CD3-zeta-1XX.
  • MNC2 CARs SEQ ID NO:1618-1625, 5041-5044, 1784-1785
  • MNE6 CARs SEQ ID NO:
  • a patient is treated with immune cells that express both a CAR having a wild type CD3-zeta signaling domain and a CAR having a 1XX signaling domain.
  • CAR T cells bearing the 1XX mutations in the CD3-zeta are more effective than CAR T cells with wild type CD3-zeta at preventing tumor recurrence.
  • Figure 218 shows graphs of tumor volume measured by an IVIS instrument wherein the tumor cells have been genetically modified to express Luciferase. The substrate Luciferin was injected 10 minutes before the photo emissions were measured in the sedated animal. On Day 1 of the experiment, animals were injected sub-cutaneously with 250,000 human breast tumor cells.
  • Tumors were made heterogeneous, comprised of two different tumor cell types.
  • a first tumor cell population was T47D-wt, a breast cancer cell line that expresses both full- length MUC1 and the growth factor receptor form MUC1*, which we engineered to express mCherry fluorescence.
  • the second tumor cell population was the same T47D breast cancer cells, except that they had been stably transduced to express even more MUC1* and GFP fluorescence, referred to here as T47D-MUC1*.
  • animals were implanted with T47D-wt plus T47D-MUC1*, wherein the population of T47D-MUC1* made up 30%, 15% or 7.5% of the tumor population.
  • FIG. 219A-219B shows IVIS photographs and graphs of IVIS tumor volume measurement.
  • Fig. 219A shows photographs of the mice that had been implanted with tumors in which 30% of the cancer cell population was T47D-MUC1*, referred to here as high antigen expressing cells.
  • a significant problem for the treatment of cancers are the tumor cells that express low levels of tumor-associated antigen, especially with regard to cellular therapies where, to date, the killing of tumor cells has been dependent on the antigen density of the tumor cells.
  • Tumor cells expressing low levels of the target antigen escape CAR T cells as well as engineered CAR NK cells.
  • Essentially all solid tumors are heterogeneous and comprised of cells that express different levels of the target antigen.
  • Fig. 251 shows that in animals that were implanted with 250,000 tumor cells, then treated once with either 2.5M CAR T cells or 250,000 CAR T cells, tumors recur and the timing and degree of recurrence is greater in animals treated with CARs bearing standard CD3z than in animals bearing CD3z with 1XX mutations.
  • the excised tumors were fluorescently photographed at mCherry wavelength and at GFP wavelength.
  • tumor recurrence is minimal for the tumors of animals treated with huMNC2-CD28-1XX.
  • the figure shows that tumor recurrence is dominated by the low antigen expressing cells that are missed by the standard CAR T cells.
  • Fig. 253 and Fig. 256 when tumors expressing low percentage of high antigen expressing cells are coupled with treating with low dose CAR T cells, a combination of CAR T cell exhaustion plus escape of low antigen cells drives tumor resistance.
  • Anti-MUC1* CAR T cells persist longer and stave off T cell exhaustion when the CD3-zeta signaling domain is mutated to slow signaling as in mutating some of the Tyrosines, for example as we have done with the huMNC2-CD28-1XX, see Tables in Fig. 257 – Fig. 282.
  • Excised tumors from the test animals were analyzed by flow cytometry for the presence of human CAR T cells and their expression of exhaustion markers.
  • mice treated with huMNC2-CD28-1XX had an average of 1,516 CAR T cells in the excised tumor, compared to only 196 CAR T cells for the huMNC2-41BB-3z and 395 CAR T cells for the huMNC2-CD28-3z treated mice.
  • the CAR T cells expressed the lowest percentages of exhaustion markers. That means that for tumors with a significant amount of high antigen expressing cells and at high dose of CAR T cells, the mutated CD3z, as in 1XX mutations, gives the CAR T cells an advantage in terms of being able to recognize tumor cells and also being able to stave off exhaustion.
  • Comparison of CAR T cell persistence among CARs, with or without mutated CD3z, is assessed by looking at these same 30% high antigen expressing tumors, but wherein animals were treated with a low dose of CAR T cells: 250,000 CAR T cells and 250,000 tumor cells, i.e.1:1 ratio.
  • the Table of Fig.262 shows that CARs with a standard CD3z signaling domain, administered to animals at low dose have almost no measurable CAR T cells in their tumors after about 70-90 days after CAR T cell injection.
  • CAR T cells with a 1XX mutated CD3z, or similar mutated signaling domain have enhanced ability to recognize and kill tumor cells that express low levels of the target antigen.
  • standard CAR T cells could not recognize nor kill tumors that expressed a low percentage, 7.5%, of high antigen expressing cells when animals were also treated with a low dose of CAR T cells.
  • FIG. 20 shows photographs of normal small intestine tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • A-D are normal small intestine tissue.
  • E-H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Fig. 21 shows photographs of cancerous small intestine tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • A-D are cancerous small intestine tissue from a patient as denoted in figure.
  • E-H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • FIG. 22 shows photographs of cancerous small intestine tissues stained with humanized MNE6-scFv-Fc anti- MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody.
  • A-D are cancerous small intestine tissue from a patient as denoted in figure.
  • E-H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • Fig. 23 shows photographs of normal colon tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti- human HRP antibody.
  • A-D are normal colon.
  • E-H are photographs of the corresponding serial sections that were stained with the secondary antibody alone.
  • One aspect of the invention is a method for treating a patient diagnosed with, suspected of having, or at risk of developing a MUC1 positive or MUC1* positive cancer, wherein a specimen is obtained from the patient’s cancer and is tested for reactivity with an antibody that binds to PSMGFR SEQ ID NO:2, or more specifically to the N-10 peptide (SEQ ID NO:3), or yet more specifically binds to N-10 peptide (SEQ ID NO:3), but does not bind to C-10 peptide (SEQ ID NO:825).
  • MUC1* the transmembrane cleavage product, not full-length MUC1
  • the growth factors that activate MUC1* bind to ectopic sites that are only exposed after cleavage and release of the tandem repeat portion of MUC1.
  • Antibodies of the invention like the activating growth factors, cannot bind to full-length MUC1. FACS analysis clearly shows that anti- MUC1* antibody MNC2 is unable to bind to HCT-116, MUC1 negative cells (Fig.
  • MUC1 can be cleaved to MUC1* by more than one cleavage enzyme and that the site of cleavage affects its fold and consequently affects which monoclonal antibody is able to recognize that form of MUC1*.
  • Different cancer cells or cancerous tissues express different cleavage enzymes.
  • cleavage enzyme inhibitors were tested on different cancer cell lines and found that an inhibitor that inhibits cleavage of MUC1 in one cancer cell line did not inhibit its cleavage in another cancer cell line. Similarly, PCR experiments showed that cleavage enzymes are expressed at different levels in different cells or cell lines. For example, hematopoietic stem cells of the bone marrow express a MUC1* that is recognized by monoclonal antibody MNC3 but not MNE6 or MNC2 (Fig. 39).
  • DU145 prostate cancer cells and T47D breast cancer cells are inhibited by the Fabs of MNC2 and MNE6 but not by the Fabs of MNC3 or MNC8, indicating that the cancer cell lines express a MUC1* that is recognized by MNE6 and MNC2 but not by MNC3 or MNC8 (Fig. 42).
  • PCR experiments show that CD34 positive cells of the bone marrow express about 2,500-times more MMP2 and about 350-times more ADAM28 than T47D breast cancer cells, while DU145 prostate cancer cells express about 2,000-times more ADAM TS16, about 400-times more MMP14 and about 100-times more MMP1 than T47D breast cancer cells (Fig. 43 and Fig. 44).
  • the expression of the second factor is controlled by an inducible promoter.
  • expression of the second factor is induced when the immune cell is activated, for example when it recognizes or engages its target.
  • a T cell is transfected or transduced with a second factor whose expression is induced when the T cell recognizes a target cancer cell.
  • One way to do this is to induce expression of the second factor when, or shortly after, an NFAT protein is expressed or translocated to the nucleus.
  • a sequence derived from an NFAT promoter region is put upstream of the gene for the second factor.
  • the transcriptional regulatory region for NFATc2 is engineered upstream of the gene encoding the second factor, which may be cleavage enzyme MMP9 (SEQ ID NO:647) or the catalytic sub-unit of MMP9 (SEQ ID NO:648).
  • MMP9 cleavage enzyme
  • SEQ ID NO:648 the catalytic sub-unit of MMP9
  • Another method for having the expression of the second factor induced when the T cell or CAR T cell is activated is to have the gene for the second factor on an inducible promoter where the NFAT protein itself binds to and induces transcription of the second factor.
  • an NFAT response element NFAT RE
  • the NFAT may bind to its responsive element upstream of the second factor alone or as part of a complex.
  • the NFAT protein may be NFATc1, NFATc2, NFATc3, NFATc4, or NFAT5.
  • the NFAT protein is NFAT2 aka NFATc1, aka NFATc.
  • the gene of the second factor or fragment thereof is cloned downstream of an NFAT-response element (SEQ ID NO:649), which may be repeats of the response element (SEQ ID NO:650) and CMV minimal promoter (mCMV) (SEQ ID NO:651) to induce expression of second factor by NFAT protein.
  • NFATs 1-4 are regulated by the calcineurin pathway
  • potential toxicities that may arise in a patient can be stopped by treatment with an immunosuppressive agent such as FK506, Cyclosporin, Cyclosporin A, or Tacrolimus that block calcineurin activity and inhibit NFAT translocation to the nucleus.
  • an immunosuppressive agent such as FK506, Cyclosporin, Cyclosporin A, or Tacrolimus that block calcineurin activity and inhibit NFAT translocation to the nucleus.
  • the T cell transduced or transfected with a cleavage enzyme on an inducible promoter may also be transfected or transduced with a CAR that recognizes a protein or molecule on the cancer cell.
  • the cleavage enzyme is one that is able to cleave MUC1 full-length and the CAR bears an antibody fragment that directs it to MUC1* on the surface of cancer cells.
  • the invention anticipates overcoming this problem by co-expressing the cleavage enzyme with its activator.
  • the cleavage enzyme is MMP9 and the co-activator is MMP3.
  • the cleavage enzyme is expressed in a form that is already active, for example by expressing a fragment of the cleavage enzyme that still has catalytic function.
  • the cleavage enzyme is an MMP9 fragment that is catalytically active.
  • MMP9 catalytic fragment is given as SEQ ID NO:645. [00655] MMP9, which must be activated by MMP3, is overexpressed in a large percentage of solid tumors.
  • MNC2 anti-MUC1* monoclonal antibody recognizes MUC1 after it is cleaved by MMP9.
  • APMA is a biochemical that activates MMPs.
  • the MUC1 and MUC1* expressing cells were stained with a red dye, CMTMR.
  • T47D MUC1 positive tumor cells were incubated with a recombinant catalytic domain of MMP9 (Enzo Life Sciences, Inc., Farmingdale, NY) at either 100ng/mL or 500ng/mL.
  • a CAR containing sequences of the antibody are expressed in a stem cell, which then may be differentiated into an immune cell.
  • the immune cell is a T cell.
  • the immune cell is an NK cell.
  • a person diagnosed with cancer or at risk of developing cancer is administered a sufficient amount of an immune cell transduced with both a CAR and a cleavage enzyme.
  • a person diagnosed with cancer or at risk of developing cancer is administered a sufficient amount of an immune cell transduced with both a CAR and a cleavage enzyme, wherein the cleavage enzyme is on an inducible promoter that is activated by proteins that are expressed when the immune cell becomes activated.
  • the anti-MUC1* antibody is MNE6scFv or a humanized form of MNE6scFv.
  • the immune cell can be a T cell, an NK cell, a mast cell, or a dendritic cell.
  • the immune cell is derived from a stem cell that has been directed to differentiate to that immune cell type in vitro.
  • a CAR containing sequences of the antibody are expressed in a stem cell, which then may be differentiated into an immune cell.
  • the immune cell is a T cell.
  • the immune cell is an NK cell.
  • expression of the cleavage enzyme is induced by constructing a plasmid where the cleavage enzyme gene is inserted downstream of a Calcineurin promoter sequence or downstream of a Calcineurin response element, then inserting the plasmid into an immune cell and then administering to a patient for the treatment or prevention of cancers.
  • a plasmid where the cleavage enzyme gene is inserted downstream of a Calcineurin promoter sequence or downstream of a Calcineurin response element, then inserting the plasmid into an immune cell and then administering to a patient for the treatment or prevention of cancers.
  • drug-inducible plasmids that can be used to induce expression of the cleavage enzyme or used to stop expression induced by an element of an activated T cell.
  • These drug inducible systems may include tetracycline-inducible systems, Tet-on, Tet-off, tetracycline response elements, doxycycline, tamoxifen inducible systems, ecdysone inducible systems and the like. [00662] It is not intended that the present invention be limited to one or two specific promoters used in the plasmids encoding the CARs or inducible cleavage enzymes.
  • promoters can be interchanged including SV40, PGK1, Ubc, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1 and U6.
  • Another solution to the problem of steric hindrance of CAR T cell access, caused by bulky cell surface proteins such as MUC1-FL, is to increase the length of the linker region of the CAR that is expressed by the T cell.
  • the length of the extracellular linker region between the transmembrane portion and the antibody fragment is about 45-50 amino acids in length.
  • the stability of these various antibody-like molecules can be increased by the introduction of Cysteines for the formation of disulfide bonds.
  • the stability of antibodies of the invention as well as of various antibody-like molecules, which may be multi-specific can be increased by the introduction of mutations as more fully described in Beckman, US9708388B2, which reference is incorporated by reference herein in its entirety, but in particular with respect to disclosures of making of bispecific antibodies and fragments of antibodies that are bispecific or multispecific.
  • the invention contemplates incorporating variable regions, or fragments thereof, of antibodies of the invention in combination with other antibodies having other binding specificities, or fragments thereof, into a variety of antibody-like formats including but not limited to the following.
  • bispecific antibody As an example of how antibodies of the invention can be incorporated into bispecific antibodies, we constructed a bispecific antibody using a knob-in-hole, also known as KIH (Spiess et al. Molecular Immunology 67, 95-106 (2015)), format.
  • a first arm of the antibody is the humanized anti-MUC1* antibody 20A10, also known as hu20A10, with a 14616 framework region; the second arm of the antibody is either the anti- CD3 antibody OKT3 or 12F6, which both bind to the same epitope on human T cells.
  • the resultant bispecific antibodies are referred to here as 20A10-OKT3-BiTE and 20A10-12F6- BiTE.
  • the bispecific antibodies are added at various concentrations to cells in culture wherein both human T cells and MUC1* positive cancer cells are present.
  • the cancer cells are T47D breast cancer cells and in the other case a MUC1* negative line HCT-116 colon cancer cells have been transduced to express MUC1*, called HCT-MUC1*.
  • a MUC1* negative line HCT-116 colon cancer cells have been transduced to express MUC1*, called HCT-MUC1*.
  • the addition of either bispecific antibody mediated the joining together of the T cells and the MUC1* positive cancer cells as evidenced by a bispecific dose-dependent cell clustering. Two control experiments were performed.
  • the adapted T cells are then expanded and administered to the donor patient who is diagnosed with, suspected of having, or is at risk of developing a MUC1* positive cancer.
  • a series of CARs were also made that had MNC2 and humanized MNC2 as the extra cellular, targeting head of the CAR.
  • the constructs for these CARs were inserted into a plasmid that was then inserted into a Lenti viral vector.
  • Human T cells were then transduced with the lenti viral vector carrying the MNC2 CARs and huMNC2 CARs.
  • MNC2-scFv-CARs that were mouse sequence or humanized were generated.
  • IFN-g interferon gamma
  • IL-2 interleukin-2
  • Fig. 55C is a fluorescent photograph of huMNC2-CAR44 T cells co-cultured with the prostate cancer cells, wherein granzyme B is stained with a red fluorophore.
  • Fig. 55D is the DAPI and granzyme B merge.
  • Fig. 55E is a FACS scan for fluorescently labeled granzyme B for untransduced T cells incubated with the cancer cells.
  • Fig. 55F is a FACS scan showing a positive increase in fluorescently labeled granzyme B for huMNC2-CAR44 T cells incubated with the cancer cells.
  • Fig. 55G is a graph of the mean fluorescent intensity. Fig.
  • Fig. 55H is an xCELLigence scan tracking the real-time killing of DU145 cancer cells by huMNC2-CAR44 T cells (blue trace) but not by untransduced T cells (green).
  • Figs. 56A-56H show the cytotoxic effect of huMNC2-CAR44 T cells on MUC1* positive CAPAN-2 pancreatic cancer cells as measured by a variety of assays.
  • Fig. 56A is a fluorescent photograph of untransduced T cells co-cultured with the pancreatic cancer cells, wherein granzyme B is stained with a red fluorophore.
  • Fig. 56B is the DAPI and granzyme B merge.
  • FIG. 56C is a fluorescent photograph of huMNC2-CAR44 T cells co-cultured with the pancreatic cancer cells, wherein granzyme B is stained with a red fluorophore.
  • Fig. 56D is the DAPI and granzyme B merge.
  • Fig. 56E is a FACS scan for fluorescently labeled granzyme B for untransduced T cells incubated with the cancer cells.
  • Fig. 56F is a FACS scan showing a positive increase in fluorescently labeled granzyme B for huMNC2-CAR44 T cells incubated with the cancer cells.
  • Fig. 56G is a graph of the mean fluorescent intensity. Fig.
  • Fig. 56H is an xCELLigence scan tracking the real-time killing of CAPAN-2 cancer cells by huMNC2-CAR44 T cells (blue trace) but not by untransduced T cells (green).
  • Figs. 57A-57C show xCELLigence scans tracking the real-time killing of MUC1* positive cancer cells, but not MUC1* negative cells, by huMNC2-CAR44 T cells.
  • Fig. 57A shows that huMNC2-CAR44 T cells effectively kill HCT colon cancer cells that have been stably transfected with MUC1*.
  • Figure 61 shows an experiment in which huMNC2-scFv-CAR44 transduced human T cell that were bead stimulated (Protocol 1) or cancer cell stimulated (Protocol 2) were tested for their ability to inhibit tumor growth in animals.
  • Human cancer cells that had been stably transfected with Luciferase were injected into female NOD/SCID/GAMMA (NSG) mice between 11 and 15 weeks of age.
  • 500,000 BT-20 breast cancer cells were injected sub-cutaneously into a rear flank. Tumor engraftment was verified by injecting the animals with Luciferin and then imaging the fluorescent cancer cells using an IVIS instrument. IVIS images taken Day 5 post implantation showed the presence of tumor cells.
  • One method used to identify stem specific antibodies is as follows: supernatants from monoclonal hybridomas were separately adsorbed onto 2 multi-well plates. Stem cells, which are non-adherent cells, were put into one plate and cancer cells which are adherent were put into an identical plate. After an incubation period, the plates were rinsed and inverted. If the non-adherent stem cells stuck to the plate, then the monoclonal antibody in that particular well recognizes stem cells and will not recognize cancer cells. Antibodies that did not capture stem cells or antibodies that captured cancer cells were identified as cancer specific antibodies. FACS analysis has confirmed this method works. [00692] Antibodies MNE6 and MNC2 are examples of cancer-specific antibodies.
  • Another method for identifying antibodies that are cancer specific is to immunize with a peptide having the sequence of the PSMGFR peptide minus the 10 N-terminal amino acids or use that peptide to screen for antibodies or antibody fragments that will be cancer specific.
  • Antibodies that bind to a peptide with a sequence of PSMGFR peptide minus the N-terminal 10 amino acids, referred to herein as N-10 peptide, but do not bind to a peptide with a sequence of PSMGFR peptide minus the C-terminal 10 amino acids, C-10 peptide, are cancer specific antibodies for use in the treatment or prevention of cancers.
  • the extracellular domain of MUC1 is also cleaved on stem cells and some progenitor cells, where activation of cleaved MUC1 by ligands NME1 in dimer form or NME7 promotes growth and pluripotency and inhibits differentiation.
  • Antibodies that recognize different cleavage site conformations may be cancer sub-type specific or patient specific, depending on which cleavage enzyme their tumor expresses.
  • a patient diagnosed with a certain type of cancer is treated with an antibody of the invention that recognizes a cleaved MUC1 wherein the antibody is specific for cleavage by a specific enzyme that is known to be typically expressed by that sub-type of cancer.
  • a patient tumor is analyzed to determine which enzyme his or her tumor expresses and an antibody that recognizes a MUC1 cleaved by that enzyme is then administered to the patient for the treatment of their cancer.
  • the antibody may be in the form of a CAR, a BiTE, an ADC, a bispecific antibody, with or without an FC region or a portion of an Fc region, a bi-scFv, a di-scFv, a tandem di-scFv, a diabody, triabody, tribody, tetrabody and other antibody-like molecules that are multi-valent and multi-specific.
  • MUC1* MUC1 transmembrane cleavage product
  • MUC1* MUC1 transmembrane cleavage product
  • Fig. 1A ligand induced dimerization of its short extra cellular domain
  • Dimerization of the MUC1* extra cellular domain activates the MAP kinase signaling cascade and stimulates growth and survival of cancer cells (Fessler et al 2009).
  • Bivalent antibodies that dimerize the MUC1* extra cellular domain stimulate cancer cell growth while the monovalent Fab of the same antibody, which cannot dimerize, inhibits cancer cell growth.
  • NME1 dimers multimerize and form hexamers, which do not bind to MUC1*, but likely bind to some unknown receptor, as the addition of NME1 hexamers turns off growth.
  • NME1 is an adult form.
  • the embryonic form is NME7AB (Carter et al 2016).
  • Each NME7AB monomer has two binding sites for MUC1* so as a monomer it dimerizes MUC1* (Fig. 1D), stimulates growth and cannot turn itself off.
  • BRD4 turns off NME7 and its co-factor JMJD6 turns on the self- regulating form, NME1.
  • NME7 which should be silenced in adult life, is aberrantly expressed again, where is renders the MUC1* growth factor receptor constitutively active.
  • NME1 SEQ ID NO:4
  • NME7AB SEQ ID NO:827
  • Both growth factors can bind to the PSMGFR peptide (SEQ ID NO:2) even if the 10 N-terminal amino acids are deleted, referred to herein as N-10 (SEQ ID NO:3).
  • N-10 SEQ ID NO:3
  • neither NME1 nor NME7 AB can bind to the PSMGFR peptide if the 10 membrane proximal amino acids are deleted (Fig.
  • NME1 and NME7AB bind to the epitope to which NME1 and NME7AB bind includes all or part of the 10 membrane proximal amino acids: PFPFSAQSGA (SEQ ID NO:1743).
  • PFPFSAQSGA SEQ ID NO:1743
  • MNC2 and MNE6 monoclonal anti- MUC1* antibodies Two other monoclonal antibodies that were generated from immunizing animals with the PSMGFR peptide are MNC3 and MNC8.
  • MNC2, MNE6, MNC3 and MNC8 all bind to the PSMGFR peptide, like NME1 and NME7 AB , MNC2 and MNE6 bind strongly to the N-10 peptide but not to the C-10 peptide (Fig. 2B-2C). In fact, MNC2 and MNE6 competitively inhibit the binding of NME1 and NME7 AB to PSMGFR (Fig. 3A- 3C). Conversely, MNC3 and MNC8 bind to the C-10 peptide, bind less well to the N-10 peptide and do not compete with NME1 nor NME7 AB for binding to MUC1* peptides, including PSMGFR (Fig. 2E-2F).
  • MUC1* is generated by enzymatic cleavage of MUC1
  • cleavage enzymes cleave MUC1 to a MUC1* and whether or not we could identify antibodies that would recognize a MUC1* generated by a first cleavage enzyme but not MUC1* generated by a second cleavage enzyme.
  • MNC2 and MNE6 recognized a MUC1* generated by cleavage of MUC1 by MMP9 but not by cleavage by other enzymes such as MMP2 (Fig. 37 and Fig. 75).
  • MMP9 is overexpressed in cancers and is a predictor of poor prognosis (vant Veer et al 2002; Dufour et al 2011) and has been implicated in metastasis (Owyong et al, 2019), whereas MMP2 is expressed in bone marrow.
  • One antibody binding to a MUC1* generated by cleavage by a first enzyme but not by cleavage by a second enzyme implies that the antibody recognizes a conformational epitope rather than a linear epitope.
  • MNC2 and MNE6 are cancer specific. [00713] Our experiments show that both MNC2 and MNE6: a) Bind to tumor cells; b) monovalent forms block tumor growth in vitro and in vivo; c) have minimal to no binding of normal tissue while having robust binding to a wide panel of tumor tissues; d) when incorporated into CAR T cells, MNC2 and MNE6 directed CAR T cells do not recognize full-length MUC1 and do not kill cells that only express full-length MUC1; e) MNC2 and MNE6 directed CAR T cells cluster then kill tumor cells expressing MUC1*; and f) MNC2 and MNE6 recognize a MUC1 cleavage product when it is cleaved by MMP9.
  • MNC2 directed CAR T cells do not recognize normal, healthy cells that are MUC1* positive.
  • MNC2 and MNE6 Characterization of MNC2 and MNE6
  • Our gold standard, cancer-specific antibodies MNC2 and MNE6 1) bind to N-10 peptide but not to the C-10 peptide; 2) compete with NME7 AB and dimeric NME1 for the same binding site near the C-terminus of the PSMGFR peptide, which is the membrane proximal portion of MUC1* on cells; 3) do not work in a Western blot assay indicating that they recognize a conformational rather than linear epitope; 4) recognize a MUC1* generated when MUC1 is cleaved by MMP9; 5) do not bind to full-length MUC1 but only to the cleaved form, MUC1*, in model cell lines as well as cancer cell lines; 6) show little to no binding to normal tissues but robustly stain a wide variety of tumor tissues; and 7) share some consensus sequences in their Complementarity Determining Regions, CDRs.
  • Monoclonal antibodies were produced by immunizing animals with peptides derived from a MUC1 that is devoid of tandem repeats. These antibodies included PSMGFR and peptides that were extended at the N-terminus of PSMGFR.
  • MNC2, MNE6, MNC3 and MNC8 all bind to the PSMGFR peptide, like NME1 and NME7 AB , MNC2 and MNE6 bind strongly to the N-10 peptide but not to the C-10 peptide. In fact, MNC2 and MNE6 competitively inhibit the binding of NME1 and NME7 AB to PSMGFR. Conversely, MNC3 and MNC8 are able to bind to the C-10 peptide, bind less well to the N-10 peptide and do not compete with NME1 nor NME7 AB for binding to MUC1* peptides, including PSMGFR (Fig. 70). MNC3 and MNC8 are less cancer specific than MNC2 and MNE6.
  • the rank order of potency for disrupting binding of NME7AB to PSMGFR according to their cognate epitope is as follows: FPFS> ASRYNLT> QFNQYKTEA>GTINVHDVET.
  • Antibodies that bind to epitopes outside of the PSMGFR peptide, such as 45C11, 8A9 and 17H6 did not compete with NME7 AB for binding.
  • Western blot assay to determine linear versus conformational cognate epitope [00736] Antibodies were tested to determine whether they recognize a linear or a conformational epitope. Only antibodies that recognize a linear epitope work in Western blots when using denaturing gels.
  • Antibody 45C11 which binds to the SNIKFRPGSVV epitope, which is outside of the PSMGFR portion of MUC1, does not recognize a MUC1 cleavage product after cleavage by MMP9 or MMP2 (Fig. 75K).
  • antibodies 8A9 and 17H6 bind to the VQLTLAFRE epitope, which is also outside of the PSMGFR sequence, and they do not bind to a MUC1 cleaved by MMP9 or MMP2. This result is consistent with the idea that MMP9 cleaves MUC1 such that the extra cellular domain of the remaining transmembrane cleavage product comprises essentially the amino acids of the PSMGFR peptide.
  • Anti-MUC1* antibody 3C2B1 is an antibody that like MNC2, MNE6 and 20A10, binds to N-10 but not to C-10. More refined epitope mapping shows that like these three other highly cancer-specific antibodies, 3C2B1 requires the FPFS sequence for binding to a MUC1* extra cellular domain peptide.
  • Figure 121 shows the photograph of the FDA normal array MNO1021.
  • Figure 122A-122X shows photographs of specific tissues from FDA normal tissue array MNO1021 stained with the anti-PSMGFR antibody 3C2B1 at 20ug/mL. As can be seen, there is no binding of 3C2B1 to any critical normal organs.
  • Antibody 8A9 is an N+9/C-9 antibody that binds to epitope VQLTLAFRE which is outside of the PSMGFR sequence. Antibody 8A9 cannot bind to the N-10 peptide. Normal tissue array FDA MNO1021 was stained with 8A9 (Fig. 189-190). As can be seen in the figures, like antibody 45C11, which also binds an epitope that is N-terminal beyond the PSMGFR sequence, antibody 8A9 shows strong binding to many normal tissues, including adrenal, brain, heart, lung, liver, spleen, skeletal muscle and bone marrow.
  • an antibody that is not purely cancer-specific can be made more cancer-specific if it is incorporated into a bispecific antibody where a first side of the molecule binds to a first cancer antigen and the second side of the molecule binds to a second antigen that may be a tissue specific antigen, another cancer specific antigen or even an antigen on a cell such as a T cell, which are called BiTES, bispecific T cell engagers.
  • the less cancer-specific antibody can be incorporated into a cell-based therapy where its expression is induced only after the cell recognizes a tumor.
  • a CAR T cell can express a first CAR that recognizes a first antigen which recognition induces expression of a second antibody, or CAR incorporating the second antibody.
  • the cell expresses a CAR directed by an antibody fragment that is cancer-specific and a second antibody or CAR expressing the second antibody is induced to be expressed in an NFAT inducible system.
  • the nucleic acids encoding the second antibody or second CAR are down stream of NFAT response elements.
  • the NFAT inducible gene may be inserted into a Foxp3 enhancer or promoter.
  • test criteria 1-4 or even 1-5 provide a set of rapid, multiplexed and inexpensive tests that can be performed on hundreds or thousands of impure hybridoma clone supernatants to identify antibodies that are highly selective for cancer-specific forms of MUC1*.
  • Satisfies test criteria [00777] In a preferred embodiment an antibody is chosen for the treatment, prevention or diagnosis of a MUC1* positive cancer based on satisfying four (4) of the seven (7) criteria set out in Table 4.
  • the antibody or antibody fragment is conjugated to a toxin or an ADC, antibody drug conjugate, then administered to a patient who has been diagnosed with or is at risk of developing a MUC1* positive cancer.
  • Bind to N-10 [00779] We have demonstrated that a MUC1 transmembrane protein, devoid of tandem repeats and having an extra cellular domain of 45 amino acids of PSMGFR sequence, is sufficient to function as a growth factor receptor and confers oncogenic characteristics to the cell (Mahanta et al 2008).
  • Antibodies that bind to the PSMGFR peptide or portion of a transmembrane MUC1 cleavage product can be cancer specific but may also bind to stem or progenitor cells. Antibodies that bind to the N-10 peptide are more cancer-specific. In a preferred embodiment an antibody is chosen for the treatment, prevention or diagnosis of cancer based on the ability of the antibody to bind to the N-10 peptide. [00780] Do not bind to C-10 [00781] We have demonstrated that the MUC1 extra cellular domain contains an ectopic binding site that is only exposed if the tandem repeat domain is missing, which can occur as a consequence of alternative splice variant or cleavage and release of the extra cellular domain.
  • MNC2 and MNE6 will not bind to full-length MUC1, but do bind to the remaining portion when MUC1 is cleaved and the tandem repeat domain is shed.
  • MNC2 and MNE6 will bind to a MUC1*-like protein if it is devoid of tandem repeats, for example if a MUC1 negative cell is transfected or transduced with an engineered MUC1 that is devoid of tandem repeats, especially if extra cellular domain comprises the PSMGFR.
  • the ectopic site to which MNC2 and MNE6 bind is unmasked when tandem repeat domain is missing or removed.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on the ability of the antibody to disrupt the binding of NME1, NME7 AB , or NME7-X1 to the PSMGFR peptide, the N-10 peptide, or to the surface of a MUC1* positive cancer cell.
  • Antibodies that are cancer-specific will be chosen based on their ability to bind to a MUC1 that is devoid of tandem repeats and for their inability to bind to full-length MUC1.
  • MUC1* is generated when MUC1 is cleaved by a cleavage enzyme and the tandem repeat domain is released from the cell surface. Cleavage and release of the tandem repeat domain may also unmask portions of MUC1*-like cleavage products that exist on normal tissues.
  • antibodies that recognize a conformation, rather than a linear epitope are more selective. Antibodies that recognize a conformational epitope rather than a linear epitope can be identified by a variety of means. In particular, antibodies that recognize a conformational epitope will not work in a denaturing Western blot assay.
  • Cleavage by first enzyme may produce a conformation or a fold that is not the same as that produced by cleavage by a second enzyme. Support for this can be found in this application and is illustrated in Figures 39-41. These figures show that although a polyclonal antibody that binds to PSMGFR recognizes a cleaved MUC1 on hematopoietic stem cells, some monoclonal antibodies that bind to the PSMGFR peptide can bind to this MUC1*-like form on hematopoietic stem cells while others cannot. For example, MNC3 readily recognizes this cleaved form of MUC1 on hematopoietic stem cells, but MNC2 and MNE6 do not.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on the ability of the antibody to recognize a MUC1 cleavage product generated when MUC1 is cleaved by MMP14.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on the ability of the antibody to recognize a MUC1 cleavage product generated when MUC1 is cleaved by MMP9.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on the ability of the antibody to recognize a MUC1 cleavage product generated when MUC1 is cleaved by MMP9 and also recognizes a conformational epitope.
  • a traditional approach to identifying antibodies that are cancer-specific involves testing a panel of antibodies against a panel of different cancer cell lines and determining, by FACS, IF, immunoprecipitation or other method, if the antibody binds to cancer cells. Although this approach is traditional, it is sequential and time-consuming, and thus limits the analysis of large numbers of monoclonal antibody clones, which is required to find an ideal antibody suitable for cancer therapeutic or diagnostic. In addition, there are no real normal cell lines and the selection of normal primary cells is limited. The selection criteria presented above provide a rapid, multiplexed method for identifying monoclonal antibody clones that are specific for MUC1* positive cancers.
  • hybridoma supernatants can be used. This provides a huge advantage over state of the art methods for identifying antibodies that are specific for MUC1* positive cancers.
  • the ability to select antibodies from assay performed using the impure hybridoma supernatants means that much of the selection can be done on hundreds or thousands of clones rapidly and at very little cost. Methods such as FACS analysis ans IHC tissue studies require the use of purified antibodies which limits the number of clones that can be tested to tens, not even hundreds. [00789]
  • selecting an antibody based on its ability to bind to cancer cells, or a cancer cell type or to a cell engineered to express a certain antigen is important for antibody selection.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on the ability of the antibody to bind to MUC1* positive cancer cells.
  • Binds to tumor tissue more than normal tissue [00791] Immunohistochemistry, IHC, tissue studies of cancerous versus normal tissues is a more stringent test of the cancer specificity of antibodies than FACS analysis. Cancer cell lines are a single cell from a single patient that have been expanded in a lab for decades and are not representative of a cross section of the human population. Further, analysis of cell lines is blind to the heterogeneity of actual tumors. Tissue studies require purified antibody, are very expensive, time-consuming and require a skilled pathologist to analyze each stained tissue specimen.
  • an antibody, or fragments thereof, that binds to a peptide comprising the sequence SDVSVSDVPFPFSAQSGA are incorporated into anti- cancer therapeutics or diagnostics for the diagnosis, treatment or prevention of a MUC1* positive cancer.
  • an antibody, or fragments thereof, that binds to a peptide comprising the sequence SVSDV are incorporated into anti-cancer therapeutics or diagnostics for the diagnosis, treatment or prevention of a MUC1* positive cancer.
  • an antibody, or fragments thereof, that binds to a peptide comprising some or all of the sequence PFPFSAQSGA are incorporated into anti-cancer therapeutics or diagnostics for the diagnosis, treatment or prevention of a MUC1* positive cancer.
  • the selected antibodies or fragments thereof are incorporated into a CAR, BiTE, ADC or bi-specific and then administered to a patient diagnosed with or at risk of developing a MUC1* positive cancer.
  • Consensus Sequences [00800] Antibodies of the invention were categorized according to cognate epitope. Sequences of their respective heavy chain CDRs are shown in Table 5.
  • Heavy Chain CDR1 for MNC2 is FTFSGYAMS, with the amino acids numbered from left to right 1 through 9, the consensus of other antibodies that bind to that portion of PSMGFR is: F or I at position 1, T at position 2, F at position 3, S at position 4, T, G, or R at position 5, Y at position 6, A, G or T at position 7, M at position 8 and S at position 9.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a heavy chain CDR2 that is at least 90% identical to a CDR2 comprising the following amino acids at the specified positions: T at position 1, I or S at position 2, I or S at position 3, G or R at position 5, G or A at position 6, T or I at position 9, Y at position 10, Y at position 11, P or S at position12 and DSVKG for positions 13-17.
  • Light Chain CDR1 for MNC2 is RASKS--VSTSGYSYMH, with the amino acids numbered from left to right 1 through 17, the consensus of other antibodies that bind to that portion of PSMGFR is: K or R at position 1, A or S at position 2, S at position 3, K or Q at position 4, S at position 5, V at position 6, L at position 7, T or S at position 10, Y at position 15, and I, L or M at position 16.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a light chain CDR1 that is at least 90% identical to a CDR1 comprising the following amino acids at the specified positions: K or R at position 1, A or S at position 2, S at position 3, K or Q at position 4, S at position 5, L or V at position 6, L at position 7, T or S at position 10, Y at position 15, and I, L or M at position 16.
  • Light Chain CDR2 for MNC2 is LASNLES, with the amino acids numbered from left to right 1 through 7, the consensus of other antibodies that bind to that portion of PSMGFR is: L or W, or S at position 1, A or T at position 2, S at position 3, N or T at position 4, L or R at position 5, E or A at position 6, and S at position 7.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a light chain CDR3 that is at least 90% identical to a CDR3 comprising the following amino acids at the specified positions: Q at position 1, H or Q at position 2, S, Q or R at position 3, R, S or Y at position 4, E, L, or S at position 5, L or S at position 6, P or S at position 7, F or L at position 8 and T at position 9.
  • Another set of antibodies was generated and resultant clones were tested for their ability to bind to PSMGFR, N-10 and C-10 peptides. Antibody clones that bound to PSMGFR and N-10 peptides, but not to the C-10 peptide were selected.
  • Table 7 shows the sequences of the heavy chain CDRs for cancer-specific antibodies MNC2, MNE6, 20A10, 3C2B1, plus new antibodies B2, B7, 8C7F3, H11 and B9.
  • Table 8 shows the sequences of the light chain CDRs for cancer-specific antibodies MNC2, MNE6, 20A10, 3C2B1, plus new antibodies B2, B7, 8C7F3, H11 and B9. Consensus sequences for the heavy and light chain CRDs were generated and are shown in Table 7 and Table 8.
  • antibodies 5C6F3 and 25E6 showed great cancer specificity in IHC tissue studies and they both bound to the PSMGFR and N-10 peptides, but not to the C-10 peptide, epitope mapping showed that they bound to epitopes that were a bit N-terminal to the epitopes to which MNC2, MNE6, 20A10 and 3C2B1 bound. For this reason, consensus sequences were generated for MNC2, MNE6, 20A10, 3C2B1 and the new antibodies plus consensus sequences were generated for all the antibodies that bound to N-10 but not to C-10.
  • Heavy Chain CDR1 for MNC2 is FTFSGYAMS, with the amino acids numbered from left to right 1 through 9, the consensus sequence of MNC2, MNE6, 20A10, 3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is: F or I at position 1, T or A at position 2, F at position 3, S at position 4, T, G, or R at position 5, Y or F at position 6, A, G or T at position 7, M at position 8 and S at position 9.
  • the underlined amino acids at positions 2 and 6 are the only additional variants to the consensus sequence generated for cancer-specific antibodies MNC2, MNE6, 20A10, 3C2B1 alone.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a heavy chain CDR1 that is at least 90% identical to a CDR1 comprising the following amino acids at the specified positions: F or I at position 1, T or A at position 2, F at position 3, S at position 4, T, G, or R at position 5, Y or F at position 6, A, G or T at position 7, M at position 8 and S at position 9.
  • Heavy Chain CDR2 for MNC2 is TISSGGTYIYYPDSVKG, with the amino acids numbered from left to right 1 through 17, the consensus sequence of MNC2, MNE6, 20A10, 3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is: [00822] T or A at position 1, I or S at position 2, I or S at position 3, N, S, T or G at position 4, G or R at position 5, G or A at position 6, G, T, or D at position 7, Y, K or S at position 8, T or I at position 9, Y at position 10, Y at position 11, P or S at position12 and D at position 13, S or T at position 14, V or L at position 15 and KG for positions 16-17.
  • the underlined amino acids indicate how this more inclusive consensus sequence differs from the consensus sequence generated for MNC2, MNE6, 20A10 and 3C2B1 alone.
  • the consensus sequence for all nine antibodies differs from the consensus sequence for the original cancer-specific four by only 4 amino acids. Note that 2 of the 4 variants are homologous changes, T for S and L for V, which generally do not significantly impact the structure or specificity of a protein.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a heavy chain CDR2 that is at least 90% identical to a CDR2 comprising the following amino acids at the specified positions: T or A at position 1, I or S at position 2, I or S at position 3, N, S, T or G at position 4, G or R at position 5, G or A at position 6, G, T, or D at position 7, Y, K, H or S at position 8, T or I at position 9, Y or F at position 10, Y at position 11, P or S at position12 and D at position 13, S or T at position 14, V or L at position 15 and KG for positions 16-17.
  • Heavy Chain CDR3 for MNC2 is LGGDNYYEYFDV, with the amino acids numbered from left to right 2 through 13, the consensus sequence of MNC2, MNE6, 20A10, 3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is: [00826] G, L, or N at position 2, G, T, or Y at position 3, G or T at position 4, A, D, P, R, or S at position 5, Y, M, I or S at position 6, Y at position 7, D, Y, or N at position 8, E, D, Y, L or H at position 9, Y, A, or G at position 10, M, D or F at position 11, D or E at position 12, V, F, Y or L at position 13, and AY at position 14-15.
  • the underlined amino acids indicate how this more inclusive consensus sequence differs from the consensus sequence generated for MNC2, MNE6, 20A10 and 3C2B1 alone.
  • the consensus sequence for all nine antibodies differs from the consensus sequence for the original cancer-specific four by 7 amino acids, with 3 of the 7 substitutions at position 6. For this reason, we conclude that the amino acid at position 6 can be varied without altering the specificity of the antibody.
  • Analysis of the consensus sequence generated with the inclusion of antibodies 5C6F3 and 25E6 highlighted which amino acids were conserved among all eleven antibodies. For this reason, our preferred consensus sequence for heavy chain CDR3 defines amino acids at positions 2, 3, 4, 7, 10, 11, 12, 14 and 15, where for 11 antibodies, there were 3 or less variants at these positions.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a heavy chain CDR3 that is at least 90% identical to a CDR3 comprising the following amino acids at the specified positions: G, L, or N at position 2, G, T, or Y at position 3, G or T at position 4, Y at position 7, Y, A, or G at position 10, M, D or F at position 11, D or E at position 12 and AY at position 14-15.
  • the underlined amino acids indicate how this more inclusive consensus sequence differs from the consensus sequence generated for MNC2, MNE6, 20A10 and 3C2B1 alone.
  • the consensus sequence for all nine antibodies differs from the consensus sequence for the original cancer-specific four by 13 amino acids. 4 of the 13 are homologous substitutions, which in general do not significantly alter the structure or specificity of the protein. Of the remaining 9 substitutions, 1 is at position 4, 1 is at position 5, 3 are at position 6, 1 is at position 7, 1 is at position 11, and 2 are at is at position 17.
  • the inclusion of the 5 new antibodies did not alter the amino acids, excluding homologous substitutions, at positions 1, 2, 3, 8, 9, 10, 12, 13, 14, 15 or 16.
  • the conserved consensus sequence for light chain CDR1 that defines a MUC1* cancer-specific antibody comprises the amino acids given above for positions 1, 2, 3, 8, 10, 12, 13, 14, 15 and 16.
  • Analysis of the consensus sequence generated with all the antibodies, including 5C6F3 and 25E6 further altered the consensus sequence for light chain CDR1 with amino acid substitutions as follows: L at position 6; D at position 9; D at position 11 and N at position 17.
  • a conserved consensus sequence for light chain CDR1 that defines at least 90% identity of a cancer-specific antibody comprises amino acids defined above at positions 1, 2, 3, 8, 10, 12, 13, 14, 15 and 16.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a light chain CDR1 that is at least 90% identical to a CDR1 comprising K or R at position 1, A or S at position 2, S or R at position 3, S, Y, I or V at position 8, T or S at position 10, G, S, D, or Q at position 12, V, Y, K or N at position 13, N, S, or T at position 14, Y or F at position 15, and I, L or M at position 16.
  • Light Chain CDR2 for MNC2 is LASNLES, with the amino acids numbered from left to right 1 through 7, the consensus sequence of MNC2, MNE6, 20A10, 3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is: L, W, S, T or K at position 1, A, T or V at position 2, S at position 3, N or T at position 4, L or R at position 5, E, A, F or D at position 6, and S at position 7.
  • the underlined amino acids indicate how this more inclusive consensus sequence differs from the consensus sequence generated for MNC2, MNE6, 20A10 and 3C2B1 alone.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a light chain CDR2 that is at least 90% identical to a CDR2 comprising the following amino acids at the specified positions: L, W, S, T or K at position 1, A, T or V at position 2, S at position 3, N or T at position 4, L or R at position 5, E, A, F or D at position 6, and S at position 7.
  • L, W, S, T or K at position 1
  • A, T or V at position 2 S at position 3
  • N or T at position 4 L or R at position 5 substitutions of which only 2 were not homologous substitutions.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a light chain CDR2 that is at least 90% identical to a CDR2 comprising: A, T or V at position 2, S at position 3, N, T, or K at position 4, L or R at position 5, E, A, F or D at position 6, and S at position 7.
  • Light Chain CDR3 for MNC2 is QHSRELPFT, with the amino acids numbered from left to right 1 through 9, t the consensus sequence of MNC2, MNE6, 20A10, 3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is: Q or F at position 1, H or Q at position 2, S, Q, R, D or N at position 3, R, S, Y or N at position 4, E, L, S or H at position 5, L, S, V, D or Y at position 6, P or S at position 7, F, L or P at position 8 and T at position 9.
  • the underlined amino acids indicate how this more inclusive consensus sequence differs from the consensus sequence generated for MNC2, MNE6, 20A10 and 3C2B1 alone. [00838] Analysis of the consensus sequence generated with all the antibodies, including 5C6F3 and 25E6 further altered the consensus sequence for light chain CDR2 with amino acid substitutions as follows: W at position 1; G at position 3; T at position 4; F at position 5; Q at position 8.
  • an antibody is chosen for the treatment, prevention or diagnosis of cancer based on having a light chain CDR3 that is at least 90% identical to a CDR2 comprising: Q, F or W at position 1, H or Q at position 2, R, S, T, Y or N at position 4, E, L, S or H at position 5, L, S, V, D or Y at position 6, P or S at position 7, and T at position 9.
  • the invention is directed to a composition that includes at least two different plasmids transfected into the same immune cell, wherein the first encodes a CAR comprising an antibody fragment, scFv, or peptide that binds to a tumor antigen and the other encodes a gene that is not a CAR, wherein the gene that is not a CAR is expressed from an inducible promoter that is activated by elements of an activated immune cell.
  • the immune cell is a T cell or an NK cell.
  • the immune cell is derived from a stem cell that has been directed to differentiate to that immune cell type in vitro.
  • a CAR containing sequences of the antibody are expressed in a stem cell, which then may be differentiated into an immune cell.
  • the CAR comprises an antibody fragment, scFv or peptide that binds to the extra cellular domain of MUC1*.
  • the CAR comprises an scFv derived from MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11.
  • the non-CAR species is a cleavage enzyme.
  • the cleavage enzyme is MMP2, MMP3, MMP9, MMP13, MMP14, MMP16, ADAM10, ADAM17, ADAM28 or catalytically active fragments thereof.
  • the non-CAR species is a cytokine.
  • the Cytokine is IL-7.
  • the cytokine is IL-15.
  • the cytokine is IL-12.
  • the cytokine is IL-18.
  • the sequence of an activated IL-18 is given (SEQ ID NOS:1637-1638). Two examples of NFAT-inducible IL-18 embedded in the Foxp3 enhancer region are given (SEQ ID NOS:1639-1640).
  • NFAT-inducible IL-18 embedded in the IL-2 enhancer region Two examples of NFAT-inducible IL-18 embedded in the IL-2 enhancer region are given (SEQ ID NOS:1641-1642). In one case, there are three (3) NFAT response elements and in the other acse there are six (6) NFAT response elements. The number of NFAT response elements can be varied in order to get the desired amount of IL-18 expressed upon CAR T cell recognition of the target.
  • Examples of antibodies of the invention incorporated into CARS with inducible IL-18 are shown as: murine or human MNC2 in a CAR with a 4-1BB or CD28 co-stimulatory domain plus inducible IL-18 (SEQ ID NOS:1643-1646), or also with a 1XX mutated CD3-zeta (SEQ ID NOS:1647-1650); murine or human MNE6 in a CAR with a 4-1BB or CD28 co-stimulatory domain plus inducible IL-18 (SEQ ID NOS:1651-1654), or also with a 1XX mutated CD3- zeta (SEQ ID NOS:1655-1658); murine or human 20A10 in a CAR with a 4-1BB or CD28 co-stimulatory domain plus inducible IL-18 (SEQ ID NOS:1659-1662), or also with a 1XX mutated CD3-zeta (SEQ ID NOS:1663-1666); murine or human 25E
  • the cytokine is IL-7 and IL-15.
  • expression of the non-CAR species is induced by elements of an activated immune cell.
  • the element of an activated immune cell is an NFAT.
  • the NFAT is NFATc1, NFATc3 or NFATc2.
  • Cytokines IL-7, IL-15, IL-12 and IL- 18 are known to promote T cell persistence.
  • an immune cell described above is administered to a patient for the treatment or prevention of cancer.
  • the cancer is a MUC1 positive cancer or a MUC1* positive cancer.
  • CAR T cells that also induce expression of a cleavage enzyme
  • CAR T cells that also induce local and transient expression of IL-18.
  • Many of the T cell based inducible systems reported insert the gene to be inducibly expressed into an IL-2 promoter or enhancer.
  • human T cells were transduced with both huMNC2-CAR44 and an NFAT inducible IL-18, wherein the Il-18 gene was either inserted into an IL-2 promoter or the Foxp3 enhancer region.
  • FIG. 211A- 211C show graphs of an ELISA experiment measuring the amount of IL-18 secreted into the condition media of huMNC2-CAR44 T cells, which also bear an NFAT inducible IL-18, co-cultured with MUC1* positive cancer cells.
  • Fig. 211A shows the graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with untransduced human T cells.
  • 211B shows the graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T cells that also bore an NFAT inducible IL-18 gene inserted into a portion of the Foxp3 enhancer.
  • Fig. 211C shows the graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T cells that also bore an NFAT inducible IL-18 gene inserted into a portion of the IL-2 enhancer.
  • the Foxp3 system induces rapid and robust expression of IL-18, which is significantly faster and higher than that of the same construct in an IL-2 promoter.
  • the IL-18 gene is inserted downstream of six (6) NFAT response elements, however one can attenuate the amount of the second factor by using a lesser number of response elements or enhance the amount by increasing the number of NFAT response elements.
  • Figure 212A- 212X shows photographs of T47D breast cancer cells (red) doped with varying percentages of T47D cells engineered to express more MUC1* (green).
  • the target cancer cells have been co-cultured with huMNC2-CAR44 T cells with NFAT inducible IL-18 wherein the IL-18 gene has been inserted into either the Foxp3 enhancer/promoter or the IL-2 enhancer/promoter.
  • Fig. 212A-212C, 212I-212K, and 212Q- 212S show the cancer cells co-cultured with untransduced T cells.
  • 212D-212F, 212L- 212N, and 212T-212V show the cancer cells co-cultured with hiMNC2-CAR44 T cells with the NFAT inducible IL-18 gene inserted into the Foxp3 enhancer/promoter.
  • Fig.212G-212H, 212O-212P, and 212W-212X show the cancer cells co-cultured with hiMNC2-CAR44 T cells with the NFAT inducible IL-18 gene inserted into the IL-2 enhancer/promoter.
  • the low antigen density T47D-wt type cells red are being killed when doped with higher percentages of cells that express more MUC1* and thus secrete more IL- 18.
  • Figure 213A- 213B shows graphs of ELISA experiments in which levels of IL-18 secreted into the conditioned media are measured for huMNC1-CAR44 T cells with NFAT inducible IL-18 gene, inserted into the Foxp3 enhancer or promoter, co-cultured with either MUC1* positive cancer cells or MUC1 negative non-cancerous cells.
  • Fig. 213A shows IL-18 secretion from huMNC2-CAR44 T cells with NFAT inducible IL-18 in co-culture with T47D breast cancer cells where the population has been doped with 5%, 10% or 30% T47D cells that had been transfected with even more MUC1*.
  • 213B shows IL-18 secretion from huMNC2-CAR44 T cells with NFAT inducible IL-18 in co-culture with non-cancerous, MUC1 negative HEK293 cells where the cell population has been doped with 5%, 10% or 30% T47D cells that had been transfected with more MUC1*.
  • the amount of IL-18 secreted into the media can be attributed to the MUC1* positive cells that the population was doped with.
  • Time course fluorescent photographs of the experiment show that even when doped with significant percentages of high antigen density MUC1* positive cells, the MUC1 negative cells are not killed by the MUC1* targeting CAR T cells.
  • Figure 214A-214X shows photographs of T47D breast cancer cells (red) or non-cancerous HEK293 cells (also red), where both cell types have been doped with varying percentages of T47D cells engineered to express more MUC1* (green). These target cancer cells have been co-cultured with huMNC2-CAR44 T cells with NFAT inducible IL-18 wherein the IL-18 gene has been inserted into the Foxp3 enhancer/promoter.
  • Fig. 214A-214F shows either T47D cells or HEK293 cells that have not been doped with T47D cells engineered to express high MUC1* density.
  • Fig.214G-214L shows either T47D cells or HEK293 cells that have been doped with 5% T47D cells engineered to express high MUC1* density.
  • Fig. 214M-214R shows either T47D cells or HEK293 cells that have been doped with 10% T47D cells engineered to express high MUC1* density.
  • Fig. 214S-214X shows either T47D cells or HEK293 cells that have been doped with 30% T47D cells engineered to express high MUC1* density.
  • Fig. 214A-B, G-H, M-N, and S-T show T47D breast cancer cells.
  • Fig.214C-F, I-L, O-R, and U-X show HEK293 cells.
  • the invention is directed to a composition that includes at least two different plasmids transfected into the same immune cell, wherein the first encodes a CAR comprising an antibody fragment, scFv or peptide that binds to the extra cellular domain of an antigen on the surface of a B cell and the other encodes a gene that is not a CAR, wherein the gene that is not a CAR is expressed from an inducible promoter that is activated by elements of an activated immune cell.
  • the immune cell is a T cell or an NK cell.
  • the immune cell is derived from a stem cell that has been directed to differentiate to that immune cell type in vitro.
  • a CAR containing sequences of the antibody are expressed in a stem cell, which then may be differentiated into an immune cell.
  • the CAR comprises an antibody fragment, scFv or peptide that binds to CD19.
  • the antibody fragment, scFv or peptide binds to a surface antigen of a B cell or a B cell precursor, or binds to CD19, CD20, CD22, BCMA, CD30, CD138, CD123, CD33 or LeY antigen.
  • the non-CAR species is a cleavage enzyme.
  • the non-CAR species is a cytokine.
  • the Cytokine is IL-7.
  • the cytokine is IL-15. In another aspect the cytokine is IL-7 and IL-15. In one case expression of the non-CAR species is induced by elements of an activated immune cell. In one aspect the element of an activated immune cell is an NFAT. In one aspect the NFAT is NFATc1, NFATc3 or NFATc2. that is not a CAR, wherein the gene that is not a CAR is expressed from an inducible promoter wherein expression is induced by elements of an activated immune cell. In one aspect the immune cell transfected or transduced with the composition is administered to a patient for the treatment or prevention of cancer. In one case the cancer is a leukemia, lymphoma or blood cancer.
  • the gene or plasmid comprising a sequence encoding a CAR or activated T cell inducible protein or peptide there encoded.
  • the gene encoding the CARs and activated T cell induced genes described herein can be virally transduced into an immune cell using viruses, which may or may not result in the CAR gene being integrated into the genome of the recipient cell.
  • Virus delivery systems and viral vectors include but are not limited to retroviruses, including gamma-retroviruses, lentivirus, adenoviruses, adeno- associated viruses, baculoviruses, poxvirus, herpes simplex viruses, oncolytic viruses, HF10, T-Vec and the like.
  • retroviruses including gamma-retroviruses, lentivirus, adenoviruses, adeno- associated viruses, baculoviruses, poxvirus, herpes simplex viruses, oncolytic viruses, HF10, T-Vec and the like.
  • CARs and activated T cell induced genes decribed herein can be directly spliced into the genome of the recipient cell using methods such as CRISPR technology, CRISPR-Cas9 and -CPF1, TALEN, Sleeping Beauty transposon system, and SB 100X.
  • BiTE Bulky cell surface proteins such as MUC1-FL can also cause a steric hindrance problem for BiTEs.
  • a BiTE is a two-headed bispecific antibody wherein one head binds to a T cell and the other head binds to a tumor-associated antigen. In this way, the BiTE links together the T cell and the tumor cells.
  • the antibody that binds to the T cell should be an antibody that activates the T cell, such as an antibody against CD3 or CD28. To solve the steric hindrance problem, the linker between the T cell specific antibody and the tumor specific antibody is lengthened.
  • an anti-MUC1* single chain molecule is fused to a cleavage enzyme or a catalytically active fragment of a cleavage enzyme.
  • the cleavage enzyme is MMP9 (SEQ ID NO:643).
  • the enzyme is a catalytically active fragment of MMP9 (SEQ ID NO:645).
  • the antibody fragment of the CAR is chosen for its ability to recognize MUC1* when cleaved by that specific cleavage enzyme.
  • the cleavage enzyme is MMP9, MMP3, MMP14, MMP2, ADAM17, ADAM TS16, and/or ADAM28.
  • the antibody or antibody fragment binds to a peptide having the sequence of FSAQSGA.
  • cleavage enzymes MMP9 and MMP3 are transduced into a T cell that is also transduced with a CAR with an antibody fragment that is a fragment of MNC2.
  • the cleavage enzyme expressed only after an immune cell recognizes the tumor-associated target on a solid tumor. In this way, the cleavage enzyme will not freely move throughout the body, cleaving MUC1, MUC16 or other proteins, wherein their cleavage could actually promote cancer.
  • cancers that are physically accessible to direct application of chemotherapy agents, CAR T cells and other anti-cancer agents.
  • CAR T cells have been injected directly into the brain and/or cerebral spinal fluid of glioblastoma patients. Radiation has been directed to the prostate area for the treatment of prostate cancers, including those that have metastasized. Hot chemo therapy agents have been directly injected into the intraperitoneal cavity for the treatment of ovarian cancers.
  • a cleavage enzyme is administered in the presence or absence of another anti-cancer agent, which could be a CAR T cell, an immune cell engineered to recognize a tumor-associated antigen, a BiTE, an ADC, a biological or a standard chemotherapy agent.
  • another anti-cancer agent which could be a CAR T cell, an immune cell engineered to recognize a tumor-associated antigen, a BiTE, an ADC, a biological or a standard chemotherapy agent.
  • ovarian cancer can metastasize to anywhere in the body, it usually stays in the abdomen as it spreads to adjacent organs, such as the intestines, liver and stomach.
  • ovarian cancer an ideal test case for improving the effect of anti-cancer agents by administering a cleavage enzyme in combination with other anti-cancer agents, including a platinum-based drug such as carboplatin (Paraplatin) or cisplatin, and/or a taxane such as paclitaxel (Taxol) or docetaxel (Taxotere).
  • a platinum-based drug such as carboplatin (Paraplatin) or cisplatin
  • a taxane such as paclitaxel (Taxol) or docetaxel (Taxotere).
  • Alkeran (Melphalan), Avastin (Bevacizumab), Carboplatin, Clafen (Cyclophosphamide), and Cytoxan have all been approved for the treatment of ovarian cancer.
  • MMP14 has been shown to efficiently cleave MUC1 to MUC1* (Fig. 38).
  • MMP14 is expressed in an immune cell that is also engineered to express a CAR.
  • the CAR is an anti-MUC1* CAR.
  • it can be an MNC2- CAR44 transduced T cell.
  • the MMP14 is directly administered to the patient either in the location of the tumor or by i.v.
  • the cancer is an ovarian cancer and either MMP9 or MMP14 is directly injected into the abdominal area along with an anti-cancer agent, which can be a chemotherapy agent, a biological, an anti-MUC1* CAR T or an anti- MUC16 CAR T.
  • an anti-cancer agent which can be a chemotherapy agent, a biological, an anti-MUC1* CAR T or an anti- MUC16 CAR T.
  • an immune cell which may be a CAR T cell, which further may be expressed off of an inducible promoter is contemplated.
  • Methods used in carrying out experimentation in relation to the present invention [00855] 1.
  • HEK293 or HEK293T cells were used to produce lentivirus.
  • the day prior transfection plates (6well plate) were coated with poly-D-lysine and cells seeded so that cell density reaches 90-95% at the time of transfection and cultures in a 5% CO2 atmosphere.
  • the next day cells were transfected with Lipofectamine 3000 (life technologies) and Opti- MEM® I Reduced Serum Medium according to the manufacturer instructions (0.75ug of lentiviral expression vector and 2.25ug of pPACKH1 packaging mix was used). After 6h incubation, the media was changed and media containing lentivirus was harvested after 24 and 48 hours.
  • Lentivirus was concentrated with Lenti-X concentrator (Clontech) and titer was calculated using the Lenti-X p@4 Rapid Titer Kit (Clontech). Lentivirus was store at -80C in single-use aliquots.
  • Transduction of immune cells with constructs including CARs [00858] Human T cells, if frozen, were thawed and pre-warmed in 100-200 units IL-2 and TexMACS medium, 20 ml, and pelleted by centrifugation.
  • Cells were resuspended in 10 ml of medium and cultured at 37°C, 5% CO2 at 1x10 6 cells/ml in complete medium with anti- CD3/anti-CD28 beads (TransAct kit). [00859] After 4 days in culture, cells were counted and 450 ul of cell suspension was placed in single well of a 24-well plate at a density of approximately 1x10 6 cells/ml. Cells were allowed to settle. 150 ul was carefully removed from the top of each well.
  • Transduced cells were removed, pelleted by centrifugation, and resuspended in fresh medium, adjusting cell density, not to exceed 1.0 x 10 6 cells/ml.
  • Transduced T cells can be expanded and frozen or used directly. Typically transduced T cells are used or frozen between Day 7 and Day 20 post activation with IL-2 and TransAct media. [00860] 2.
  • Human T cells (ALLCELLS) were transduced with huMNC2-CAR44 or huMNC2-CAR50.
  • CAR44 is huMNC2-scFv-CD8-CD8 (transmembrane-41BB-3z).
  • CAR50 is the same as CAR44 except that CAR50 has a murine MNC2-scFv and a CD4 transmembrane domain.
  • the CAR T cells were incubated for 18 hours with target and non- target cells that have been dyed red using CMTMR. When T cells recognize a target cell, they cluster the target cells and begin to kill them.
  • FIG. 45 shows huMNC2-CAR44 or huMNC2-CAR50 T cells being co-cultured with HCT-116 cells transduced to express MUC1*, “HCT-MUC1*” or with HCT-116 cells transduced with a full-length MUC1, “HCT-MUC1-41TR”.
  • MNC2 recognizes an ectopic epitope that is only revealed after cleavage and release of the MUC1 tandem repeat domain.
  • huMNC2-CAR44 nor huMNC2-CAR50 T cells recognize the cells expressing full-length MUC1 (Fig. 45F-45H).
  • Figure 47 shows the contrast between huMNC2-CAR44 recognition of HCT-MUC1* cells, T47D-wt breast cancer cells, and T47D cells with added MMP9 which presumably cleaves the full-length MUC1 to an MNC2 recognizable MUC1*.
  • Figure 55 shows fluorescent images of the huMNC2-CAR44 T cells secreting Granzyme B when co-cultured with the prostate cancer cells, FACS analysis showing increased expression of Granzyme B by the CAR T cells and an xCELLigence experiment showing that the target prostate cancer cells were in fact killed.
  • [00864] 5 Analysis of CAR T cell induced killing of MUC1* positive cancer cells by FACS analysis [00865]
  • the killing effect of the huMNC2-CAR44 T cells increases as the amount of target MUC1* expressed on the cells increases.
  • the xCELLigence instrument uses electrode arrays upon which cancer cells are plated.
  • the adherent cancer cells insulate the electrode and so cause an increase in impedance as they grow.
  • T cells are not adherent and remain in suspension so do not contribute to insulation of the electrode which would increase impedance.
  • the T cells or CAR T cells kill the cancer cells on the electrode plate, the cancer cells ball up and float off as they die, which causes the impedance to decrease.
  • the xCELLigence instrument measures impedance as a function of time, which is correlated to cancer cell killing.
  • the electrode plates also have a viewing window.
  • Fig. 48, Fig. 49, Fig. 55H, Fig. 56H, Figs. 57A-57C all show results of CAR T and cancer cell experiments performed on an xCELLigence instrument.
  • Fig. 48, Fig. 49, Fig. 55H, Fig. 56H, Figs. 57A-57C all show results of CAR T and cancer cell experiments performed on an xCELLigence instrument.
  • Fig. 48, Fig. 49, Fig. 55H, Fig. 56H, Figs. 57A-57C all show results of CAR T and cancer cell experiments performed on an xCELLigence instrument.
  • 7 Anti-MUC1* CAR T cell therapy in mice bearing human tumors [00873] Female NOD/SCID/GAMMA (NSG) mice between 8-12 weeks of age were implanted with 500,000 human cancer cells, wherein the cancer cells had previously been stably transfected with Luciferase. Mice bearing Luciferase positive cells can
  • FIG. 58A-58F show fluorescent photographs of mice taken on an IVIS instrument. 10 minutes prior to IVIS photographs, mice were injected intraperitoneally (IP) with Luciferin, which fluoresces after cleavage by Luciferase, thus making tumor cells fluoresce. NSG (NOD/SCID/GAMMA) immune compromised mice that on Day 0 were subcutaneously implanted on the flank with 500,000 human MUC1* positive cancer cells that had been stably transfected with Luciferase.
  • Figure 58F shows a table summarizing the characteristics of the human T cells that were collected from the test mice upon sacrifice.
  • the starting Car T cell population was 50% CD4 positive helper T cells and 50% CD8 positive killer T cells.
  • the percent of CD8 positive cells has increased in the CAR T treated group, indicating in vivo expansion of that group of cells, which is an indicator of efficacy.
  • the CAR T cells express higher levels of PD1 which is a marker of T cell exhaustion.
  • NSG mice were sub-cutaneously implanted into the flank with 500,000 tumor cells then injected on Day 7 and again on Day 14 with either saline solution, PBS, or 10M huMNC2-CAR44 T cells (Fig. 59A-59C).
  • the amount of MUC1* expressed on the tumor cells was varied.
  • the tumor cells that were implanted were T47D-wildtype (Fig. 59B).
  • the T47D cells were doped with 95% T47D cells that had been transfected to express even more MUC1* (Fig. 59C).
  • the tumors comprised of cells expressing more MUC1* were eliminated more quickly and did not recur.
  • FIG. 60A-60C shows NSG mice implanted with T47D-wt breast cancer cells that have been doped with 30% of T47D cells transfected to express more MUC1*. As can be seen, even a small percentage of cells expressing high levels of MUC1* is sufficient to trigger CAR T cell mediated killing of the entire tumor.
  • Naturally occurring tumors are heterogeneous and are comprised of both high and low antigen expressing cells. This experiment indicates that huMNC2-CAR44 T cells would be effective in eradicating naturally occurring tumors.
  • Figures 61A-61J show fluorescent photographs of mice taken on an IVIS instrument.
  • NSG (NOD/SCID/GAMMA) immune compromised mice that on Day 0 were subcutaneously injected into the flank with 500K human BT-20 cells which are a MUC1* positive triple negative breast cancer cell line. The cancer cells had been stably transfected with Luciferase. Tumors were allowed to engraft. On Day 6 after IVIS measurement, animals were given a one-time injection of 10 million of either human T cells transduced with huMNC2-scFv-CAR44 or untransduced T cells. 5 million T cells were injected intra-tumor and 5 million were injected into the tail vein. 10 minutes prior to IVIS photographs, mice were IP injected with Luciferin.
  • the huMNC2-CAR44 T cells were first incubated with beads to which was attached the PSMGFR peptide to pre-stimulate the T cells and in the figure is marked Protocol 1.
  • Protocol 2 the huMNC2-CAR44 T cells were pre-stimulated with live tumor cells, which likely injected more tumor cells into the animals’ circulation.
  • Figures 62A-62M show fluorescent photographs of mice taken on an IVIS instrument. NSG (NOD/SCID/GAMMA) immune compromised mice that on Day 0 were injected into the intraperitoneal cavity (IP) with 500K human SKOV-3 cells which are a MUC1* positive ovarian cancer cell line. The cancer cells had been stably transfected with Luciferase.
  • the pGL4-14 3xIL2 NFAT and pGL4-14 3xFoxP3 NFAT were digested with XhoI and HindIII restriction enzymes (New England Biolabs).
  • the purified plasmids and the synthesized IL18 sequences were assembled using the Gibson assembly cloning kit (New England Biolab).
  • the resulting constructs contains 3 repeats of NFAT response element (IL2 or FoxP3) followed by a minimum promoter (mCMV: SEQ ID NO:1634) and IL18 (SEQ ID NOS:1752-1753) with CD8 leader sequence.
  • MNC2 CAR sequence was amplified from previously made vector by polymerase chain reaction (PCR) using the following primers: 5’- agggagacccaagctggctagttaagcttggatggccttaccagtgaccgccttgc-3’ (SEQ ID NO:1754) and 5’- taggccagagaaatgttctggcattatcagcgagggggcagggcctgc-3’ (SEQ ID NO:1755).
  • IL18 sequence including NFAT response element was amplify from pGL4-14 3xNFAT-IL18 by polymerase chain reaction (PCR) using the following primers: 5’- tgccagaacatttctctgg-3’ (SEQ ID NO:1756) and 5’- acagtcgaggctgatcagcgggtttaaacttatcagtcctcgttctgcacgg-3’ (SEQ ID NO: 1757).
  • the purified PCR fragments and digested pCDNA 3.1 V5 were assembled using the Gibson assembly cloning kit (New England Biolab) to create the construct pCDNA MNC2CAR-3xIL2NFAT-IL18 and pCDNA MNC2CAR-3xFoxP3NFAT-IL18.
  • MNC2 CAR-NFAT-IL18 sequence was amplified from pCDNA MNC2CAR- 3xIL2NFAT-IL18 and pCDNA MNC2CAR-3xFoxP3NFAT-IL18.by polymerase chain reaction (PCR) using the following primers: 5’- atgcaggccctgcccctcgctgataagtttaaactgccagaacatttctctggcctaac-3’ (SEQ ID NO:1758) and 5’- accggagcgatcgcagatccttcgcggccgcttatcagtcctcgttctgcacggtgaac-3’ (SEQ ID NO:1759).
  • the purified PCR fragments and digested pCDH Dual Hygro (System Biosciences, CA) were assembled using the Gibson assembly cloning kit (New England Biolab) to create the construct pCDH MNC2CAR-3xIL2NFAT-IL18 and pCDH MNC2CAR-3xFoxP3NFAT- IL18.
  • MSCV promoter sequence was amplified from pCDH-MSCV-MCS-EF1a-GFP (System Biosciences).by polymerase chain reaction (PCR) using the following primers: 5’- attgcactagttgaaagaccccacctgtagg-3’ (SED ID NO:1760) and 5’- aatgctctagaatacgggtatccagg- 3’ (SEQ ID NO:1761).
  • MNC2 CAR-IL2NFAT-IL18 sequence was amplified from pCDNA MNC2CAR- 3xIL2NFAT-IL18 by polymerase chain reaction (PCR) using the following primers: 5’ atagcgaattcgtaccgagggccaccatgg-3’ (SEQ ID NO:1762) and 5’- taggcctcccaccgtacacgcctaggtaccacgccttctgtatg-3’ (SEQ ID NO:1763) MNC2 CAR- IL2NFAT-IL18 sequence was amplified from pCDNA MNC2CAR-3xFoxP3NFAT-IL18 by polymerase chain reaction (PCR) using the following primers: 5’ atagcgaattcgtaccgagggccaccatgg -3’ (SEQ ID NO:176
  • 6xNFAT response elements [00891] 6xNFAT (IL2 and FoxP3) response element were synthesized followed by different minimal promoter: mCMV (SEQ ID NO:1634), mIL2P (SEQ ID NO:1635) and miniP (SEQ ID NO:1636).
  • 6xNFAT sequences were amplified by polymerase chain reaction (PCR) using the following primers: 5’-tgccagaacatttctctgg-3’ (SEQ ID NO:1756) and 5’- taaggccatggtggctagc-3’ (SEQ ID NO:1765).
  • the purified PCR fragments and digested (KpnI and XhoI) pCDNA MNC2CAR 3XNFAT IL18 were assembled using the Gibson assembly cloning kit (New England Biolab) to create constructs with 6x NFAT response elements in place of the 3x NFAT response elements.
  • 6xNFAT sequences were amplified, from the pCDNA vector created above, by polymerase chain reaction (PCR) using the following primers: 5’- aataagtttaaactgccagaacatttctctgg-3’ (SEQ ID NO:1766) and 5’- atatagcggccgcttatcagtcctcgttctgcacgg-3’ (SEQ ID NO:1767).

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US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12006366B2 (en) 2021-01-26 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes

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US20180112007A1 (en) * 2015-02-10 2018-04-26 Minerva Biotechnologies Corporation Humanized anti-muc1* antibodies
WO2020146902A2 (en) * 2019-01-11 2020-07-16 Minerva Biotechnologies Corporation Anti-variable muc1* antibodies and uses thereof

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US20180112007A1 (en) * 2015-02-10 2018-04-26 Minerva Biotechnologies Corporation Humanized anti-muc1* antibodies
WO2020146902A2 (en) * 2019-01-11 2020-07-16 Minerva Biotechnologies Corporation Anti-variable muc1* antibodies and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12006366B2 (en) 2021-01-26 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes

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