Anti-glyco-MUC1 antibody or antigen-binding fragment, fusion protein, chimeric antigen receptor, antibody-drug conjugate, pharmaceutical composition, and use of anti-glyco-MUC1 antibody or antigen-binding fragment.

Abstract

This description refers to anti-glyco-muc1 antibodies and antigen-binding fragments thereof that specifically bind to a cancer-specific glycosylation variant of muc1 and related fusion proteins and antibody-drug conjugates, as well as nucleic acids encoding such biomolecules. This description further refers to the use of the antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates, and nucleic acids for cancer therapy.

Description

172 Anti-glyco-MUC1 antibody or antigen-binding fragment, fusion protein, chimeric antigen receptor, antibody-drug conjugate, pharmaceutical composition, and use of anti-glyco-MUC1 antibody or antigen-binding fragment. 1. LISTING OF SEQUENCES

[001] This application contains a Sequence Listing that was submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 24, 2017, is named GOT-001WO_Sequence_Listing.txt and is 33,265 bytes in size. 2. FUNDAMENTALS

[002] Human mucin MUC1 is a polymorphic transmembrane glycoprotein expressed on the apical surfaces of simple and glandular epithelia (Taylor-Papadimitriou et al., 1999). MUC1 is highly overexpressed and aberrantly O-glycosylated in adenocarcinomas. The extracellular domain of mucin contains a variable number of tandem repeats (TRs) (25-125) of 20 amino acid residues with five potential sites for O-glycosylation. O-glycans are incompletely processed in cancer cells resulting in the expression of pancarcinoma carbohydrate antigens Tn (GalNAca1-O-Ser / Thr) (Springer, 1984). Simple mucin-type O-glycans, Tn, are widely expressed in adenocarcinomas (including breast and ovarian cancers) and show limited distribution in normal adult tissues (Springer, 1984).The expression of these O-glycans in cancer correlates with poor prognosis, and natural antibodies to these carbohydrate haptens increase in cancer patients (Miles et al., 1995; Soares et al., 1996; Werther et al., 1996). There is a need in the technique for modalities. Petition 870260076091, dated 07 / 30 / 2026, page 12 / 24 / 172, therapeutics that utilize glyco-MUC1 epitopes that are overexpressed in cancer cells. 3. SUMMARY

[003] The description captures the tumor specificity of glycopeptide variants by providing therapeutic and diagnostic agents based on antibodies and antigen-binding fragments that are selective for glyco-MUC1 epitopes.

[004] The present description provides anti-glyco-MUC1 antibodies and antigen-binding fragments thereof that bind to a cancer-specific glycosylation variant of MUC1. The present description further provides fusion proteins and antibody-drug conjugates comprising anti-glyco-MUC1 antibodies and antigen-binding fragments and nucleic acids encoding the anti-glyco-MUC1 antibodies, antigen-binding fragments and fusion proteins.

[005] The present description further provides methods of using anti-glyco-MUC1 antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates and nucleic acids for cancer therapy.

[006] In certain respects, the description provides bispecific and multispecific anti-glycoMUC1 antibodies and antigen-binding fragments that bind to a cancer-specific glycosylation variant of MUC1 and to a second epitope. The second epitope may be in MUC1 itself, in another protein co-expressed in MUC1-containing cancer cells, or in another protein presented in a different cell, such as an activated T cell. In addition, nucleic acids encoding such antibodies are also described, including nucleic acids comprising coding regions optimized in the codon and nucleic acids comprising coding regions that are not optimized in the codon for expression in a particular host cell. Petition 870200050086, dated 04 / 22 / 2020, page 7 / 209 / 172

[007] Anti-glyco-MUC1 antibodies and binding fragments may be in the form of fusion proteins containing a fusion partner. The fusion partner may be useful in providing a second function, such as a signaling domain function of a T cell signaling protein, a peptide modulator of T cell activation, or an enzymatic component of a labeling system. Exemplary T cell signaling proteins include 4-1BB, CO3C, and fusion peptides, for example, CD28-CD3-zeta and 4-1BB-CD3-zeta. 4-1BB, or CD137, is a co-stimulatory receptor for T cells; CD3-zeta is a signal transduction component of the T cell antigen receptor. The portion providing a secondary function may be a modulator of T cell activation, such as IL-15, IL-15Ra, or an IL-15 / IL-15Ra fusion, or it may encode a label or an enzymatic component of a labeling system useful in monitoring the degree and / or location of binding in vivo or in vitro.Constructs encoding these prophylactically and therapeutically active biomolecules placed in the context of T cells, such as autologous T cells, provide a powerful platform for recruiting adoptively transferred T cells to prevent or treat a variety of cancers in some of the modalities described.

[008] In certain respects, an anti-glucose-MUC1 antibody or antigen-binding fragment of the description comprises variable heavy and / or light chain sequences (or nucleotide-encoded sequences) presented in Table 1. For clarity, when the term “anti-glucose-MUC1 antibody” is used in this document, it is intended to include monospecific and multispecific (including bispecific) anti-glucose-MUC1 antibodies, antigen-binding fragments of monospecific and multispecific antibodies, and fusion proteins and conjugates containing the antibodies and their antigen-binding fragments, unless the context otherwise requires. Similarly, when the term Petition 870200050086, dated 04 / 22 / 2020, page 8 / 209 / 172 “anti-glyco-MUC1 antibody or antigen-binding fragment” is used, the same is also intended to include monospecific and multispecific (including bispecific) anti-glyco-MUC1 antibodies and their antigen-binding fragments, together with fusion proteins and conjugates containing such antibodies and antigen-binding fragments, unless the context otherwise requires.

[009] In other respects, an anti-glyco-MUC1 antibody or antigen-binding fragment of the description comprises heavy and / or light chain CDR sequences (or nucleotide-encoded sequences) presented in Tables 1 to 3. The CDR sequences presented in Table 1 include CDR sequences defined according to the IMGT schemes (Lefranc et al., 2003, Dev Comparat Immunol 27:55-77, Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) and Chothia (AlLazikani et al., 1997, J. Mol. Biol 273: 927-948) to define the CDR boundaries. The CDR sequences presented in Table 2 are the combined overlapping regions for the sequences of CDRs are shown in Table 1, with IMGT, Kabat and Chothia sequences shown in bold underlined text.The CDR sequences presented in Table 3 are the common overlapping regions for the CDR sequences shown in Table 1. The structural sequences for such anti-glyco-MUC1 antibody and antigen-binding fragment may be the native murine structural sequences in Table 1 or may be non-native structural sequences (e.g., humanized or human). Table 1 Description Sequence SEQ ID NO: Amino acid sequence VH (incl. signal sequence) MGWSGIFLFFLSVTTGVHSQVQLQQSDAELVKPGASVKI SCKAS GYTFTDHAIHWVKQRPEQGLEWIGYFSPGNDDI HYNEKFEGKATLTADKSSSTAYMQLNSLTSEDSAVYFC KRSYDKDFDCWGQGTTLTVSS 1 Amino acid sequence VL (incl. signal sequence) MVLILLLLWVSGTCGDIVMSQSPSSLGVSVGEKVTMSC KSSQSLLYSTNQKNYQSLLYSTNQKNYLAWYQQKPGQ SPKLLIYWVSNRKSGVPDRFTGSGSGTDFTLTISSVKAED LAVYYC QQYYRYPLTFGAGTKLELK 2 Petition 870200050086, dated 04 / 22 / 2020, page 9 / 209 / 172 Table 1 Description Sequence SEQ ID NO: VH amino acid sequence (predicted maturity) QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVK QRPEQGLEWIGYFSPGNDDIHYNEKFEGKATLTADKSSS TAYMQLNSLTSEDSAVYFCKRSYDKDFDCWGQGTTLT VSS 3 VL amino acid sequence (predicted maturity) DIVMSQSPSSLGVSVGEKVTMSCKSSQSLLYSTNQKNY QSLLYSTNQKNYLAWYQQKPGQSPKLLIYWVSNRKSG VPDRFTGSGSGTDFTLTISSVKAEDLAVYYC QQYYRYPLTFGAGTKLELK 4 CDRH1 amino acid sequence (IMGT definition) GYTFTDHA 5 CDRH2 amino acid sequence (IMGT definition) FSPGNDDI 6 Amino acid sequence of CDRH3 (IMGT definition) KRSYDKDFDC 7 Amino acid sequence of CDRL1 (IMGT definition) QSLLYSTNQKNY 8 Amino acid sequence of CDRL2 (IMGT definition) WVS 9 Amino acid sequence of CDRL3 (IMGT definition) QQYYRYPLT 10 Nucleotide sequence VH (incl.sequência de sinal) ATGGGATGGAGCGGGATCTTTCTCTTCTTCCTGTCAGT AACTACAGGTGTCCACTCCCAGGTTCAGCTGCAGCAG TCTGACGCGGAGTTGGTGAAACCTGGGGCTTCAGTGA AGATATCCTGCAAGGCTTCTGGCTACACTTTCACTGA CCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAG GGCCTGGAATGGATTGGATATTTTTCTCCCGGAAATG ATGACATTCACTACAATGAGAAGTTCGAGGGCAAGG CCACACTGACTGCAGACAAATCCTCCAGCACTGCCTA CATGCAGCTCAACAGCCTGACATCTGAAGATTCTGCA GTGTATTTCTGTAAAAGATCTTACGACAAGGACTTTG ACTGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTC A 11 Sequência de nucleotídeo VL (incl. sequência de sinal) ATGGTTCTTATCTTACTGCTGCTATGGGTATCTGGTAC CTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCC CTAGGTGTGTCAGTTGGAGAGAAGGTTACTATGAGCT GCAAGTCCAGTCAGAGCCTTTTATACAGTACCAATCA AAAGAACTACCTGGCCTGGTACCAGCAGAAACCAGG GCAGTCTCCTAAGTTGCTGATTTACTGGGTATCTAATA GGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGG ATCTGGGACAGATTTCACTCTCACCATCAGTAGTGTG AAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAAT ATTATAGGTATCCGCTCACGTTCGGTGCTGGGACCAA GCTGGAGCTGAAA 12 Sequência de nucleotídeo VH (excl.(signal sequence) CAGGTTCAGCTGCAGCAGTCTGACGCGGAGTTGGTGA AACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTC TGGCTACACTTTCACTGACCATGCTATTCACTGGGTG AAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGA TATTTTTCTCCCGGAAATGATGACATTCACTACAATG AGAAGTTCGAGGGCAAGGCCACACTGACTGCAGACA AATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCT GACATCTGAAGATTCTGCAGTGTATTTCTGTAAAAGA TCTTACGACAAGGACTTTGACTGCTGGGGCCAAGGCA CCACTCTCACAGTCTCCTCA 13. Petition 870200050086, dated 04 / 22 / 2020, page 10 / 209 / 172 Table 1 Description Sequence SEQ ID NO: VL nucleotide sequence (excl. signal sequence) GACATTGTGATGTCACAGTCTCCATCCTCCCTAGGTGT GTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCC AGTCAGAGCCTTTTATACAGTACCAATCAAAAGAACT ACCTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCC TAAGTTGCTGATTTACTGGGTATCTAATAGGAAATCT GGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGA CAGATTTCACTCTCACCATCAGTAGTGTGAAGGCTGA AGACCTGGCAGTTTATTACTGTCAGCAATATTATAGG TATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGC TGAAA 14 FR-H1 QVQLQQSDAELVKPGASVKISCKAS 15 FR-H2 IHWVKQRPEQGLEWIGY 16 FR-H3 HYNEKFEGKATLTADKSSSTAYMQLNSLTSEDSAVYFC 17 FR-H4 WGQGTTLTVSS 18 FR-L1 DIVMSQSPSSLGVSVGEKVTMSCKSS 19 FR-L2 LAWYQQKPGQSPKLLIY 20 FR-L3 NRKSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC 21 FR-L4 FGAGTKLELK 22 Amino acid sequence of CDRH1 (Kabat definition) DHAIH 23 Amino acid sequence of CDRH2 (Kabat definition) YFSPGNDDIHYNEKFEG 24 Amino acid sequence of CDRH3 (Kabat definition) SYDKDFDC 25 Amino acid sequence of CDRL1 (Kabat definition) KSSQSLLYSTNQKNYLA 26 Sequence ofAmino acid sequence of CDRL2 (Kabat definition) WVSNRKS 27 Amino acid sequence of CDRL3 (Kabat definition) QQYYRYPLT 10 Amino acid sequence of CDRH1 (Chothia definition) GYTFTDH 28 Amino acid sequence of CDRH2 (Chothia definition) SPGNDD 29 Amino acid sequence of CDRH3 (Chothia definition) SYDKDFDC 25 Amino acid sequence of CDRL1 (Chothia definition) SQSLLYSTNQKNY 30 Petition 870200050086, dated 04 / 22 / 2020, page 11 / 209 / 172 Table 1 Description Sequence SEQ ID NO: Amino acid sequence of CDRL2 (Chothia definition) WVS 9 Amino acid sequence of CDRL3 (Chothia definition) YYRYPLT 31 Table 2 Description Sequence SEQ ID NO: Amino acid sequence of CDR-H1 (matched overlap) GYTFTDHAIH (IMGT) GYTFTDHAIH (Kabat) GYTFTDHAIH (Chothia) 32 Amino acid sequence of CDR-H2 (matched overlap) YFSPGNDDIHYNEKFEG (IMGT) YFSPGNDDIHYNEKFEG (Kabat) YFSPGNDDIHYNEKFEG (Chothia) 24 Amino acid sequence of CDR-H3 (matched overlap) KRSYDKDFDC (IMGT) KRSYDKDFDC (Kabat) KRSYDKDFDC (Chothia) 7 Amino acid sequence of CDRLI (matched overlap) KSSQSLLYSTNQKNYLA (IMGT) KSSQSLLYSTNQKNYLA (Kabat) KSSQSLLYSTNQKNYLA (Chothia) 26 Sequence Amino acid sequence CDRL2 (matched overlap) WVSNRKS (IMGT) WVSNRKS (Kabat) WVSNRKS (Chothia) 27 Amino acid sequence CDRL3 (matched overlap) QQYYRYPLT (IMGT) QQYYRYPLT (Kabat) QQYYRYPLT (Chothia) 10 Table 3 Description Sequence SEQ ID NO: Amino acid sequence of CDR-H1 (common sequence) DH 33 Amino acid sequence of CDR-H2 (common sequence) SPGNDD 29 Amino acid sequence of CDR-H3 (common sequence) SYDKDFDC 25 Amino acid sequence of CDRL1 (common sequence) QSLLYSTNQKNY 8 Petition 870200050086, dated 04 / 22 / 2020, page 12 / 209 8 / 172 Table 3 Description Sequence SEQ ID NO: Amino acid sequence CDRL2 (common sequence) wvs 9 Amino acid sequence CDRL3 (common sequence) YYRYPLT 31

[0010] In certain aspects, the description provides that an anti-glycoMUCl antibody or antigen-binding fragment of the description comprises CDRs comprising the amino acid sequences of any of the CDR combinations presented in embodiments numbered 3 to 17. Thus, in certain embodiments, an anti-glycoMUCl antibody or antigen-binding fragment of the description comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID N—: 5, 23, 28, or 32. In some embodiments, CDR-H2 comprises the amino acid sequence SEQ ID N—: 6 or 24.In some embodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30 or 26. In some embodiments, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10. In other aspects, an anti-glyco-MUCl antibody or antigen-binding fragment of the description comprises heavy chain CDRs of SEQ ID NOS: 5 to 7 and light chain CDRs of SEQ ID NOS: 8 to 10. In other aspects, an anti-glyco-MUCl antibody or antigen-binding fragment of the description. Petition 870200050086, dated 04 / 22 / 2020, page. 13 / 209 / 172 comprises heavy chain CDRs of SEQ IDs NOS: 23 to 25 and light chain CDRs of SEQ IDs NOS: 26, 27 and 10. In other aspects, an anti-glucone-MUC1 antibody or antigen-binding fragment of the description comprises heavy chain CDRs of SEQ IDs NOS: 28, 29 and 25 and light chain CDRs of SEQ IDs NOS: 30, 9 and 31. In other aspects, an anti-glucone-MUC1 antibody or antigen-binding fragment of the description comprises heavy chain CDRs of SEQ IDs NOS: 32, 24 and 7 and light chain CDRs of SEQ IDs NOS: 26, 27 and 10. In other aspects, an anti-glucone-MUC1 antibody or antigen-binding fragment of the description comprises heavy chain CDRs of SEQ IDs NOS: 33, 29 and 25 and CDRs of the light chain of SEQ IDs NOS: 8, 9 and 31. The antibody or antigen-binding fragment may be murine, chimeric, humanized or human.

[0011] In additional aspects, an anti-glyco-MUC1 antibody or antigen-binding fragment of the description competes with an antibody or antigen-binding fragment comprising variable heavy and light chain regions of SEQ IDs NOS: 3 and 4, respectively. In other aspects, the description provides an anti-MUC1 antibody or antigen-binding fragment having variable heavy and light chain regions having at least 95%, 98%, 99%, or 99.5% sequence identity of SEQ IDs NOS: 3 and 4, respectively.

[0012] In other respects, an anti-glyco-MUC1 antibody or antigen-binding fragment of the description is a single-chain variable fragment (scFv). An exemplary scFv comprises the variable heavy chain fragment to the variable light chain N-terminal fragment. In some embodiments, the variable heavy chain fragment and variable light chain fragment of scFv are covalently linked to a linker sequence of 4 to 15 amino acids. The scFv may be in the form of a bispecific T cell engager or within a chimeric antigen receptor (CAR). Petition 870200050086, dated 04 / 22 / 2020, page 14 / 209 / 172

[0013] Anti-glyco-MUC1 antibodies and antigen-binding fragments may be in the form of a single-chain variable fragment multimer, a bispecific single-chain variable fragment, and a bispecific single-chain variable fragment multimer. In some embodiments, the single-chain variable fragment multimer is selected from a bivalent single-chain variable fragment, a tribody, or a tetrabody. In some of these embodiments, the bispecific single-chain variable fragment multimer is a bispecific T cell engager.

[0014] Other aspects of the description are outlined for the nucleic acids encoding the anti-glyco-MUC1 antibodies and antibody-binding fragments of the description. In some embodiments, the portion of the nucleic acid encoding an anti-glyco-MUC1 antibody or antigen-binding fragment is optimized at the codon for expression in a human cell. In certain respects, the description provides an anti-glyco-MUC1 antibody or antigen-binding fragment having variable heavy and light chain regions encoded by a heavy chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 11 or SEQ ID NO: 13 and a light chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 14. Vectors (e.g., a viral vector such as a lentiviral vector) and host cells comprising nucleic acids are also within the scope of the description.The coding sequences for heavy and light chains can be present in a single vector or in separate vectors.

[0015] In another aspect of the description, it is a pharmaceutical composition comprising an anti-glyco-MUC1 antibody, antigen-binding fragment, nucleic acid (or pair of nucleic acids), vector (or pair or vectors) or host cell as described, and a buffer, Petition 870200050086, dated 04 / 22 / 2020, page 15 / 209 / 172 physiologically suitable adjuvant or diluent.

[0016] Yet another aspect of the description is a method of manufacturing a chimeric antigen receptor comprising incubating a cell comprising a nucleic acid or a vector according to the description, under conditions suitable for the expression of the coding region and collecting the chimeric antigen receptor.

[0017] Another aspect of the description is a method of detecting cancer comprising contacting a cell or tissue sample with an anti-glyco-MUC1 antibody or antigen-binding fragment of the description and detecting whether the antibody is bound to the cell or tissue sample.

[0018] Yet another aspect of the description is a method of treating cancer comprising administering a prophylactically or therapeutically effective amount of an anti-glyco-MUC1 antibody, antigen-binding fragment, nucleic acid, vector, host cell or pharmaceutical composition as described to a subject in need thereof. 4. BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1: ELISA assay results showing GO2 binding specificity to glyco-MUC1 relative to MUC1.

[0020] FIG. 2: Binding of GO2 to colon cancer tissue. Immunohistochemical labeling of invasive colonic carcinoma tissue and adjacent healthy tissue using GO2 mAbs. The GO2 mAb shows distinct binding to colonic cancer tissue with high reactivity to both intracellular and surface structures in cancer cells. In contrast, no reactivity is observed in surface structures in healthy colonic cells.

[0021] FIG. 3: Binding of GO2 to pancreatic cancer tissue. Immunohistochemical labeling of pancreatic cancer tissue using GO2 mAbs. The GO2 mAb shows distinct binding to pancreatic cancer cells. Petition 870200050086, dated 04 / 22 / 2020, page 16 / 209 / 172 In contrast, no or limited reactivity is observed in the surrounding healthy tissue.

[0022] FIG. 4: Binding of GO2 to breast cancer tissue. Immunohistochemical labeling of breast cancer tissue using GO2 mAbs. The GO2 mAb showed distinct binding to invasive breast cancer cells.

[0023] FIG. 5: Results of an antibody-dependent cellular cytotoxicity assay with GO2 antibody and a secondary antibody conjugated to the anti-tubulin agent monomethyl auristatin F (MMAF).

[0024] FIG. 6: Results of an ELISA assay quantifying circulating tumor cells using GO2. The X-axis shows the number of cells and the Y-axis shows OD450 values.

[0025] FIGS. 7A-E: Representative images of MUC1-positive TMA tumor nuclei. FIG. 7A: breast cancer; FIG. 7B: non-small cell lung cancer; FIG. 7C: ovarian cancer; FIG. 7D: colorectal cancer; FIG. 7E: prostate cancer.

[0026] FIG. 8: Schematic of an exemplary anti-glyco-MUC1 and bispecific T cell (TCB) anti-CD3 antibody.

[0027] FIGS. 9A-B: Jurkat-NFAT activation assay with undigested patient-derived tumor samples (malignant neoplasm of bronchus and lung: intermediate lobe, bronchus or lung, squamous cell carcinoma) and different TCBs at 50 nM (FIG. 9A) or 5 nM (FIG. 9B).

[0028] FIG. 10: Jurkat-NFAT activation assay with undigested patient-derived tumor samples (malignant neoplasm of bronchus and lung: lower lobe, bronchus or lung, non-keratinizing squamous cell carcinoma) and different TCBs at 50 nM.

[0029] FIG. 11: Jurkat-NFAT activation assay with undigested patient-derived tumor samples (bronchial malignant neoplasm and Petition 870200050086, dated 04 / 22 / 2020, page 17 / 209 / 172 lung: upper lobe, bronchus or lung, adenocarcinoma with acinar type) and different TCBs at 50 nM.

[0030] FIGS. 12A-12B: Binding of TCB GO2 to MUC1 expressed in MCF7 cs (FIG. 12A) and T3M4 pzfv (FIG. 12B) cells measured by flow cytometry.

[0031] FIGS. 13A-X: Induction of tumor cell killing and T cell activation mediated by upregulation of CD25 and CD69 in CD4 T cells and CD8 T cells as well as release of IL6, IL8, IL10, IFNγ, TNFα and Granzyme B with TCB GO2 in T3M4 pzfv in the presence of PBMCs from two healthy donors (donor 1 FIG. 13A-13L; donor 2 FIG. 13M-13X). The same legend applies to each of FIGS. 13A-13X.

[0032] FIGS. 14A-14F: Induction of tumor cell killing (FIGS. 14A-14B) and T cell activation measured by upregulation of CD25 and CD69 in CD8 T cells and CD4 T cells (FIGS. 14C-14F, respectively) with TCB GO2 in MCF7 cs in the presence of PBMCs. The same legend applies to each of FIGS. 14A-14F.

[0033] FIG. 15A-B: Binding of TCB GO2 and TCB HMFG1 to MCF10A (human non-tumorigenic mammary epithelial cell line) (FIG. 15A) and HBEpiC (human bronchial epithelial cells) (FIG. 15B).

[0034] FIG. 16A-C: Induction of tumor cell extermination (FIG. 16A) and T cell activation measured by CD25 upregulation in CD4 T cells (FIG. 16B) and CD8 T cells (FIG. 16C) with TCB GO2 and TCB HMFG1 in MCF10A cells in the presence of PBMCs.

[0035] FIG. 17: Illustration of GO2 and TCB GO2 flowing through a flow cell having glycopeptides attached.

[0036] FIG. 18A-B: Sensory graphs showing the binding of GO2 (FIG. 18A) and TCB GO2 (FIG. 18B) to human and cynomolgus glycopeptides.

[0037] FIG. 19A-D: Binding (avidity) of GO2 antibody (FIG. 19A-19B) and TCB GO2 (FIG. 19C-19D) to human glycopeptides and cynomolgo Petition 870200050086, dated 04 / 22 / 2020, page 18 / 209 / 172 estimate of the “apparent” KD. 5. DETAILED DESCRIPTION 5.1 Antibodies

[0038] The inventor has developed novel antibodies that target a glycoform of MUC1 present in tumor cells. These are exemplified by antibody 5F7, here referred to as “GO2”. GO2 was identified in a screening for antibodies that bind to a glycosylated 60-mer representing 3 copies of one of the tandem repeats present in MUC1, VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 50), glycosylated with purified recombinant human glycosyltransferase polypeptides GalNAc-T2, GalNAc-T4 and GalNAc-T1 in order to mimic the MUC1 glycosylation pattern present in tumor cells.

[0039] The anti-glyco-MUC1 antibodies described, exemplified by the GO2 antibody, are useful as tools in the diagnosis and therapy of cancer.

[0040] Thus, in certain respects, the description provides antibodies and antigen-binding fragments that bind to a glycoform of MUC1 present in tumor cells (hereinafter referred to as “glyco-MUC1”) and preferably to the 60-mer peptide (VTSAPDTRPAPGSTAPPAHG)3 (SEQ ID NO: 47) glycosylated with GalNAc-T2, GalNAc-T4 and GalNAc-T1 as described in US Patent No. 6,465,220.

[0041] The anti-glyco-MUC1 antibodies described may be polyclonal, monoclonal, genetically engineered and / or otherwise modified in nature, including but not limited to chimeric antibodies, humanized antibodies, human antibodies, primatized antibodies, single-chain antibodies, bispecific antibodies, dual variable domain antibodies, etc. In various embodiments, the antibodies comprise all or a portion of a constant region of an antibody. In some embodiments, the constant region is a selected isotype of: Petition 870200050086, dated 04 / 22 / 2020, p. 19 / 209 / 172 IgA (e.g., IgAi or IgA2), IgD, IgE, IgG (e.g., IgGi, IgG2, IgG3, or IgG4), and IgM. In specific embodiments, the anti-glycoMUC1 antibodies in the description comprise an isotype of the IgG1 constant region.

[0042] The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. A monoclonal antibody is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Useful monoclonal antibodies of the present description can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. In many uses of the present description, including in vivo use of anti-glyco-MUC1 antibodies in humans, chimeric, primatized, humanized, or human antibodies can be appropriately used.

[0043] The term “chimeric” antibody as used herein refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and constant regions of human immunoglobulin, typically chosen from a human immunoglobulin pattern. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719): 1202-7; Oi et al., 1986, BioTechniques 4: 214-221; Gillies et al., 1985, J. Immunol. Methods 125: 191-202; U.S. Pat. Nos. 5,807,715, 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety.

[0044] The “humanized” form of non-human antibodies (e.g., murine) are chimeric immunoglobulins containing minimal sequences derived from non-human immunoglobulin. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, where all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. Petition 870200050086, dated 22 / 04 / 2020, p. 20 / 209 / 172 human and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of a constant (Fc) region of the immunoglobulin, typically that of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art. See, for example, Riechmann et al., 1988, Nature 332: 323-7; US Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762 and 6,180,370 granted to Queen et al.; EP239400; PCT publication WO 91 / 09967; Pat. US No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Imunol., 28: 489-498; Studnicka et al., 1994, Prot. Eng. 7: 805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91: 969-973 and US Pat. No. 5,565,332, all of which are hereby incorporated by reference in their entirety.

[0045] “Human antibodies” include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from transgenic animals for one or more human immunoglobulins and that do not express endogenous immunoglobulins. Human antibodies can be manufactured by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111 and PCT publications WO 98 / 46645; WO 98 / 50433; WO 98 / 24893; WO 98 / 16654; WO 96 / 34096; WO 96 / 33735 and WO 91 / 10741, each of which is incorporated herein by reference in its entirety. Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins, but which can express human immunoglobulin genes.See, for example, publications PCT WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771 and 5,939,598, which are... Petition 870200050086, dated 22 / 04 / 2020, p. 21 / 209 / 172, incorporated herein by reference in its entirety. Fully human antibodies that recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this method, a selected non-human monoclonal antibody, for example, a mouse antibody, is used to guide the selection of a fully human antibody recognizing the same epitope (see Jespers et al., 1988, Biotechnology 12: 899-903).

[0046] “Primate-like antibodies” comprise variable monkey regions and constant human regions. Methods for producing primate-like antibodies are known in the art. See, for example, U.S. Pat. Nos. 5,658,570, 5,681,722 and 5,693,780, which are incorporated herein by reference in their entirety.

[0047] Anti-glyco-MUC1 antibodies in the description include both full-size (intact) antibody molecules as well as antigen-binding fragments that are capable of binding glyco-MUC1. Examples of antigen-binding fragments include, by way of example and not limitation, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv fragments, and single-domain fragments.

[0048] A Fab fragment contains the constant domain of the light chain (CL) and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. F(ab') fragments are produced by cleavage of the disulfide bond in the hinge cysteines of the pepsin digestion product of F(ab')2. Additional chemical junctions of antibody fragments are known to those of common skill in the art. Fab and F(ab')1 fragments lack the intact antibody Fc fragment, clear more rapidly from animal circulation, and may have less non-specific tissue binding than an intact antibody (see, for example). Petition 870200050086, dated 04 / 22 / 2020, page 22 / 209 / 172 example, Wahl et al., 1983, J. Nucl. Med. 24: 316).

[0049] A “Fv” fragment is the smallest fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of a heavy and a light variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Often, all six CDRs confer target binding specificity to the antibody. However, in some cases even a single variable domain (or half of an Fv comprising only three target-specific CDRs) may have the ability to recognize and bind the target, albeit at a lower affinity than the entire binding site.

[0050] “Single-chain Fv” or antigen-binding fragments “scFv” comprise the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for target binding.

[0051] “Single-domain antibodies” are composed of single VH or VL domains that exhibit sufficient affinity for glyco-MUC1. In one specific embodiment, the single-domain antibody is a camelized antibody (See, for example, Riechmann, 1999, Journal of Immunological Methods 231: 25-38).

[0052] The anti-glyco-MUC1 antibodies described may also be bispecific and other multispecific antibodies. Bispecific antibodies are monoclonal antibodies, often human or humanized, that have binding specificities for two different epitopes on the same or different antigens. In the present description, one of the binding specificities may be directed to glyco-MUC1, the other Petition 870200050086, dated 22 / 04 / 2020, page 23 / 209 / 172, may be for any other antigen, for example, for a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc. In certain preferred embodiments, bispecific and other multispecific anti-glycoMUC1 antibodies and antigen-binding fragments specifically bind to a second MUC1 epitope, an epitope on another protein co-expressed on MUC1-containing cancer cells, or an epitope on another protein presented on a different cell, such as an activated T cell. The bispecific antibodies described include bispecific antibodies in IgG format and single-chain-based bispecific antibodies.

[0053] The bispecific IgG-format antibodies described may be any of several types of bispecific IgG-format antibodies known in the art, such as quadroma bispecific antibodies, “knobs-in-holes” bispecific antibodies, CrossMab bispecific antibodies, bispecific antibodies paired in charge, common light chain bispecific antibodies, single-arm single-immunoglobulin single-chain Fab gamma bispecific antibodies, disulfide-stabilized bispecific Fv antibodies, DuetMabs, controlled Fab branch exchange bispecific antibodies, engineered domain body filament exchange bispecific antibodies, two-branch leucine zipper heterodimeric monoclonal bispecific antibodies, κλ body bispecific antibodies, double variable domain bispecific antibodies, and cross-linked double variable domain bispecific antibodies.See, for example, Kohler and Milstein, 1975, Nature 256: 495-497; Milstein and Cuello, 1983, Nature 305: 537-40; Ridgway et al., 1996, Protein Eng. 9: 617-621; Schaefer et al., 2011, Proc Natl Acad Sci USA 108: 11187-92; Gunasekaran et al., 2010, J Biol Chem 285: 19637-46; Fischer et al., 2015 Nature Commun 6: 6113; Schanzer et al.,. Petition 870200050086, of 22 / 04 / 2020, p. 24 / 209 20 / 172 2014, J Biol Chem 289:18693–706; Metz et al., 2012 Protein Eng Des Sei 25: 571-80; Mazor et al., 2015 MAbs 7: 377-89; Labrijn et al., 2013 Proc Natl Acad Sci USA 110: 5145-50; Davis et al., 2010 Protein Eng Des Sei 23: 195-202; Wranik et al., 2012, J Biol Chem 287: 43331-9; Gu et al., 2015, PLoS One 10(5): e0124135; Steinmetz et al., 2016, MAbs 8(5): 867-78; Klein et al., 2016, mAbs, 8(6): 1010-1020; Liu et al., 2017, Front. Immunol. 8: 38 and Yang et al., 2017, Int. J. Mol. Sci. 18: 48, which are here incorporated by reference in their entirety.

[0054] In some embodiments, the bispecific antibodies in the description are CrossMabs. CrossMab technology is described in detail in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2013 / 026833, WO 2016 / 020309 and Schaefer et al., 2011, Proc Natl Acad Sci USA 108: 11187-92, which are incorporated herein by reference in their entirety. In summary, CrossMab technology is based on a domain cross-linking between heavy and light chains within a bispecific Fab branch IgG, which promotes correct chain association. A bispecific CrossMab antibody of the description may be a “CrossMabFAB” antibody, in which the heavy and light chains of the Fab portion of one arm of a bispecific IgG antibody are exchanged. In other embodiments, a bispecific CrossMab antibody of the description may be a “CrossMabVH’VL” antibody, in which the only variable domains of the heavy and light chains of the Fab portion of one branch of a bispecific IgG antibody are exchanged.In other embodiments, a bispecific CrossMab antibody from the description may be a “CrossMabCH1'CL” antibody, in which only the constant domains of the heavy and light chains of the Fab portion of a branch of a bispecific IgG antibody are exchanged. CrossMabCH1'CL antibodies, unlike CrossMabFAB and CrossMabVH'VL, do not have predictive byproducts and, therefore, in some embodiments, bispecific CrossMabCH1'CL antibodies are preferred. See Klein et al., 2016, mAbs, 8(6): 1010. Petition 870200050086, dated 22 / 04 / 2020, p. 25 / 209 / 172. 1020. Additional CrossMabs modalities described are described below in Section 5.2.

[0055] In some embodiments, the bispecific antibodies described are Fab-controlled branch-switch bispecific antibodies. Methods for manufacturing Fab-controlled branch-switch bispecific antibodies are described in PCT Publication No. WO2011 / 131746 and Labrijn et al., 2014 Nat Protoc. 9(10): 2450-63, which are incorporated herein by reference in their entirety. Briefly, Fab-controlled branch-switch bispecific antibodies can be manufactured by separately expressing two precursor IgG1s containing single-pair point mutations in the CH3 domain, mixing the precursor IgG1s under redox conditions in vitro to allow recombination of half molecules, and removing the reductant to allow reoxidation of the interchain disulfide bond, thereby forming the bispecific antibodies.

[0056] The bispecific antibodies described may comprise an Fc domain composed of a first and a second subunit. In one embodiment, the Fc domain is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at the S228 position (Kabat EU index numbering), particularly the S228P amino acid substitution. This amino acid substitution reduces the in vivo Fab branch exchange of IgG4 antibodies (see Stubenrauch et al., 2010, Drug Metabolism and Disposition 38: 84-91). In a further particular embodiment, the Fc domain is a human Fc domain. In a still more particular embodiment, the Fc domain is a human IgG1 Fc domain. An exemplary sequence of a human IgG1 Fc region is given in SEQ ID NO: 42.

[0057] In particular modalities, the Fc domain comprises a Petition 870200050086, dated 04 / 22 / 2020, page 26 / 209 / 172 modification promoting the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain. Thus, in one embodiment, said modification is in the CH3 domain of the Fc domain.

[0058] In a specific embodiment, the said modification promoting the association of the first and second subunits of the Fc domain is a modification called “knob-into-hole”, comprising a “knob” modification in one of the two subunits of the Fc domain and a “whole” modification in the other of the two subunits of the Fc domain. The knob-into-hole technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9: 617-621 and Carter, J, 2001, Immunol Meth 248: 7-15. Generally, the method involves introducing a protrusion (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) at the interface of a second polypeptide, such that the protrusion can be positioned in the cavity so as to promote heterodimer formation and hindered homodimer formation.The bumps are constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the bumps are created at the interface of the second polypeptide by replacing the large amino acid side chains with small ones (e.g., alanine or threonine).

[0059] Consequently, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion within the CH3 domain of the first subunit that is positionable in a cavity within the CH3 domain of the second subunit and an amino acid residue in the CH3 domain of the Petition 870200050086, dated 22 / 04 / 2020, page 27 / 209 / 172 The second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thus generating a cavity within the CH3 domain of the second subunit within which the protrusion within the CH3 domain of the first subunit is positionable. Preferably, said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be made by altering the nucleic acid encoding the polypeptides, for example by site-specific mutagenesis or by peptide synthesis. An exemplary replacement is the Y470T.

[0060] In one such specific embodiment, in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU Kabat index).In a further embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is additionally substituted with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine ​​residue (E356C) (particularly the serine residue at position 354 is substituted with a cysteine ​​residue) and in the second subunit of the Fc domain, the tyrosine residue at position 349 is additionally substituted with a cysteine ​​residue (Y349C) (numbering according to the EU Kabat index). In a particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W and the second subunit of the... Petition 870200050086, dated 22 / 04 / 2020, page 28 / 209 / 172 Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU Kabat index).

[0061] In some embodiments, the electrostatic direction (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used to promote the association of the first and second subunits of the Fc domain.

[0062] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0063] In a particular embodiment the Fc receptor is an Fcy receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activator of the human Fcy receptor, more specifically FcYRIIIa, FcyRI or FcYRIIa, more specifically human FcYRIIIa. In one embodiment the effector function is one or more selected from the group of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-phagocytosis (ADCP) and cytokine secretion. In one particular embodiment, the effector function is ADCC.

[0064] Typically, the same one or more amino acid substitution(s) is / are present in each of the two subunits of the Fc domain. In one embodiment, the one or more amino acid substitution(s) reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the one or more amino acid substitution(s) reduces the binding affinity of the Fc domain to an Fc receptor by at least 2 times, at least 5 times, or at least 10 times.

[0065] In one embodiment, the Fc domain comprises an amino acid substitution at a selected position of the E233, L234, L235, N297, P331 and P329 group (numbering according to the EU index of Petition 870200050086, dated 04 / 22 / 2020, p. 29 / 209 / 172 In a more specific embodiment, the Fc domain comprises an amino acid substitution at a selected position of the d group L234, L235, and P329 (numbering according to the EU Kabat index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to the EU Kabat index). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In another embodiment, the Fc domain comprises an amino acid substitution at the P329 position. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the EU Kabat index). In one embodiment, the Fc domain comprises an amino acid substitution at the P329 position and an additional amino acid substitution at a selected position of E233, L234, L235, N297, and P331 (numbering according to the EU Kabat index).In a more specific embodiment, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In particular embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the EU Kabat index). In even more particular embodiments, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”). Specifically, in particular embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (EU Kabat index numbering), that is, in each of the first and second subunits of the Fc domain the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A) and the proline residue at position 329 is substituted by a glycine residue (P329G) (numbering according to the EU Kabat index).In such a embodiment, the Fc domain is an IgG1 Fc domain, specifically a human IgG1 Fc domain. Petition 870200050086, dated 04 / 22 / 2020, page 30 / 209 / 172

[0066] The single-chain-based bispecific antibodies described may be any of several types of single-chain-based bispecific antibodies known in the art, such as bispecific T-cell engagers (BiTEs), diabodies, tandem diabodies (tandabs), dual-affinity re-bleaching molecules (DARTs), and bispecific killer cell engagers. See, for example, Loffler et al., 2000, Blood 95: 2098-103; Holliger et al., 1993, Proc Natl Acad Sci USA, 90: 6444-8; Kipriyanov et al., 1999, Mol Biol 293: 41-56; Johnson et al., 2010, Mol Biol 399: 436-49; Wiernik et al., 2013, Clin Cancer Res 19: 3844-55; Liu et al., 2017, Front. Immunol. 8: 38 and Yang et al., 2017, Int. J. Mol. Sci. 18: 48, which are incorporated herein by reference in their entirety.

[0067] In some embodiments, the bispecific antibodies of the description are bispecific T-cell engagers (BiTEs). BiTEs are single-chain polypeptide molecules having two antigen-binding domains, one of which binds to a T-cell antigen and the second of which binds to an antigen present on the surface of a target (See, Publication PCT WO 05 / 061547; Baeuerle et al., 2008, Drugs of the Future 33: 137-147; Bargou, et al., 2008, Science 321: 974-977, incorporated herein by reference in their entirety). Thus, the BiTEs of the description have one antigen-binding domain that binds to a T-cell antigen and a second antigen-binding domain that is directed to glyco-MUC1.

[0068] In some embodiments, the bispecific antibodies described are dual-affinity re-bleaching molecules (DARTs).DARTs comprise at least two polypeptide chains that associate (especially through a covalent interaction) to form at least two epitope-binding sites, which may recognize the same or different epitopes. Each of the polypeptide chains of a DART comprises a variable immunoglobulin light chain region and a variable immunoglobulin heavy chain region, but these regions do not... Petition 870200050086, dated 22 / 04 / 2020, p. 31 / 209 / 172 interact to form an epitope binding site. Instead, the immunoglobulin heavy chain variable region of one (e.g., the first) of the DART polypeptide chains interacts with the immunoglobulin light chain variable region of a different DART® polypeptide chain (e.g., the second) to form an epitope binding site. Similarly, the immunoglobulin light chain variable region of one of the DART polypeptide chains (e.g., the first) interacts with the immunoglobulin heavy chain variable region of a different DART polypeptide chain (e.g., the second) to form an epitope binding site. DARTs can be monospecific, bispecific, trispecific, etc., thus being able to simultaneously bind one, two, three or more different epitopes (which may be from the same or different antigens).DARTs can additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., thus being capable of simultaneously binding one, two, three, four, five, six or more molecules. These two attributes of DARTs (i.e., the degree of specificity and valence) can be combined, for example to produce bispecific antibodies (i.e., capable of binding two epitopes) that are tetravalent (i.e., capable of binding four sets of epitopes), etc. DART molecules are described in PCT Publications WO 2006 / 113665, WO 2008 / 157379 and WO 2010 / 080538, which are incorporated herein by reference in their entirety.

[0069] In some embodiments of the bispecific antibodies described, one of the binding specificities is directed toward glyco-MUC1 and the other is directed toward an antigen expressed on immune effector cells. The terms “immune effector cell” or “effector cell” as used herein refer to a cell within the natural repertoire of cells in the mammalian immune system that can be activated to affect the viability of a target cell. Immune effector cells include cells of the lineage Petition 870200050086, dated 22 / 04 / 2020, page 32 / 209 / 172 lymphoid cells such as natural killer (NK) cells, T cells including cytotoxic T or B cells, but also cells of the myeloid lineage can be considered as immune effector cells, such as monocytes or macrophages, dendritic cells and neutrophilic granulocytes. Consequently, said effector cell is preferably an NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell or a neutrophilic granulocyte. The recruitment of effector cells to aberrant cells means that immune effector cells are brought into immediate vicinity to aberrant target cells such that the effector cells can directly kill or indirectly initiate the extermination of the aberrant cells to which they are recruited.In order to avoid nonspecific interactions, it is preferred that the bispecific antibodies described specifically recognize antigens on immune effector cells that are at least overexpressed by these immune effector cells compared to other cells in the body. Target antigens present on immune effector cells may include CD3, CD8, CD16, CD25, CD28, CD64, CD89, NKG2D, and NKp46. Preferably, the antigen on immune effector cells is CD3 expressed on T cells.

[0070] As used herein, “CD3” refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed “full-size” CD3 as well as any form of CD3 resulting from processing in the cell. The term also encompasses naturally occurring variants of CD3, for example, junction variants or allelic variants. The most preferred antigen on an immune effector cell is the epsilon chain of CD3. This antigen has proven to be very effective in recruiting T cells to aberrant cells. Consequently, a bispecific antibody of the description Petition 870200050086, dated 22 / 04 / 2020, p. 33 / 209 / 172 preferably specifically recognizes CD3 epsilon. The amino acid sequence of human CD3 epsilon is shown in UniProt accession (www.uniprot.org) no. P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. The amino acid sequence of cynomolgus [Macaca fascicularis] CD3 epsilon is shown in NCBI GenBank no. BAB71849.1. For human therapeutic use, bispecific antibodies in which the CD3-binding domain specifically binds to human CD3 (e.g., the human CD3 epsilon chain) are used. For preclinical testing in non-human animals and cell lines, bispecific antibodies in which the CD3-binding domain specifically binds to CD3 in the species used for preclinical testing (e.g., cynomolgus CD3 for primate testing) can be used.

[0071] As used herein, a binding domain that “specifically binds to” or “specifically recognizes” a target antigen of a particular species does not preclude binding to or recognition of an antigen of another species and thus encompasses antibodies in which one or more of the binding domains have interspecies cross-reactivity. For example, a CD3 binding domain that “specifically binds to” or “specifically recognizes” human CD3 may also bind to or recognize cynomolgus CD3 and vice versa.

[0072] In some embodiments, a bispecific antibody from the description may compete with the H2C monoclonal antibody (described in PCT publication no. WO2008 / 119567) for binding to a CD3 epitope. In other embodiments, a bispecific antibody from the description may compete with the V9 monoclonal antibody (described in Rodrigues et al., 1992, Int J Cancer Suppl 7: 45-50 and US Pat. No. 6,054,297) for binding to a CD3 epitope. In still other embodiments, a bispecific antibody from the description may compete with the FN18 monoclonal antibody (described in Nooij et al., 1986, Eur J Immunol 19: 981-984) for binding to an epitope. Petition 870200050086, dated 04 / 22 / 2020, p. 34 / 209 / 172 of CD3. In other embodiments, a bispecific antibody as described may compete with the monoclonal antibody SP34 (described in Pessano et al., 1985, EMBO J 4: 337-340) for binding to a CD3 epitope.

[0073] The anti-glyco-MUC1 antibodies described include derivatized antibodies. For example, but not by way of limitation, derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known guarding / blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein. Any of numerous chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-natural amino acids, for example, using ambrx technology (See, for example, Wolfson, 2006, Chem. Biol. 13(10): 1011-2).

[0074] Anti-glucone-MUC1 antibodies or binding fragments may be antibodies or fragments whose sequences have been modified to alter at least one biological effector function mediated by the constant region. For example, in some embodiments, an anti-glucone-MUC1 antibody may be modified to reduce at least one biological effector function mediated by the constant region relative to the unmodified antibody, for example, reduced binding to the Fc receptor (FcyR). FcyR binding may be reduced by mutation of the constant segment of the antibody's immunoglobulin region in particular regions required for FcyR interactions (See, for example, Canfield and Morrison, 1991, J. Exp. Med. 173: 1483-1491 and Lund et al., 1991, J. Immunol. 147: 2657-2662).A reduction in the antibody's FcyR binding capacity may also reduce other effector functions that rely on FcyR interactions, such as opsonization, phagocytosis, and antigen-dependent cellular cytotoxicity (“ADCC”). Petition 870200050086, dated 04 / 22 / 2020, p. 35 / 209 / 172

[0075] The anti-gluc-MUCl antibody or binding fragments described herein include antibodies and / or binding fragments that have been modified to acquire or enhance at least one biological effector function mediated by the constant region relative to an unmodified antibody, for example, to enhance FcyR interactions (See, for example, US 2006 / 0134709). For example, an anti-gluc-MUC1 antibody in the description may have a constant region binding FcyRIIA, FcyRIIB, and / or FcyRIIIA with higher affinity than the corresponding wild-type constant region.

[0076] Thus, the antibodies described may have alterations in biological activity that result in increased or decreased opsonization, phagocytosis, or ADCC. Such alterations are known in the art. For example, modifications in antibodies that reduce ADCC activity are described in U.S. Pat. No. 5,834,597. An exemplary ADCC-decreasing variant corresponds to “mutant 3” (shown in FIG. 4 of U.S. Pat. No. 5,834,597) in which residue 236 is deleted and residues 234, 235, and 237 (using EU numbering) are replaced with alanines.

[0077] In some embodiments, the anti-glyco-MUC1 antibodies described have low levels or lack fucose. Antibodies lacking fucose have been correlated with enhanced ADCC activity, especially at low antibody doses. See Shields et al., 2002, J. Biol. Chem. 277: 26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278: 346673. Methods of preparing antibodies with less fucose include growth in mouse myeloma YB2 / 0 cells (ATCC CRL 1662). YB2 / 0 cells express low levels of FUT8 mRNA, which encodes α1,6-fucosyltransferase, an enzyme required for polypeptide fucosylation.

[0078] In another aspect, anti-glyco-MUC1 antibodies or binding fragments include modifications that increase or decrease the Petition 870200050086, dated 22 / 04 / 2020, p. 36 / 209 / 172 its binding affinities to the fetal Fc receptor, FcRn, for example, by mutation of the constant segment of the immunoglobulin region in particular regions involved in FcRn interactions (see, for example, WO 2005 / 123780). In particular embodiments, an anti-glyco-MUC1 antibody of the IgG class is mutated such that at least one of the amino acid residues 250, 314 and 428 of the constant region of the heavy chain is substituted alone or in any combination thereof, such as at positions 250 and 428 or at positions 250 and 314 or at positions 314 and 428 or at positions 250, 314 and 428, with positions 250 and 428 being a specific combination.For position 250, the substituent amino acid residue may be any amino acid residue other than threonine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine. For position 314, the substituent amino acid residue may be any amino acid residue other than leucine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine.For position 428, the substituting amino acid residues may be any amino acid residue other than methionine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. Specific combinations of suitable amino acid substitutions are identified in Table 1 of U.S. Pat. No. 7,217,797, which is incorporated herein by reference. Such mutations increase binding to FcRn, which protects the antibody from degradation and increases its half-life.

[0079] In other respects, an anti-glyco-MUC1 antibody of the antigen-binding fragment described has one or more amino acids Petition 870200050086, dated 04 / 22 / 2020, p. 37 / 209 / 172 inserted in one or more of its hypervariable regions, for example, as described in Jung and Pluckthun, 1997, Protein Engineering 10: 9, 959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17(5): 481-9. Epub August 17, 2004 and US Petition Pat. No. 2007 / 0280931.

[0080] In other respects, particularly useful for diagnostic applications, an anti-glyco-MUC1 antibody of the antigen-binding fragment described is attached to a detectable portion. Detectable portions include a radioactive portion, a colorimetric molecule, a fluorescent portion, a chemiluminescent portion, an antigen, an enzyme, a detectable granule (such as a magnetic or electron-dense granule (e.g., gold)) or a molecule that binds to another molecule (e.g., biotin or streptavidin)).

[0081] Radioisotopes or radionuclides may include 3H, 14C, 15N, 35S, 90Y, 99Tc, mIn, 1251, 131I.

[0082] Fluorescent labels may include rhodamine, lanthanide phosphors, fluorescein and its derivatives, fluorochrome, GFP (GFP for “Green Fluorescent Protein”), dansil, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.

[0083] Enzyme labels may include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose-6-phosphate dehydrogenase (“G6PDH”), alpha-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase.

[0084] Chemiluminescent or chemiluminescent labels, such as isoluminol, luminol and dioxetanes.

[0085] Other detectable portions include molecules such as biotin, digoxigenin, or 5-bromodeoxyuridine.

[0086] In certain respects, an anti-glyco-MUC1 antibody or antigen-binding fragment of the description competes with GO2 or an antibody or antigen-binding fragment comprising regions Petition 870200050086, dated 04 / 22 / 2020, page 38 / 209 / 172 heavy and light chain variables of GO2 (SEQ ID NOS: 3 and 4, respectively).

[0087] The competition can be tested in cells expressing the GO2-bound glyco-MUC1 epitope or in a glycosylated MUC1 peptide containing the GO2-bound epitope, for example, the 60-mer peptide (VTSAPDTRPAPGSTAPPAHG)3 glycosylated with GalNAc-T2, GalNAc-T4, and GalNAc-T1 as described in U.S. Patent No. 6,465,220. Cells that do not express the epitope or non-glycosylated peptides can be used as controls.

[0088] Cells in which the competition assay can be performed include, but are not limited to, MCF7 or T47D breast cancer cell lines and recombinant cells that are engineered to express the glyco-MUC1 epitope. In a non-limiting example, CHO IdID cells, which lack UDP-Gal / GalNAc epimerase and are deficient in GalNAc O-glycosylation and galactosylation in the absence of exogenous addition of GalNAc and Gal, respectively, are engineered to express MUC1 and grow in the absence or presence of GalNAc, the latter cells producing the expression of the Tn glycoform of MUC1 to which GO2 binds. Cells expressing the non-glycosylated form of MUC1 can be used as a negative control.

[0089] Assays for competition include, but are not limited to, a radioactive material labeled immunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell sorting assay in intercalated ELISA (FACS), and Biacore assays.

[0090] In conducting an antibody competition assay between a reference antibody and a test antibody (regardless of species or isotype), the reference antibody may first be labeled with a detectable label, such as a fluorophore, biotin, or an enzymatic label (or Petition 870200050086, dated 22 / 04 / 2020, p. 39 / 209 / 172 (even radioactive) to allow subsequent identification. In this case, cells expressing glyco-MUC1 are incubated with unlabeled test antibody, labeled reference antibody is added, and the intensity of the bound label is measured. If the test antibody competes with the labeled reference antibody for binding to an overlapping epitope, the intensity will be decreased relative to a control reaction performed without test antibody.

[0091] In a specific embodiment of this assay, the concentration of labeled reference antibody that produces 80% maximum binding (“conc80%”) under the assay conditions (e.g., a specific cell density) is first determined and a competition assay performed with 10 x conc80% of unlabeled test antibody and conc80% of labeled reference antibody.

[0092] Inhibition can be expressed as an inhibition constant or Ki, which is calculated according to the following formula: Ki = IC50 / (1+[reference concentration Ab] / Kd), where IC50 is the concentration of test antibody that produces a 50% reduction in the binding of the reference antibody and Kd is the dissociation constant of the reference antibody, a measure of its affinity for glyco-MUC1. Antibodies that compete with anti-glyco-MUC1 antibodies described herein may have a Ki of 10 pM to 10 nM under the assay conditions described herein.

[0093] In several embodiments, a test antibody is considered to compete with a reference antibody if it diminishes the binding of the reference antibody by at least about 20% or more, for example, by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even more or by a percentage ranging between any of the preceding values, at a reference antibody concentration that is 80% of the maximum binding under the specific assay conditions used and a Petition 870200050086, dated 04 / 22 / 2020, p. 40 / 209 / 172 test antibody concentration that is 10 times higher than the reference antibody concentration.

[0094] In an example of a competition assay, the glycosylated 60-mer MUC1 peptide is adhered to a solid surface, for example, a microwell plate, by contacting the plate with a solution of the peptide (e.g., at a concentration of 1 μg / mL in PBS overnight at 4°C). The plate is washed (e.g., 0.1% Tween 20 in PBS) and blocked (e.g., in Superblock, Thermo Scientific, Rockford, IL). A mixture of a subsaturating amount of biotinylated GO2 (e.g., at a concentration of 80 ng / mL) and unlabeled GO2 (the “reference” antibody) or competing antibody, the anti-glucose-MUC1 antibody (the “test” antibody) in serial dilution (e.g., at a concentration of 2.8 μg / mL, 8.3 μg / mL, or 25 μg / mL) in ELISA buffer (e.g., 1% BSA and 0.1% Tween 20 in PBS) is added to the wells, and the plates are incubated for 1 hour with gentle shaking.The plate is washed, 1 μg / mL of HRP-conjugated streptavidin diluted in ELISA buffer is added to each well, and the plates are incubated for 1 hour. The plates are washed again, and bound antibodies are detected by the addition of substrate (e.g., TMB, Biofx Laboratories Inc., Owings Mills, MD). The reaction is terminated by the addition of stop buffer (e.g., Bio FX Stop Reagents, Biofx Laboratories Inc., Owings Mills, MD), and absorbance is measured at 650 nm using a microplate reader (e.g., VERSAmax, Molecular Devices, Sunnyvale, CA).

[0095] Variations in this competition assay can also be used to test the competition between GO2 and another anti-glycoMUC1 antibody. For example, in certain aspects, the anti-glycoMUC1 antibody is used as a reference antibody and GO2 is used as a test antibody. Additionally, instead of glycosylated 60-mer MUC1 peptide, membrane-bound glycoMUC1 expressed on the cell surface (by Petition 870200050086, dated 22 / 04 / 2020, page 41 / 209 / 172 example on the surface of one of the cell types mentioned above) in culture can be used. Generally, about 10⁴ to 10⁶ transfectants, for example, about 10⁵ transfectants, are used. Other formats for competition assays are known in the art and can be used.

[0096] In several embodiments, an anti-glucose-MUC1 antibody of the description reduces the binding of labeled GO2 by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by a percentage ranging between any of the preceding values ​​(e.g., an anti-glucose-MUC1 antibody of the description reduces the binding of labeled GO2 by 50% to 70%) when the anti-glucose-MUC1 antibody is used at a concentration of 0.08 μg / mL, 0.4 μg / mL, 2 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, or at a concentration ranging between any of the preceding values ​​(e.g., at a concentration ranging from 2 μg / mL to 10 μg / mL). μg / mL).

[0097] In other embodiments, GO2 reduces the binding of a labeled anti-glyco-MUC1 antibody from the description by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by a percentage ranging between any of the preceding values ​​(e.g., GO2 reduces the binding of a labeled anti-glyco-MUC1 antibody from the description by 50% to 70%) when GO2 is used at a concentration of 0.4 μg / mL, 2 μg / mL, 10 μg / mL, 50 μg / mL, 250 μg / mL, or at a concentration ranging between any of the preceding values ​​(e.g., at a concentration ranging from 2 μg / mL to 10 μg / mL).

[0098] In previous assays, the GO2 antibody can be replaced by any antibody or antigen-binding fragment comprising the CDRs or the variable heavy and light chain regions of GO2, such as a humanized or chimeric counterpart of GO2.

[0099] In certain respects, an anti-glyco-MUC1 antibody or antigen-binding fragment of the description comprises sequences Petition 870200050086, dated 04 / 22 / 2020, page 42 / 209 / 172 heavy and / or light chain variables (or encoded by nucleotide sequences) presented in Table 1. In other respects, an anti-glyco-MUC1 antibody or antigen-binding fragment of the description comprises heavy and / or light chain CDR sequences (or encoded by nucleotide sequences) presented in Table 1. The structural sequences for such an anti-glyco-MUC1 antibody and antigen-binding fragment may be the native murine structural sequences in Table 1 or may be non-native structural sequences (e.g., humanized or human).

[00100] In other respects, the description provides an anti-MUC1 antibody or antigen-binding fragment having variable heavy and light chain regions having at least 95%, 98%, 99% or 99.5% sequence identity of SEQ IDs NOS: 3 and 4, respectively.

[00101] In other respects, an anti-glyco-MUC1 antibody or antigen-binding fragment of the description is a single-chain variable fragment (scFv). An exemplary scFv comprises the N-terminal heavy chain variable fragment to the light chain variable fragment. In some embodiments, the heavy chain variable fragment and light chain variable fragment of scFv are covalently linked to a linker sequence of 4 to 15 amino acids. The scFv may be in the form of a bispecific T cell engager or within a chimeric antigen receptor (CAR). 5.2 Anti-glyco-MUCl and Anti-CD3 Bispecific Antibodies

[00102] In some respects, bispecific antibodies of the description may comprise a first antigen-binding domain that specifically binds to CD3 (e.g., comprising the CDRs or VH and VL presented in Table 4) and a second antigen-binding domain that specifically binds to glyco-MUC1. The second antigen-binding domain may comprise, individually or in combination, the Petition 870200050086, dated 22 / 04 / 2020, page 43 / 209 / 172 resources described for the glyco-MUC1 antibodies above (for example, comprising a combination of CDRs identified in Tables 1 to 3, for example CDRs comprising the amino acid sequences of any of the CDR combinations presented in the embodiments numbered 3 to 17, below, or the VH and VL sequences identified in Table 1). Table 4 Description Sequence SEQ ID NO: CD3 CDR-H1 (Kabat) TYAMN 34 CD3 CDR-H2 (Kabat) RIRSKYNNYATYYADSVKG 35 CD3 CDR-H3 (Kabat) HGNFGNSYVSWFAY 36 CD3 CDR-L1 (Kabat) GSSTGAVTTSNYAN 37 CD3 CDR-L2 (Kabat) GTNKRAP 38 CD3 CDR-L3 (Kabat) ALWYSNLWV 39 CD3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG LEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLILQMNSLR AEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 40 CD3 VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQ AFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYY CALWYSNLWVFGGGTKLTVL 41 região Fc of hIgG1 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 42 MUC1 VL- CL(RK) DIVMSQSPSSLGVSVGEKVTMSCKSSQSLLYSTNQKNYQSLLYSTN QKNILAWYQQKPGQSPKLLIYWVSNRKSGVPDRFTGSGSGTDFTL TISSVKAEDLAVYYC QQYYRYPLTFGAGTKLELK RTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 CD3 VH-CL EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG LEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLILQMNSLR AEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 44 MUC1 VH- CH1(EE)-Fc (hole, P329G LALA) QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQRPEQGL EWIGYFSPGNDDIHYNEKFEGKATLTADKSSSTAYMQLNSLTSEDS AVYFCKRSYDKDFDCWGQGTTLTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQ VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Petição 870200050086, de 22 / 04 / 2020, pág. 44 / 209 / 172 MUC1 VH- CH1(EE)-CD3 VL-CH1-Fc (knob, P329G LALA) QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQRPEQGL EWIGYFSPGNDDIHYNEKFEGKATLTADKSSSTAYMQLNSLTSEDS AVYFCKRSYDKDFDCWGQGTTLTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGS GGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQ EKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDE AEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQ VSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 46

[00103] In some embodiments, the first antigen-binding domain comprises a variable heavy chain region comprising the CDR-H1 heavy chain of SEQ ID NO: 34, the CDR-H2 of SEQ ID NO: 35 and the CDR-H3 of SEQ ID NO: 36 and a variable light chain region comprising the CDR-L1 light chain of SEQ ID NO: 37, the CDR-L2 of SEQ ID NO: 38 and the CDR-L3 of SEQ ID NO: 39.

[00104] In some embodiments, the second antigen-binding domain comprises, for example, CDRs comprising the amino acid sequences of any of the CDR combinations presented in embodiments numbered 3 to 17, for example (i) a variable heavy chain region comprising the CDR-H1 heavy chain of SEQ ID NO: 5, the CDRH2 of SEQ ID NO: 6 and the CDR-H3 of SEQ ID NO: 7 and a variable light chain region comprising the CDR (CDR-L) 1 light chain of SEQ ID NO: 8, the CDR-L2 of SEQ ID NO: 9 and the CDR-L3 of SEQ ID NO: 10.

[00105] In a particular embodiment, the bispecific antibody comprises (i) a first antigen-binding domain that specifically binds to CD3 and comprises a variable heavy chain region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 and a variable light chain region Petition 870200050086, dated 04 / 22 / 2020, p. 45 / 209 / 172 comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39;and (ii) a second antigen-binding domain that specifically binds to glyco-MUC1 and comprises (i) a variable heavy chain region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, more preferably a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, more preferably a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 6 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, more preferably a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain region comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31, more preferably a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.;

[00106] In some embodiments, the first antigen-binding domain comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41.

[00107] In some embodiments, the first antigen-binding domain comprises the variable heavy chain region sequence of SEQ ID NO: 40 and the variable light chain region sequence of SEQ ID NO: 41.

[00108] In some embodiments, the second antigen-binding domain Petition 870200050086, dated 04 / 22 / 2020, page 46 / 209 / 172 antigen comprises a variable heavy chain region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3 and a variable light chain region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4.

[00109] In some embodiments, the second antigen-binding domain comprises the variable heavy chain region sequence of SEQ ID NO: 3 and the variable light chain region sequence of SEQ ID NO: 4.

[00110] In some embodiments, the first and / or second antigen-binding domain is a Fab molecule. In some embodiments, the first antigen-binding domain is a cross-linked Fab molecule in which the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged. In such embodiments, the second antigen-binding domain is preferably a conventional Fab molecule.

[00111] In some embodiments where the first and second antigen-binding domains of the bispecific antibody are both Fab molecules and in one of the antigen-binding domains (particularly the first antigen-binding domain) the variable VL and VH domains of the Fab light chain and the Fab heavy chain are substituted for one another, i) in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is replaced by a positively charged amino acid (numbering according to Kabat) and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (numbering according to Kabat's EU index); or ii) in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is replaced by an amino acid Petition 870200050086, dated 22 / 04 / 2020, page 47 / 209 / 172 positively charged (numbering according to Kabat) and in which in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (numbering according to the EU Kabat index).

[00112] The bispecific antibody does not comprise both modifications mentioned under i) and ii). The constant CL and CH1 domains of the antigen-binding domain having the VH / VL exchange are not replaced by each other (i.e., they remain unexchanged).

[00113] In a more specific modality, i) In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index); or ii) In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index).

[00114] In such an embodiment, in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 or the amino acid at position Petition 870200050086, dated 04 / 22 / 2020, page 48 / 209 / 172 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index).

[00115] In a further embodiment, in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index).

[00116] In a particular embodiment, in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index).

[00117] In a more particular embodiment, in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by lysine (K) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to Kabat's EU index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to Kabat's EU index). Petition 870200050086, dated 04 / 22 / 2020, page 49 / 209 / 172

[00118] In a further particular embodiment, in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by arginine (R) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to Kabat's EU index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to Kabat's EU index).

[00119] In particular embodiments, if amino acid substitutions according to the embodiments above are made in the CL constant domain and in the CH1 constant domain of the second antigen-binding domain, the CL constant domain of the second antigen-binding domain is of the kappa isotype.

[00120] In some embodiments, the first and second antigen-binding domains are fused together, optionally via a peptide linker.

[00121] In some embodiments, the first and second antigen-binding domains are each a Fab molecule and (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain.

[00122] In some embodiments, the bispecific antibody provides monovalent binding to CD3.

[00123] In particular embodiments, the bispecific antibody comprises a single antigen-binding domain that specifically binds to CD3 and two antigen-binding domains that specifically bind to CD3. Petition 870200050086, dated 22 / 04 / 2020, p. 50 / 209 / 172 bind to glyco-MUC1. Thus, in some embodiments, the bispecific antibody comprises a third antigen-binding domain that binds specifically to glyco-MUC1. In some embodiments, the third antigen portion is identical to the first antigen-binding domain (e.g., it is also a Fab molecule and comprises the same amino acid sequence).

[00124] In particular embodiments, the bispecific antibody further comprises an Fc domain composed of a first and a second subunit. In one embodiment, the Fc domain is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at the S228 position (EU Kabat index numbering), particularly the S228P amino acid substitution. In a further particular embodiment, the Fc domain is a human Fc domain. In a still more particular embodiment, the Fc domain is a human IgG1 Fc domain. An exemplary sequence of a human IgG1 Fc region is given in SEQ ID NO: 42.

[00125] In some embodiments where the first, second, and, where present, third antigen-binding domain are each a Fab molecule, (a) (i) the second antigen-binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain and the first antigen-binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the first Fc domain subunit or (ii) the first antigen-binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain and the second antigen-binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the first Fc domain subunit and (b) the third Petition 870200050086, dated 04 / 22 / 2020, page 51 / 209 / 172 antigen-binding domain, where present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[00126] In particular embodiments, the Fc domain comprises a modification promoting the association of the first and second subunits of the Fc domain, for example, as described in Section 0.

[00127] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function, for example as described in Section 0.

[00128] In a particular embodiment of the bispecific antibody, it comprises (i) a first antigen-binding domain that specifically binds to CD3, wherein the first antigen-binding domain is a cross-linked Fab molecule in which the variable or constant regions, particularly the variable regions, of the Fab light chain and the Fab heavy chain are exchanged; (ii) a second and a third antigen-binding domains that specifically bind to glyco-MUC1, comprising a variable heavy chain region comprising the CDR-H1 heavy chain of SEQ ID NO: 5, the CDR-H2 of SEQ ID NO: 6 and the CDR-H3 of SEQ ID NO: 7 and a variable light chain region comprising the CDR-L1 light chain of SEQ ID NO: 8, the CDR-L2 of SEQ ID NO: 9 and the CDR-L3 of SEQ ID NO: 10, wherein the second and third antigen-binding domains are each a Fab molecule, particularly a conventional Fab molecule; (iii) an Fc domain composed of a first and a second subunit capable of stable association, wherein the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Petition 870200050086, dated 04 / 22 / 2020, page 52 / 209 / 172 first subunit of the Fc domain and in which the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[00129] In one embodiment, the first antigen-binding domain comprises a variable heavy chain region comprising the CDR-H1 heavy chain of SEQ ID NO: 34, the CDR-H2 of SEQ ID NO: 35 and the CDR-H3 of SEQ ID NO: 36 and a variable light chain region comprising the CDR-L1 light chain of SEQ ID NO: 37, the CDR-L2 of SEQ ID NO: 38 and the CDR-L3 of SEQ ID NO: 39.

[00130] In one embodiment, the first antigen-binding domain comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41.

[00131] In one embodiment, the first antigen-binding domain comprises the heavy chain variable region sequence of SEQ ID NO: 40 and the light chain variable region sequence of SEQ ID NO: 41.

[00132] In one embodiment, the second and third antigen-binding domains comprise a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4. Preferably, the antigen-binding domain comprises CDRs comprising the amino acid sequences of any of the CDR combinations presented in embodiments numbered 3 to 17. In one embodiment, the second and third antigen-binding domains comprise the variable heavy chain region of SEQ ID NO: 3 and the region Petition 870200050086, dated 04 / 22 / 2020, page 53 / 209 / 172 light chain variable of SEQ ID NO: 4.

[00133] The Fc domain according to the above modalities may incorporate, individually or in combination, all of the features described above in relation to Fc domains.

[00134] In some embodiments, the antigen-binding domains and the Fc region are fused to each other by peptide linkers, for example by peptide linkers as in SEQ ID NO: 45 and SEQ ID NO: 46.

[00135] In one embodiment, in the constant domain CL of the second and third Fab molecules under (ii) the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat) and in the constant domain CH1 of the second and third Fab molecules under (ii) the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the EU Kabat index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the EU Kabat index).

[00136] In one embodiment, the bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 43 (and preferably comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31), a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 44 (and preferably comprises the CD3 heavy and light chain CDR sequences shown in Table 4), a polypeptide comprising a A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the SEQ ID sequence. Petition 870200050086, dated 04 / 22 / 2020, p. 54 / 209 / 172 NO: 45 (and preferably comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25) and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 46 (and preferably comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39).

[00137] In one embodiment, the bispecific antibody comprises a polypeptide (particularly two polypeptides) comprising the sequence SEQ ID NO: 43, a polypeptide comprising the sequence SEQ ID NO: 44, a polypeptide comprising the sequence SEQ ID NO: 45 and a polypeptide comprising the sequence SEQ ID NO: 46. 5.3 Antibody-Drug Conjugates

[00138] Another aspect of the description concerns antibody-drug conjugates (ADCs), including the anti-glucose-MUC1 antibodies and antigen-binding fragments described herein. ADCs generally comprise an anti-glucose-MUC1 antibody and / or binding fragment as described herein having one or more cytotoxic and / or cytostatic agents linked to it via one or more linkers. In specific embodiments, ADCs are composed according to the structural formula (I): [DL-XY]n-Ab or salts thereof, where each “D” represents, independently of the others, a cytotoxic and / or cytostatic agent. Petition 870200050086, dated 22 / 04 / 2020, p. 55 / 209 / 172 (“drug”); each “L” represents, independently of the others, a linker; “Ab” represents an anti-glyco-MUC1 antigen-binding domain, such as an anti-glyco-MUC1 antibody or binding fragment described herein; each “XY” represents a link formed between a functional group Rx in the linker and a “complementary” functional group Ryno in the antibody; en represents the number of drugs bound to or drug-to-antibody ratio (DAR) of the ADC.

[00139] The specific modalities of the various antibodies (Ab) that may comprise ADCs include the various modalities of anti-glyco-MUC1 antibodies and / or binding fragments described above.

[00140] In some specific embodiments of the ADCs and / or salts of the structural formula (I), each D is the same and / or each L is the same.

[00141] The specific types of cytotoxic and / or cytostatic agents (D) and linkers (L) that may comprise the anti-glycoMUC1 ADCs described, as well as the number of cytotoxic and / or cytostatic agents linked to the ADCs, are described in more detail below. 5.3.1. Cytotoxic and / or cytostatic agents

[00142] Cytotoxic and / or cytostatic agents may be any agents known to inhibit the growth and / or replication of dead cells and / or cancer cells and in particular tumor cells. Numerous agents having cytotoxic and / or cytostatic properties are known in the literature. Non-limiting examples of classes of cytotoxic and / or cytostatic agents include, by way of example and not limitation, radionuclides, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA intercalating agents (e.g., groove-linking agents such as minor groove binders), RNA / DNA antimetabolites, cell cycle modulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase inhibitors, mitochondrial inhibitors, and antimitotic agents. Petition 870200050086, dated 04 / 22 / 2020, page 56 / 209 / 172

[00143] Specific non-limiting examples of agents within certain of these various classes are provided below.

[00144] Alkylating agents: asaley ((L-Leucine, N-[N-acetyl-4-[bis-(2chloroethyl)amino]-DL-phenylalanyl]-, ethyl ester; NSC 167780; CAS Registration No. 3577897)); AZQ ((1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1aziridinyl)-3,6-dioxo-, diethyl ester; NSC 182986; CAS Registration No 57998682)); BCNU ((N,N'-Bis(2-chloroethyl)-N-nitrosourea; NSC 409962; CAS Registration No 154938)); busulfan (1,4-butanediol dimethanesulfonate; NSC 750; CAS Registration No 55981); (carboxyphthalate)platinum (NSC 27164; CAS Registration No 65296813); CBDCA ((cis-(1,1-cyclobutanedicarboxylate)diaminoplatin(II)); NSC 241240; CAS Registration No 41575944)); CCNU ((N(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea; NSC 79037; CAS Registration No 13010474)); CHIP (iproplatin; NSC 256927); chlorambucil (NSC 3088; CAS Registration No 305033); chlorozotocin ((2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose; NSC 178248; CAS Registry No. 54749905)); cisplatin (cisplatin; NSC 119875; CAS Registry No. 15663271);clomesson (NSC 338947; CAS Registration No 88343720); cyanomorpholinodoxorubicin (NCS 357704; CAS Registration No 88254073); cyclodyssone (NSC 348948; CAS Registration No 99591738); dianhydrogalactitol (5,6-diepoxydulcitol; NSC 132313; CAS Registration No. 23261203); Fluorodopan ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyluracil; NSC 73754; CAS Registry No. 834913); Hepsulfam (NSC 329680; CAS Registry No. 96892578); Hycantone (NSC 142982; CAS Registry No. 23255938); Melphalan (NSC 8806; CAS Registry No. 3223072); Methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea; NSC 95441; 13909096); Mitomycin C (NSC 26980; CAS Registry No. 50077); Mitozolamide (NSC 353451; CAS Registry No. 85622953); Nitrogen mustard ((bis(2chloroethyl)methylamine hydrochloride; NSC 762; CAS Registration No 55867); PCNU ((1-(2chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea; NSC 95466; CAS Registration; Petition 870200050086, dated 04 / 22 / 2020, page 57 / 209 / 172 No 13909029)); piperazine alkylator ((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dichloride; NSC 344007)); piperazinedione (NSC 135758; CAS Registry No 41109802); pipobroman ((N,N-bis(3bromopropionyl)piperazine; NSC 25154; CAS Registry No 54911)); porfiromycin (N-methylmitomycin C; NSC 56410; CAS Registry No 801525); spiro-hydantoin shown (NSC 172112; CAS Registry No 56605164); teroxirone (triglycidyl isocyanurate; NSC 296934; CAS Registry No 2451629); tetraplatin (NSC 363812; CAS Registry No. 62816982); thio-tepa (N,N',N'' tri-1,2-ethanediylthiophosphoramide; NSC 6396; CAS Registry No. 52244); triethylenemelamine (NSC 9706; CAS Registry No. 51183); uracil nitrogen mustard (desmethyldopan; NSC 34462; CAS Registry No. 66751); Yoshi-864 (bis(3-mesyloxypropyl)amine(hydrochloride); NSC 102627; CAS Registry No. 3458228).

[00145] Topoisomerase I inhibitors: camptothecin (NSC 94600; CAS Registry No. 7689-03-4); various camptothecin derivatives and analogues (e.g., NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC 295500, NSC 249910, NSC 606985, NSC 74028, NSC 176323, NSC 295501, NSC 606172, NSC 606173, NSC 610458, NSC 618939, NSC 610457, NSC 610459, NSC 606499, NSC 610456, NSC 364830 and NSC 606497); morpholinisoxorubicin (NSC 354646; CAS Registry No. 89196043); SN-38 (NSC 673596; CAS Registry No. 86639-52-3).

[00146] Topoisomerase II inhibitors: doxorubicin (NSC 123127; CAS Registry No. 25316409); amonafide (benzisoquinolinedione; NSC 308847; CAS Registry No. 69408817); m-AMSA ((4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide; NSC 249992; CAS Registry No. 51264143)); antrapyrazole derivative ((NSC 355644); etoposide (VP-16; NSC 141540; CAS Registration No 33419420); pyrazoloacridine ((pyrazolo[3,4,5-kl]acridine2(6H)-propanamine, 9-methoxy-N,N-dimethyl-5-nitro-, monomethanesulfonate; NSC 366140; CAS Registration No. 99009219; Petition 870200050086, dated 04 / 22 / 2020, pages 58 / 209 / 172 337766; Register CAS No 71439684); daunorubicin (NSC 821151; CAS Registry No 23541506); desoxidoxorubicin (NSC 267469; CAS Registry No 63950061); mitoxantrone (NSC 301739; CAS Registry No 70476823); menogaril (NSC 269148; CAS Registry No 71628961); N,N-dibenzyl daunomycin (NSC 268242; CAS Registry No 70878512); oxantrazol (NSC 349174; CAS Registry No 105118125); rubidazone (NSC 164011; CAS Registry No 36508711); teniposide (VM-26; NSC 122819; CAS Registry No. 29767202).

[00147] DNA Intercalating Agents: anthramycin (CAS Registration No. 4803274); chicamycin A (CAS Registration No. 89675376); Tomaimycin (CAS Registration No. 35050556); DC-81 (CAS Registration No. 81307246); sibiromycin (CAS Registration No. 12684332); pyrrolobenzodiazepine derivative (CAS Registration No 945490095); SGD-1882 ((S)-2-(4aminophenyl)-7-methoxy-8-(3-4(S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11adihydro-1H-benzo[e] pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-1Hbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one); SG2000 (SJG-136; (11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one); NSC 694501; CAS Registry No. 232931576).

[00148] RNA / DNA Antimetabolites: L-alanosine (NSC 153353; CAS Registry No. 59163416); 5-azacytidine (NSC 102816; CAS Registry No. 320672); 5-fluorouracil (NSC 19893; CAS Registry No. 51218); acivicin (NSC 163501; CAS Registry No. 42228922); aminopterin derivative N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC 132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC 184692); aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acid monohydrate (NSC 134033); um antifo ((Na-(4-amino-4-deoxypteroyl)-N7-hemiftaloyl-L-ornithine; NSC Petition 870200050086, dated 04 / 22 / 2020, page. 59 / 209 / 172 623017)); Baker's antifol soluvel (NSC 139105; CAS Registry No 41191042); dichloroallyl lawsone ((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone; NSC 126771; CAS Registry No. 36417160); brequinar (NSC 368390; CAS Registry No. 96201886); ftorafur ((pro-drug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil; NSC 148958; CAS Registry No. 37076689); 5,6-dihydro-5-azacytidine (NSC 264880; CAS Registry No. 62402317); methotrexate (NSC 740; CAS Registry No. 59052); methotrexate acid derivative (N-[[4[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]Lglutamic; NSC 174121); PALA ((N-(phosphonoacetyl)-L-aspartate; NSC 224131; CAS Registry No 603425565); pyrazofurin (NSC 143095; CAS Registry No 30868305); trimetrexate (NSC 352122; CAS Registry No 82952645).

[00149] DNA antimetabolites: 3-HP (NSC 95678; CAS Registry No. 3814797); 2'-deoxy-5-fluorouridine (NSC 27640; CAS Registry No. 50919); 5-HP (NSC 107392; CAS Registry No. 19494894); α-TGDR (α-2'-deoxy-6-thioguanosine; NSC 71851; CAS Registry No. 2133815); aphidicolin glycinate (NSC 303812; CAS Registry No. 92802822); ara C (cytosine arabinoside; NSC 63878; CAS Registry No. 69749); 5-aza-2'-deoxycytidine (NSC 127716; CAS Registry No. 2353335); β-TGDR (e-2'-deoxy-6thioguanosine; NSC 71261; CAS Registration No 789617); cyclocytidine (NSC 145668; CAS Registration No 10212256); guanazole (NSC 1895; CAS Registration No. 1455772); hydroxyurea (NSC 32065; CAS Registration No 127071); inosine glycodialdehyde (NSC 118994; CAS Registration No 23590990); macbecin II (NSC 330500; CAS Registration No. 73341738); pyrazoloimidazole (NSC 51143; CAS Registration No 6714290); thioguanine (NSC 752; CAS Registration No. 154427); thiopurine (NSC 755; CAS Registration No 50442).

[00150] Cell cycle modulators: silibinin (CAS Registration No. 22888-70-6); epigallocatechin gallate (EGCG; CAS Registration No 989515); procyanidin derivatives (e.g., procyanidin A1 [CAS Registry No 103883030], procyanidin B1 [CAS Registry No 20315257], procyanidin B4 Petition 870200050086, dated 04 / 22 / 2020, pages 60 / 209 / 172 [CAS Registry No. 29106512], arecatanin B1 [CAS Registry No. 79763283]); isoflavones (e.g., genistein [4%5,7-trihydroxyisoflavone; CAS Registry No 446720], daidzein [4',7-dihydroxyisoflavone, CAS Registry No 486668]); indole-3-carbinol (CAS Registry No 700061); quercetin (NSC 9219; CAS Registry No 117395); estramustine (NSC 89201; CAS Registration No. 2998574); nocodazole (CAS Registration No. 31430189); podophyllotoxin (CAS Registration No. 518285); 124689652).

[00151] Kinase inhibitors: afatinib (CAS Registry No. 850140726); axitinib (CAS Registry No. 319460850); ARRY-438162 (binimetinib) (CAS Registry No. 606143899); bosutinib (CAS Registry No. 380843754); cabozantinib (CAS Registry No. 1140909483); ceritinib (CAS Registry No. 1032900256); crizotinib (CAS Registry No. 877399525); dabrafenib (CAS Registry No. 1195765457); dasatinib (NSC 732517; CAS Registry No. 302962498); erlotinib (NSC 718781; CAS Registration No 183319699); everolimus (NSC 733504; CAS Registration No. 159351696); fostamatinib (NSC 745942; CAS Registration No 901119355); gefitinib (NSC 715055; CAS Registration No. 184475352); ibrutinib (CAS Registration No. 936563961); imatinib (NSC 716051; CAS Registration No 220127571); lapatinib (CAS Registration No. 388082788); lenvatinib (CAS Registration No 857890392); mubritinib (CAS 366017096); nilotinib (CAS Registration No 923288953); nintedanib (CAS Registration No. 656247175); palbociclib (CAS Registration No 571190302); Pazopanib (NSC 737754; CAS Registration No. 635702646);pegaptanib (CAS Registry No. 222716861); ponatinib (CAS Registry No. 1114544318); rapamycin (NSC 226080; CAS Registry No. 53123889); regorafenib (CAS Registry No. 755037037); AP 23573 (ridaforolimus) (CAS Registry No. 572924540); INCB018424 (ruxolitinib) (CAS Registry No. 1092939177); ARRY-142886 (selumetinib) (NSC 741078; CAS Registry No. 606143-52-6); sirolimus (NSC 226080; CAS Registry No. 53123889); sorafenib (NSC 724772; CAS Registry No. Petition 870200050086, dated 04 / 22 / 2020, page 61 / 209 / 172 No. 475207591); sunitinib (NSC 736511; CAS Registry No. 341031547); tofacitinib (CAS Registry No. 477600752); temsirolimus (NSC 683864; CAS Registry No. 163635043); trametinib (CAS Registry No. 871700173); vandetanib (CAS Registry No. 443913733); vemurafenib (CAS Registry No. 918504651); SU6656 (CAS Registry No. 330161870); CEP-701 (lesaurtinib) (CAS Registry No. 111358884); XL019 (CAS Registry No. 945755566); PD325901 (CAS Registration No 391210109); PD-98059 (CAS Registration No 167869218);ATP-competitive TORC1 / TORC2 inhibitors including PI-103 (CAS Registry No. 371935749), PP242 (CAS Registry No. 1092351671), PP30 (CAS Registry No. 1092788094), Torin 1 (CAS Registry No. 1222998368), LY294002 (CAS Registry No. 154447366), XL-147 (CAS Registry No. 934526893), CAL-120 (CAS Registry No. 870281348), ETP-45658 (CAS Registry No. 1198357797), PX 866 (CAS Registry No. 502632668), GDC-0941 (CAS Registry No. 957054307), BGT226 (CAS Registration No. 1245537681), BEZ235 (CAS Registration No. 915019657), XL-765 (CAS Registration No. 934493762).;

[00152] Protein synthesis inhibitors: acriflavine (CAS Registry No 65589700); amikacin (NSC 177001; CAS Registry No 39831555); arbecacin (CAS Registry No 51025855); astromycin (CAS Registry No 55779061); azithromycin (NSC 643732; CAS Registry No 83905015); becanamycin (CAS Registry No 4696768); chlortetracycline (NSC 13252; CAS Registry No 64722); clarithromycin (NSC 643733; CAS Registry No 81103119); clindamycin (CAS Registry No 18323449); clomocycline (CAS No. 1181540); cycloheximide (CAS No. 66819); dactinomycin (NSC 3053; CAS No. 50760); dalfopristin (CAS No. 112362502); demeclocycline (CAS No. 127333); dibecacin (CAS No. 34493986); dihydrostreptomycin (CAS No. 128461); dirithromycin (CAS No. 62013041); doxycycline (CAS No. 17086281); emetine (NSC 33669; CAS No. 483181); Petition 870200050086, dated 04 / 22 / 2020, page 62 / 209 / 172 erythromycin (NSC 55929; CAS Registry No. 114078); flurithromycin (CAS Registry No. 83664208); framycetin (neomycin B; CAS Registry No. 119040); gentamicin (NSC 82261; CAS Registry No. 1403663); glycylcyclines, such as tigecycline (CAS Registry No. 220620097); hygromycin B (CAS Registry No. 31282049); isepamycin (CAS Registry No. 67814760); josamycin (NSC 122223; CAS Registry No. 16846245); kanamycin (CAS Registration No 8063078); ketolides such as telithromycin (CAS Registry No. 191114484), cethromycin (CAS Registry No. 205110481) and solithromycin (CAS Registry No. 760981837); lincomycin (CAS Registry No. 154212); lymecycline (CAS Registration No 992212); meclocycline (NSC 78502; CAS Registration No 2013583); methacycline (rondomycin; NSC 356463; CAS Registration No 914001); midecamycin (CAS Registration No. 35457808); minocycline (NSC 141993; CAS Registration No 10118908); myocamycin (CAS Registration No. 55881077); neomycin (CAS Registry No 119040);netilmicin (CAS Registry No. 56391561); oleandomycin (CAS Registry No. 3922905); oxazolidinones, such as eperezolid (CAS Registry No. 165800044), linezolid (CAS Registry No. 165800033), posizolid (CAS Registry No. 252260029), radezolid (CAS Registry No. 869884786), ranbezolid (CAS Registry No. 392659380), sutezolid (CAS Registry No. 168828588), tedizolid (CAS Registry No. 856867555); oxytetracycline (NSC 9169; CAS Registry No. 2058460); paromomycin (CAS Registry No 7542372); penimepicycline (CAS Registry No 4599604); peptidyl transferase inhibitors, for example, chloramphenicol (NSC 3069; CAS Registry No 56757) and derivatives such as azidanphenicol (CAS Registry No 13838089), florfenicol (CAS Registry No 73231342) and thiamphenicol (CAS Registry No 15318453) and pleuromutilins such as retapamulin (CAS Registry No 224452668), tiamulin (CAS Registry No 55297955), valnemulin (CAS Registry No 101312929); pirlimicin (CAS Registry No. 79548735); puromycin (NSC 3055; CAS Registry No. 53792);quinupristin (CAS Registry No. 120138503); ribostamycin (CAS Registry No. 53797356); Petition 870200050086, dated 04 / 22 / 2020, page. 63 / 209 / 172 roquitamycin (CAS Registry No 74014510); rolitetracycline (CAS Registry No 751973); roxithromycin (CAS Registry No 80214831); sisomycin (CAS Registry No 32385118); spectinomycin (CAS Registry No 1695778); spiramycin (CAS Registry No 8025818); Streptogramins such as pristinamycin (CAS Registry No. 270076603), quinupristin / dalfopristin (CAS Registry No. 126602899) and virginiamycin (CAS Registry No. 11006761); streptomycin (CAS Registry No. 57921); tetracycline (NSC 108579; CAS Registry No. 60548); tobramycin (CAS Registry No. 32986564); troleandomycin (CAS Registry No. 2751099); tylosin (CAS Registry No. 1401690); verdamycin (CAS Registry No. 49863481).

[00153] Histone deacetylase inhibitors: abexinostat (CAS Registry No. 783355602); belinostat (NSC 726630; CAS Registry No. 414864009); chidamide (CAS Registry No. 743420022); entinostat (CAS Registry No. 209783802); givinostat (CAS Registry No. 732302997); mocetinostat (CAS Registry No. 726169739); panobinostat (CAS Registry No. 404950807); quisinostat (CAS Registry No. 875320299); resminostat (CAS Registry No. 864814880); romidepsin (CAS Registry No. 128517077); sulforaphane (CAS Registration No 4478937); thioureidobutyronitrile (Kevetrin®; CAS Registration No. 6659890); valproic acid (NSC 93819; CAS Registration No 99661); vorinostat (NSC 701852; CAS Registration No. 149647789); ACY-1215 (rocilinostat; CAS Registration No. 1316214524); CUDC-101 (CAS Registration No. 1012054599); CHR-2845 (tefinostat; CAS Registration No. 914382608); CHR3996 (CAS Registration No. 1235859138); 4SC-202 (CAS Registration No. 910462430); CG200745 (CAS Registry No. 936221339); SB939 (pracinostat; CAS Registry No. 929016966).

[00154] Mitochondrial Inhibitors: pancratistatin (NSC 349156; CAS Registry No. 96281311); rhodamine-123 (CAS Registry No. 63669709); edelfosine (NSC 324368; CAS Registry No. 70641519); d-alpha-tocopherol succinate (NSC 173849; CAS Registry No. 4345033); compound 11β (Registration Petition 870200050086, dated 04 / 22 / 2020, page 64 / 209 / 172 CAS No. 865070377); aspirin (NSC 406186; CAS Registration No 50782); ellipticine (CAS Registration No519233); berberine (CAS Registration No633658); cerulenin (CAS Registry No. 17397896); GX015-070 (Obatoclax®; 1HIndole, 2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrole-5-yl)-; NSC 729280; CAS Registration No 803712676); Celastrol (tripterin; CAS Registration No. 34157830); metformin (NSC 91485; CAS Registration No 1115704); Brilliant green (NSC 5011; CAS Registry No. 633034); ME-344 (CAS Registry No. 1374524556).

[00155] Antimitotic Agents: allocolchicine (NSC 406042); auristatins, such as MMAE (monomethyl auristatin E; CAS Registry No. 474645-27-7) and MMAF (monomethyl auristatin F; CAS Registry No. 74501794-1); halicondrin B (NSC 609395); colchicine (NSC 757; CAS Registry No. 64868); colchicine derivative (N-benzoyl-desacetylbenzamide; NSC 33410; CAS Registry No. 63989753); dolastatin 10 (NSC 376128; CAS Registry No. 110417-88-4); maytansine (NSC 153858; CAS Registry No. 35846-53-8); rozoxin (NSC 332598; CAS Registry No. 90996546); taxol (NSC 125973; CAS Registration No 33069624); taxol derivative ((2'-N-[3(dimethylamino)propyl]glutaramate taxol; NSC 608832); thiocolchicine (3demethylthiocolchicine; NSC 361792); trityl cysteine ​​(NSC 49842; CAS Registration No 2799077); vinblastine sulfate (NSC 49842; CAS Registration No 143679); vincristine sulfate (NSC 67574; CAS Registration No 2068782).

[00156] Any of these agents that include or can be modified to include an antibody attachment site may be included in the ADCs described herein.

[00157] In one specific embodiment, the cytotoxic and / or cytostatic agent is an antimitotic agent.

[00158] In another specific embodiment, the cytotoxic and / or cytostatic agent is an auristatin, for example, monomethyl auristatin E (“MMAE”) or monomethyl auristatin F (“MMAF”). Petition 870200050086, dated 04 / 22 / 2020, p. 65 / 209 / 172 5.3.2. Connectors

[00159] In the anti-glyco-MUC1 ADCs described, cytotoxic and / or cytostatic agents are linked to the antibody by means of linkers. The linker linking a cytotoxic and / or cytostatic agent to the antibody of an ADC may be short, long, hydrophobic, hydrophilic, flexible or rigid, or may be composed of segments that each independently has one or more of the properties mentioned above, such that the linker may include segments having different properties. Linkers may be polyvalent, such that they covalently link more than one agent to a single site on the antibody, or monovalent, such that they covalently link a single agent to a single site on the antibody.

[00160] As will be assessed by experienced professionals, linkers bind cytotoxic and / or cytostatic agents to antibodies by forming a covalent bond to the cytotoxic and / or cytostatic agent at one location and a covalent bond to the antibody at another. The covalent bonds are formed by the reaction between functional groups on the linker and functional groups on the agents and antibody.As used herein, the term “linker” is intended to include (i) unconjugated forms of the linker that include a functional group capable of covalently linking the linker to a cytotoxic and / or cytostatic agent and a functional group capable of covalently linking the linker to an antibody; (ii) partially conjugated forms of the linker that include a functional group capable of covalently linking the linker to an antibody and that is covalently linked to a cytotoxic and / or cytostatic agent or vice versa; and (iii) fully conjugated forms of the linker that are covalently linked to both a cytotoxic and / or cytostatic agent and an antibody. In some specific embodiments of linkers and anti-glyco-MUC1 ADCs of the description, as well as synthons used to conjugate linking agents to antibodies, portions comprising the functional groups in the linker and covalent bonds formed between the linker and... Petition 870200050086, dated 22 / 04 / 2020, page 66 / 209 / 172 antibody are specifically illustrated as Rxe XY, respectively.

[00161] The linkers are preferably, but need not be, chemically stable to conditions outside the cell and may be designed to cleave, immolate and / or otherwise specifically degrade within the cell. Alternatively, linkers that are not designed to specifically cleave or degrade within the cell may be used. The choice of stable versus unstable linker may depend on the toxicity of the cytotoxic and / or cytostatic agent. For agents that are toxic to normal cells, stable linkers are preferred. For agents that are selective or targeted and have lower toxicity to normal cells, chemical stability of the linker to the extracellular medium is less important. A wide variety of linkers useful for binding drugs to antibodies in the context of ADCs are known in the art.Any of these linkers, as well as other linkers, can be used to bind cytotoxic and / or cytostatic agents to the antibody of the antiglyco-MUC1 ADCs described.

[00162] Exemplary multivalent linkers that can be used to link many cytotoxic and / or cytostatic agents to a single antibody molecule are described, for example, in WO 2009 / 073445; WO 2010 / 068795; WO 2010 / 138719; WO 2011 / 120053; WO 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379; WO 2014 / 093394; WO 2014 / 093640, the content of which is incorporated herein by reference in its entirety. For example, the Fleximer linker technology developed by Mersana et al. has the potential to enable ADCs with high DAR with good physicochemical properties. As shown below, Mersana technology is based on incorporating drug molecules into a solubilizing polyacetal backbone via an ester linkage sequence. The methodology makes the ADCs highly charged (DAR up to 20) while maintaining good physicochemical properties. Petition 870200050086, dated 04 / 22 / 2020, page 67 / 209 / 172

[00163] Additional examples of dendritic-type linkers can be found in US 2006 / 116422; US 2005 / 271615; de Groot et al. (2003) Angew. Chem. Int. Ed. 42: 4490-4494; Amir et al. (2003) Angew. Chem. Int. Ed. 42: 4494-4499; Shamis et al. (2004) J. Am. Chem. Soc. 126: 1726-1731; Sun et al.(2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768; King et al. (2002) Tetrahedron Letters 43: 1987-1990, each of which is incorporated herein by reference.

[00164] Exemplary monovalent ligands that can be used are described, for example, in Nolting, 2013, Antibody-Drug Conjugates, Methods in Molecular Biology 1045:71-100; Kitson et al., 2013, CROs / CMOs--Chemica Oggi--Chemistry Today 31(4):30-38; Ducry et al., 2010, Bioconjugate Chem. 21:5-13; Zhao et al., 2011, J. Med. Chem. 54:3606-3623; US Pat. No 7,223,837; US Pat. No 8,568,728; US Pat. No 8,535,678; and WO2004010957, each of which is incorporated herein by reference.

[00165] By way of example and not limitation, some cleavable and non-cleavable ligands that may be included in the anti-glyco-MUC1 ADCs described are described below. 5.3.3. Cleavable Connectors

[00166] In certain embodiments, the selected linker is cleavable in vivo. Cleavable linkers may include chemically or enzymatically unstable or degradable linkages. Cleavable linkers generally rely on processes within the cell to release the drug, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes within the cell. Cleavable linkers generally incorporate one or more chemical linkages that are chemically or enzymatically cleavable while the rest of the linker is not cleavable. In certain embodiments, a linker comprises a group Petition 870200050086, dated 04 / 22 / 2020, page 68 / 209 / 172, chemically unstable such as hydrazone and / or disulfide groups. Linkers comprising chemically unstable groups exploit differential properties between plasma and some cytoplasmic compartments. The intracellular conditions to facilitate drug release for hydrazone-containing linkers are the acidic environment of endosomes and lysosomes, while disulfide-containing linkers are reduced in the cytosol, which contains high concentrations of thiol, for example, glutathione. In certain embodiments, the plasma stability of a linker comprising a chemically unstable group can be increased by introducing steric hindrance using substituents close to the chemically unstable group.

[00167] Acid-unstable groups, such as hydrazones, remain intact during systemic circulation in the neutral pH environment of blood (pH 7.3 to 7.5) and undergo hydrolysis and release the drug once the ADC is internalized within the mildly acidic endosome (pH 5.0 to 6.5) and lysosomal compartments (pH 4.5 to 5.0) of the cell. These pH-dependent release mechanisms have been associated with nonspecific drug release. To increase the stability of the hydrazone group of the linker, the linker can be varied by chemical modification, for example, substitution, allowing tuning to achieve more efficient release in the lysosome with minimized loss in circulation.

[00168] Hydrazone-containing linkers may contain additional cleavage sites, such as cleavage sites unstable to additional acid and / or enzymatically unstable cleavage sites. Exemplary ADCs including hydrazone-containing linkers include the following structures: Petition 870200050086, dated 04 / 22 / 2020, pages 69 / 209 / 172 where D and Ab represent the cytotoxic and / or cytostatic agent (drug) and Ab, respectively, represents the number of drug-binding ligands bound to the antibody. In certain ligands such as (Ig) ligand, the ligand comprises two cleavable groups – a disulfide moiety and a hydrazone moiety. For such ligands, effective release of the unmodified free drug requires acidic pH or disulfide reduction and acidic pH. Ligands such as (Ih) and (li) have been shown to be effective with a single hydrazone cleavage site.

[00169] Additional linkers that remain intact during systemic circulation and undergo hydrolysis and release the drug when the ADC is internalized within acidic cellular compartments include carbonates. Such linkers may be useful in cases where the cytotoxic and / or cytostatic agent can be covalently bound via an oxygen atom.

[00170] Other acid-stable groups that can be included in linkers include cis-aconityl-containing linkers. The chemistry of cis-aconityl Petition 870200050086, dated 22 / 04 / 2020, p. 70 / 209 / 172 uses a carboxylic acid juxtaposed to an amide bond to accelerate amide hydrolysis under acidic conditions.

[00171] Cleavable linkers may also include a disulfide group. Disulfides are thermodynamically stable at physiological pH and are designed to release the drug upon internalization within cells, where the cytosol provides a significantly more reducing environment compared to the extracellular environment. Cleavage of disulfide bonds generally requires the presence of a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH), such that disulfide-containing linkers are reasonably stable in circulation, selectively releasing the drug into the cytosol. The intracellular protein disulfide isomerase enzyme or similar enzymes capable of cleaving disulfide bonds may also contribute to the preferential cleavage of disulfide bonds within cells.GSH is reported to be present in cells in the concentration range of 0.5 to 10 mM compared to a significantly lower concentration of GSH or cysteine, the most abundant low molecular weight thiol, in circulation in approximately 5 tumor cells, where irregular blood flow leads to a hypoxic state, resulting in enhanced activity of reductive enzymes and therefore even higher glutathione concentrations. In certain embodiments, the in vivo stability of a disulfide-containing linker can be enhanced by chemical modification of the linker, for example, the use of steric hindrance adjacent to the disulfide bond.

[00172] ADCs including disulfide-containing linkers include the following structures: Petition 870200050086, dated 22 / 04 / 2020, page 71 / 209 / 172 (Ij) (II) where D and Ab represent the drug and antibody, respectively, n represents the number of drug-linkers linked to the antibody, and R is independently selected from each occurrence of hydrogen or alkyl, for example. In certain embodiments, increasing the steric hindrance adjacent to the disulfide bond increases the stability of the ligand. Structures such as (Ij) and (Il) show increased in vivo stability when one or more R groups are selected from a lower alkyl such as methyl.

[00173] Another type of cleavable linker that can be used is a linker that is specifically cleaved by an enzyme. Such linkers are typically peptide-based or include peptide regions that act as substrates for enzymes. Peptide-based linkers tend to be more stable in plasma and extracellular media than chemically unstable linkers. Peptide linkers generally have good serum stability, whereas lysosomal proteolytic enzymes have very low activity in the blood due to endogenous inhibitors and the unfavorably high pH value of blood compared to lysosomes. The release of a drug from a Petition 870200050086, dated 04 / 22 / 2020, page 72 / 209 / 172: antibody production occurs specifically due to the action of lysosomal proteases, for example, cathepsin and plasmin. These proteases may be present at elevated levels in certain tumor cells.

[00174] In exemplary embodiments, the cleavable peptide is selected from tetrapeptides such as Gly-Phe-Leu-Gly (SEQ ID NO: 128), Ala-LeuAla-Leu (SEQ ID NO: 129) or dipeptides such as Val-Cit, Val-Ala, Met(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D)Asp 5, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Asn(D)Lys, AM Met-(D)Lys, Asn-(D)Lys, AW Met-(D)Lys and Asn-(D)Lys. In certain embodiments, dipeptides are preferred over longer polypeptides due to the hydrophobicity of the longer peptides.

[00175] A variety of cleavable dipeptide-based linkers useful for linking drugs such as doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepine, thalissomycin, and auristatin / auristatin family members to antibodies has been described (see, Dubowchik et al., 1998, J. Org. Chem. 67: 1866-1872; Dubowchik et al., 1998, Bioorg. Med. Chem. Lett. 8(21): 3341-3346; Walker et al., 2002, Bioorg. Med. Chem. Lett. 12: 217-219; Walker et al., 2004, Bioorg. Med. Chem. Lett. 14: 4323-4327; Sutherland et al., 2013, Blood 122: 1455-1463 and Francisco et al., 2003, Blood 102: 1458-1465, each of which is incorporated herein by reference). All of these dipeptide linkers, or modified versions of these dipeptide linkers, can be used in the anti-glyco-MUC1 ADCs described.Other dipeptide linkers that can be used include those found in ADCs such as Brentuximab Vendotin SGN-35 from Seattle Genetics (Adcetris®), SGN-75 from Seattle Genetics (anti-CD-70, Val-Cyt-monomethyl auristatin F (MMAF)), SGN-CD33A from Seattle Genetics (anti-CD33, Val-Ala-(SGD-1882)), glembatumumab from Celldex Therapeutics (CDX011) (anti-NMB, Val-Cyt-monomethyl auristatin E (MMAE)) and PSMA-ADC from [unclear]. Petition 870200050086, dated 04 / 22 / 2020, p. 73 / 209 / 172 Cytogen (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).

[00176] Enzymatically cleavable linkers may include a self-immolating spacer to spatially separate the drug from the enzymatic cleavage site. Direct binding of a drug to a peptide linker can result in proteolytic release of an amino acid adduct from the drug, thereby impairing its activity. The use of a self-immolating spacer allows for the elimination of the fully active, chemically unmodified drug upon hydrolysis of the amide bond.

[00177] A self-immolating spacer is the bifunctional para-aminobenzyl alcohol group, which is linked to the peptide via the amino group, forming an amide bond, while amine-containing drugs can be attached via carbamate functionalities to the benzyl hydroxyl group of the linker (PABC). The resulting prodrugs are activated by protease-mediated cleavage, leading to a 1,6 elimination reaction releasing the unmodified drug, carbon dioxide, and linker group remnants. The following diagram represents the fragmentation of the para-aminobenzyl ether and the release of the drug: Petition 870200050086, dated 04 / 22 / 2020, pp. 74 / 209 / 172 HN where XD represents the unmodified drug.

[00178] Heterocyclic variants of this autoimmolating group have also been described. See, for example, U.S. Pat. No. 7,989,434, incorporated herein by reference.

[00179] In some embodiments, the enzymatically cleavable linker is a β-glucuronic acid-based linker. Easy drug release can be achieved through cleavage of the β-glucuronide glycosidic bond by the lysosomal enzyme β-glucuronidase. This enzyme is abundantly present within lysosomes and is overexpressed in some tumor types, although enzymatic activity outside cells is low. β-glucuronic acid-based linkers can be used to circumvent the tendency of an ADC to undergo aggregation due to the hydrophilic nature of β-glucuronides. In some embodiments, β-glucuronic acid-based linkers are preferred as linkers for ADCs bound to hydrophobic drugs. The following scheme represents the drug release from a Petition 870200050086, dated 04 / 22 / 2020, page 75 / 209 / 172 linker based on β-glucuronic acid and containing ADC:

[00180] A variety of cleavable linkers based on β-glucuronic acid useful for linking drugs such as auristatins, camptothecin and doxorubicin analogs, CBI minor groove binders and psimberin to antibodies has been described (see, see Nolting, Chapter 5 “Linker Technology in Antibody-Drug Conjugates”, In: Antibody-Drug Conjugates: Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), Springer Science & Business Medica, LLC, 2013; Jeffrey et al., 2006, Bioconjug. Chem. 17: 831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280; and Jiang et al., 2005, J. Am. Chem. Soc. 127:11254-11255, Petition 870200050086, dated 22 / 04 / 2020, pp. 76 / 209 / 172, each of which is incorporated herein by reference). All of these β-glucuronic acid-based ligands can be used in the anti-glycoMUC1 ADCs described.

[00181] Additionally, cytotoxic and / or cytostatic agents containing a phenol group can be covalently linked to a linker via the phenolic oxygen. One such linker, described in WO 2007 / 089149, employs a methodology in which a diaminoethane “SpaceLink” is used in conjunction with traditional “PABO”-based self-immolating groups to release phenols. The linker cleavage is schematically represented below, where D represents a cytotoxic and / or cytostatic agent having a The lysosomal enzyme in SpaceLink is a cyclic urea.

[00182] Cleivable linkers may include non-cleivable portions or segments Petition 870200050086, dated 22 / 04 / 2020, page 77 / 209 / 172 Cleivable and / or cleivable segments or portions may be included in an otherwise non-cleivable linker to make it cleivable. By way of example only, polyethylene glycol (PEG) and related polymers may include cleivable groups in the polymer chain. For example, a polyethylene glycol or polymer linker may include one or more cleivable groups such as a disulfide, a hydrazone, or a dipeptide.

[00183] Other degradable linkages that may be included in the linkers include ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups in a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulting from the reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; acetal linkages which are the product of the reaction of an aldehyde and an alcohol; orthoester linkages which are the product of the reaction of a formate and an alcohol; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.

[00184] In certain embodiments, the linker comprises an enzymatically cleavable peptide portion, for example, a linker comprising the structural formulas (IVa) or (IVb):. Petition 870200050086, dated 04 / 22 / 2020, pp. 78 / 209 74 / 172 or a salt thereof, wherein: peptide represents a peptide (illustrated C—>N and not showing the carboxy and amino “terminals”) cleavable by a lysosomal enzyme; T represents a polymer comprising one or more ethylene glycol units or an alkylene chain or combinations thereof; Ra is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; p is an interior number ranging from 0 to 5; q is O₁₀; x is O₁₀; y is O₁₀; represents the point of attachment of the linker to a cytotoxic and / or cytostatic agent and * represents the point of attachment to the rest of the linker.

[00185] In certain embodiments, the peptide is selected from a tripeptide or a dipeptide. In particular embodiments, the dipeptide is selected from: Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; CitAsn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Val-Lys; Ala-Lys; Phe-Cit; Leu-Cit; IleCit; Phe-Arg and Trp-Cit. In certain embodiments, the dipeptide is selected from: Cit-Val and Ala-Val.

[00186] The specific exemplary forms of linkers according to the structural formula (IVa) that may be included in the anti-glycoMUC1 ADCs of the description include the linkers illustrated below (as illustrated, Petition 870200050086, dated 04 / 22 / 2020, page 79 / 209 / 172 (the linkers include a group suitable for covalently linking the linker to an antibody): (IVa.2)0(IVa.1) Petition 870200050086, dated 04 / 22 / 2020, pages 80 / 209 / 172

[00187] Specific exemplary forms of connectors according to with the structural formula (IVb) that can be included in the anti-glycoMUC1 ADCs of the description include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody): Petition 870200050086, dated 04 / 22 / 2020, p. 81 / 209 / 172 Petition 870200050086, dated 04 / 22 / 2020, p. 82 / 209 / 172 (IVb.9) (IVb.10) HO—S=O O (IVb.11) THE HN NH NH THE NH2 HO—S=O O (IVb.12) THE HN NH NH THE NH2 Petition 870200050086, dated 04 / 22 / 2020, p. 83 / 209 / 172 OH O J. ° ι^0 ° \ ιτ < ' I η \^Ν / ''Μ. / 'Μ. f Ν Τΐ Ν II Η = Η ο Ο ΝΗ O^^NHj Ο Λ-Ζ° Τ 1 υ ΓΤ ' If ν Τΐ γ ν II η 1 Η Ο ο L ΗΝ^ HjN^^O ο θ ο ÃJJ\Â ÁJ / / Ί< f ΊΤ Ο Η 1 Η • ..... ΝΗ 9 Η \\ Η ? Η / Ν Tf ^f Ν >f ^f Ν ν / I Η 1 I Η V-Αν ο L o L Ό SO3 A 'NH o^^nh2 (IVb.13) 0 (IVb.14) z (IVb.15) Ju (IVb.16) 0 pA Petition 870200050086, dated 04 / 22 / 2020, page 84 / 209 / 172 In certain forms, the linker comprises a portion of

[00188] enzymatically cleavable peptide, for example, a linker comprising the structural formula (IVc) or (IVd): THE Raou a salt of the same kind, in which: Peptide represents a peptide (illustrated C > N and not showing the carboxy and amino "terminals") that can be cleaved by a lysosomal enzyme; T represents a polymer. Petition 870200050086, dated 22 / 04 / 2020, p. 85 / 209 / 172 comprising one or more ethylene glycol units or an alkylene chain or combinations thereof; Ra is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; p is an integer ranging from 0 to 5; q is Ooul; x is Ooul; y is Ooul; x represents the attachment point of the linker to a cytotoxic and / or cytostatic agent and * represents the attachment point to the rest of the linker.

[00189] Specific exemplary forms of linkers according to the structural formula (IVc) that may be included in the anti-glycoMUC1 ADCs of the description include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody): Petition 870200050086, dated 04 / 22 / 2020, p. 86 / 209 / 172 Specific exemplary types of connectors according to

[00190] with structural formula (IVd) that may be included in the anti-glycoMUC1 ADCs of the description include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody): Petition 870200050086, dated 04 / 22 / 2020, p. 87 / 209 / 172 Petition 870200050086, dated 04 / 22 / 2020, pp. 88 / 209 / 172

[00191] In certain embodiments, the ligand comprising the structural formulas (IVa), (IVb), (IVc) or (IVd) additionally comprises a carbonate moiety that is cleavable by exposure to an acidic medium. In particular embodiments, the ligand is fixed via an oxygen atom to a cytotoxic and / or cytostatic agent. 5.3.4. Non-cleavable ligands

[00192] Although cleavable linkers may provide certain advantages, linkers comprising the anti-glyco-MUC1 ADC described need not be cleavable. For non-cleavable linkers, drug release does not depend on the differential properties between plasma and some cytoplasmic compartments. Drug release is postulated to occur after internalization of the ADC via antigen-mediated endocytosis and delivery to the lysosomal compartment, where the antibody is degraded to the amino acid level through intracellular proteolytic degradation. This process releases a drug derivative, which is formed by the drug, the linker, and the amino acid residue to which the linker was covalently attached. Amino acid drug metabolites of conjugates with non-cleavable linkers are more hydrophilic and generally Petition 870200050086, dated 22 / 04 / 2020, p. 89 / 209 / 172 less permeable in the membrane, which leads to fewer bystander effects and less non-specific toxicities compared to conjugates with a cleavable linker. Overall, ADCs with non-cleavable linkers have greater stability in circulation than ADCs with cleavable linkers. Non-cleavable linkers can be alkylene chains or can be polymeric in nature, such as, for example, based on polyalkylene glycol polymers, amide polymers, or may include alkylene chain segments, polyalkylene glycols and / or amide polymers.

[00193] A variety of non-cleavable linkers used to bind drugs to antibodies have been described. See Jeffrey et al., 2006, Bioconjug. Chem. 17; 831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17: 2278-2280 and Jiang et al., 2005, J. Am. Chem. Soc. 127: 11254-11255, each of which is incorporated herein by reference. All of these linkers may be included in the anti-glyco-MUC1 ADCs described.

[00194] In certain embodiments, the linker is not cleavable in vivo, for example a linker conforming to the structural formulas (VIa), (VIb), (VIc) or (VId) (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody: (Via)

[00195] or salts thereof, wherein: Ra is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; Rx is a portion including a group Petition 870200050086, dated 04 / 22 / 2020, pp. 90-209 86 / 172 functional group capable of covalently binding the linker to an antibody and represents the attachment point of the linker to a cytotoxic and / or cytostatic agent.

[00196] The specific exemplary embodiments of linkers according to structural formula (Vla)-(VId) that may be included in the antiglyco-MUCl ADCs of the description include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody and / represents the attachment point to an agent). (Vld.3) 5.3.5. Groups Used to Attach Ligands to Antibodies Petition 870200050086, dated 04 / 22 / 2020, pp. 91 / 209 / 172

[00197] A variety of groups can be used to attach drug-linker syntheses to antibodies to produce ADCs. The attachment groups can be electrophilic in nature and include: maleimide groups, activated disulfides, activated esters such as NHS esters and HOBt esters, haloformates, acid halides, alkyl and benzyl halides such as haloacetamides. As discussed below, there are also emerging technologies related to “self-stabilizing” and “bridge-linking disulfide” maleimides that can be used as described. The specific group used will depend, in part, on the antibody attachment site.

[00198] An example of a “self-stabilizing” maleimide group that spontaneously hydrolyzes under antibody conjugation conditions to give an ADC species with improved stability is represented in the schematic below. See US20130309256 A1; also Lyon et al., Nature Biotech published online, doi: 10.1038 / nbt.2968. Normal system: Petition 870200050086, dated 04 / 22 / 2020, pp. 92 / 209 / 172 This leads to "loss of DAR" over time. SGN MalDPR (maleimido dipropylamido) system: Spontaneous at pH 7.4 stable in plasma (slow retro-hetero-Michael reaction shown above)

[00199] Politherics described a method for bridging a Petition 870200050086, dated 04 / 22 / 2020, pp. 93-209 89 / 172 pair of sulfhydryl groups derived from the reduction of a native hinge disulfide bond. See, Badescu et al., 2014, Bioconjugated Chern. 25: 1124-1136. The reaction is represented in the schematic below. An advantage of this methodology is the ability to synthesize DAR4-enriched ADCs by the total reduction of IgGs (to give 4 sulfhydryl pairs) followed by reaction with 4 equivalents of the alkylating agent. ADCs containing “bridged disulfides” are also claimed to have increased stability. "bridged disulfide"

[00200] Similarly, as represented below, a maleimide derivative (1, below) that is capable of bridging a pair of groups Petition 870200050086, dated 04 / 22 / 2020, pp. 94 / 209 90 / 172 sulfhydryl has been developed. See WO2013 / 085925. 5.3.6. Ligature Selection Considerations

[00201] As is known to experienced practitioners, the linker selected for a particular ADC can be influenced by a variety of factors, including but not limited to the antibody binding site (e.g., lys, cys, or other amino acid residues), structural constraints of the drug pharmacophore, and the drug's lipophilicity. The specific linker selected for an ADC must seek to balance these different factors for the specific antibody / drug combination. For a review of the factors that are influenced by the choice of linkers in ADCs, see Nolting, Chapter 5 “Linker Technology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates: Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), Springer Science & Business Medica, LLC, 2013.

[00202] For example, ADCs have been observed to effect the extermination of antigen-negative bystander cells present in the vicinity of antigen-positive tumor cells. The mechanism of bystander cell extermination by ADCs has indicated that metabolic products formed during the intracellular processing of ADCs may play a role. Neutral cytotoxic metabolites generated by the metabolism of ADCs in antigen-positive cells appear to play a role in bystander cell extermination, while charged metabolites may be prevented from diffusing across the membrane into the medium and therefore may not affect bystander extermination. In certain Petition 870200050086, dated 04 / 22 / 2020, page 95 / 209 / 172 In certain modes, the linker is selected to attenuate the bystander killing effect caused by ADC cellular metabolites. In certain modes, the linker is selected to increase the bystander killing effect.

[00203] The properties of the linker can also impact ADC aggregation under conditions of use and / or storage. Typically, ADCs reported in the literature contain no more than 3 to 4 drug molecules per antibody molecule (see, for example, Chari, 2008, Acc Chem Res 41: 98-107). Attempts to obtain higher drug-to-antibody ratios (“DAR”) have frequently failed, particularly if both the drug and the linker were hydrophobic, due to ADC aggregation (King et al., 2002, J Med Chem 45: 4336-4343; Hollander et al., 2008, Bioconjugate Chem 19: 358-361; Burke et al., 2009, Bioconjugate Chem 20: 1242-1250). In many cases, DARs higher than 3 to 4 would be beneficial as a means of increasing power.In cases where the cytotoxic and / or cytostatic agent is hydrophobic in nature, it may be desirable to select linkers that are relatively hydrophilic as a means of reducing ADC aggregation, especially in cases where DARS greater than 3 to 4 are desired. Thus, in certain embodiments, the linker incorporates chemical moieties that reduce ADC aggregation during storage and / or use. A linker may incorporate polar or hydrophilic groups such as charged groups or groups that become charged under physiological pH to reduce ADC aggregation. For example, a linker may incorporate charged groups such as salts or groups that deprotonate, for example, carboxylates or protonate, for example, amines, at physiological pH.

[00204] Exemplary polyvalent linkers that have been reported to produce DARs as high as 20 that can be used to link numerous cytotoxic and / or cytostatic agents to a single antibody are described in WO 2009 / 073445; WO 2010 / 068795; WO 2010 / 138719; WO 2011 / 120053; WO Petition 870200050086, dated 04 / 22 / 2020, pages 96 / 209 / 172 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379; WO 2014 / 093394; WO 2014 / 093640, the contents of which are incorporated herein by reference in their entirety.

[00205] In particular embodiments, the aggregation of ADCs during storage or use is less than about 10% as determined by size exclusion chromatography (SEC). In particular embodiments, the aggregation of ADCs during storage or use is less than 10%, such as less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1% or even lower, as determined by size exclusion chromatography (SEC). 5.3.7. MANUFACTURING METHODS OF ANTI-GLYCO-MUC1 ADCS

[00206] The anti-glyco-MUC1 ADCs described can be synthesized using well-known chemistries. The chemistries selected will depend, among other things, on the identity of the cytotoxic and / or cytostatic agent(s), the linker, and the groups used to attach the linker to the antibody. Generally, ADCs according to formula (I) can be prepared according to the following scheme: DL-Rx+Ab-RyMD-L-XY]n-Ab (I) where D, L, Ab, XY and n are as previously defined and Rx and Ry represent complementary groups capable of forming a covalent bond with each other, as discussed above.

[00207] The identities of the Rx and Rx groups will depend on the chemistry used to link the DL-Rx synton to the antibody. Generally, the chemistry used should not alter the integrity of the antibody, for example its ability to bind its target. Preferably, the binding properties of the conjugated antibody closely resemble those of the unconjugated antibody. A variety of chemistries and techniques for conjugating molecules to biological molecules such as antibodies are known in the art and in particular to Petition 870200050086, of 04 / 22 / 2020, p. 97 / 209 / 172 antibodies, are well known. See, for example, Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. Eds., Alan R. Liss, Inc., 1985; Hellstrom et al., “Antibodies For Drug Delivery,” in: Controlled Drug Delivery, Robinson et al. Eds., Marcel Dekker, Inc., 2nd Ed. 1987; Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in: Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., Eds., 1985; “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” in: Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al., Eds., Academic Press, 1985; Thorpe et al., 1982, Immunol. Rev. 62:119-58; publication PCT WO 89 / 12624. Any of these chemistries can be used to bind the synthons to an antibody.

[00208] Several useful chemical Rxe functional groups for linking synthons to accessible lysine residues are known and include, by way of example and not limitation, NHS esters and isothiocyanates.

[00209] Several chemical functional groups useful for attaching synthons to accessible free sulfhydryl groups of cysteine ​​residues are known and include, by way of example and not limited to, haloacetyls and maleimides.

[00210] However, conjugation chemistries are not limited to available side chain groups. Side chains such as amines can be converted to other useful groups, such as hydroxyls, by attaching an appropriate small molecule to the amine. This strategy can be used to increase the number of available binding sites on the antibody by conjugating multifunctional small molecules to the side chains of accessible amino acid residues of the antibody. Rx functional groups suitable for covalently linking the synthons to these “converted” functional groups are then included in the synthons.

[00211] The antibody can also be engineered to include Petition 870200050086, dated 04 / 22 / 2020, p. 98 / 209 / 172 amino acid residues for conjugation. An approach to engineering antibodies to include non-genetically encoded amino acid residues useful for conjugating drugs in the context of ADCs is described by Axup et al., 2012, Proc Natl Acad Sci USA. 109(40): 16101-16106, as are the chemistries and functional groups useful for binding synthons to non-encoded amino acids.

[00212] Typically, synthons are linked to the side chains of antibody amino acid residues, including, for example, the primary amino group of accessible lysine residues or the sulfhydryl group of accessible cysteine ​​residues. Free sulfhydryl groups can be obtained by reducing the interchain disulfide bond.

[00213] For linkages where Ry is a sulfhydryl group (for example, when Rx is a maleimide), the antibody is usually first fully or partially reduced to break the interchain disulfide bridges between cysteine ​​residues.

[00214] Cysteine ​​residues that do not participate in disulfide bridges can be engineered into an antibody by mutating one or more codons. Reduction of these unpaired cysteines produces a sulfhydryl group suitable for conjugation. Preferred positions for incorporating engineered cysteines include, by way of example and not limitation, positions S112C, S113C, A114C, S115C, A176C, S180C, S252C, V286C, V292C, S357C, A359C, S398C, S428C (Kabat numbering) on ​​the human IgG1 heavy chain and positions V110C, S114C, S121C, S127C, S168C, V205C (Kabat numbering) on ​​the human Ig kappa light chain (see, for example, U.S. Pat. No. 7,521,541, U.S. Pat. No. 7,855,275 and U.S. Pat. No. 8,455,622).

[00215] As will be assessed by experienced professionals, the number of cytotoxic and / or cytostatic agents bound to an antibody molecule can vary, such that a collection of ADCs may be heterogeneous in nature, Petition 870200050086, dated 22 / 04 / 2020, pp. 99 / 209 / 172 where some antibodies contain one bound agent, some two, some three, etc. (and some none). The degree of heterogeneity will depend, among other things, on the chemistries used to bind the cytotoxic and / or cytostatic agents. For example, where antibodies are reduced to produce sulfhydryl groups for fixation, heterogeneous mixtures of antibodies having zero, 2, 4, 6, or 8 bound agents per molecule are frequently produced. Furthermore, limiting the molar ratio of fixation compound, antibodies having zero, 1, 2, 3, 4, 5, 6, 7, or 8 bound agents per molecule are frequently produced. Thus, it will be understood that depending on the context, established DARs may be averages for a collection of antibodies.For example, “DAR4” may refer to an ADC preparation that has not been purified to isolate specific DAR peaks and may comprise a heterogeneous mixture of ADC molecules having different numbers of antibody-bound cytostatic and / or cytotoxic agents (e.g., 0, 2, 4, 6, 8 agents per antibody), but has an average drug-to-antibody ratio of 4. Similarly, in some embodiments, “DAR2” refers to a heterogeneous ADC preparation in which the average drug-to-antibody ratio is 2.

[00216] When enriched preparations are desired, antibodies having defined numbers of bound cytotoxic and / or cytostatic agents can be obtained by purifying heterogeneous mixtures, for example, via column chromatography, for example, hydrophobic interaction chromatography.

[00217] Purity can be assessed by a variety of methods, as is known in the art. As a specific example, an ADC preparation can be analyzed by means of HPLC or other chromatography and the purity assessed by analyzing areas under the resulting peak curves. 5.4. Chimeric Antigen Receptors

[00218] The present description provides chimeric antigen receptors. Petition 870200050086, dated 22 / 04 / 2020, pp. 100 / 209 / 172 (CARs) comprising the anti-glyco-MUC1 antibodies or antigen-binding fragments described herein.

[00219] The CARs described typically comprise an extracellular domain operably linked to a transmembrane domain which is in turn operably linked to an intracellular domain for signaling.

[00220] The extracellular domains of the CARs described comprise the sequence of an anti-glyco-MUC1 antibody or antigen-binding fragment (e.g., as described in Section 0 or embodiments 1 to 0).

[00221] Exemplary transmembrane domain sequence and intracellular domain sequences are described in Sections 0 and 0, respectively.

[00222] Several fusion proteins described herein (e.g., embodiments 0 and 0 to 0) are CARs, and the descriptions related to CARs apply to such fusion proteins. 5.4.1. Transmembrane Domain

[00223] With respect to the transmembrane domain, the CAR can be designed to comprise a transmembrane domain that is operably linked (e.g., fused) to the extracellular domain of the CAR.

[00224] The transmembrane domain may be derived from a natural or synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane regions of particular use in this description may be derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some cases, a variety of human hinges may also be used, including the Petition 870200050086, dated 04 / 22 / 2020, page 101 / 209 / 172 hinge of human Ig (immunoglobulin).

[00225] In one embodiment, the transmembrane domain is synthetic (i.e., non-naturally occurring). Examples of synthetic transmembrane domains are peptides comprising predominantly hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[00226] In one embodiment, the transmembrane domain in the CAR of the description is the CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence ILHLGALGRDLWGPSPVTGYHPLL.

[00227] In one embodiment, the transmembrane domain in the CAR of the description is the CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV.

[00228] In some cases, the transmembrane domain of the CAR described comprises the CD8a hinge domain. In one embodiment, the CD8a hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC. 5.4.2. Intracellular Domain

[00229] The CAR intracellular signaling domain described is responsible for activating at least one of the normal effector functions of the immune cell in which CAR is expressed. The term “effector function” refers to a specialized function of a cell. The effector function of a T cell, for example, may be cytolytic activity or helper activity including Petition 870200050086, dated 04 / 22 / 2020, pp. 102 / 209 / 172 cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. Although usually the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[00230] Examples of preferred intracellular signaling domains for use in the CAR description include cytoplasmic sequences of the T cell receptor (TCR) and coreceptors that act together to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[00231] The signals generated through the TCR alone may be insufficient for full T cell activation, and a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[00232] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory mode. The primary cytoplasmic signaling sequences that Petition 870200050086, dated 04 / 22 / 2020, p. 103 / 209 / 172 act in a stimulatory manner and may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[00233] Examples of primary cytoplasmic signaling sequences containing ITAMs that are of particular use in the CARs described include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR described comprises a CD3zeta cytoplasmic signaling sequence.

[00234] In a preferred embodiment, the cytoplasmic domain of the CAR is designed to include a domain of primary cytoplasmic signaling sequences containing ITAM (e.g., that of CD3-zeta) alone or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR description. For example, the cytoplasmic domain of the CAR may include a portion of the CD3 zeta chain and a co-stimulatory signaling region.

[00235] The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA1), CD2, CD7, LEVE, NKG2C, B7-H3, and a ligand that binds specifically to CD83 and similar molecules.

[00236] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR description can be linked together in a random or specified order. Optionally, an oligolinker Petition 870200050086, dated 04 / 22 / 2020, pp. 104 / 209 A 100 / 172 or short polypeptide, preferably between 2 and 10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[00237] In one embodiment, the cytoplasmic domain comprises the CD3-zeta signaling domain and the CD28 signaling domain. In another embodiment, the cytoplasmic domain comprises the CD3-zeta signaling domain and the 4-1BB signaling domain. 5.5 Nucleic Acids, Recombinant Vectors, and Host Cells

[00238] The present description covers nucleic acid molecules encoding immunoglobulin light and heavy chain genes for anti-glyco-MUC1 antibodies, vectors comprising such nucleic acids, and host cells capable of producing the anti-glyco-MUC1 antibodies of the description. In certain aspects, the nucleic acid molecules encode, and the host cells are capable of expressing, the anti-glyco-MUC1 antibodies and antibody-binding fragments of the description (e.g., as described in Section 0 and embodiments 0 to 0) as well as fusion proteins (e.g., as described in embodiments 0 to 0) and chimeric antigen receptors (e.g., as described in Section 0 and embodiments 0 to 0) containing the same. Exemplary vectors of the description are described in embodiments 0 to 0 and exemplary host cells are described in embodiments 0 to 0.

[00239] An anti-glyco-MUC1 antibody of the description can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express a recombinant antibody, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, optionally, secreted into the medium in which the host cells are cultured, from which the antibodies can be recovered. Petition 870200050086, dated 04 / 22 / 2020, pages 105 / 209 101 / 172 Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes within recombinant expression vectors, and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM et al., eds., Greene Publishing Associates, 1989) and in Pat. US No. 4,816,397.

[00240] To generate nucleic acids encoding such anti-glyco-MUC1 antibodies, DNA fragments encoding the variable region of light and heavy chains are first obtained. These DNAs can be obtained by amplifying and modifying germline DNA or cDNA encoding variable sequences of heavy and light chains, for example using the polymerase chain reaction (PCR). The germline DNA sequences for the human heavy and light chain variable region genes are known in the art (See, for example, the human germline sequence database “VBASE”; see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J. Mol. Biol. 22T: 116-198 and Cox et al., 1994, Eur. J. Immunol. 24: 827-836; the contents of each of which are incorporated herein by reference).

[00241] Once the VH and VL segments related to the anti-glyco-MUC1 antibody-encoding DNA fragments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes, or to an scFv gene. In these manipulations, a VH or VL-encoding DNA fragment is operatively ligated to another Petition 870200050086, dated 04 / 22 / 2020, pages 106 / 209 102 / 172 DNA fragment encoding another protein, such as a constant region of an antibody or a flexible linker. The term “operatively linked,” as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in the frame.

[00242] Isolated DNA encoding the VH region can be converted to a full-size heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, CH3, and optionally CH4). The sequences of the human heavy chain constant region genes are known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but in certain embodiments it is an IgG1 or IgG4 constant region.For a Fab fragment heavy chain gene, the DNA encoding VH can be operatively linked to another DNA molecule encoding only the CH1 constant region of the heavy chain.

[00243] Isolated DNA encoding the VL region can be converted to a full-size light chain gene (as well as a Fab light chain gene) by operatively ligating the VL-encoding DNA to another DNA molecule encoding the constant light chain region, CL. The sequences of the human constant light chain region genes are known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by PCR amplification. Petition 870200050086, dated 04 / 22 / 2020, pages 107 / 209 103 / 172 standard. The constant light chain region can be a constant kappa or lambda region, but in certain embodiments it is a constant kappa region.

[00244] To create an scFv gene, the DNA fragments encoding VH and VL can be operatively linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4~Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-stranded protein, with the VH and VL regions joined by the flexible linker (See, for example, Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., 1990, Nature 348: 552-554).

[00245] To express the anti-glyco-MUC1 antibodies described, DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to the transcriptional and translational control sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is linked into a vector such that the transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and the expression control sequences are chosen to be compatible with the expression of the host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.

[00246] Antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector or blunt-end ligation if neither restriction site is present). Before insertion of the light or heavy chain sequences related to Petition 870200050086, dated 04 / 22 / 2020, pp. 108 / 209 104 / 172 anti-glyco-MUC1 antibody, the expression vector may already carry the antibody constant region sequences. For example, one approach to converting the VH and VL sequences related to the anti-glyco-MUC1 monoclonal antibody into full-size antibody genes is to insert them into expression vectors already encoding the heavy chain constant and light chain constant regions, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates the secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is frame-linked to the amino terminus of the antibody chain gene.The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (that is, a signal peptide from a protein other than immunoglobulin).

[00247] In addition to antibody chain genes, the recombinant expression vectors described carry regulatory sequences that control the expression of antibody chain genes in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990. It will be assessed by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, etc. The appropriate regulatory sequences for expression Petition 870200050086, dated 04 / 22 / 2020, pp. 109 / 209 105 / 172 in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus late major promoter (AdMLP)), and polyome. For further description of viral regulatory elements and their sequences, see, for example, U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al., and U.S. Pat. No. 4,968,615 by Schaffner et al.

[00248] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors described may carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (See, for example, U.S. Pats. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate in a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofoliate reductase (DHFR) gene (for use in DHFR-selected / methotrexate-amplified host cells) and the neo gene (for G418 selection).For the expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is / are transfected into a host cell using standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, for example, electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and so on. Petition 870200050086, dated 04 / 22 / 2020, pages 110 / 209 106 / 172 similar.

[00249] It is possible to express the antibodies described in prokaryotic or eukaryotic host cells. In certain embodiments, antibody expression is carried out in eukaryotic cells, for example, mammalian host cells, ideally secreting an appropriately folded and immunologically active antibody. Exemplary mammalian host cells for expressing the recombinant antibodies described include Chinese Hamster Ovary cells (CHO cells) (including DHFR-CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a selectable DHFR marker, for example, as described in Kaufman and Sharp, 1982, Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells.When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow antibody expression on the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce intact antibody portions, such as Fab fragments or scFv molecules. It is understood that variations on the above procedure are within the scope of the present description. For example, it may be desirable to transfect a host cell with DNA encoding the light chain or the heavy chain (but not both) of an anti-glyco-MUC1 antibody of this description.

[00250] For the expression of a CAR of the description, for example as described in Section 0 and in embodiments 0 and 0, it is preferable that the host cell be a T cell, preferably a human T cell. In some embodiments, the host cell exhibits antitumor immunity. Petition 870200050086, dated 04 / 22 / 2020, pp. 111 / 209 107 / 172 when the cell is crosslinked with MUC1 in a tumor cell. Detailed methods for producing the T cells of the description are described in Section 0

[00251] Recombinant DNA technology can also be used to remove some or all of the DNA encoding each or both of the light and heavy chains that are not required for binding to glyco-MUC1. Molecules expressed from such truncated DNA molecules are also covered by the antibodies of the description.

[00252] For recombinant expression of an anti-glycoMUC1 antibody as described, the host cell can be co-transfected with two expression vectors as described, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. The two vectors may contain identical selectable markers or they may each contain a separate selectable marker. Alternatively, a single vector may be used that encodes both heavy and light chain polypeptides.

[00253] Once a nucleic acid encoding one or more portions of an anti-glyco-MUC1 antibody is established, further alterations or mutations can be introduced within the coding sequence, for example to generate nucleic acids encoding antibodies with different CDR sequences, antibodies with reduced affinity for the Fc receptor, or antibodies of different subclasses.

[00254] The anti-glyco-MUC1 antibodies described can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Variant antibodies can also be generated using a cell-free platform (See, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology, 17: 420-426).

[00255] Once an anti-glyco-MUC1 antibody from the description was Petition 870200050086, dated 04 / 22 / 2020, pp. 112 / 209 108 / 172 produced by recombinant expression, it can be purified by any method known in the art for the purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity and size-sorting column chromatography), centrifugation, differential solubility or by any other standard technique for protein purification. Furthermore, the anti-glyco-MUC1 antibodies of the present description and / or binding fragments can be fused to the heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[00256] Once isolated, the anti-glyco-MUC1 antibody can, if desired, be further purified, for example by high-performance liquid chromatography (see, for example, Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980) or by gel filtration chromatography on a Superdex® 75 column (Pharmacia Biotech AB, Uppsala, Switzerland). 5.5.1. Recombinant Production of CARs in T Cells

[00257] In some embodiments, nucleic acids encoding the anti-glyco-MUC1 CARs described are released into cells using a retroviral or lentiviral vector. Retroviral and lentiviral vectors expressing CARs can be released into different types of eukaryotic cells as well as into tissues and whole organisms using transduced cells as carriers or local or systemic cell-free delivery of encapsulated, bound, or naked vectors. The method used can be for any purpose where stable expression is required or sufficient.

[00258] In other modalities, CAR sequences are delivered into cells using mRNA transcribed in vitro. CAR from mRNA transcribed in vitro can be delivered into different types of eukaryotic cells as well as into tissues and whole organisms using transfected cells as carriers or free local or systemic delivery. Petition 870200050086, dated 04 / 22 / 2020, pp. 113 / 209 109 / 172 encapsulated, bound or naked mRNA cell. The method used can be for any purpose where transient expression is required or sufficient.

[00259] In another embodiment, the desired CAR can be expressed in cells by means of transpons.

[00260] One advantage of the RNA transfection methods described is that RNA transfection is essentially transient and vector-free: an RNA transgene can be delivered to a lymphocyte and expressed there following brief in vitro cell activation, as a minimal expression cassette without the need for any additional viral sequence. Under these conditions, integration of the transgene into the host cell genome is unlikely. Cell cloning is not necessary because of the efficiency of RNA transfection and its ability to uniformly modify the entire lymphocyte population.

[00261] Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) makes use of two different strategies, both of which have been successively tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. Preferably, it is desirable to stabilize IVT-RNA using various modifications in order to obtain prolonged expression of the transferred IVT-RNA.

[00262] Some IVT vectors are known in the literature that are used in a standardized way as a template for in vitro transcription and that have been genetically modified in such a way that stabilized RNA transcripts are produced. Currently used protocols in the technique are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter allowing RNA transcription, followed by a gene of interest that is flanked 3' and / or 5' by untranslated regions (UTRs) and a 3' polyadenyl cassette containing 50-70 A nucleotides. Before in vitro transcription, the circular plasmid is linearized to Petition 870200050086, dated 04 / 22 / 2020, pp. 114 / 209 110 / 172 downstream of the polyadenyl cassette by type II restriction enzymes (the recognition sequence corresponds to the cleavage site). The polyadenyl cassette thus corresponds to the last poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and extend or mask the poly(A) sequence at the 3' end. It is unclear whether this non-physiological projection affects the amount of protein produced intracellularly from such a construct.

[00263] RNA has several advantages over more traditional plasmid or viral approaches. Gene expression from an RNA source does not require transcription, and the protein product is produced rapidly after transfection. Furthermore, since RNA gains access only to the cytoplasm, rather than the nucleus, typical transfection methods result in an extremely high transfection rate. Additionally, plasmid-based approaches require that the promoter directing the expression of the gene of interest be active in the cells under study.

[00264] In another aspect, RNA constructs can be delivered into cells by electroporation. See, for example, the formulations and methodology for electroporation of nucleic acid constructs within mammalian cells as described in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1. The various parameters, including the electric field strength required for electroporation of any known cell type, are generally known in the relevant research literature as well as numerous patents and applications in the field. See, for example, US Pat. No. 6,678,556, US Pat. No. 7,171,264, and US Pat. No. 7,173,116. Devices for the therapeutic application of electroporation are commercially available, for example, the MedPulser® DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.) and are described in patents such as Petition 870200050086, dated 04 / 22 / 2020, pages 115 / 209 111 / 172 U.S. Pat. No. 6,567,694, U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701 and U.S. Pat. No. 6,233,482; electroporation can also be used for in vitro cell transfection as described for example in U.S. 20070128708A1. Electroporation can also be used to deliver nucleic acids into cells in vitro. Consequently, electroporation-mediated delivery into cells of nucleic acids including expression constructs using any of the many available devices and electroporation systems known to those skilled in the art presents an exciting new means of delivering an RNA of interest to a target cell. 5.5.1.1 T-Cell Sources

[00265] Prior to genetic expansion and modification, a source of T cells is obtained from a subject. The term “subject” is intended to include living organisms in which an immune response can be evoked (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subjects are humans.

[00266] T cells can be obtained from various sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. In certain embodiments of the present description, any number of T cell lineages available in the art can be used. In certain embodiments of the present description, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled practitioner, such as Ficoll® separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, and other white blood cells. Petition 870200050086, dated 04 / 22 / 2020, pages 116 / 209 112 / 172 nucleated, red blood cells and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in a buffer or medium appropriate for subsequent processing steps. In one embodiment of the description, the cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the washing solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Again, surprisingly, initial activation steps in the absence of calcium lead to enhanced activation. As those of ordinary skill in the art would readily assess, a washing step can be performed by methods known to those in the art, such as using a semi-automated “direct flow” centrifuge (e.g., the Cobe 2991 cell processor, the Baxter CitoMate, or the Haemonetics Cell Saver 5) according to the manufacturer’s instructions.After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free PBS, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, undesirable components of the apheresis sample can be removed and the cells directly resuspended in culture media.

[00267] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysis of red blood cells and depletion of monocytes, for example, by centrifugation through a PERCOLL® gradient or by counter-flow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28', CD4+, CD8+, CD45RA+ and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 conjugated beads (i.e., 3 x 28), such as DYNABEADS® M-450 CD3 / CD28 T, for a period of time sufficient for positive selection. Petition 870200050086, dated 04 / 22 / 2020, pp. 117 / 209 113 / 172 of the desired T cells. In one embodiment, the time period is approximately 30 minutes. In an additional embodiment, the time period ranges from 30 minutes to 36 hours or longer, and all integer values ​​in between. In another embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In a preferred embodiment, the incubation time period is 24 hours. For the isolation of T cells from leukemia patients, the use of longer incubation times, such as 24 hours, may increase cell production. Longer incubation times may be used to isolate T cells in any situation where there are few T cells compared to other cell types, such as in the isolation of tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals.Furthermore, the use of longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, by simply shortening or lengthening the time T cells are allowed to bind to CD3 / CD28 beads and / or increasing or decreasing the bead-to-T cell ratio (as further described herein), T cell subpopulations can be preferentially selected for or against the start of culture or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, T cell subpopulations can be preferentially selected for or against the start of culture or at other desired time points. The skilled practitioner would recognize that multiple rounds of selection can also be used in the context of this description.In certain modes, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. The "unselected" cells may also be subjected to additional rounds of selection. Petition 870200050086, dated 04 / 22 / 2020, pages 118 / 209 114 / 172

[00268] Enrichment of a T cell population by negative selection can be achieved with a combination of antibodies targeting surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain modalities, it may be desirable to enrich or positively select for regulatory T cells that typically express CD4+, CD25+, CD62Lhl, GITR+, and FoxP3+. Alternatively, in certain modalities, the regulated T cells are depleted by anti-C25 conjugated beads or another similar selection method.

[00269] For the isolation of a desired cell population by positive or negative selection, the concentration of cells and surface (e.g., particles such as granules) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which granules and cells are mixed together (i.e., increase the cell concentration) to ensure maximum cell-granule contact. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml are used. In yet another embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In additional modalities, concentrations of 125 or 150 million cells / ml can be used.The use of concentrations. Petition 870200050086, dated 04 / 22 / 2020, pages 119 / 209 High concentrations of 115 / 172 cells can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells or samples containing many tumor cells (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and would be desirable to obtain. For example, using high cell concentrations allows for more efficient selection of CD8+ T cells that typically have weaker CD28 expression.

[00270] In a related embodiment, it may be desirable to use lower cell concentrations. By significantly diluting the mixture of T cells and surface particles (e.g., particles such as beads), the interactions between the particles and the cells are minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells at diluted concentrations. In one embodiment, the cell concentration used is 5 x 10⁶ / ml. In other embodiments, the concentration used may be from about 1 x 10⁵ / ml to 1 x 10⁶ / ml and any integer value between these.

[00271] In other modes, cells can be incubated in a rotator for varying durations of time at varying speeds from 2 to 10°C, or at room temperature.

[00272] T cells for stimulation can also be frozen after a washing step. Not wishing to be bound by theory, the subsequent freeze-thaw step provides a more uniform product by removing granulocytes and to some extent monocytes from the cell population. After the washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are Petition 870200050086, dated 04 / 22 / 2020, pages 120 / 209 116 / 172 known in the art and will be useful in this context, a method involving the use of PBS containing 20% ​​DMSO and 8% human serum albumin or culture medium containing 10% Dextran 40 and 5% Dextrose, 20% human serum albumin and 7.5% DMSO or 31.25% Plasmalyte-A, 31.25% Dextrose at 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% human serum albumin and 7.5% DMSO or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, the cells are then frozen at -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used, as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[00273] In certain embodiments, cryopreserved cells are thawed and washed as described herein and left to stand for one hour at room temperature before activation using the methods described herein.

[00274] Also considered in the context of the description is the collection of blood samples or apheresis product from a subject at a time prior to when the expanded cells as described herein would be needed. As such, the source of the cells to be expanded can be collected at any point in time and the necessary and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or an apheresis sample is collected from a generally healthy subject. In certain embodiments, a blood sample or an apheresis sample is collected from a generally healthy subject who is at risk of developing a disease but has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain Petition 870200050086, dated 04 / 22 / 2020, pp. 121 / 209 In 117 / 172 modalities, T cells can be expanded, frozen, and used at a later time. In certain modalities, samples are collected from a patient soon after diagnosis of a particular disease as described herein, but before any treatment. In an additional modality, cells are isolated from a blood sample or apheresis of a subject before any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and irradiation.These drugs inhibit calcium-dependent calcineurin phosphatase (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signaling (rapamycin). (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun. 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun. 5: 763-773, 1993). In an additional modality, cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously with, or following) bone marrow or stem cell transplantation or ablative T-cell therapy using chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide.

[00275] In a further embodiment of the present description, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from treatment, the quality of the T cells obtained may be optimal or improved with respect to their ability to expand ex vivo. Petition 870200050086, dated 04 / 22 / 2020, pages 122 / 209 118 / 172 Similarly, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced grafting and in vivo expansion. Thus, within the context of this description, collecting blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase is considered. Furthermore, in certain modalities, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject where repopulation, recirculation, regeneration, and / or expansion of particular cell types are favored, especially during a defined time window following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other immune system cells. 5.5.1.2 T Cell Activation and Expansion

[00276] T cells are activated and expanded generally using methods as described, for example, in U.S. Pats. Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041 and U.S. Patent Application Publication No. 20060121005.

[00277] Generally, the T cells described are expanded by contact with a surface having fixed to it an agent that stimulates a signal associated with the CD3 / TCR complex and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations can be stimulated as described herein, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For the co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be Petition 870200050086, dated 04 / 22 / 2020, pp. 123 / 209 119 / 172 contacted with an anti-CD3 antibody and an anti-CD28 antibody, under appropriate conditions to stimulate T cell proliferation. To stimulate the proliferation of CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8): 3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 1319-1328, 1999; Garland et al., J. Immunol Meth.

[00278] In certain embodiments, the primary stimulatory signal and the co-stimulatory signal to the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in the “cis” formation) or to separate surfaces (i.e., in the “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents may be in solution. In another embodiment, the agents may be in soluble form and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent that will bind to the agents.In this regard, see, for example, US Patent Application Publications Nos. 20040101519 and 20060034810 concerning artificial antigen-presenting cells (aAPCs) that are considered for use in the activation and expansion of T cells in the present description.

[00279] In one embodiment, the two agents are immobilized in the granules, either in the same granule, i.e., “cis,” or in separate granules, i.e., “trans.” By way of example, the agent providing the primary activation signal is Petition 870200050086, dated 04 / 22 / 2020, pages 124 / 209 120 / 172 an anti-CD3 antibody or an antigen-binding fragment thereof and the co-stimulatory signaling agent is an anti-CD28 antibody or an antigen-binding fragment thereof, and both agents are co-immobilized on the same bead in equivalent molecular amounts. In one embodiment, a 1:1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell culture is used. In certain aspects of the present description, a ratio of anti-CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed when compared with the expansion observed using a 1:1 ratio. In a particular embodiment, an increase of about 1 to about 3 times is observed when compared with the expansion observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values ​​in between.In one aspect of the present description, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, that is, the CD3:CD28 ratio is less than one. In certain embodiments of the description, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the granules is greater than 2:1. In one particular embodiment, a CD3:CD28 ratio of 1:100 of antibody bound to the granules is used. In another embodiment, a CD3:CD28 ratio of 1:75 of antibody bound to the granules is used. In a further embodiment, a CD3:CD28 ratio of 1:50 of antibody bound to the granules is used. In another embodiment, a CD3:CD28 ratio of 1:30 of antibody bound to the granules is used. In a preferred embodiment, a CD3:CD28 ratio of 1:10 of antibody bound to the granules is used. In one embodiment, a CD3:CD28 ratio of 1:3 of antibody bound to the granules is used. In yet another embodiment, a CD3:CD28 ratio of 3:1 of antibody bound to the granules is used.

[00280] Particle-to-cell ratios of 1:500 to 500:1 and any integer values ​​in between can be used to stimulate T cells or Petition 870200050086, dated 04 / 22 / 2020, pp. 125 / 209 121 / 172 other target cells. As those of common skill in the technique can easily assess, the particle-to-cell ratio can depend on the particle size relative to the target cell. For example, small-sized granules would only bind a few cells, while larger granules would bind many. In certain embodiments, the cell-to-particle ratio varies from 1:100 to 100:1, and any integer values ​​between these, and in additional embodiments the ratio comprises 1:9 to 9:1, and any integer values ​​between these, can also be used to stimulate T cells. The ratio of particles coupled to anti-CD3 and anti-CD28 to T cells that results in T cell stimulation can vary as mentioned above; however, certain preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with a preferred ratio being... at least 1:1 particles per T cell.In one embodiment, a particle-to-cell ratio of 1:1 or less is used. In a particular embodiment, a preferred particle-to-cell ratio is 1:5. In additional embodiments, the particle-to-cell ratio may be varied depending on the day of stimulation. For example, in one embodiment, the particle-to-cell ratio is 1:1 to 10:1 on the first day, and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of 1:1 to 1:10 (based on cell counts on the day of addition). In a particular embodiment, the particle-to-cell ratio is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis at a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation.In another modality, the particle-to-cell ratio is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In yet another modality, particles are added on a daily or every-other-day basis to a final ratio of... Petition 870200050086, dated 04 / 22 / 2020, pages 126 / 209 122 / 172 1:1 on the first day and 1:10 on the third and fifth days of stimulation. A person skilled in the art will judge that a variety of other ratios may be suitable for use in the present description. In particular, the ratios will vary depending on the particle size and the cell size and type.

[00281] In further embodiments of the present description, cells, such as T cells, are combined with agent-coated beads, the beads and cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased binding of cell surface markers, thereby inducing cell stimulation.

[00282] By way of example, cell surface proteins can be linked by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3 x 28 beads) to contact T cells. In one embodiment, the cells (e.g., 10⁴ to 10⁹ T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). Again, those of average skill in the art can easily assess any cell concentration that can be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample, or the entire sample (i.e., 100%) may comprise the target cell of interest. Consequently, any number of cells is within the scope of the present description.In certain embodiments, it may be desirable to significantly decrease the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum cell-particle contact. For example, in one embodiment, a concentration of approximately 2. Petition 870200050086, dated 04 / 22 / 2020, pages 127 / 209 In one modality, concentrations of 123 / 172 billion cells / ml are used. In another modality, more than 100 million cells / ml are used. In yet another modality, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another modality, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In additional modalities, concentrations of 125 or 150 million cells / ml may be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and would be desirable to obtain in certain modalities.For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which typically have weaker CD28 expression.

[00283] In one embodiment of the present description, the mixture can be cultured for several hours (about 3 hours) to about 14 days or any whole hourly value between these. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the description, the granules and T cells are cultured together for about eight days. In another embodiment, the granules and T cells are cultured together for 2 to 3 days. Multiple stimulation cycles may also be desired such that T cell culture times can be 60 days or more.Appropriate conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI 1640 Medium or X-vivo 15 (Lonza)) which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFe, and TNF-α, or any other additives for cell growth known to the skilled practitioner. Other additives for growth... Petition 870200050086, dated 04 / 22 / 2020, pages 128 / 209 124 / 172 cells include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and XVivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or a sufficient amount of cytokine(s) for T cell growth and expansion. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cell cultures intended for infusion into a subject. The target cells are maintained under conditions necessary to sustain growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[00284] T cells that have been exposed to varying stimulation times may exhibit different characteristics.For example, typical peripheral blood mononuclear cell products, or those subjected to apheresis, have a population of helper T cells (TH, CD4+) that is larger than the population of cytotoxic or suppressor T cells (TC, CD8+). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a T cell population that before about 8 to 9 days consists predominantly of TH cells, while after about 8 to 9 days, the T cell population comprises an increasingly larger population of TC cells. Consequently, depending on the purpose of the treatment, infusing a subject with a T cell population comprising predominantly TH cells may be advantageous. Similarly, if a specific antigen subset of TC cells has been isolated, it may be beneficial to expand this subset to a greater degree.

[00285] In addition, besides the CD4 and CD8 markers, other phenotypic markers vary significantly, but in larger, reproducible parts during the course of the cell expansion process. Thus, Petition 870200050086, dated 04 / 22 / 2020, pages 129 / 209 125 / 172 This reproducibility makes it possible to adapt an activated T-cell product for specific purposes. 5.6 COMPOSITIONS

[00286] The anti-glucose-MUC1 antibodies and / or anti-glucose-MUC1 ADCs described may be in the form of compositions comprising the anti-glucose-MUC1 antibody and / or ADC and one or more carriers, excipients and / or diluents. The compositions may be formulated for specific uses, such as for veterinary or pharmaceutical uses in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and of the excipients, diluents and / or carriers used will depend on the intended uses of the antibody and / or ADC and, for therapeutic uses, on the route of administration.

[00287] For therapeutic uses, the compositions may be supplied as part of a sterile pharmaceutical composition that includes a pharmaceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administering it to a patient). The pharmaceutical composition may be administered to a patient by a variety of routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically or locally. The most suitable route of administration in any given case will depend on the particular antibody and / or ADC, the subject, and the nature and severity of the disease and the subject's physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[00288] Pharmaceutical compositions may conveniently be presented in unit dosage forms containing a predetermined amount of an anti-glucone-MUC1 antibody and / or anti-glucone-MUC1 ADC as described per dose. The amount of antibody and / or ADC included in a unit dose will depend on the disease being treated, as well as other factors as are well known in the art. Such unit dosages may Petition 870200050086, dated 04 / 22 / 2020, pp. 130 / 209 126 / 172 may be in the form of a lyophilized dry powder containing an amount of antibody and / or ADC suitable for a single administration or in the form of a liquid. Unit dose forms of dry powder may be packaged in a kit with a syringe, an appropriate amount of diluent and / or other components useful for administration. Unit doses in liquid form may be conveniently supplied in the form of a pre-filled syringe with an amount of antibody and / or ADC suitable for a single administration.

[00289] Pharmaceutical compositions may also be supplied in bulk containing quantities of ADC suitable for multiple administrations.

[00290] Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing an antibody and / or ADC having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers typically used in the art (all of which are herein referred to as “carriers”), i.e., buffering agents, stabilizing agents, preservatives, isotonic agents, non-ionic detergents, antioxidants, and other assorted additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be non-toxic to receptors at the dosages and concentrations used.

[00291] Buffering agents help maintain pH in a range approximating physiological conditions. They can be present in a wide variety of concentrations, but typically will be present in concentrations ranging from about 2 mM to about 50 mM. Buffering agents suitable for use with the present description include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-citrate mixture of Petition 870200050086, dated 04 / 22 / 2020, pages 131 / 209 127 / 172 monosodium, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide ... gluconic acid-potassium gluconate, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.Lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.) can be used. Additionally, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.

[00292] Preservatives may be added to retard microbial growth and may be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use with the present description include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide and iodide), hexamethonium chloride and alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonic agents sometimes known as “stabilizers” may be added to ensure the isotonicity of liquid compositions of the present description and include Petition 870200050086, dated 04 / 22 / 2020, pp. 132 / 209 128 / 172 Polyhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients that can vary in function from a bulking agent to an additive that solubilizes the therapeutic agent or helps to prevent denaturation or adhesion to the container wall. Typical stabilizers may be polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thiotic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; Disaccharides such as lactose, maltose, sucrose and trehalose, and trisaccharides such as raffinose and polysaccharides such as dextran. Stabilizers may be present in amounts ranging from 0.5 to 10% by weight of ADC.

[00293] Non-ionic surfactants or detergents (also known as “wetting agents”) may be added to help solubilize the glycoprotein as well as to protect the glycoprotein against agitation-induced aggregation, which also allows the formulation to be exposed to stressed surface shear without causing protein denaturation. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.) and pluronic polyols. Non-ionic surfactants may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL. Petition 870200050086, dated 04 / 22 / 2020, pp. 133 / 209 129 / 172 mg / mL, for example approximately 0.07 mg / mL to approximately 0.2 mg / mL.

[00294] Various additional excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and cosolvents. 5.7 Methods of Use

[00295] The anti-glyco-MUC1 antibody or binding fragments described herein can be used in various diagnostic assays. For example, the antibodies and binding fragments can be used in immunoassays, such as competitive binding assays, direct and indirect intercalated assays, and immunoprecipitation assays, including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and Western blots.

[00296] The anti-glyco-MUC1 antibody or binding fragments described herein are also useful for in vivo radiographic imaging, whereby an antibody labeled with a detectable portion such as a radiopaque agent or radioisotope is administered to a subject, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. This imaging technique is useful in the staging and treatment of malignancies.

[00297] The anti-glyco-MUC1 antibody or binding fragments, ADCs and CARs described herein are useful for the treatment of cancers expressing glyco-MUC1, particularly epithelial cancers such as breast cancer, ovarian cancer, pancreatic cancer and lung cancer.

[00298] When using the CARs described for therapy, the therapeutic methods described comprise administering to a subject with a tumor expressing glyco-MUC1 a quantity of a genetically modified cell engineered to express a CAR described, for example as described in Section 0 or in embodiment 0 or embodiment 0. Methods of modifying cells, particularly T cells, to Petition 870200050086, dated 04 / 22 / 2020, pp. 134 / 209 130 / 172 express a CAR, are described in Section 0. 6. Examples 6.1 Example 1: Identification of Anti-Glyco-MUCl Antibodies 6.1.1. Overview

[00299] Chemoenzymatic synthesis of multi-repeat MUC1 glycopeptides with different O-glycan densities and Tn glycoforms (GalNAca1-O-Ser / Thr) was developed using recombinant glycosyltransferases. Different polypeptide GalNAc-transferase isoforms were used to target O-glycan occupancy sites (Bennett et al., 1998). The ideal vaccine design was found to be Tn glycoforms with high O-glycan density, and KLH-conjugated glycopeptides were found to overcome tolerance. In wild-type Balb / c mice, glycopeptides with complete O-glycan occupancy evoked the strongest antibody response reacting with MUC1 expressed in breast cancer cell lines, thus representing the most effective vaccine design. The evoked humoral immune response showed marked specificity for cancer cells. 6.1.2 Materials and Methods 6.1.2.1. Chemoenzymatic synthesis of multimeric Tn MUC1 glycopeptides

[00300] 60-mer MUC1 peptide (VTSAPDTRPAPGSTAPPAHG)n = 3 (SEQ ID NO: 47) was synthesized as originally reported by Fontenot et al., 1993. The control peptides used were derived from tandem repeats (TRs) of MUC2 (PTTTPISTTTMVTPTPTPTC) (SEQ ID NO: 51) and MUC4 (CPLPVTDTSSASTGHATPLPV) (SEQ ID NO: 52). The peptides were glycosylated in vitro using purified recombinant human glycosyltransferase polypeptides GalNAc-T2, GalNAc-T4, and GalNAc-T1 (Bennett et al., 1998; Schwientek et al., 2002) as described in US Patent No. Petition 870200050086, dated 04 / 22 / 2020, pages 135 / 209 131 / 172 6,465,220. Glycosylation of the peptides with GalNAc was performed in a reaction mixture (1 mg peptide / mL) containing 25 mM cacodylate buffer (pH 7.4), 10 mM MnCE, 0.25% Triton X-100, and 2 mM UDP-GalNAc. Glycosylation of 1 mg of 60-mer peptide with two GalNAc per TR (MUC160Tn6) was achieved using GalNAc-T1. Incorporation of three GalNAc per TR (MUC160Tn9) was achieved using GalNAc-T2. Substitution of all five putative O-glycosylation sites in MUC1 TR (MUC160Tn15) was performed using MUC160Tn9 as substrate in a reaction with GalNAc-T4. Glycosylation was monitored using nanoscale reversed-phase columns (Poros R3, PerSeptive Biosystems, Framingham, MA) and MALDI-TOF mass spectrometry.Glycopeptides were purified by high-performance liquid chromatography (HPLC) on a Zorbax 300SB-C3 column (9.4 mm x 25 cm) (Agilent Technologies, Palo Alto, CA) in a Hewlett Packard 1100 system (Avondale, PA) using 0.1% trifluoroacetic acid (TFA) and a 0-80% acetonitrile gradient. Quantification and yield estimation of glycosylation reactions were performed by comparing HPLC peaks with UV 210 absorbance using 10 pg of weighed peptide as a standard. GalNAc glycosylation of peptides generally yielded 80-90% recovery. The purified glycopeptides were distinguished by MALDI-TOF mass spectrometry on a Voyager DE or Voyager DE Pro MALDI-TOF mass spectrometer (PerSeptive Biosystems) equipped with long extraction. The MALDI matrix was 2,5-dihydroxybenzoic acid 10 g / L (Aldrich, Milwaukee, WI) dissolved in a 2:1 mixture of 0.1% TFA in 30% aqueous acetonitrile.Samples dissolved in 0.1% TFA at a concentration of ~1 pmol / pL were prepared for analysis by placing 1 pL of sample solution on a probe tip followed by 1 pL of matrix. All mass spectra were obtained in linear mode. Data processing was performed using software. Petition 870200050086, dated 04 / 22 / 2020, pages 136 / 209 132 / 172 GRAMS / 386 (Galactic Industries, Salem, NH). 6.1.2.2 Immunization Protocol

[00301] Glycopeptides were coupled to KLH (Pierce, Rockford, IL) using glutaraldehyde. Conjugation efficiency was assessed by analyzing the reaction using size exclusion chromatography on a PD-10 column using anti-MUC1 ELISA of the fractions. Essentially all reactivity was found with the excluded fraction and negligible reactivity in the included fractions expected to contain peptides. Further evaluation included comparative titration analysis of the conjugated KLH with the corresponding glycopeptide in the ELISA. Both analyses indicated that conjugation was nearly complete, which should result in a KLH to glycopeptide ratio of 1:300. Female wild-type Balb / c mice were injected subcutaneously with 10 or 15 pg of (glyco)peptide in a total volume of 200 pL (1:1 mixture with Freunds adjuvant, Sigma).The mice received four immunizations 14 days apart, and blood samples were obtained by bleeding from the tail or eye 1 week after the third and fourth immunizations. 6.1.2.3 Generation of anti-Tn-MUC1 MAb mice

[00302] A MAb was produced from a wild-type Balb / c mouse immunized with the fully glycosylated 60-mer MUC1 glycopeptide coupled with KLH-coupled GalNAc. Screening was based on glycopeptide ELISA followed by immunocytology with breast cancer cell lines (MCF7 and T47D) and immunohistology with cancerous tissues. Selection was based on the similar reactivity pattern to whole sera from the same mouse. 6.1.2.4 ELISA

[00303] ELISA tests were performed using 96-well MaxiSorp plates (Nunc, Denmark). The plates were coated overnight at 4°C with 1 pg / mL of glycopeptides in bicarbonate-carbonate buffer (pH 9.6). Petition 870200050086, dated 04 / 22 / 2020, pp. 137 / 209 133 / 172 blocked with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), followed by incubation with sera (diluted in PBS) or MAbs for 2 h at room temperature. Bound antibodies were detected with peroxidase-conjugated rabbit anti-mouse immunoglobulins (DakoCytomation, Glostrup, Denmark) or peroxidase-conjugated goat anti-mouse isotype-specific antibodies IgM, IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology Associates, Birmingham, AL). Plates were developed with O-phenylenediamine tablets (DakoCytomation) and read at 492 nm. Control antibodies included anti-MUC1 HMFG2 and SM3 antibodies (Burchell et al., 1987) and anti-carbohydrate 5F4 (Tn) and 3F1 (STn) antibodies (Mandel et al., 1991). Control sera included mice immunized with KLH-linked mucin peptide MUC4. 6.1.3 Results

[00304] Glycopeptide-specific mAbs were produced for GalNAc-MUC1 using KLH-conjugated 60-mer GalNAc-MUC1 glycopeptide as immunogen. Using an ELISA assay, the generated GO2 (5F7) mAb reacted specifically with the tandem repeat MUC1 (VTSAPDTRPAPGSTAPPAHG)3 (SEQ ID NO: 47) which was glycosylated in vitro using purified recombinant human glycosyltransferases GalNAcT1, GalNAc-T2 and GalNAc-T4, without any reaction with the corresponding MUC1 peptide without GalNAc residues or irrelevant glycopeptides with the same Tn glycoform type. The ELISA assay results are shown in Figure 1. 6.2 Example 2: Characterization of Anti-Glyco-MUCl Antibodies 6.2.1 Overview

[00305] The monoclonal antibody GO2 (5F7) was distinguished for the specificity of its binding to the Tn glycoforms of MUC1 associated with cancer cells. Petition 870200050086, dated 04 / 22 / 2020, pp. 138 / 209 134 / 172 6.2.2 Materials and Methods 6.2.2.1 Immunocytochemistry

[00306] Cell lines were fixed for 10 min in ice-cooled acetone or methanol:acetone. Fixed cells were incubated overnight at 5°C with mouse sera (1:200 / 1:400 / 1:800) or MAbs, followed by incubation for 45 min at room temperature with fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulins (FITC) (DakoCytomation). Slides were mounted in glycerol-containing p-phenylenediamine and examined on a Zeiss fluorescence microscope (FluoresScience, Hallbergmoos, Germany). 6.2.2.2 Immunohistochemistry

[00307] Formalin-fixed, paraffin-embedded tissues of breast carcinoma were obtained. All cases were conventionally classified by histological type. The avidin-biotin peroxidase complex method was used for immunostaining. Paraffin sections were deparaffinized, rehydrated, and treated with 0.5% H2O2 in methanol for 30 min. Sections were rinsed with TBS and incubated for 20 min with non-immune rabbit serum. Sections were rinsed and incubated overnight at 5°C with primary antibody. Sections were rinsed and incubated with biotin-labeled rabbit anti-mouse serum (DakoCytomation) diluted 1:200 in TBS for 30 min, rinsed with TBS, and incubated for 1 h with avidin-biotin peroxidase complex (DakoCytomation). The sections were rinsed with TBS and developed with 0.05% freshly prepared 3,3'-diaminobenzidine tetrachloride in 0.05 M TBS containing 0.1% H2O2.The sections were stained with hematoxylin, dehydrated, and mounted. 6.2.3 Results

[00308] Immunohistochemistry of colorectal carcinoma, carcinoma Petition 870200050086, dated 04 / 22 / 2020, pages 139 / 209 135 / 172 pancreatic and invasive breast adenocarcinomas were stained with GO2. Staining of colorectal cancer tissue (Figure 2) demonstrated strong labeling of intracellular and surface structures in a large proportion of cancer cells. In contrast, no or significantly lower reactivity was observed for healthy columnar epithelial cells. Labeling in healthy cells was restricted to intracellular structures, which is expected due to the presence of large amounts of biosynthetic intermediates (GalNAc-modified glycoproteins) in cells with high secretory capacity such as colonic columnar epithelia. A similar pattern was observed with pancreatic (Figure 3) and breast (Figure 4) cancer tissue, with strong reactivity with cancer cells and no or limited reactivity with intracellular structures in the surrounding healthy epithelia or connective tissue cells. 6.3 Example 3: Anti-Glyco-MUCl Antibody Sequence Analysis

[00309] The mRNA of the GO2 (5F7) hybridoma-producing monoclonal antibody was prepared, reverse transcribed, and sequenced.

[00310] The nucleotide sequences encoding the variable heavy and light chain regions with their signal sequences are presented in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The variable heavy and light chain regions encoded by SEQ ID NO: 11 and SEQ ID NO: 12 are presented in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The sequences of the mature prognosticated variable region (following the signal peptide truncation) are presented in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and are encoded by SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The prognosticated heavy chain sequences of CDR (IMGT definition) are presented in SEQ ID Nos: 5 to 7, respectively, and the prognosticated light chain sequences of CDR (IMGT definition) are presented in SEQ ID Nos: 8 to 10, respectively. 6.4 Example 4: Drug Delivery to Cancer Cells with Petition 870200050086, dated 04 / 22 / 2020, pages 140 / 209 136 / 172 Anti-Glyco-MUC1 Antibodies 6.4.1 Overview

[00311] GO2 (5F7) monoclonal antibody was tested for its ability to release a cytotoxic agent to target cells. 6.4.2 Materials and Methods

[00312] Human ovarian carcinoma (OVCar) ​​cells were added to a 24-well cell culture plate at 1,000 cells / well. Monoclonal antibody GO2 and a secondary antibody conjugated to the anti-tubulin agent monomethyl auristatin F (MMAF) (antimFc-NC-MMAF) (Moradec catalog no. AM-101-AF) were added to the plate to give the following concentrations (in pg / ml) of GO2 and ADC: Table 2 Column 1 2 3 4 5 6 Row^^^^ A GO2: 5 GO2: 1 GO2: 0.2 GO2: 0.04 GO2: 0.008 GO2: 0 ADC: 2.0 ADC: 2.0 ADC: 2.0 ADC: 2.0 ADC: 2.0 ADC: 2.0 B GO2: 5 GO2: 1 GO2: 0.2 GO2: 0.04 GO2: 0.008 GO2: 0 ADC: 0.6 ADC: 0.6 ADC: 0.6 ADC: 0.6 ADC: 0.6 ADC: 0.6 C GO2: 5 GO2: 1 GO2: 0.2 GO2: 0.04 GO2: 0.008 GO2: 0 ADC: 0.2 ADC: 0.2 ADC: 0.2 ADC: 0.2 ADC: 0.2 ADC: 0.2 D GO2: 5 GO2: 1 GO2: 0.2 GO2: 0.04 GO2: 0.008 GO2: 0 ADC: 0 ADC: 0 ADC: 0 ADC: 0 ADC: 0 ADC: 0

[00313] The plate was incubated at 37°C for 48 hours. After 48 hours of incubation, AlamarBlue® (Invitrogen) was added to each well and the fluorescence at 600 nm was measured. 6.4.3 Results

[00314] The results are shown in Figure 5. The results show that cellular toxicity is dependent on the concentration of the primary antibody (GO2) and the presence of the antibody and the concentration and presence of the antibody conjugated to the secondary ADC. In other words, GO2 induces cellular toxicity in its cancer cell line when coupled with a secondary antibody carrying a cytotoxic agent MMAF. 6.5 Example 5: Quantification of Circulating Tumor Cells with Anti-MUC1 Antibodies 6.5.1 Overview Petition 870200050086, dated 04 / 22 / 2020, pp. 141 / 209 137 / 172

[00315] The monoclonal antibody GO2 (5F7) was tested for its ability to be used to quantify circulating tumor cells. 6.5.2 Materials and Methods

[00316] GO2 was conjugated to a magnetic separation bead and allowed to interact with samples of different concentrations of tumor cells. Cells and beads were pulled from the solution with a magnet and washed several times to remove unbound material. GO2 that was conjugated to horseradish peroxidase was then applied to the magnetic separation beads containing bound cancer cells, incubated, and then the unbound conjugated GO2 was washed away. A colorimetric reaction was performed using TNB as a substrate. The reaction was terminated with sulfuric acid, and then readings at OD 450 were taken on the samples. 6.5.3 Results

[00317] The results are shown in Figure 6. The assay results demonstrated the binding of GO2 to tumor cells. 6.6 Example 6: Immunohistochemical staining of tumor tissue using anti-glyco-MUC1 antibodies 6.6.1 Materials and Methods

[00318] Sections of six formalin-fixed, paraffin-embedded (FFPE) tissue microarrays (TMAs) were cut to a thickness of 2.5 µm. TMAs from breast cancer (BC), colorectal cancer (CRC), ovarian cancer (OVC), non-small cell lung cancer (NSCLC), and prostate cancer (PrC) tumors were used in the study. 25 to 47 tumor tissue cores per TMA were available for evaluation. Core size was 1 mm, 2 mm, or 3 mm, depending on the TMA. Each tissue core represented one patient.

[00319] Staining was performed using a Discovery XT autostainer (Ventana Medical Systems). Following antigen recovery with cell conditioning solution 1 (CC1) (Ventana Medical Systems), the Petition 870200050086, dated 04 / 22 / 2020, pp. 142 / 209 138 / 172 GO2 was applied at a concentration of 1 pg / mL in Dako green antibody diluent and incubated for 60 min at 37°C. Binding of GO2 to tumor cells was detected using the Optiview DAB IHC detection kit (Ventana Medical Systems) visualized in DAB (brown precipitate). 6.6.2 Results

[00320] Representative images of MUC1-positive TMA tumor nuclei are shown in Figure 7. In BC and OVC TMAs, most stains (>90%) showed moderate or strong GO2 binding to tumor cells. 70% and 51% of NSCLC and CRC cases showed moderate to strong antibody binding to tumor cells, respectively. In prostate cancer, the antigen appeared to be less expressed. Only 28% of TMA 1 stains revealed moderate or strong staining intensity upon GO-2 application. Staining patterns were always cytoplasmic and in many cases membrane-bound. An apical membrane staining pattern was observed in a few nuclei. 6.7 Example 7: Production and purification of an anti-MUCl antibody in the T-cell bispecific (TCB) format. 6.7.l Materials and Methods 6.7.ll Production of expression vector

[00321] The GO2 antibody was converted into the TCB format, including knob-into-holes and P329G / L234A / L235A (“PGLALA”) mutations in the Fc region and charged residues in the MUC1 CH1 (147E / 213E; “EE”) and CL (123R / 124K; “RK”) regions (see SEQ ID Nos: 43 to 46). The TCB antibody is illustrated in Figure 8. In summary, variable heavy and variable light chains of GO2 mAb were synthesized (Geneart, Regensburg, Germany) and inserted into suitable expression vectors in which they are fused to the appropriate human constant heavy or human constant light chains. The expression cassettes in these vectors consist of the CMV promoter, Intron A with 5' UTR and a BGH polyadenylation site. In addition, the plasmids contain the Petition 870200050086, dated 04 / 22 / 2020, pp. 143 / 209 139 / 172 region oriP of the Epstein-Barr virus for stable maintenance in HEK293 cells harboring the EBV nuclear antigen (EBNA). 6.7.1.2 Transient transfection and production

[00322] Antibodies were transiently produced in HEK293 EBNA cells using a PEI-mediated transfection procedure as follows. HEK293 EBNA cells are cultured in serum-free suspension in Excell culture medium containing 6 mM L-Glutamine. For antibody production in a 500 ml shaken flask, 300 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all quantities were adjusted accordingly). For transfection, cells are centrifuged for 10 min at 210 x eO, and the supernatant is replaced with 20 ml of pre-warmed CD CHO medium. Expression vectors are mixed in 20 ml of CD CHO medium to a final amount of 200 μg of DNA. After adding 540 μl of PEI, the solution is swirled for 15 min and subsequently incubated for 10 min at room temperature.After this, the cells are mixed with the DNA / PEI solution, transferred to a 500 ml shaken flask and incubated for 3 hours at 37°C in an incubator with a 5% CO2 atmosphere. After incubation, 160 ml of Ex-cell® medium (Sigma-Aldrich), containing 6 mM glutamine, 1.25 mM valproic acid and 12.5% ​​Pepsoy, are added and the cells are cultured for 24 hours. One day after transfection, 12% Feed 7 (48 mL) + 3 g / L glucose are added. After 7 days, the culture supernatant is collected for purification by centrifugation for 45 min at 3000 x g. The solution is filtered sterile (0.22 μm filter) and sodium azide at a final concentration of 0.01% w / v is added. The solution is then stored at 4°C. 6.7.1.3 Antibody purification

[00323] The secreted proteins were purified by affinity chromatography using Protein A, followed by exclusion chromatography Petition 870200050086, dated 04 / 22 / 2020, pages 144 / 209 140 / 172 size. For affinity chromatography, the supernatant was loaded onto a MabSelect SuRe Protein A (GE Healthcare) column equilibrated with 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Unbound protein was removed by washing with equilibration buffer. Bound protein was eluted using a step-up elution (standard IgG) or a gradient (bispecific antibody) created with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. The pH of the collected fractions was adjusted by adding 1 / 10 (v / v) of 0.5 M sodium phosphate, pH 8.0. The protein was concentrated and filtered before loading onto a HiLoad Superdex 200 (GE Healthcare) column equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[00324] The aggregate content of the eluted fractions was analyzed by analytical size exclusion chromatography. Therefore, 30 µl of each fraction were applied to a TSK G3000 SWXL column (TOSOH, 7.8 mm x 30 cm) equilibrated with 200 mM Arginine, 25 mM K2PO4, 125 mM sodium chloride, 0.02% NaN3, pH 6.7. Fractions containing less than 2% oligomers were pooled and concentrated to a final concentration of 1 to 1.5 mg / ml using centrifugal concentrators (Millipore, Amicon® ULTRA - 15, 30k MWCO). The purified proteins were stored at -80°C. 6.7.2 Results

[00325] The yield and quality of TCB GO2 antibody production are shown in Table 5. Table 5 Molecule Yield [mg / L] Monomer [%] SEC HMW [%] LMW [%] Purity [%] CE-SDS TCB GO2 0.51 94.16 0.00 5.84 85.61 6.8 Example 8: Jurkat-NFAT reporter assay to monitor target expression ex vivo in undigested patient-derived tumor samples 6.8.1 Overview

[00326] A Jurkat NFAT reporter essay was used to monitor the Petition 870200050086, dated 04 / 22 / 2020, pages 145 / 209 141 / 172 target expression (glucose-MUCl) ex vivo in undigested primary human tumor samples using a TCB GO2. 6.8.2 Materials and Methods 6.8.2.1 Materials • TCB GO2 (see Example 7) • DP47 TCB (untargeted, negative control) • Matrigel (Item no. 734-1101, Corning / VWR, Switzerland) • Corning® Costar® Ultra-low Fixation Multi-Pocket Plates (Item no. CLS7007-24EA, Sigma) • Cell culture microplate, 96 wells (Item no. 655098, Greiner Bio-A, Switzerland) • RPMI1640 medium (Item no. 42401-018, FisherScientific, Switzerland) • Jurkat medium: RPMI1640 medium with 2 g / l D-Glucose, 2 g / l NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-Glutamine, 1 x NEAA, 1 Sodium Pyruvate, 200 pg / ml Hygromycin B • Jurkat NFAT luciferase reporter cells (Promega) • Tumor samples were received from Indivumed GmbH, Germany. Samples were shipped overnight in transport medium. Approximately 24 hours after surgery, the samples were cut into small pieces. 6.8.2.2 Methods

[00327] 96-well cell culture microplates were prepared by adding 17 μl of cold Matrigel. The plate was incubated for 2 min at 37°C before tumor fragments were added (in triplicate). 33 μl of cold Matrigel were added per well and the plate was incubated again for 2 min at 37°C. 100 μl (50 nM or 5 nM) of TCB antibody dilution (diluted in Jurkat medium without Hygromycin, but with 2X Penicillin / Streptomycin) was added per well. Reporter cells Petition 870200050086, dated 04 / 22 / 2020, pages 146 / 209 142 / 172 Jurkat-NFAT cells were harvested and viability was assessed using ViCell. Cells were centrifuged at 350 x g for 7 min before being resuspended in Jurkat medium without hygromycin. 50 µl of the cell suspension were added per well (50,000 cells / well). The plate was incubated for 4–5 ha at 37°C in a humidified incubator before being collected for luminescence reading. 50 µl of ONE-Glo solution were added to each well and incubated for 10 min at room temperature in the dark. Luminescence was detected using a WALLAC Victor3 ELISA reader (PerkinElmer 2030), with a detection time of 5 s / well. 6.8.3 Results

[00328] The results of tumor samples from three patients are shown in Figures 9 to 11. The results shown in Figure 9 are from tumor samples obtained from a patient having a malignant neoplasm of the bronchus and lung: intermediate lobe, bronchus or lung, squamous cell carcinoma. The results shown in Figure 10 are from tumor samples obtained from a patient having a malignant neoplasm of the bronchus and lung: lower lobe, bronchus or lung, non-keratinizing squamous cell carcinoma. The results shown in Figure 11 are from tumor samples obtained from a patient having a malignant neoplasm of the bronchus and lung: upper lobe, bronchus or lung, acinar-type adenocarcinoma. Each bar in Figures 9 to 11 represents the average of triplicates. The standard error is indicated by the error bars. The dashed line indicates luminescence for Jurkat NFA T cells incubated with tumor samples without any TCB.A two-tailed, unpaired t-test was used for statistical analysis. P-values ​​below 0.05 were considered significant and were indicated with asterisks (* P < 0.05; ** P < 0.001; *** P < 0.001). In each of Figures 9 to 11, tumor samples incubated with TCB GO2 demonstrated significantly more luminescence. Petition 870200050086, dated 04 / 22 / 2020, pages 147 / 209 143 / 172 than the samples incubated with the negative control TCB DP47. 6.9 Example 9: In vitro characterization of TCB GO2 6.9.1 Overview

[00329] TCB GO2 (Example 7) which recognized aberrantly tumor-specific glycosylated MUC1 was functionally distinguished in tumor cells expressing MUC1. 6.9.2 Materials and Methods 6.9.2.1 Cell lines and PBMCs

[00330] Engineered tumor cell lines T3M4 pfzv and MCF7 cs were cultured in DMEM with 10% FCS and 2 mM Glutamine. MCF10A is a non-tumorigenic human mammary epithelial cell line (ATCC® CRL-10317). HBEpiC are human bronchial epithelial cells (Sciencell #3210). PBMCs were isolated by gradient centrifugation using whole blood from healthy volunteers. 6.9.2.2 Target binding by flow cytometry

[00331] Target cells as indicated were harvested with Cell Dissociation Buffer, washed with PBS, and resuspended in FACS buffer. Antibody staining was performed in a 96-well round-bottom plate. 200,000 cells were seeded per well. The plate was centrifuged for 4 min at 400g and the supernatant was removed. Test antibodies were diluted in FACS buffer and 30 µl of the antibody solution was added to the cells for 30 min at 4°C. To remove unbound antibody, the cells were washed twice with FACS buffer before the addition of the diluted secondary antibody (Goat IgG Fcy-Specific Fragment anti-human F(ab')2 AffiniPure PE-conjugated, Jackson ImmunoResearch #109-116-170). After 30 min of incubation at 4°C, the unbound secondary antibody was washed away. Before measurement, the cells were resuspended in 200 µl of FACS buffer and analyzed by flow cytometry using BD Fortessa. The assays Petition 870200050086, dated 04 / 22 / 2020, pp. 148 / 209 144 / 172 were performed in triplicate. 6.9.2.3 T-cell mediated tumor killer cells and T-cell activation

[00332] Target cells were harvested with Trypsin / EDTA, counted, and viability checked. The cells were resuspended in their respective media with a final concentration of 300,000 cells per ml. Then, 100 µl of the target cell suspension were transferred into each well of a 96-well flat-bottomed plate. The plate was incubated overnight at 37°C in an incubator to allow cell adhesion to the plate. The following day, PBMCs were isolated from whole blood. Blood was diluted 2:1 with PBS and topped with 15 ml of Histopaque-1077 (#10771, Sigma-Aldrich) in Leucosep tubes and centrifuged for 30 min at 450g without brake. After centrifugation, the cell-containing strip was collected with a 10 ml pipette and transferred into 50 ml tubes. The tubes were filled with PBS to 50 ml and centrifuged (400 g, 10 min, room temperature). The supernatant was removed and the pellet resuspended in PBS.After centrifugation (300g, 10 min, room temperature), the supernatants were discarded, 2 tubes were pooled, and the washing step was repeated (this centrifugation time 350g, 10 min, room temperature). After that, the cells were resuspended and the pellets pooled in 50 ml of PBS for cell counting. After counting, the cells were centrifuged (350g, 10 min, room temperature) and resuspended at 6 million cells per ml in RPMI with 2% FCS and 2 nM Glutamine. The medium was removed from the plated target cells, and the test antibodies diluted in RPMI with 2% FCS and 2 nM Glutamine were added. 300,000 cells from the effector cell solution were transferred to each well, resulting in an E:T ratio of 10:1. To determine maximum release, target cells were lysed with Triton X-100. LDH release was then determined. Petition 870200050086, dated 04 / 22 / 2020, pages 149 / 209 145 / 172 of 24 h and 48 h using Cytotoxicity Detection Kit (1644793, Roche Applied Science). Upregulation of activation markers in T cells after tumor cell elimination was measured by flow cytometry. In summary, PBMCs were harvested, transferred into a 96-well round-bottom plate, and stained with CD4 APC (300514, BioLegend), CD8 FITC (344704, BioLegend), CD25 BV421 (302630, BioLegend), and CD69 PE (310906, BioLegend) antibodies diluted in FACS buffer. After 30 min of incubation at 4°C, the cells were washed twice with FACS buffer. Before measuring fluorescence using BD Fortessa II, the cells were resuspended in 200 µl of FACS buffer. The tests were performed in triplicate. 6.9.2.4 Cytokine / chemokine release by cytometric granule arrangement

[00333] Cytokine / chemokine secretion in the supernatant was measured by flow cytometry using the cytometric bead array (CBA) according to the manufacturer's guidelines. T-cell supernatant-mediated kill assays were collected and stored at -20°C. The supernatants were subsequently thawed and tested according to the manufacturer's instructions. The following CBA kits (BD Biosciences) were used: human interferon gamma CBA (IFNy) Flex Set (E7), human granzyme B CBA Flex Set (D7), human IL6 CBA Flex Set (A7), human IL8 CBA Flex Set (A9), human IL10 CBA Flex Set (B7), and human tumor necrosis factor CBA (TNF) Flex Set (D9). Samples were measured using the BD FACS Canto II and analysis was performed using Diva software (BD Biosciences). Assays were performed in triplicate. 6.9.3 Results

[00334] The binding of TCB GO2 to the MCF7 breast cancer cell line and the T3M4 pancreatic cancer cell line, both engineered to express aberrantly glycosylated MUC1, was Petition 870200050086, dated 04 / 22 / 2020, pages 150 / 209 146 / 172 confirmed (Figure 12). Subsequently, TCB GO2 activity was tested in both tumor cell lines, MCF7 and T3M4, using freshly isolated PBMCs (Figure 13 and Figure 14). Tumor cell extermination of both cell lines was detected after 24 h and even stronger after 48 h. This was accompanied by strong activation of CD4 T cells and CD8 T cells determined by the upregulation of the two activation markers CD25 and CD69 and the release of IL6, IL8, IL10, IFNγ, TNFα and Granzyme B into the supernatant. The respective untargeted TCB was included as a negative control.

[00335] To prove that TCB GO2 does not bind to normally glycosylated MUC1 in epithelial cells, binding to MCF10A, a non-tumorigenic human mammary epithelial cell line, and HBEpiC, primary human bronchial epithelial cells, was tested. As a positive control, HMFG1 TCB, which does not discriminate between MUC1 expressed in normal and tumor cells, was included. HMFG1 TCB was found to bind to both cell types tested, confirming MUC1 expression, but TCB GO2 was not stable enough to bind to these cells (Figure 15). In addition, TCB GO2 was tested to see if it would induce T cell killing or activation in the presence of normal epithelial cells expressing MUC1. This was tested in MCF10A cells and there was no detectable T cell killing or activation with TCB GO2, whereas HMFG1 TCB induced both killing and T cell activation (Figure 16). 6.10 Example 10: Functional characterization of GO2 and TCB GO2 antibodies by surface plasmon resonance 6.10.1 Overview

[00336] GO2 and TCB GO2 (Example 7) were distinguished by surface plasmonic resonance. 6.10.2 Materials and Methods 6.10.2.1 Binding of GO2 and TCB GO2 to immobilized glycopeptides Petition 870200050086, dated 04 / 22 / 2020, pp. 151 / 209 147 / 172

[00337] The binding of GO2 and TCB GO2 antibodies to human and cynomolgus glycopeptides (Table 6) was evaluated by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25°C with HBS-EP as conducting buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg / Germany). Table 6 Glycopeptide Sequence Concentration in PBS Human peptide PDTSAAPGSTAPPAHVVTSAP (SEQ ID NO: 48) 0.9 mg / ml Cynomolgus peptide PDTSAAPGSTGPPAHVVTSAP (SEQ ID NO: 49) 1.8 mg / ml

[00338] Biotinylated glycopeptides were dissolved in PBS and the final concentration was between 0.9 and 1.8 mg / ml (Table 6). Biotinylated glycopeptides were directly coupled to a flow cell of a streptavidin (SA) sensor chip. Immobilization levels up to 880 resonance units (RU) were used. GO2 antibody or TCB GO2 was injected at a flow rate of 30 pL / minute through the flow cells over 240 seconds at a concentration of 1000 nM (Figure 17). Dissociation was monitored for 500 s. Differences in mass refractive index were corrected by subtracting the response obtained in a reference flow cell where no protein was immobilized. 6.10.2.2 Avidity of GO2 and TCB GO2 to immobilized glycopeptides

[00339] The avidity of GO2 and TCB GO2 was evaluated by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25°C with HBS-EP as the conducting buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg / Germany). Biotinylated glycopeptides (Table 6) were directly coupled to a flow cell of a streptavidin (SA) sensor chip. Immobilization levels up to 200 resonance units (RU) were used.

[00340] The GO2 antibody or the GO2 TCB was injected with a Petition 870200050086, dated 04 / 22 / 2020, pages 152 / 209 148 / 172 flow rate of 30 μL / minute through flow cells for 120 seconds and in a concentration range of 3.9 to 1000 nM (1:2 dilution). Dissociation was monitored for 400 s. Mass refractive index differences were corrected by subtracting the response obtained in a reference flow cell where no protein was immobilized. KDs were derived, despite the bivalence of the interaction, by fitting the curve to a 1:1 Langmuir linkage using Biaeval software (GE Healthcare). The “apparent” KG can therefore only be used for comparison purposes. 6.10.3 Results

[00341] As can be seen in the sensorgrams in Figure 18, the GO2 (Figure 18A) and TCB GO2 (Figure 18B) antibodies bind to both human and cynomolgus glycopeptides. The GO2 and TCB GO2 antibodies bind with higher avidity to the cynomolgus glycopeptide than to the human glycopeptide.

[00342] As can be seen in Figure 19, the binding of a bivalent GO2 binder (IgG, TCB) to the human glycopeptide is in the three nanomolar digits (Figures 19A and 19C), whereas the binding to the cynomolgus glycopeptide is in the two nanomolar digits (Figures 19B and 19D). 6.11 Example 11: Exploratory single-dose pharmacokinetic and tolerability study of TCB GO2 in cynomolgus monkeys 6.11.1 Overview

[00343] The objectives of this study are to determine the pharmacokinetics and tolerability of TCB GO2 described in Example 7, when given by a single intravenous injection to cynomolgus monkeys. 6.11.2 Materials and Methods 6.11.2.1 Preparation of TCB GO2

[00344] Thawing of frozen stock solution of TCB GO2 (2.12 mg / mL) and formulation buffer (20 mM Histidine, 140 mM Petition 870200050086, dated 04 / 22 / 2020, pages 153 / 209 149 / 172 of NaCl, 0.01% Tween 20; pH 6.0) is made overnight in a refrigerator to maintain 4°C. Dosage formulations of the test item are prepared under sterile conditions at concentrations appropriate to meet dose level requirements by dilution with formulation buffer.

[00345] The dosage formulation is prepared within 2 hours before injection and stored at room temperature until use. Polypropylene containers are used for the preparation and storage of the dosage formulation to prevent adsorption. Dosage formulations should not be filtered, agitated, or shaken. Any mixing is done by gentle pipetting or gentle shaking of the container. 6.11.2.2 Animals

[00346] Cynomolgus macaques (Macaca fascicularis) aged 2 to 4 years and weighing less than 4 kg are used in the study. The animals are allowed to acclimatize to the primate toxicology accommodation of the testing facility for at least 6 weeks before dosing begins.

[00347] During the week prior to the start of dosing, animals are approved for entry into the experiment based on a satisfactory veterinary examination (performed soon after arrival), clinical observation records, body weight profile, and clinical pathology investigations.

[00348] The animals selected for the study are randomly allocated to cages based on the supplied group compatibility information and then allocated with individual study numbers. The animals are allocated to a cage in groups of up to 5. 6.11.2.3 Creation

[00349] The animals are socially housed where possible, in groups of up to 5 per sex in two group cages on different levels measuring 1.61 x 1.66 x 2.5 m on the lower level and 1.61 x 1.66 x 2.03 m on the upper level. The bedding material is sawdust. No known contaminants were found in Petition 870200050086, dated 04 / 22 / 2020, pp. 154 / 209 150 / 172 forage that would interfere with the objectives of the study.

[00350] The target conditions for the animal room environment should be as follows: Temperature: 18 to 24°C Humidity: 40 to 70% Ventilation: a minimum of 10 air changes per hour. Light Cycle: 12 hours of light and 12 hours of darkness (except when interrupted by study procedures / activities).

[00351] There is automatic temperature and humidity control that is continuously monitored and recorded. There is automatic light cycle control.

[00352] Special Diet Services (SDS) MP(E) Short SQC Diet is provided as a daily ration throughout the study. Approximately 200 grams of feed per animal is provided once daily. There are no contaminants in the feed that interfere with the study objectives. The animals had ad libitum access to public water supplies. There are no known contaminants in the water that would interfere with the study objectives.

[00353] The animal's home environment is enriched to promote social interaction, play, and exploration. The animals have perches and materials such as plastic toys, balls, climbing frames, and stainless steel mirrors. These are rotated frequently to reduce familiarity. Before rotation, all toys and climbing frames are thoroughly cleaned to prevent cross-contamination. The animals are also offered a range of other treats such as forage mix, vegetables, nuts, biscuits, and fruit, usually on a daily basis.

[00354] Veterinary care is available throughout the course of the study and animals are examined by the veterinary staff as warranted by clinical signs or other changes. Petition 870200050086, dated 04 / 22 / 2020, pages 155 / 209 151 / 172 6.11.2.4 Experimental project

[00355] One male and one female animal are administered TCB GO2 Table 7 Group No. Dose Level (μg / kg) Dose Volume (mL / kg) Dose Concentration (μg / kg) 1 (1 male and 1 female) 100 1 100 2 (1 male and 1 female) 300 1 300

[00356] TCB GO2 is administered to appropriate animals by a single slow intravenous bolus injection (1 to 2 min) into the saphenous vein or tail vein at least 8 days apart. It is staggered so that only one male and one female receive a new dose level on any given day. Based on observations from the previous dose level (including clinical pathology data), doses are increased or decreased for the next dose group. Naive animals are used for each dose level. Doses are given using a syringe with a Vygon infusion needle attached.

[00357] Animals are necropsied approximately 72 hours after dosing (after the last scheduled sample has been collected). For all animals that had to be culled before the scheduled date due to severe clinical symptoms, a full panel of clinical pathology (additional sampling before termination, if practicable) and histopathology are analyzed.

[00358] The intravenous injection route of administration was selected for this study as this route is a possible route for clinical application. The low and high dose levels were chosen to cover a clinically relevant dose range and to minimize potential harm to the animals. The low dose was selected based on experience in cynomolgus monkeys with similar potency bispecific T-cell antibodies, and the high dose represents a 3-fold increase of the same. 6.11.3 Results

[00359] TCB GO2 is tolerated at the doses tested. 7. SPECIFIC MODALITIES, CITATION OF REFERENCES Petition 870200050086, dated 04 / 22 / 2020, pages 156 / 209 152 / 172

[00360] Although several specific modalities have been illustrated and described, it will be considered that various changes may be made without departing from the spirit and scope of the description(s). The present description is exemplified by the numbered modalities presented below. 1. An anti-glyco-MUC1 antibody or antigen-binding fragment that: a. preferentially binds to a glyco-MUC1 epitope that is overexpressed in cancer cells when compared to normal cells and b. competes with an antibody or antigen-binding fragment comprising a variable heavy chain (VH) sequence of SEQ ID NO: 3 and a variable light chain (VL) sequence of SEQ ID NO: 4 for binding to the MCF7 or T47D breast cancer cell line. 2. An anti-glyco-MUC1 antibody or antigen-binding fragment that a. binds to the tandem repeat MUC1 (VTSAPDTRPAPGSTAPPAHG)3 which has been glycosylated in vitro using purified recombinant human glycosyltransferases GalNAc-T1, GalNAc-T2 and GalNAc-T4 and (hereinafter referred to as the “first epitope”) and b. competes with an antibody or antigen-binding fragment comprising a variable heavy chain (VH) sequence of SEQ ID NO: 3 and a variable light chain (VL) sequence of SEQ ID NO: 4 for binding to the MCF7 or T47D breast cancer cell line. 3. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 1 or modality 2 comprising a complementarity-determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, a CDR-H3 comprising the sequence Petition 870200050086, dated 04 / 22 / 2020, pp. 157 / 209 153 / 172 of amino acid from SEQ ID NO: 25, a CDR-L1 comprising the amino acid sequence from SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence from SEQ ID NO: 9 and a CDR-L3 comprising the amino acid sequence from SEQ ID NO: 31. 4. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5. 5. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 23. 6. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 28. 7. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 32. 8. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the 0 to 0 embodiments, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6. 9. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the 0 to 0 embodiments, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24. 10. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the 0 to 0 embodiments, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7. 11. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the 0 to 0 embodiments, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30. 12. The anti-glyco-MUC1 antibody or binding fragment to Petition 870200050086, dated 04 / 22 / 2020, pp. 158 / 209 154 / 172 antigen of any of the 0 to 0 modalities, where CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26. 13. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the 0 to 0 embodiments, wherein CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27. 14. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the 0 to 0 embodiments, wherein CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10. 15. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 1 or modality 2 wherein VH comprises the complementarity-determining regions (CDRs) of SEQ ID NOS: 5 to 7 and VL comprises CDRs of SEQ ID NOS: 8 to 10. 16. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 1 or modality 2 wherein VH comprises the complementarity-determining regions (CDRs) of SEQ ID NOS: 23 to 25 and VL comprises CDRs of SEQ ID NOS: 26, 27 and 10. 17. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 1 or modality 2 wherein VH comprises the complementarity-determining regions (CDRs) of SEQ ID NOS: 28, 29 and 25 and VL comprises CDRs of SEQ ID NOS: 30, 9 and 31. 18. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the embodiments 1 to 0 that is a chimeric or humanized antibody. 19. The anti-glyco-MUC1 antibody or antigen-binding fragment of any embodiment 1 to 0 wherein VH comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3 and VL comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4. Petition 870200050086, dated 04 / 22 / 2020, pp. 159 / 209 155 / 172 20. The anti-glyco-MUCl antibody or antigen-binding fragment of any embodiment 1 to 0 wherein VH comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 3 and VL comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 4. 21. The anti-glyco-MUC1 antibody or antigen-binding fragment of any embodiment 1 to 0 wherein VH comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 3 and VL comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 4. 22. The anti-glyco-MUC1 antibody or antigen-binding fragment of embodiment 1 or embodiment 2 wherein VH comprises the amino acid sequence of SEQ ID NO: 3 and VL comprises the amino acid sequence of SEQ ID NO: 4. 23. The anti-glyco-MUC1 antibody or antigen-binding fragment of either modality 1 to 0 that is multivalent. 24. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the embodiments 1 to 0 that is in the form of a single-chain variable fragment (scFv). 25. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0 in which the scFv comprises the N-terminal heavy chain variable fragment relative to the light chain variable fragment. 26. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0 in which the variable heavy chain fragment and variable light chain fragment of scFv are covalently linked to a linker sequence of 4 to 15 amino acids. 27. The anti-glyco-MUC1 antibody or binding fragment to Petition 870200050086, dated 04 / 22 / 2020, pp. 160 / 209 156 / 172 antigen of any of the modalities 1 to 0 that is in the form of a multispecific antibody. 28. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the multispecific antibody is a bispecific antibody that binds to a second epitope that is different from the first epitope. 29. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, wherein the bispecific antibody is a CrossMab, a Fab arm exchange antibody, a bispecific T cell engager (BiTE), or a dual-affinity rebleaching molecule (DART). 30. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the bispecific antibody is a CrossMab. 31. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the bispecific antibody is a CrossMabFAB. 32. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the bispecific antibody is a CrossMabVHVL. 33. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the bispecific antibody is a CrossMabCH1-CL. 34. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the bispecific antibody is a Fab arm exchange antibody. 35. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the bispecific antibody is a dual-affinity rebleaching molecule (DART). 36. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the bispecific antibody is an engager of Petition 870200050086, dated 04 / 22 / 2020, pp. 161 / 209 157 / 172 bispecific T cell (BiTE). 37. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the 0 to 0 embodiments, wherein the second epitope is a MUC1 epitope. 38. Anti-glyco-MUC1 antibody with antigen-binding fragment of any of the 0 to 0 modes, wherein the second epitope is a MUC1 epitope that is overexpressed in cancer cells when compared to normal cells. 39. The anti-glyco-MUC1 antibody or antigen-binding fragment of either 0 to 0 embodiment, wherein the second epitope is a T-cell epitope. 40. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the T cell epitope comprises a CD3 epitope, a CD8 epitope, a CD16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2D epitope. 41. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, in which the T-cell epitope comprises a CD3 epitope, which is optionally an epitope present in human CD3. 42. The anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0, wherein the CD3 epitope comprises a CD3 gamma epitope, a CD3 delta epitope, a CD3 epsilon epitope, or a CD3 zeta epitope. 43. The anti-glyco-MUC1 antibody or antigen-binding fragment of any of the embodiments 1 to 0 that is conjugated to a detectable moiety. 44. Anti-glyco-MUC1 antibody or antigen-binding fragment of modality 0 in which the detectable marker is an enzyme, a radioisotope, or a fluorescent label. 45. A bispecific antibody comprising a first Petition 870200050086, dated 04 / 22 / 2020, pp. 162 / 209 158 / 172 antigen-binding domain that binds to CD3 (optionally human CD3) and a second antigen-binding domain that binds to glycoMUC1, wherein the bispecific antibody competes with an antibody or antigen-binding fragment comprising a variable heavy chain (VH) sequence of SEQ ID NO: 3 and a variable light chain (VL) sequence of SEQ ID NO: 4 for binding to the MCF7 or T47D breast cancer cell line and wherein the first antigen-binding domain comprises a variable heavy chain region comprising the CDR-H1 heavy chain of SEQ ID NO: 34, the CDR-H2 of SEQ ID NO: 35 and the CDR-H3 of SEQ ID NO: 36 and a variable light chain region comprising the CDR-L1 light chain of SEQ ID NO: 37, the CDR-L2 of SEQ ID NO: 38 and the CDR-L3 of SEQ ID NO: 39. 46. ​​The bispecific antibody of embodiment 0, wherein the second antigen-binding domain comprises (i) a variable heavy chain region comprising CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6 and CDR-H3 of SEQ ID NO: 7 and a variable light chain region comprising CDR-L1 light chain of SEQ ID NO: 8, CDRL2 of SEQ ID NO: 9 and CDR-L3 of SEQ ID NO: 10. 47. The bispecific antibody of modality 0 or modality 0, wherein the first antigen-binding domain comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41. 48. The bispecific antibody of modality 0, the first antigen-binding domain comprises the variable region sequence of the heavy chain of SEQ ID NO: 40 and the variable region sequence of the light chain of SEQ ID NO: 41. Petition 870200050086, dated 04 / 22 / 2020, pages 163 / 209 159 / 172 49. A bispecific antibody of any of the 0 to 0 embodiments, wherein the second antigen-binding domain comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4. 50. The bispecific antibody of modality 0, wherein the second antigen-binding domain comprises the variable heavy chain region sequence of SEQ ID NO: 3 and the variable light chain region sequence of SEQ ID NO: 4. 51. The bispecific antibody of either of the 0 to 0 modes, in which the first and / or second antigen-binding domain is a Fab molecule. 52. The bispecific antibody of modality 0, in which the first antigen-binding domain is a cross-linked Fab molecule, where the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged. 53. The bispecific antibody of embodiment 0, in which the first and second antigen-binding domains of the bispecific antibody are both Fab molecules and in one of the antigen-binding domains (particularly the first antigen-binding domain) the variable VL and VH domains of the Fab light chain and the Fab heavy chain are substituted for one another, in which a. In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is replaced by a positively charged amino acid (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is replaced by Petition 870200050086, dated 04 / 22 / 2020, pp. 164 / 209 160 / 172 a negatively charged amino acid (numbering according to the EU Kabat index); or b. in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is replaced by a positively charged amino acid (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (numbering according to Kabat's EU index), wherein the constant domains CL and CH1 of the antigen-binding domain having the VH / VL exchange are not replaced by each other. 54. The bispecific antibody of modality 0, in which a. In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index); or b. in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index). 55. The bispecific antibody of modality 0, in which the constant CL domain of the second antigen-binding domain is... Petition 870200050086, dated 04 / 22 / 2020, pages 165 / 209 161 / 172 amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index). 56. The bispecific antibody of embodiment 0, in which in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index). 57. In the bispecific antibody of modality 0, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the EU Kabat index). 58. The bispecific antibody of modality 0, in which in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by lysine (K) (numbering according to Kabat) and in the constant domain CH1 of the second Petition 870200050086, dated 04 / 22 / 2020, pp. 166 / 209 162 / 172 antigen-binding domain the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the EU Kabat index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the EU Kabat index). 59. The bispecific antibody of embodiment 0, in which in the constant domain CL of the second antigen-binding domain the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by arginine (R) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding domain the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the EU Kabat index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the EU Kabat index). 60. The bispecific antibody of either of the 0 to 0 embodiments, in which the constant CL domain of the second antigen-binding domain is of the kappa isotype. 61. The bispecific antibody of either of the 0 to 0 embodiments, in which the first and second antigen-binding domains are fused together, optionally via a peptide linker. 62. The bispecific antibody of embodiment 0, wherein the first and second antigen-binding domains are each a Fab molecule and (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. 63. The bispecific antibody of any of the 0 to 0 embodiments, in which the bispecific antibody provides monovalent binding to CD3. 64. The bispecific antibody of modality 0, which comprises Petition 870200050086, dated 04 / 22 / 2020, pp. 167 / 209 163 / 172 two antigen-binding domains that specifically bind to glycoMUC1. 65. The bispecific antibody of modality 0, in which the two antigen-binding domains that specifically bind to glyco-MUC1 comprise the same amino acid sequence. 66. The bispecific antibody of any of the 0 to 0 embodiments, wherein the bispecific antibody additionally comprises an Fc domain composed of a first and a second subunit. 67. The bispecific antibody of modality 0, in which the Fc domain is an IgG Fc domain. 68. The bispecific antibody of modality 0, in which the Fc domain is an Fc domain of IgG1. 69. The bispecific antibody of modality 0, in which the Fc domain is an Fc domain of IgG4. 70. The bispecific antibody of modality 0, in which the Fc domain of IgG4 comprises an amino acid substitution at the S228 position (Kabat EU index numbering), preferably the S228P amino acid substitution. 71. The bispecific antibody of modality 0, in which the Fc domain is a human Fc domain. 72. The bispecific antibody of modality 0, in which the Fc domain is a human IgG1 Fc domain, optionally comprising SEQ ID NO: 42. 73. The bispecific antibody of any of the 0 to 0 embodiments, wherein the first, second, and, where present, third antigen-binding domains are each a Fab molecule and (a) (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain and the first antigen-binding domain is fused at the C-terminus Petition 870200050086, dated 04 / 22 / 2020, pages 168 / 209 164 / 172 of the Fab heavy chain to the N-terminus of the first Fc domain subunit or (ii) the first antigen-binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain and the second antigen-binding domain is fused from the C-terminus of the Fab heavy chain to the N-terminus of the first Fc domain subunit and (b) the third antigen-binding domain, where present, is fused from the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit. 74. The bispecific antibody of either of the 0 to 0 forms, in which the Fc domain comprises a modification promoting the association of the first and second subunits of the Fc domain. 75. A bispecific antibody of any of the 0 to 0 forms, in which the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function. 76. The bispecific antibody of modality 0, comprising a. a first antigen-binding domain that specifically binds to CD3, wherein the first antigen-binding domain is a cross-linked Fab molecule in which the variable or constant regions, preferably the variable regions, of the Fab light chain and the Fab heavy chain are exchanged; b. a second and a third antigen-binding domain that specifically bind to glyco-MUC1, comprising a variable heavy chain region comprising the CDR-H1 heavy chain of SEQ ID NO: 5, the CDR-H2 of SEQ ID NO: 6 and the CDR-H3 of SEQ ID NO: 7 and a variable light chain region comprising the CDR-L1 light chain of SEQ ID NO: 8, the CDR-L2 of SEQ ID NO: 9 and the CDR-L3 of SEQ ID NO: 10, wherein the second and third antigen-binding domains are each a Fab molecule; c. an Fc domain composed of a first and a second Petition 870200050086, dated 04 / 22 / 2020, pages 169 / 209 165 / 172 subunits capable of stable association, wherein the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first Fc domain subunit and wherein the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit. 77. The bispecific antibody of embodiment 0, wherein the first antigen-binding domain comprises a variable heavy chain region comprising the CDR-H1 heavy chain of SEQ ID NO: 34, the CDR-H2 of SEQ ID NO: 35 and the CDR-H3 of SEQ ID NO: 36 and a variable light chain region comprising the CDR-L1 light chain of SEQ ID NO: 37, the CDR-L2 of SEQ ID NO: 38 and the CDR-L3 of SEQ ID NO: 39. 78. The bispecific antibody of embodiment 0, wherein the first antigen-binding domain comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41. 79. The bispecific antibody of modality 0, wherein the first antigen-binding domain comprises the variable heavy chain region sequence of SEQ ID NO: 40 and the variable light chain region sequence of SEQ ID NO: 41. 80. A bispecific antibody of any of the 0 to 0 embodiments, wherein the second and third antigen-binding domains comprise a variable heavy chain region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of Petition 870200050086, dated 04 / 22 / 2020, pages 170 / 209 166 / 172 amino acid of SEQ ID NO: 3 and a variable light chain region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4. 81. The bispecific antibody of modality 0, wherein the second and third antigen-binding domains comprise the variable heavy chain region of SEQ ID NO: 3 and the variable light chain region of SEQ ID NO: 4. 82. The bispecific antibody of any of the 0 to 0 embodiments, in which the Fc domain incorporates, individually or in combination, all the features described in Sections 0 and 0 in relation to the Fc domains. 83. The bispecific antibody of either of the 0 to 0 modes, in which the antigen-binding domains and the Fc region are fused together by peptide linkers. 84. The bispecific antibody of modality 0, in which the peptide linkers comprise the peptide linkers as in SEQ ID NO: 45 and / or SEQ ID NO: 46. 85. The bispecific antibody of either of the 0 to 0 embodiments, wherein in the constant domain CL of the second and third Fab molecules, the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by lysine (K) or arginine (R), preferably by arginine (R) (numbering according to Kabat) and in the constant domain CH1 of the second and third Fab molecules under (ii) the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the EU Kabat index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the EU Kabat index). 86. A bispecific antibody of any of the 0 to 0 embodiments, comprising a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to Petition 870200050086, dated 04 / 22 / 2020, pp. 171 / 209 167 / 172 sequence of SEQ ID NO: 43, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to sequence of SEQ ID NO: 44, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to sequence of SEQ ID NO: 45 and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to sequence of SEQ ID NO: 46. 87. The bispecific antibody of embodiment 0, wherein the bispecific antibody comprises a polypeptide comprising the sequence SEQ ID NO: 43, a polypeptide comprising the sequence SEQ ID NO: 44, a polypeptide comprising the sequence SEQ ID NO: 45, and a polypeptide comprising the sequence SEQ ID NO: 46. 88. The bispecific antibody of modality 0, comprising two polypeptides comprising the sequence SEQ ID NO: 43. 89. The bispecific antibody of any of the 0 to 0 modes that is conjugated to a detectable moiety. 90. The bispecific antibody of modality 0 in which the detectable marker is an enzyme, a radioisotope, or a fluorescent label. 91. A fusion protein comprising the amino acid sequence of the anti-glyco-MUC1 antibody or antigen-binding fragment of either embodiment 1 to 0 or the bispecific antibody of either embodiment 0 to 0 operably linked to at least one second amino acid sequence. 92. The fusion protein of modality 0, where the second amino acid sequence is that of 4-1BB, CD3-zeta, or a fragment thereof. 93. The fusion protein of modality 0, in which the second amino acid sequence is that of a fusion peptide. Petition 870200050086, dated 04 / 22 / 2020, pp. 172 / 209 168 / 172 94. Fusion protein of modality 0, where the fusion peptide is a CD28-CD3-zeta or 4-IBB (CD137)-CD3-zeta fusion peptide. 95. The fusion protein of modality 0, in which the second amino acid sequence is that of a T cell activation modulator or a fragment thereof. 96. The fusion protein of modality 0, in which the modulator of T cell activation is IL-15 or IL-15Ra. 97. A chimeric antigen receptor (CAR) comprising scFv of either of the 0 to 0 modes. 98. The CAR of modality 0, comprising, in order from amino terminus to carboxy terminus: a human CD8 leader peptide, scFv, a human CD8 hinge domain, a human CD8 transmembrane domain, and a CD3-zeta signaling domain. 99. An antibody-drug conjugate comprising the anti-glyco-MUC1 antibody or antigen-binding fragment of any of the 1 to 0 embodiments or the bispecific antibody of any of the 0 to 0 embodiments or the fusion protein of any of the 0 to 0 embodiments conjugated to a cytotoxic agent. 100. The antibody-drug conjugate of embodiment 0, wherein the cytotoxic agent is an auristatin, a minor groove DNA binding agent, an alkylating agent, an enediine, a lexitropsin, a duocarmycin, a taxane, a dolastatin, a maytansinoid, or a vinca alkaloid. 101. The antibody-drug conjugate of modality 0, in which the anti-glyco-MUC1 antibody or antigen-binding fragment or bispecific antibody is conjugated to the cytotoxic agent via a linker. 102. The antibody-drug conjugate of modality 0, in which the ligand is cleavable under intracellular conditions. 103. The antibody-drug conjugate of modality 0, in Petition 870200050086, dated 04 / 22 / 2020, pp. 173 / 209 169 / 172 that the cleavable linker is cleavable by an intracellular protease. 104. The antibody-drug conjugate of modality 0, in which the ligand comprises a dipeptide. 105. The antibody-drug conjugate of modality 0, in which the dipeptide is val-cit or phe-lys. 106. The antibody-drug conjugate of embodiment 0, in which the cleavable linker is hydrolyzable at a pH of less than 5.5. 107. The antibody-drug conjugate of modality 0, in which the hydrolyzable linker is a hydrazone linker. 108. The antibody-drug conjugate of embodiment 0, in which the cleavable linker is a disulfide linker. 109. A nucleic acid comprising a coding region for an anti-glyco-MUC1 antibody or antigen-binding fragment of any of the embodiments 1 to 0 or the bispecific antibody of any of the embodiments 0 to 0, the fusion protein of any of the embodiments 0 to 0 or the CAR of embodiment 0 or embodiment 0. 110. The nucleic acid of modality 0 in which the coding region is optimized at the codon for expression in a human cell. 111. A vector comprising nucleic acid of modality 0 or modality 0. 112. The vector of modality 0 is a viral vector. 113. The vector of modality 0 in which the viral vector is a lentiviral vector. 114. A host cell engineered to express nucleic acid of modality 0 or modality 0. 115. The host cell of modality 0, which is a human T cell engineered to express the CAR of modality 0 or modality 0. 116. A host cell comprising the vector of Petition 870200050086, dated 04 / 22 / 2020, pages 174 / 209 170 / 172 either of the modalities 0 to 0. 117, The host cell of mode 0 is a T cell and the vector encodes the CAR of mode 0 or mode 0. 118. A pharmaceutical composition comprising (a) the anti-glyco-MUC1 antibody or antigen-binding fragment of any of the embodiments 1 to 0, the bispecific antibody of any of the embodiments 0 to 0, the fusion protein of any of the embodiments 0 to 0, the CAR of embodiment 0 or embodiment 0, the antibody-drug conjugate of any of the embodiments 0 to 0, the nucleic acid of embodiment 0 or embodiment 0, the vector of any of the embodiments 0 to 0 or the host cell of any of the embodiments 0 to 0 and (b) a physiologically suitable buffer, adjuvant or diluent. 119. A method for treating cancer comprising administering to a subject in need thereof an effective amount of anti-glyco-MUC1 antibody or antigen-binding fragment of any of the embodiments 1 to 0, bispecific antibody of any of the embodiments 0 to 0, fusion protein of any of the embodiments 0 to 0, CAR of embodiment 0 or embodiment 0, antibody-drug conjugate of any of the embodiments 0 to 0, nucleic acid of embodiment 0 or embodiment 0, vector of any of the embodiments 0 to 0, host cell of any of the embodiments 0 to 0 or pharmaceutical composition of embodiment 0. 120. The modality 0 method, in which the subject is suffering from breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, or colorectal cancer. 121. A method of detecting cancer in a biological sample, comprising contacting a sample with an anti-glyco-MUC1 antibody or antigen-binding fragment according to any of embodiments 1 to 0 and detecting the binding of the anti-glyco-MUC1 antibody or Petition 870200050086, dated 04 / 22 / 2020, pages 175 / 209 171 / 172 antigen-binding fragment. 122. The method of modality 0 additionally comprises quantifying the binding of the anti-glyco-MUC1 antibody or antigen-binding fragment. 123. The modality 0 method, or modality 0, in which the linkage is compared to a normal tissue control as a negative / baseline control and / or to a cancerous tissue control as a positive control.

[00361] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same degree as if each individual publication, patent, patent application or other document were individually indicated for incorporation by reference for all purposes. In the event of any inconsistency between the descriptions of one or more of the references incorporated herein and the present description, the descriptions in this descriptive report shall prevail. 8. REFERENCES 1. Bennett, E.P., Hassan, H., Mandel, U., Mirgorodskaya, E., Roepstorff, P., Burchell, J., Taylor-Papadimitriou, J., Hollingsworth, M.A., Merkx, G., van Kessel, A.G., and others. (1998) Cloning of a human UDP-Nacetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl- transferase that complements other GalNAc-transferases in complete Oglycosylation of the MUC1 tandem repeat. J. Biol. Chem., 273, 30472-30481. 2. Fontenot, J.D., Finn, O.J., Dales, N., Andrews, P.C., and Montelaro, R.C. (1993) Synthesis of large multideterminant peptide immunogens using a poly-proline beta-turn helix motif. Pept. Res., 6, 330336. 3. Mandel, U., Petersen, O.W., Sorensen, H., Vedtofte, P., Petição 870200050086, de 22 / 04 / 2020, pág. 176 / 209 172 / 172 Hakomori, S.I., Clausen, H., and Dabelsteen, E. (1991) Simple mucin-type carbohy- drates in oral stratified squamous and salivary-gland epithelia. J. Invest. Dermatol., 97, 713-721. 4. Miles, D.W., Linehan, J., Smith, P., and Filipe, I. (1995) Expression of sialyl-Tn in gastric cancer: correlation with known prognostic factors. Br. J. Cancer., 71, 1074-1076. 5. Schwientek, T., Bennett, E.P., Flores, C., Thacker, J., Hollmann, M., Reis, C.A., Behrens, J., Mandel, U., Keck, B., Schafer, M.A., and others. (2002) Functional conservation of subfamilies of putative UDP-Nacetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila. J. Biol. Chem., 277, 22623-22638. 6. Soares, R., Marinho, A., and Schmitt, F. (1996) Expression of Sialyl-Tn in breast cancer. Correlation with prognostic parameters. Pathol. Res. Pract., 192, 1181-1186. 7. Springer, G.F. (1984) T and Tn, general carcinoma autoantigens. Science, 224, 1198-1206. 8. Werther , JL , Tatematsu , M , Klein , R , Kurihara , M , Kumagai , K , Llorens , P , Guidugli , NJ , Bodian , C , Pertsemlidis , D , Yamachika , T , et al. (1996) Sialosyl-Tn antigen as a marker of gastric cancer progression: an international study. Int. J. Cancer., 69, 193–199. 9. Taylor-Papadimitriou, J., Burchell, J., Miles, DW, and Dalziel, M. (1999) MUC1 and cancer. Biochim. Biophys. Acta, 1455, 301313. Petition 870200050086, 4 / 22 / 2020, p. 177 / 2

Claims

1 / 4 CLAIMS:

1. Anti-glyco-MUCl antibody or antigen-binding fragment, characterized in that: a. it comprises a complementarity-determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31; b.preferentially binds to a glyco-MUC1 epitope that is overexpressed in cancer cells when compared to normal cells and / or binds to the MUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG)3 (SEQ ID NO: 47) that has been glycosylated in vitro using purified recombinant human glycosyltransferases GalNAcT1, GalNAc-T2 and GalNAc-T4 and (hereinafter referred to as the “first epitope”); optionally, wherein the anti-glyco-MUC1 antibody or antigen-binding fragment is a chimeric or humanized antibody.

2. Anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 1, characterized in that it comprises a VH comprising the amino acid sequence encoded by SEQ ID NO: 13 and a VL comprising the amino acid sequence encoded by SEQ ID NO:

14.

3. Anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that: a) it is in the form of a single-chain variable fragment (scFv), optionally wherein: i. the scFv comprises the N-terminal variable heavy chain fragment to the variable light chain fragment; and / or ii. the variable heavy chain fragment and variable light chain fragment of scFv are covalently linked to a linker sequence of 4 to 15 amino acids.

4. Anti-glyco-MUC1 antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that it is conjugated to a detectable moiety, which is optionally an enzyme, a radioisotope or a fluorescent label.

5. Fusion protein, characterized in that it comprises the amino acid sequence of the anti-glyco-MUC1 antibody or antigen-binding fragment as defined in any one of claims 1 to 3 operably linked to at least one second amino acid sequence, optionally wherein the second amino acid sequence is: a. that of 4-1BB, CD3-zeta or a fragment thereof; b. that of a CD28-CD3-zeta or 4-1BB (CD137)-CD3-zeta fusion peptide; or c. that of IL-15 or IL-15Ra.

6. Chimeric antigen receptor (CAR), characterized in that it comprises scFv as defined in claim 3, optionally wherein CAR comprises, in amino-to-carboxy-terminal order: a human CD8 leader peptide, scFv, a human CD8 hinge domain, a human CD8 transmembrane domain, and a CD3-zeta signaling domain.

7. Antibody-drug conjugate, characterized in that it comprises the anti-glyco-MUC1 antibody or antigen-binding fragment as defined in any one of claims 1 to 4, conjugated to a cytotoxic agent, optionally wherein: a. the cytotoxic agent is an auristatin, a minor groove DNA binding agent, an alkylating agent, an enediine, a lexitropsin, a duocarmycin, a taxane, a dolastatin, a maytansinoid or a vinca alkaloid; and / or b. the anti-glyco-MUC1 antibody or antigen-binding fragment is conjugated to the cytotoxic agent via a linker, optionally wherein the linker is cleavable under intracellular conditions, and optionally wherein: i. the cleavable linker is clivable by an intracellular protease, optionally wherein the linker comprises a dipeptide, optionally wherein the dipeptide is Val-cit ouphe-lys; ii.The linker is hydrolyzable at a pH of less than 5.5, optionally where the linker is a hydrazone linker; or iii. the linker is a disulfide linker.

8. Pharmaceutical composition, characterized in that it comprises (a) the anti-glyco-MUC1 antibody or antigen-binding fragment as defined in any one of claims 1 to 4, the fusion protein as defined in claim 5, the CAR as defined in claim 6, or the antibody-drug conjugate as defined in claim 7, and (b) a physiologically suitable buffer, adjuvant or diluent.

9. Use of the anti-glyco-MUC1 antibody or antigen-binding fragment as defined in any of claims 1 to 4, the fusion protein as defined in claim 5, the CAR as defined in claim 6, the antibody-drug conjugate as defined in claim 7, characterized in that it is for the manufacture of a medicament to treat cancer, optionally wherein the cancer is breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer or colorectal cancer.

10. Use of anti-glucose-MUC1 antibody or antigen-binding fragment as defined in any of claims 1 to 4 in a method for detecting cancer in a biological sample, characterized in that said method comprises the steps of: a) contacting a sample with an anti-glucose-MUC1 antibody or antigen-binding fragment; b) detecting the binding of the anti-glucose-MUC1 antibody or antigen-binding fragment; c) optionally, quantifying the binding of the anti-glucose-MUC1 antibody or antigen-binding fragment; d) optionally, comparing the binding with a normal tissue control as a negative / baseline control and / or to a cancerous tissue control as a positive control. Petition 870260076091, dated July 30, 2026, pp. 16 / 24

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