BR112020008001A2 - anti-glyco-muc1 antibody or antigen-binding fragment, fusion protein, chimeric antigen receptor, antibody-drug conjugate, nucleic acid, vector, host cell, pharmaceutical composition, and methods of treating cancer and detecting cancer in a biological sample. - Google Patents

Abstract

The present description relates 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. The present description further relates to the use of antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates and nucleic acids for cancer therapy.

Description

ANTI-GLICO-MUC1 ANTIBODY OR ANTIGEN BINDING FRAGMENT, FUSION PROTEIN, CHEMICAL ANTIGEN RECEPTOR, ANTIBODY-PHARMACEUTICAL, NUCLEIC ACID, VECTOR, HOSPITAL, EATER, COTERIC, COMPOSITION, ETC

DETECTING CANCER IN A BIOLOGICAL SAMPLE

1. SEQUENCE LISTING

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

2. GROUNDS

[002] The human mucin MUCI is a polymorphic transmembrane glycoprotein expressed on the apical surfaces of simple and glandular epithelia (Taylor-Papadimitriou er al., 1999). MUCI is highly overexpressed - and aberrantly - O-glycosylated in adenocarcinomas. The extracellular mucin domain 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 carbohydrate antigens pancreatic Tn (GalNAcal-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 insufficient 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 art for therapeutic modalities using glyco-MUCI1 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-MUCI epitopes.

[004] The present description provides anti-glyco-MUCI1I antibodies and antigen-binding fragments thereof that bind to a cancer-specific glycosylation variant of MUCI. The present description further provides fusion proteins and antibody-drug conjugates comprising anti-glyco-MUCI antibodies and antigen-binding fragments and nucleic acids encoding anti-glyco-MUCI antibodies, antigen-binding fragments and fusion proteins.

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

[006] In certain respects, the description provides bispecific and other multispecific anti-glyco-MUCI antibodies and antigen-binding fragments that bind to a MUCI cancer-specific glycosylation variant and a second epitope. The second epitope may be in the MUCI itself, in another protein coexpressed in cancer cells with MUC1, 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 codon-optimized coding regions and nucleic acids comprising coding regions that are not codon-optimized for expression in a particular host cell.

[007] Anti-glyco-MUCI antibodies and binding fragments can be in the form of fusion proteins containing a fusion partner. The fusion partner can be useful to provide a second function, such as a signaling function of the signaling domain 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-IBB-CD3-zeta. 4-1BB or CD137, are a co-stimulatory T cell receptor; CD3-zeta is a signal transduction component of the T cell antigen receptor. The portion providing a second function may be a modulator of T cell activation, such as IL-15, IL-15Ra or an IL-15 / fusion. IL-15Ra or the same can encode a label or enzyme component of a labeling system useful in monitoring the degree and / or location of the binding in vivo or in vitro. Prophylactically and therapeutically active constructs - encoding - these - 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 modalities of the description.

[008] In certain respects, an anti-glyco-MUCI antibody or antigen-binding fragment of the description comprises heavy and / or light chain variable sequences (or encoded by the nucleotide sequences) shown in Table 1. For clarity, when the the term "anti-glyco-MUCI antibody" is used in this document, it is intended to include monospecific and multispecific anti-glyco-MUCI antibodies (including bispecifics), antigen-binding fragments of monospecific and multispecific antibodies and fusion proteins and conjugates containing antibodies and their antigen-binding fragments, unless the context otherwise requires. Likewise, when the term

“Anti-glyco-MUCI antibody or antigen-binding fragment” is used, it is also intended to include monospecific and multispecific (including bispecific) anti-glyco-MUCI 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-MUCI1I antibody or antigen-binding fragment of the description comprises CDR sequences of the heavy and / or light chain (or encoded by the nucleotide sequences) shown in Tables | to 3. The CDR sequences shown 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, Sa Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) And Chothia (Al-Lazikani et al., 1997, J. Mol. Biol 273: 927-948) to define the boundaries of The CDR sequences shown in Table 2 are the combined overlapping regions for the CDR sequences shown in Table 1, with the IMGT, Kabat and Chothia sequences shown in bold underlined text The CDR sequences shown in Table 3 are the common overlapping regions for the CDR sequences shown in Table 1. The structural sequences for such anti-glyco-MUCI1 antibody and antigen binding fragment may be the murine native structural sequences in Table 1 or they may be non-structural sequences native (for example , humanized or human).

[String ofMGWSGIFLFFLSVTTGVHSQVQLQQSDAELVKPGASVKI | 1 lamino acid VH (incl SCKAS GYTFTDHAIHWVKQRPEQGLEWIGYFSPGNDDI signal frequency) - HYNEKFEGKATLTADKSTSYYNS

IKRSYDKDFDCWGQGTTLTVSS [String of MVLILLLLWVSGTCGDIVMSQSPSSLGVSVGEKVTMSC Pp Lamino Acid VL (incl. 1LAVYYC QQYYRYPLTFGAGTKLELK

Sequence deQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVK B laminoácido VHQRPEQGLEWIGYFSPGNDDIHYNEKFEGKATLTADKSSS mature 'AYMQLNSLTSEDSAVYFCKRSYDKDFDCWGQGTTLT iprognosticada) SS [deDIVMSQSPSSLGVSVGEKVTMSCKSSQSLLYSTNQKNY sequence amino acid VLQSLLYSTNQKNYLAW YQQKPGQSPKLLIYWVSNRKSG mature IVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC | predicted) IQQY YRYPLTFGAGTKLELK sequence deGYTFTDHA 5 laminoácido CDR + | H1 (IMGT definition) [Sequence delFSPGNDDI 6 laminoácido of CDR-2 (definition IMGT) Sequence deKRSYDKDFDC laminoacid of CDR- | H3 (IMGT definition) [Sequence delQSLLYSTNQKNY B laminoacid of CDR +] 1 (IMGT definition) Sequence of WVS Pp laminoacid of CDR- | L2 (IMGT definition Sequence of QQYYRYPLT 10-laminoid | ) ATGGGATGGAGCGGGATCTTTCTCTTCTTCCTGTCAGT sequence |! 1 VH hucleotide (incl. CTACAGGTGTCCACTCCCAGGTTCAGCTGCAGCAG signal sequence) CTGACGCGGAGTTGGTGAAACCTGGGGCTCTAGTGA

AGATATCCTGCAAGGCTTCTGGCTACACTTTCACTGA CATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAG IGGCCTGGAATGGATTGGATATTTTTCTCCCGGAAATG TGACATTCACTACAATGAGAAGTTCGAGGGCAAGG CACACTGACTGCAGACAAATCCTCCAGCACTGCCTA ATGCAGCTCAACAGCCTGACATCTGAAGATTCTGCA IGTGTATTTCTGTAAAAGATCTTACGACAAGGACTTTG

ACTGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTC Sequence of ATGGTTCTTATCTTACTGCTGCTATGGGTATCTGGTAC | 12 ucleotide VL (incl CTGTGGGGACATTGTGATGTCACAGTCTGATGTGTGTGTGG

IGCAAGTCCAGTCAGAGCCTTTTATACAGTACCAATCA | AAAGAACTACCTGGCCTGGTACCAGCAGAAACCAGG IGCAGTCTCCTAAGTTGCTGATTTACTGGGTATCTAATA; |

IGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGG | ATCTGGGACAGATTTCACTCTCACCATCAGTAGTGTG

IAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAAT I | ATTATAGGTATCCGCTCACGTTCGGTGCTGGGACCAA

GCTGGAGCTGAAA Sequence ofCAGGTTCAGCTGCAGCAGTCTGACGCGGAGTTGGTGA | 13 VH ucleotide (excl. CCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTC signal sequence) GGCTACACTTTCACTGACCATGCTATTCACTGGGG

GCAGAGGCCTGAACAGGGCCTGGAATGGATTGGA 'ATTTTTCTCCCGGAAATGATGACATTCACTACAATG GAAGTTCGAGGGCAAGGCCACACTGACTGCAGACA TCCTCCAGCACTGCCTACATGCAGCTCAACAGCCT IGACATCTGAAGATTCTGCAGTGTATTTCTGTAAAAGA CTTACGACAAGGACTTTGACTGCTGGGGCCAAGGCA CACTCTCACAGTCTCCTCA

DelGACATTGTGATGTCACAGTCTCCATCCTCCCTAGGTGT | 14 hucleotide sequence VL (excl GTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCC signal sequence) GTCAGAGCCTTTTATACAGTACCAATCAAAAGAACT

CCTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCC 'AAGTTGCTGATTTACTGGGTATCTAATAGGAAATCT GGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGA CAGATTTCACTCTCACCATCAGTAGTGTGAAGGCTGA GACCTGGCAGTTTATTACTGTCAGCAATATTATAGG 'ATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGC

GAAA ERHIO - - OVOLQQSDAELVKPGASVKISCKAS ERL2 ——— LAWYOQOKPGOSPRIEZ bo Sequence of DHAIH 3 laminoacid from CDR- HI (definition of IKabat) [Sequence of YFSPGNDDIHYNEKFEG p4 lamina from (CDR +) definition of KRD (3) | (IKabat sequence) [DSSQSLLYSTNQKNYLA p6 CDR + lamino acid sequence] 11 (definition of | Kabat) DefWVSNRKS sequence 7 CDR-12 lamino acid (IKabat definition) [QQYYRYPLT sequence 1st lamino acid of CDR +] 13 (definition of | of CDR- HI (definition of “hothia) [Sequence of desPGNDD by CDR + lamino acid] H2 (definition of hothia) Sequence of YDKDFDC 5 lamino acid of CDR-H3 (definition of“ hothia) [Sequence ofSQSLLYSTNQKNY BO lamino acid of CDR + 11 (definition of | Chothia)

TI17T2 CDR-12 lamino acid defWVS sequence (definition of 'hothia) CDY-13 amino acid sequence YYRYPLT Bl (definition of [| Chothia) dellGYTFTDHAIH (IMGT) B2 amino acid da GYTFTDHAIH (Kabat) -DR-H1HTHHAID -H1HTDHHHHIDDHH ) equation delYFSPGNDDIHY NEKFEG (IMGT) 2) imino acid daYFSPGNDDIHYNEKFEG (Kabat) DR-H2 FSPGNDDIHYNEKFEG (Chothia) combined overlapping 6 laminoacid - CDR-KSSOSLLYSTNQKNYLA (Kabat) 11 (overlapKSKSLLYSTNQKNYLA (Chothia) combined) equis delWVSNRKS (IMGT) 7 amino acid CDR- | WVSNRKS (Kabat) 12 - (overlapWVSNRQYTTH) (10) -JOQY YRYPLT (Kabat) 13 (QQYYRYPLT overlay (Chothia) combined) debDH B3 imino acid d: -DR-H1 common sequence) PSPGNDD 29 DR-H2 imino acid (common sequence) YD equation KDFDC pS amino acid d: CDR-H3 common sequence) QSLLYSTNQKNY imino acid equation —CDR-LI (sequence | common)

delWVS equation p lamino acid = CDR + 1L2 (omum sequence) Dely YRYPLT B1 sequence incincid CDR-L3 (.omum sequence)

[0010] In certain aspects, the description provides an anti-glyco-MUCI antibody or antigen-binding fragment of the description comprises CDRs comprising the amino acid sequences of any of the CDR combinations presented in the numbered 3 to 17 modalities. certain embodiments, an anti-glyco-MUCI1 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-HI1 comprises the amino acid sequence of SEQ ID NO: 5, 23, 28 or 32. In some embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6 or 24. In a 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 respects, an anti-glyco-MUC! 1 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 respects, an anti-glyco-MUCI1 antibody or antigen-binding fragment of the description comprises CDRs of heavy chain of SEQ ID NOS: 23 to 25 and light chain CDRs of SEQ ID NOS: 26, 27 and 10. In other aspects, an anti-glyco-MUC1I antibody or antigen binding fragment of the description comprises heavy chain CDRs of SEQ ID NOS: 28, 29 and 25 and light chain CDRs of SEQ ID NOS: 30, 9 and 31. In other respects, an antibody anti-glyco-MUC1 or antigen binding fragment of the description comprises heavy chain CDRs of SEQ ID NOS: 32, 24 and 7 and light chain CDRs of SEQ ID NOS: 26, 27 and 10. In other respects, an antibody anti-glyco-MUCI1 or antigen binding fragment of the description comprises heavy chain CDRs of SEQ ID NOS: 33, 29 and 25 and light chain CDRs of SEQ ID NOS: 8, 9 and 31. The antibody or binding fragment the antigen can be murine, chimeric, humanized or human.

[0011] In additional aspects, an anti-glyco-MUCI1 antibody or antigen binding fragment of the description competes with an antibody or antigen binding fragment comprising variable regions of heavy and light chain of SEQ ID NOS: 3 and 4, respectively . In other respects, the description provides an anti-MUCI1 antibody or antigen binding fragment having variable regions of heavy and light chain having at least 95%, 98%, 99% or 99.5% sequence identity of SEQ ID NOS: 3 and 4, respectively.

[0012] In other respects, an anti-glyco-MUCI1 antibody or antigen-binding fragment of the description is a single-chain variable fragment (scFv). An exemplary scFv comprises the heavy chain variable fragment to the N-terminal 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 can be in the form of a bispecific T-cell engagement or within a chimeric antigen receptor (CAR).

[0013] Anti-glyco-MUCI1 antibodies and antigen-binding fragments can be in the form of a multimer of a single chain variable fragment, a bispecific single chain variable fragment and a multimeter of a bispecific single chain variable fragment. In some embodiments, the multimer of a single variable chain fragment is selected from a bivalent single chain variable fragment, a tri-body or a tetrabody. In some of these embodiments, the multimer of a bispecific single-stranded variable fragment is a bispecific T-cell engagement.

[0014] Other aspects of the description are outlined for nucleic acids encoding the anti-glyco-MUCI antibodies and antibody binding fragments of the description. In some embodiments, the portion of the nucleic acid encoding an anti-glyco-MUCI1 antibody or antigen binding fragment is optimized at the codon for expression in a human cell. In certain aspects, the description provides an anti-glyco-MUCI1 antibody or antigen binding fragment having variable regions of heavy and light chain 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 (for example, a viral vector such as a lentiviral vector) and host cells comprising nucleic acids are also within the scope of the description. The heavy and light chain coding sequences 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-MUCI antibody, antigen-binding fragment, nucleic acid (or pair of nucleic acids), vector (or pair or vectors) or host cell according with description and a cap,

physiologically suitable adjuvant or diluent.

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

[0017] Another aspect of the description is a method of detecting cancer comprising contacting a cell or tissue sample with an anti-glyco-MUCI1 antibody or antigen binding fragment of the description and detecting whether the antibody is attached to the cell sample or fabric.

[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-MUCI antibody, antigen-binding fragment, nucleic acid, vector, host cell or pharmaceutical composition according to the description to a subject in need of them.

4. BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1: Results of ELISA assay showing specificity of GO2 binding to glyco-MUCI1 over MUCI.

[0020] FIG. 2: Binding of GO2 to colonic cancer tissue. Immunohistochemical labeling of invasive colonic carcinoma tissue and adjacent healthy tissue using mAbs GO2. MAb GO? 2 shows a distinct link to colonic cancer tissue with high reactivity with both intracellular and superficial structures in cancer cells. In contrast, no reactivity is observed in the surface structures in healthy colonic cells.

[0021] FIG. 3: Binding of GO2 to pancreatic cancer tissue. Immunohistochemical labeling of pancreatic cancer tissue using mAbs GO? 2. GO? 2 mAb shows distinct binding to pancreatic cancer cells.

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 mAbs GO2. MAb GO? 2 showed a distinct link to invasive breast cancer cells.

[0023] FIG. 5: Results of an antibody-dependent cell cytotoxicity assay with GO2 antibody and a secondary antibody conjugated to the antitubulin 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 TMA tumor nuclei positive in MUCI. 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-MUCI and bispecific T-cell anti-CD3 (TCB) antibody.

[0027] FIGS. 9A-B: Jurkat-NFAT activation assay with tumor samples derived from undigested patient (malignant bronchial and lung neoplasm: intermediate lobe, bronchus or lung, squamous cell carcinoma) and different TCBs at 50 nM (FIG. 94 ) or 5 nM (FIG. 9B).

[0028] FIG. 10: Jurkat-NFAT activation assay with tumor samples derived from undigested patient (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 tumor samples derived from undigested patient (malignant neoplasm of bronchus and lung: upper lobe, bronchus or lung, adenocarcinoma with acinar type) and different TCBs at 50 nM.

[0030] FIGS. 12A-12B: Binding of TC2 GO2 to MUCI expressed in cells MCF7 cs (FIG. 12A) and T3M4 pzfv (FIG. 12B) measured by flow cytometry.

[0031] FIGS. 13A-X: Induction of tumor cell extermination and T cell activation measured by CD25 and CD69 overloading on CD4 T cells and CD8 T cells as well as IL6, IL8, ILIO, IFNy, TNFa and Granzima B release with TCB GO2 in T3MA4 pzfv in the presence of PBMCs from two healthy donors (donor 1 FIG. 13A-13L; donor 2 FIG. 13M-13X). The same legend for each of the FIGS. 13A-13X.

[0032] FIGS. 14A-14F: Induction of tumor cell extermination (FIGS. 14A-14B) and T cell activation measured by the supra regulation 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 for each of the FIGS. 14A-14F.

[0033] FIG. 15A-B: Binding of TCB GO2 and TCB HMFGI1 to MCFIOA (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 overloading on CD4 T cells (FIG. 16B) and CD8 T cells (FIG. 16C) with TCB GO2 and TCB HMFGI1 in MCFIOA cells in the presence of PBMCs.

[0035] FIG. 17: Illustration of GO2 and TCB GO? 2 flowing through a flow cell having glycopeptides attached.

[0036] FIG. 18A-B: Sensorgrams showing the binding of GO2 (FIG. 18A) and TCB GO? 2 (FIG. 18B) to human and cinomolg glycopeptides.

[0037] FIG. 19A-D: Binding (avidity) of GO2 antibody (FIG. 19A-19B) and TCB GO? 2 (FIG. 19C-19D) to human glycopeptides and cinomolg and “apparent” KD estimate.

5. DETAILED DESCRIPTION

5.1 Antibodies

[0038] The inventor has developed new antibodies that target a glycoform of MUCI1 present in tumor cells. These are exemplified by the 5F7 antibody, referred to here as “GO2”. GO? 2 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, VISAPDTRPAPGSTAPPAHG (SEQ ID NO: 50), glycosylated with purified recombinant human glycosyltransferase polypeptides GalNAc-T2, GalNAc-T4 and GalNAc-T1 in order to mimic the glycosylation pattern of MUCI1 present in tumor cells.

[0039] The anti-glyco-MUCI1 antibodies of the description, exemplified by the GO? 2 antibody, are useful as tools in the diagnosis and therapy of cancer.

[0040] Thus, in certain aspects, the description provides antibodies and antigen-binding fragments that bind to a MUC1 glycoform present in tumor cells (referred to herein as "glyco-MUCI") and preferably to the 60-mer peptide (VTSAPDTRPAPGSTAPPAHG); (SEQ ID NO: 47) glycosylated with GalNAc-T2, GalNAc-T4 and GalNAc-TI1 as described in US Patent No. 6,465,220.

[0041] The anti-glyco-MUCI antibodies of the description 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, antibodies of single chain, bispecific antibodies, double variable domain antibodies, etc. In various embodiments, antibodies comprise all or a portion of an antibody constant region. In some modalities, the constant region is an isotype selected from:

IgA (for example, IgA, or IgA>), IgD, IgE, IgG (for example, IgG :, IgG ', IgG; or IgG.) And IWGM. In specific embodiments, the anti-glyco-MUCI antibodies of the description comprise an isotype of the IgG constant region.

[0042] The term "monoclonal antibody" as used herein is not limited to antibodies produced using 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. Monoclonal antibodies useful with 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-MUCI antibodies in humans, chimeric, primatized, humanized or human antibodies can be used appropriately.

[0043] The term "chimeric" antibody as used herein refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a mouse or mouse antibody and human immunoglobulin constant regions, typically chosen from a standard of human immunoglobulin. 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; Pat. No. 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 (for example, murine) are chimeric immunoglobulins that contain 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 and all or substantially all of the FR regions are those of a sequence of human immunoglobulin. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Antibody humanization methods are known in the art. See, for example, Riechmann et al., 1988, Nature 332: 323-7; Pat. No. 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. No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28: 489-498; Studnicka er al., 1994, Prot. Eng. 7: 805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91: 969-973 and Pat. No. 5,565,332, all of which are hereby incorporated by reference in their entirety.

"Human antibodies" include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or animals transgenic to one or more human immunoglobulins and which 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 Pat. No. 4,444,887 and 4716111 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 that can express human immunoglobulin genes. See, for example, PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; Pat. US. No. 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 incorporated herein by reference in their 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 completely human antibody recognizing the same epitope (see, Jespers et al., 1988, Biotechnology 12: 899-903) .

[0046] "Primatized antibodies" comprise variable regions of monkeys and constant human regions. Methods for producing primatized antibodies are known in the art. See, for example, Pat. U.S. No.

5,658,570, 5,681,722 and 5,693,780, which are incorporated herein by reference in their entirety.

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

[0048] A Fab fragment contains the light chain constant domain (CL) and the first heavy chain constant domain (CH1). 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 antibody hinge region. F (ab ') fragments are produced by cleaving the disulfide bond in the hinge cysteines of the F (ab') pepsin digestion product. Additional chemical junctions of antibody fragments are known to those of ordinary skill in the art. Fab and F (ab ') fragments lack the intact antibody Fc fragment, clear more quickly from animal circulation and may have less non-specific tissue binding than an intact antibody (see, for example, Wahl er al., 1983, J. Nucl. Med. 24: 316).

[0049] An "Fv" fragment is the minimal fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer from a heavy-chain variable domain and a light one in a firm, non-covalent association (Vu-V1 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 Vu-Vr dimer. Often, the six CDRs confer specificity of target binding to the antibody. However, in some cases even a single variable domain (or half of a Fvy 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 "scFv" antigen binding fragments comprise the Vu and Vr 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 Vu and Vr domains that allows the scFv to form the desired structure for the target binding.

[0051] "Single domain antibodies" are composed of single VH or Vr domains that exhibit sufficient affinity for glyco-MUCI1. In a 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-MUCI1 antibodies of the description can also be bispecific and other multiple specific antibodies. Bispecific antibodies are monoclonal antibodies, often human or humanized, that have binding specificities for two different or different epitopes on the same antigen. In the present description, one of the binding specificities can be directed to glyco-MUCI, the other can be to any other antigen, for example, to a cell surface protein, receptor, receptor subunit, tissue specific antigen, viral derived protein, virally encoded envelope protein, bacterially derived protein or bacterial surface protein, etc. In certain preferred embodiments, bispecific and other multispecific anti-glyco-MUCI antibodies and antigen-binding fragments that specifically bind to a second MUC1 epitope, an epitope on another protein coexpressed in cancer cells with MUC1 or an epitope on another protein displayed in a different cell, such as an activated T cell. The bispecific antibodies of the description include bispecific antibodies in IgG format and bispecific antibodies based on single chain.

[0053] The bispecific IgG-format antibodies of the description can be any one of several types of bispecific IgG-format antibodies known in the art, such as bispecific quadroma antibodies, bispecific "knobs-in-holes" antibodies, bispecific CrossMab antibodies, antibodies bispecific paired in the load, bispecific common light chain antibodies, bis-specific single-range Fab arm antibodies single-immunoglobulin, bispecific disulfide stabilized Fv antibodies, DuetMabs, bis Fab specific branch exchange antibodies, bispecific engineered domain body antibodies filament exchange, heterodimeric monoclonal bispecific monoclonal antibodies of two-branch leucine zipper, bispecific KX body antibodies, bispecific double variable domain antibodies and bispecific double variable domain antibodies. See, for example, Kôhler 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.,

2014, J Biol Chem 289: 18693-706; Metz et al., 2012 Protein Eng Des Sel 25: 571-80; Mazor et al., 2015 MAbs 7: 37789; Labrijn et al., 2013 Proc Natl Acad Sci USA 110: 5145—-50; Davis et al., 2010 Protein Eng Des Sel 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 incorporated herein by reference in their entirety.

[0054] In some embodiments, the bispecific antibodies of 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 crossing between heavy and light chains within a bispecific Fab branch IgG, which promotes the correct chain association. A CrossMab bispecific antibody of the description can be a "CrossMab" "P" antibody, in which the heavy and light chains of the Fab portion of an arm of a bispecific IgG antibody are switched in. In other embodiments, a CrossMab bispecific antibody of the description can be a "CrossMabY" -Y! I "" antibody, in which the only variable domains of the heavy and light chains of the Fab portion of a branch of a bispecific IgG antibody are exchanged. In other embodiments, a bispecific CrossMab antibody of the description may be a “CrossMabF! Cr 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 switched. CrossMabH !! - eL antibodies, unlike CrossMab" * P e CrossMabY "YL, do not have prognosticated side products and therefore, in some modalities bispecific antibodies CrossMabCºHF! | Are preferred. See, Klein er al., 2016, mAbs, 8 (6): 1010-

1020. Additional CrossMabs modalities of the description are described below in Section 5.2.

[0055] In some embodiments, the bispecific antibodies of the description are controlled Fab branch exchange bispecific antibodies. Methods for making bispecific Fab branch exchange antibodies are described in PCT Publication No. WO2011 / 131746 and Labrijn et al., 2014 Nat Protoc. 9 (10): 2450-63, hereby incorporated by reference in their entirety. Briefly, bispecific controlled Fab branch exchange antibodies can be manufactured by separately expressing two precursor IZGls containing single pairing mutations in the CH3 domain, mixing the precursor IgG1ls under redox conditions in vitro to allow recombination of half molecules and removing the reducer to allow reoxidation of the interchain disulfide bond, thereby forming bispecific antibodies.

The bispecific antibodies of the description can comprise an Fc domain composed of a first and a second subunit. In one embodiment, the Fc domain is an IZG Fc domain. In a particular embodiment, the Fc domain is an IZG Fc domain. In another embodiment, the Fc domain is an Fc IgG domain. In a more specific embodiment, the Fc domain is an Fc IgG domain, comprising an amino acid substitution at position S228 (numbering the EU index of Kabat), particularly the substitution of amino acid S228P. This amino acid substitution reduces the exchange of the Fab branch of IgG antibodies in vivo, (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 an even more particular embodiment, the Fc domain is a human IgG Fc domain. An exemplary sequence of a human IgG Fc region is given in SEQ ID NO: 42.

[0057] In particular modalities, the Fc domain comprises a 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 IZG 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 modality, 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. 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 protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protuberance can be positioned in the cavity in order to promote heterodimer formation and formation of hindered homodimer. The bulges are constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (for example tyrosine or tryptophan). Compensatory cavities of identical or similar size to the bulges are created at the interface of the second polypeptide by replacing the large amino acid side chains with the small ones (for example alanine or threonine).

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 bulge within the CH3 domain of the first subunit that it is positionable in a cavity within the CH3 domain of the second subunit and an amino acid residue in the CH3 domain of the second Fc domain subunit is replaced with an amino acid residue having a smaller side chain volume, thereby 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 bulge and cavity can be made by changing the nucleic acid encoding the polypeptides, for example by site-specific mutagenesis or by peptide synthesis. An exemplary replacement is Y470T.

[0060] In such a 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) (numbered according to index US from Kabat). In an additional embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue) and in the second subunit of the Fc domain in addition the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numbered according to the EU Kabat index ). In a particular embodiment, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the EU index of Kabat).

[0061] In some embodiments, electrostatic steering (for example, 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 receiver is an activating Fc receiver. In a specific embodiment the Fc receptor is an activator of the human Fcy receptor, more specifically FcyRIIIa, FeyRI or FeyRITa, humans more specifically human FceyRIIla. 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 a particular modality, 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, one or more amino acid substitution (s) reduces (in) the binding affinity of the Fc domain to an Fc receptor. In one embodiment, one or more amino acid substitution (s) reduces (in) 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 position selected from the group of E233, L234, L235, N297, P331 and P329 (numbers according to the EU index of

Kabat). In a more specific modality, the Fc domain comprises an amino acid substitution in a position selected from the group d L234, 1235 and P329 (numbers according to the EU index of Kabat). In some embodiments, the Fc domain comprises amino acid substitutions L1234A and L235A (numbers according to the EU index of Kabat). In such an embodiment, the Fc domain is an IgG Fc domain, particularly a human IZG Fc domain.

In one embodiment, the Fc domain comprises an amino acid substitution at the P329 position. In a more specific modality, the amino acid substitution is P329A or P329G, particularly P329G (numbers 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 (numbers 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 (numbers according to the EU index of Kabat). In more particular modalities, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in particular modalities, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A and P329G (numbering the EU index of Kabat), that is, in each of the first and second subunits of the Fc domain the leucine residue in position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced with a glycine residue (P329G) (numbering according to the EU Kabat Index). In such an embodiment, the Fc domain is an IgG Fc domain, particularly a human IZG Fc domain.

[0066] The bispecific single-chain based antibodies of the description can be any of the several types of bispecific single-chain based antibodies known in the art, such as bispecific T cell engagers (BiTEs), diabody, tandem diabody (tandabs) ), dual affinity targeting molecules (DARTs) and bispecific killer cell See, for example, Lóffler et al., 2000, Blood 95: 2098-103; Holliger et al., 1993, Proc Natl Acad Sci USA, 90: 64448; Kipriyanov et al., 1999, Mol Biol 293: 41-56; Johnson et al., 2010, Mol Biol 399: 43649; 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 polypeptide chain molecules that have 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, PCT Publication WO 05/061547; Baeuerle et al., 2008, Drugs of the Future 33: 137-147; Bargou, et al., 2008, Science 321: 974-977, hereby incorporated by reference in their entirety). Thus, the BiTEs of the description have an antigen binding domain that binds to a T cell antigen and a second antigen binding domain that is targeted to glyco-MUCI.

[0068] In some embodiments, the bispecific antibodies of the description are double affinity targeting molecules (DARTs). DARTs comprise at least two polypeptide chains that associate (especially through covalent interaction) to form at least two epitope binding sites, which can recognize the same or different epitopes. Each of the polypeptide chains of a DART comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope binding site. Instead, the immunoglobulin heavy chain variable region of one (for example, the first) of the DART polypeptide chains interacts with the immunoglobulin light chain variable region of a DARTO polypeptide chain (for example, the second) different to form an epitope binding site. Similarly, the immunoglobulin light chain variable region of one of the DART polypeptide chains (for example, the first) of DART interacts with the immunoglobulin heavy chain variable region of a different DART polypeptide chain (for example, the second) to form an epitope binding site. DARTs can be monospecific, bispecific, triespecific, etc., thus being able to simultaneously link one, two, three or more different epitopes (which can be from the same or different antigens). DARTs can additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., thus being able to simultaneously link one, two, three, four, five, six or more molecules. These two attributes of DARTs (that is, the degree of specificity and valence can be combined, for example to produce bispecific antibodies (that is, capable of binding two epitopes) that are tetravalent (that is, 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 of the description, one of the binding specificities is directed to glyco-MUC1 and the other is directed to an antigen expressed in the 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 lymphoid lineage such as natural killer cells (NK), T cells including T cells or cytotoxic B cells, but also cells of the myeloid lineage can be considered as immune effector cells, such as monocytes or macrophages, cells dendritic 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 the immune effector cells are brought into immediate vicinity to the aberrant target cells such that the effector cells can directly kill or indirectly initiate the extermination of the aberrant cells for which they are recruited. In order to avoid non-specific interactions, it is preferred that the bispecific antibodies of the description 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 can include CD3, CD8, CDI16, 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 CD3 native to any vertebrate source, including mammals such as primates (eg humans), non-human primates (eg cynomolous monkeys) and rodents (eg mice and rats), unless otherwise indicated. The term covers unprocessed “full size CD3” as well as any form of CD3 that results 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 in an immune effector cell is the epsilon chain of CD3. This antigen has been shown to be very effective in recruiting T cells to aberrant cells. Consequently, a bispecific antibody of the description preferably specifically recognizes epsilon CD3. The amino acid sequence of human CD3 epsilon is shown at UniProt (wwW.uniprot.org) at. P07766 (version 144) or NCBI (www.ncbi.nlIm.nih.gov/) RefSeq NP 000724.1. The amino acid sequence of CD3 epsilon of cinomolgo [Macaca fascicularis] 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 epsilon CD3 chain) are used. For preclinical testing on non-human animals and cell lines, bispecific antibodies in which the CD3 binding domain specifically binds to CD3 in the species used for the preclinical test (eg, cynomolgo CD3 for primate testing) can be used.

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

[0072] In some embodiments, a bispecific antibody of the description may compete with monoclonal antibody H2C (described in PCT publication No. WO2008 / 119567) for binding to a CD3 epitope. in other embodiments, a bispecific antibody of the description may compete with monoclonal antibody V9 (described in Rodrigues et al., 1992, Int J Cancer Suppl 7: 45-50 and US Pat. No. 6,054,297) for binding to an epitope of CD3. In other embodiments, a bispecific antibody of the description can compete with the monoclonal antibody FNI8 (described in Nooij et al., 1986, Eur J Immunol 19: 981-984) for binding to a CD3 epitope. In other embodiments, a bispecific antibody of the description can compete with the monoclonal antibody SP34 (described in Pessano et al., 1985, EMBO J 4: 337-340) for binding to a CD3 epitope.

The anti-glyco-MUCI antibodies of the description 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 protection / blocking groups, proteolytic cleavage, binding to a cell ligand or other protein. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivative may contain one or more unnatural amino acids, for example, using ambrx technology (See, for example, Wolfson, 2006, Chem. Biol. 13 (10): 1011-2).

[0074] Anti-glyco-MUCI1 antibodies or binding fragments can 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-glyco-MUCI antibody can be modified to reduce at least one biological effector function mediated by the constant region in relation to the unmodified antibody, for example, reduced binding to the Fc receptor (FeyR). FcyR binding can be reduced by mutating the constant segment of the immunoglobulin region of the antibody in the particular regions necessary for FcyR interactions (See, for example, Canfield and Morrison, 1991, 5. Exp. Med. 173: 1483-1491 and Lund et al., 1991, J. Immunol. 147: 2657-2662). The 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 cell cytotoxicity (“ADCC”).

The anti-glyco-MUCI1 antibody or binding fragments described herein include antibodies and / or binding fragments that have been modified to acquire or improve at least one biological effector function mediated by the constant region relative to an unmodified antibody, for example. example, to highlight FcyR interactions (See, for example, US 2006/0134709). For example, an anti-glyco-MUC1 antibody of the description may have a constant region that binds FcyRIIA, FeyRIIB and / or FcyRIIIA with greater affinity than the corresponding wild type constant region.

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

[0077] In some embodiments, the anti-glyco-MUC1 antibodies of the description have low levels or lack of fucose. Antibodies lacking fucose have been correlated with enhanced ADCC activity, especially at low doses of antibody. See Shields et al., 2002, J. Biol. Chem. 277: 26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278: 3466-

73. Methods of making antibodies with less fucose include growth in rat myeloma YB2 / 0 cells (ATCC CRL 1662). YB2 / 0 cells express low levels of FUTS mMRNA, which encodes α-1,6-fucosyltransferase, an enzyme required for polypeptide fucosylation.

[0078] In yet another aspect, anti-glyco-MUC1 antibodies or binding fragments include modifications that increase or decrease their binding affinities to the fetal Fc receptor, FcRn, for example, by mutating the constant segment of the immunoglobulin in the 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 combinations thereof, such as at positions 250 and 428 or in positions 250 and 314 or in positions 314 and 428 or in positions 250, 314 and 428, with positions 250 and 428 being a specific combination. For position 250, the substituting amino acid residue can 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 substituting amino acid residue can 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 heading 428, the substituting amino acid residues can 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 Pat. No. 7,217,797, which is incorporated herein by reference. Such mutations increase the binding to FcRn, which protects the antibody from degradation and increases its half-life.

[0079] In other respects, an anti-glyco-MUCI1I antibody of the antigen binding fragment of the description has one or more amino acids 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 Ped. Pat. No. 2007/0280931.

[0080] In other respects, particularly useful for diagnostic applications, an anti-glyco-MUCI1 antibody of the antigen binding fragment of the description is attached to a detectable portion. Detectable moieties include a radioactive moiety, a colorimetric molecule, a fluorescent moiety, a chemiluminescent moiety, an antigen, an enzyme, a detectable granule (such as a magnetic or electrode dense granule (for example, gold)) or a molecule that binds to another molecule (for example, biotin or streptavidin)).

[0081] Radioisotopes or radionuclides may include? H, * C, 'N, 358, 90 Tc, "Tn, 251, VM,

[0082] Fluorescent labels may include rhodamine, lanthanide matches, fluorescein and its derivatives, fluorochrome, GFP (GFP for "Protein - Fluorescent Green"), dansili umbeliferone, phycoerythrin, phycocyanin, allophicocyanine, o-phthaldehyde and fluorescamine.

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

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

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

[0086] In certain respects, an anti-glyco-MUCI antibody or antigen binding fragment of the description competes with GO2 or an antibody or antigen binding fragment comprising GO2 heavy and light chain variable regions (SEQ ID NOS: 3 and 4, respectively).

The competition can be run on cells expressing the GO2-linked glyco-MUCI epitope or on a glycosylated MUCI peptide containing the GO2-linked epitope, for example, the 60-mer peptide (VTSAPDTRPAPGSTAPPAHG); glycosylated with GalNAc-T2, GalNAc-T4 and GalNAc-T1 as described in US Patent No. 6,465,220. Cells that do not express the epitope or non-glycosylated peptides can be used as controls.

[0088] The cells on 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 GalNArc, the last cells producing the expression of the MUCI1 glycoform Tn to which GO? 2 binds. Cells expressing the non-glycosylated form of MUC1 can be used as a negative control.

[0089] Competition assays include, but are not limited to, a radioactive material labeled immunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell classification assay in the merged ELISA (FACS) and Biacore trials.

[0090] When conducting an antibody competition assay between a reference antibody and a test antibody (regardless of species or isotype), one can first label the reference with a detectable label, such as a fluorophore, biotin or an enzymatic (or even radioactive) label to allow subsequent identification. In this case, cells expressing glyco-MUCI1 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 reference antibody labeled for binding to an overlapping epitope, the intensity will be decreased in relation to a control reaction performed without a test antibody.

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

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

[0093] In several embodiments, a test antibody is considered to compete with a reference antibody if it decreases 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 varying between any of the preceding values, at a reference antibody concentration that is 80% of maximum binding under the specific assay conditions used and a 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 MUCI peptide is adhered to a solid surface, for example, a microwell plate, by contacting the plate with a solution of the peptide (for example, in a concentration of 1 µg / ml in PBS overnight at 4 ° C). The plate is washed (for example, 0.1% Tween 20 in PBS) and blocked (for example, in Superblock, Thermo Scientific, Rockford, IL). A mixture of subsaturant amount of biotinylated GO2 (for example, at a concentration of 80 ng / mL) and unlabeled GO2 (the "reference" antibody) or competition antibody the anti-glyco-MUCI antibody (the "test" antibody ”) In serial dilution (for example, at a concentration of 2.8 µg / mL, 8.3 µg / mL or 25 µg / mL) in ELISA buffer (for example, 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 streptavidin conjugated to HRP diluted in ELISA buffer is added to each well and the plates incubated for 1 hour. Plates are washed and bound antibodies have been detected by adding substrate (for example, TMB, Biofx Laboratories Inc., Owings Mills, MD). The reaction is terminated by adding stop buffer (for example, Bio FX Stop Reagents, Biofx Laboratories Inc., Owings Mills, MD) and absorbance is measured at 650 nm using a microplate reader (for example, VERSAmax, Molecular Devices, Sunnyvale, CA).

[0095] Variations in this competition assay can also be used to test competition between GO2 and another anti-glyco-MUCI antibody. For example, in certain respects, the anti-glyco-MUC1 antibody is used as a reference antibody and GO2 is used as a test antibody. In addition, instead of the glycosylated 60-mer MUC1 peptide, the membrane-bound glyco-MUCI expressed on the cell surface (for example on the surface of one of the cell types mentioned above) in culture can be used. Generally, about 10 to 10 transfectants, for example, about 10 transfectants, are used. Other formats for competition trials are known in the art and can be used.

[0096] In several embodiments, an anti-glyco-MUCI1 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 %, by at least 90% or by a percentage varying between any of the preceding values (for example, an anti-glyco-MUCI1 antibody of the description reduces the binding of labeled GO2 by 50% to 70%) when the anti-glyco antibody -MUC! 1 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 in a concentration ranging from one of the preceding values (for example, at a concentration ranging from 2 µg / ml to 10 µg / ml).

[0097] In other embodiments, GO2 reduces the binding of an anti-glyco-MUCI1 antibody labeled from the description by at least 40%, at least 50%, at least 60%, at least 70%, at least 80 %, by at least 90% or by a percentage varying between any of the preceding values (for example, GO2 reduces the binding of an anti-glyco-MUCI antibody labeled by the description by 50% to 70%) when GO2 is used in a concentration of 0.4 µg / ml, 2 µg / ml, 10 µg / ml, 50 µg / ml, 250 µg / ml or in a concentration varying between any of the preceding values (for example, in a concentration ranging from 2 µg / ml to 10 µg / ml).

[0098] In the preceding assays, the GO2 antibody can be replaced by any antibody or antigen binding fragment comprising the CDRs or the GO? 2 heavy and light chain variable regions, such as a humanized or chimeric GO2 counterpart.

[0099] In certain respects, an anti-glyco-MUCI antibody or antigen-binding fragment of the description comprises heavy and / or light chain variable sequences (or encoded by the nucleotide sequences) shown in Table 1. In other respects, a anti-glyco-MUC1I antibody or antigen binding fragment of the description comprises heavy and / or light chain CDR sequences (or encoded by the nucleotide sequences) shown in Table 1. The structural sequences for such anti-glyco-MUCI1 antibody and antigen binding fragment may be the native murine backbones in Table 1 or may be non-native backbones (for example, humanized or human).

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

[00101] In other respects, an anti-glyco-MUCI 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 scFv heavy chain variable and light chain variable fragment are covalently linked to a 4 to 15 amino acid linker sequence. The scFv can be in the form of a bispecific T-cell engagement or within a chimeric antigen receptor (CAR).

5.2 Bispecific Anti-glyco-MUCI1 and Anti-CD3 Antibodies

[00102] In some respects, bispecific antibodies of the description may comprise a first antigen binding domain that specifically binds to CD3 (for example, comprising the CDRs or VH and VL shown in Table 4) and a second binding domain of antigen that specifically binds to glyco-MUCI. The second antigen binding domain may comprise, alone or in combination, the features described for the glyco-MUCI antibodies above (for example, comprise 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 modalities numbered 3 to 17, infra or the VH and VL sequences identified in Table 1). script [String NO: "D3 - CDR-HIFIYAMN B4 (Kabat) Kabat D3 —CDR-H3HGNFGNSYVSWFAY B6 KKabat)" D3 - CDR-LIGSSTGAVTTSNYAN B7 KKabat) KKabat) "D3 - CDR-LQWG

LEWVSRIRSKYNNYATY Y ADS VKGRFTISRDDSKNTLILQMNSLR

EDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS "D3 VL IQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQ

FRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYY

CALWYSNLWVFGGGTKLTVL FK region of DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSA2 hIgG1 HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVQSN

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

IGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP UCI - VI DIVMSQSPSSLGVSVGEKVTMSCKSSQSLLYSTNQKNYQSLLYSTNH3 "L (RK) QRKNILAWYQYQYGYLQKLQKKLQKKLQKKLQQKK

IRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVT

IQGLSSPVTKSFNRGEC "D3 VH-CL - [EVQLLESGGGLVQPGGSLRLSCAASGFTFSTY AMNWVRQAPGKG 4

LEWVSRIRSKYNNYATY Y ADS VKGRFTISRDDSKNTLILQMNSLR EDTAVYYCVRHGNFGNSYVSWFAY WGQGTLVTVSSASVAAPS FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QRESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSP

TKSFNRGEC MUCI - VH-QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQRPEQGL / AS HI (EE) Fc - EEWIGYFSPGNDDIHYNEKFEGKATLTADKSSSTAY MQLNSLTSEDS | (hole, - P329GAVYFCKRSYDKDFDCWGQGTTLTVSSASTKGPSVFPLAPSSKSTS ALA) IGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

ILSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCP IPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV IKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG IKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQ IVSLSCAVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLVS IKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Muci - VHIQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQRPEQGLH6 "HI (EE) CD3 [EWIGYFSPGNDDIHYNEKFEGKATLTADKSSSTAY MQLNSLTSEDS | L-CHI-Fe - | AVYFCKRSYDKDFDCWGQGTTLTVSSASTKGPSVFPLAPSSKSTS (knob, - P329GGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS ALA) IL.SSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGS

IGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQ IEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDE | | AEY YCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTS

IGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS IL.SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP |

IPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV

IKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG IKEYKCKVSNKALGAPIEKTISKAKGQPREPQVY TLPPCRDELTKNQ |

IVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY ISKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

[00103] In some embodiments, the first antigen binding domain comprises a heavy chain variable region comprising the heavy chain CDR-H1 of SEQ ID NO: 34, CDR-H2 of SEQID NO: 35e and CDR-H3 of SEQ ID NO: 36 and a light chain variable region comprising the light chain CDR-LI1 of SEQ ID NO: 37, CDR-L2 of SEQ ID NO: 38 and 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 shown in embodiments numbered 3 to 17, for example (1) a heavy chain variable 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 light chain variable region comprising the CDR light chain (CDR-L ) 1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9 and CDR-L3 of SEQ ID NO: 10.

[00105] In a particular embodiment, the bispecific antibody comprises (1) a first antigen binding domain that specifically binds to CD3 and comprises a heavy chain variable region comprising a CDR-HI 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 light chain variable region comprising a CDR-L1 comprising the sequence of amino acid 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 SEQID NO: 39; and (11) a second antigen binding domain that specifically binds glyco-MUCI1 and comprises (1) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, more preferably a CDR-HI 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 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 sequence of the heavy chain variable region that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of amino acid of SEQ ID NO: 40 and a sequence of the light chain variable region 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 sequence of the heavy chain variable region of SEQ ID NO: 40 and the sequence of the light chain variable region of SEQ ID NO: 41.

[00108] In some embodiments, the second antigen binding domain comprises a sequence of the heavy chain variable region that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of amino acid of SEQ ID NO: 3 and a sequence of the light chain variable region 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 sequence of the heavy chain variable region of SEQ ID NO: 3 and the sequence of the light chain variable region of SEQ ID NO: 4.

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

[00111] In some embodiments in which the first and the second antigen binding domain of the bispecific antibody are both Fab molecules and in one of the antigen binding domains (particularly the first antigen binding domain) the VL and Fab light chain and Fab heavy chain VH are replaced by each other, 1) 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 where in the CH1 constant domain 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 (numbered according to the EU Kabat index); or ii) in the CL constant domain 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 CHI constant domain of the second antigen binding domain the amino acid in position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (numbered according to the EU Kabat index).

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

[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 replaced independently 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 are independently replaced by glutamic acid (E) or aspartic acid (D) (numbered according to the EU Kabat index ); or 11) in the CL constant domain of the second antigen binding domain, the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbered according to Kabat) and in the CHI constant domain of According to the 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) (numbered according to the EU index of Kabat).

[00114] In such a modality, 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 CH1 constant domain of the second antigen binding domain the amino acid at position 147 or the amino acid at position

44/172 213 is replaced independently by glutamic acid (E) or aspartic acid (D) (numbered according to the EU Kabat index).

[00115] In an additional 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 CH1 constant domain of the second antigen binding domain, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbered 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 replaced by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is replaced independently by lysine (K), arginine (R) or histidine (H) (numbered according to Kabat) and in the constant domain CH1 of the second antigen binding domain the amino acid at position 147 is replaced independently with glutamic acid (E) or aspartic acid (D) (numbered according to the EU Kabat index) and the amino acid at position 213 is replaced independently by glutamic acid (E) or aspartic acid (D) (numbered according to 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) (numbered according to Kabat) and the amino acid at position 123 is replaced by lysine ( K) (numbered 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) (numbered according to the EU index of Kabat) and the amino acid at position 213 is replaced by glutamic acid (E) (numbered according to the EU Kabat index).

[00118] In an even 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) (numbered according to Kabat) and the amino acid at position 123 is replaced by arginine (R) (numbered 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) (numbered according to the EU Kabat index) and the amino acid in heading 213 is replaced by glutamic acid (E) (numbered according to the EU Kabat index).

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

[00120] In some embodiments, the first and the second antigen binding domain are fused to each other, optionally via a peptide linker.

[00121] In some embodiments, the first and the second antigen binding domain are each a Fab molecule and (1) the second antigen binding domain is fused at the C terminal of the Fab heavy chain to the N terminal of the Fab heavy chain of the first antigen binding domain or (il) the first antigen binding domain is fused at the C terminal of the Fab heavy chain to the N terminal 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 CD3 and two antigen binding domains that specifically binds

46/172 bind glyco-MUCI. Thus, in some embodiments, the bispecific antibody comprises a third antigen binding domain that specifically binds glyco-MUCI1. In some embodiments, the third antigen portion is identical to the first antigen binding domain (for example, it is also a Fab molecule and comprises the same amino acid sequence).

[00124] In particular embodiments, the Dbiespecific 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 IgG Fc domain. In another embodiment, the Fc domain is an IgG Fc domain. In a more specific embodiment, the Fc domain is an IgG Fc domain, comprising an amino acid substitution at position 8228 (numbering the EU index of Kabat), particularly the substitution of amino acid S228P. In a further particular embodiment, the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IZG1 Fc domain. An exemplary sequence of a human IgG1 Fc region is given in SEQ ID NO: 42.

[00125] In some embodiments in which the first, the second and, where present, the third antigen binding domain are each a Fab molecule, (a) (1) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N terminal of the Fab heavy chain of the first antigen binding domain and the first antigen binding domain is fused at the C terminal of the Fab heavy chain to the N terminal of the first subunit of the Fc or (ii) ) the first antigen binding domain is fused at the C terminal of the Fab heavy chain to the N terminal of the Fab heavy chain of the second antigen binding domain and the second antigen binding domain is fused at the C terminal of the heavy heavy chain Fab to the N-terminus of the first subunit of the Fc domain and (b) the third antigen-binding domain, where present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit.

[00126] In particular modalities, 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 bispecific antibody embodiment it comprises (1) a first antigen binding domain that specifically binds to CD3, wherein the first antigen binding domain is a crossed Fab molecule in which the variable or constant regions , particularly the variable regions, the Fab light chain and the Fab heavy chain are switched; (11) a second and a third antigen binding domain that specifically binds glyco-MUC1, comprising a heavy chain variable region comprising the heavy chain CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6 and CDR-H3 of SEQ ID NO: 7 and a light chain variable region comprising the light chain CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9 and CDR-L3 SEQ ID NO: 10, wherein the second and third antigen binding domains are each a Fab molecule, particularly a conventional Fab molecule; (ii) 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 terminal of the Fab heavy chain to the N terminal of the Fab heavy chain of the first domain antigen binding and the first antigen binding domain is fused at the C terminal of the Fab heavy chain to the N terminal of the first subunit of the Fc domain and where the third antigen binding domain is fused at the C terminal of the Fab heavy chain to the N-terminal of the second subunit of the Fc domain.

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

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

[00132] In one embodiment, the second and third antigen binding domains comprise a sequence of the heavy chain variable region that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid sequence of SEQ ID NO: 3 and a sequence of the light chain variable region 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 shown in embodiments numbered 3 through 17. In one embodiment, the second and third antigen binding domains comprise the variable region heavy chain of SEQ ID NO: 3 and the region

49/172 light chain variable of SEQ ID NO: 4.

[00133] The Fc domain according to the above modalities can incorporate, alone or in combination, all of the resources described here above in relation to the 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) (numbered 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 CHI constant domain of the second and third Fab molecules under (1) the amino acid at position 147 is replaced by acid glutamic (E) (numbered according to the EU Kabat Index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbered 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 sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the SEQ ID string

NO: 45 (and preferably comprises a CDR-HI 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-HI 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 of SEQ ID NO: 43, a polypeptide comprising the sequence of SEQ ID NO: 44, a polypeptide comprising the sequence of SEQ ID NO: 45 and a polypeptide comprising the sequence of SEQ ID NO: 46.

5.3 Antibody-Drug Conjugates

[00138] Another aspect of the description concerns antibody-drug conjugates (ADCs) including the anti-glyco-MUCI antibodies and antigen-binding fragments of the description. ADCs generally comprise an anti-glyco-MUCI1 antibody and / or binding fragment as described herein having one or more cytotoxic and / or cytostatic agents attached thereto by means of one or more linkers. In specific modalities, ADCs are composed according to structural formula (1): [D-L-XY], - Ab or salts thereof, where each "D" represents, independently of the others, a cytotoxic and / or cytostatic agent

(“Drug”); each "L" represents, independently of the others, a linker; "Ab" represents an anti-glyco-MUCI antigen binding domain, such as an anti-glyco-MUCI1 antibody or binding fragment described herein; each "XY" represents a bond formed between a functional group R * on the linker and a "complementary" functional group R * on the antibody and n represents the number of drugs bound to or ADC drug to antibody ratio (DAR).

[00139] The specific modalities of the various antibodies (Ab) that can comprise ADCs include the various modalities of anti-glyco-MUCI1 antibodies and / or binding fragments described above.

[00140] In some specific modalities of ADCs and / or salts of structural formula (1), each D is the same and / or each L is the same.

[00141] The specific modalities of cytotoxic and / or cytostatic agents (D) and linkers (L) that can comprise the anti-glyco-MUCI ADCs of the description, as well as the number of cytotoxic and / or cytostatic agents linked to the ADC, are described in more detail below.

5.3.1. Cytotoxic and / or cytostatic agents

[00142] Cytotoxic and / or cytostatic agents can be any agents known to inhibit the growth and / or replication of and / or dead cells and in particular cancer and / or 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 without limitation, radionuclides, alkylating agents, topoisomerase 1 inhibitors, topoisomerase II inhibitors, DNA intercalating agents (for example, groove such as minor groove binders), RNA / DNA antimetabolites, cell cycle modulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase inhibitors, mitochondria inhibitors and antimitotic agents.

[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- (2-chloroethyl) amino] -DL-phenylalanyl] -, ethyl ester; NSC 167780; CAS Registry No. 3577897) ); AZQ ((1,4-cyclohexadiene-1 4-dicarbamic acid, 2,5-bis (1-aziridinyl) -3,6-dioxo-, diethyl ester, NSC 182986; CAS Registry No. 57998682)); BCNU ((N, N -Bis (2-chloroethyl) -N-nitrosourea ;, NSC 409962; CAS Registry No. 154938)); busulfan (1,4-butanediol dimethanesulfonate; NSC 750; CAS Registry No. 55981); (carboxyphthalate) platinum (NSC 27164; CAS Registry No. 65296813); CBDCA ((cis- (1,1-cyclobutanedicarboxylate) - diaminoplatin (II)); NSC 241240; CAS Registry No. 41575944)); CCNU (((N- (2-chloroethyl) -N'-cyclohexyl-N-nitrosourea; NSC 79037; CAS Registry No. 13010474)); CHIP (iproplatin; NSC 256927); chlorambucil (NSC 3088; CAS Registry No. 305033); chlorozotocin ((2 - [[[(2-chloroethyl) nitrosoamino] - carbonyl Jamino] -2-deoxy-D-glycopyranose; NSC 178248; CAS Registry No. 54749905)); cisplatin (cisplatin; NSC 119875; CAS Registry No. 15663271); clomessone (NSC 338947; CAS Registry No. 88343720); cyanomorpholinodoxorubicin (NCS 357704; CAS Registry No. 88254073); cyclodisone (NSC 348948; CAS Registry No. 99591738); dianhydrogalactitol (5,6-diepoxidulcitol; NSC 132313; CAS Registry No. 23261203); fluorodopan ((5 - [(2-chloroethyl) - (2-fluoroethyl) amino] -6-methyl-uracil ;, NSC 73754; CAS Registry No. 834913); hepsulfam (NSC 329680; CAS Registry No. 96892578); hygrantone (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 ((Dbis hydrochloride (2-chloroethyl)) methylamine; NSC 762; CAS Registry No. 55867); UNCP ((1- (2-chloroethyl) -3- (2,6-dioxo-3-piperidyl) -I-nitrosourea; NSC 95466; CAS Registry

No. 13909029)); piperazine alkylator ((1- (2-chloroethyl dihydrochloride) -4- (3-chloropropyl) -piperazine; NSC 344007)); piperazinodione (NSC 135758; CAS Registry No. 41109802); pipobroman ((N, N-bis (3-bromopropionyl) piperazine; NSC 25154; CAS Registry No. 5491])); porphyromycin (N-methylmitomycin C; NSC 56410; CAS Registry No. 801525); shown spirohydantoin (NSC 172112; CAS Registry No. 56605164); teroxirone (triglycidylisocyanurate; NSC 296934; CAS Registry No. 2451629); tetraplatin (NSC 363812; CAS Registry No. 62816982); thio-tepa (N, N ', No. ”- tri-1,2-ethanediylthio phosphoramide; NSC 6396; CAS Registry No. 52244); triethylenomelamine (NSC 9706; CAS Registry No. 51183); nitrogenous uracil mustard (desmetildopan; NSC 34462; CAS Registry No. 66751); Yoshi-864 ((bis (3-mesyloxy-propylJamine hydrochloride; NSC 102627; CAS Registry No. 3458228).

[00145] Topoisomerase 1 inhibitors: camptothecin (NSC 94600; CAS Registry No. 7689-03-4); various camptothecin derivatives and analogs (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, 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 (benzisoquinolinodione; NSC 308847; CAS Registry No. 69408817); m-AMSA ((4 '- (9-acridinylamino) -3'- methoxymethanesulfonanilide; NSC 249992; CAS Registry No. 51264143)); anthrapyrazole derivative ((NSC 355644); etoposide (VP-16; NSC 141540; CAS Registry No. 33419420); pyrazoloacridine ((pyrazolo [3,4,5-klJacridine-2 (6H) -propanamine, 9-methoxy-N, N-dimethyl-S-nitro-, monomethanesulfonate; NSC 366140; CAS Registry No. 99009219); bisanthrene hydrochloride (NSC

337766; CAS Registry No. 71439684); daunorubicin (NSC 821151; CAS Registry No. 23541506); deoxidoxorubicin (NSC 267469; CAS Registry No. 63950061); mitoxantrone (NSC 301739; CAS Registry No. 70476823); menogaryl (NSC 269148; CAS Registry No. 71628961); N, N-dibenzyl daunomycin (NSC 268242; CAS Registry No. 70878512); oxantrazole (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 Registry No. 4803274); chicamycin A (CAS Registry No. 89675376); tomaycin (CAS Registry No. 35050556); DC-81 (CAS Registry No. 81307246); sibiromycin (CAS Registry No. 12684332); pyrrolobenzodiazepine derivative (CAS Registry No. 945490095); SGD-1882 ((S) -2- (4-aminophenyl) -7-methoxy-8- (3-4 (S) -7-methoxy-2- (4-methoxyphenyl) -5-0x0-5,11a- dihydro-1 H-benzo [e] pyrrole [1,2-a] [1,4] diazepin-8-yl) oxy) propoxy) -1 H-benzo [e] pyrrole [1,2-a] [1 , 4] diazepin-S (11aH) -one); SG2000 (SJG-136; (11aS, 11a'S) -8.8 '- (propane-1,3-diylbis (6xi)) bis (7-methoxy-2-methylene-2,3- - - dihydro-1 H- benzo [e] pyrrolo [1,2-a] [1,4] diazepin-S (11aH) -one); NSC 694501; CAS Registry No. 232931576).

[00148] RNA / DNA antimetabolites: L-alanosine (NSC 153353; CAS Registry No. 59163416); 5-azacitidine (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 Jamino] benzoyl] L-aspartic acid (NSC 132483); aminopterin derivative N- [4 - [[(2,4-diamino-5-ethyl-6-quinazolinyl)] methyl Jamino] benzoyl] L-aspartic (NSC 184692); aminopterin derivative N- [2-chloro-4 - [[(2,4-diamino-6-pteridinyl)] methyl Jamino] benzoyl] L-aspartic monohydrate (NSC 134033); an antiphon ((No. * - (4-amino-4-deoxypteroil) -N'-hemiftaloil-L-ornithine; NSC

623017)); Baker soluble antifol (NSC 139105; CAS Registry No. 41191042); dichloralil lawsone ((2- (3,3-dichloroalyl) -3-hydroxy-1,4-naphthoquinone; NSC 126771; CAS Registry No. 36417160); brequinar (NSC 368390; CAS Registry No. 96201886); ftorafur ((prodrug) ; 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), derived from acid methotrexate (N - [[4- [[(2,4-diamino-6-pteridinyl)] methyl J] methylamino] -1-naphthalenyl | carbonyl] L-glutamic, 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-S-fluorouridine (NSC 27640; CAS Registry No. 50919); 5-HP (NSC 107392; CAS Registry No. 19494894); a-TGDR (0a-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); B-TGDR (B-2'-deoxy-6-thioguanosine; NSC 71261; CAS Registry No. 789617); cyclocytidine (NSC 145668; CAS Registry No. 10212256); guanazole (NSC 1895; CAS Registry No. 1455772); hydroxyurea (NSC 32065; CAS Registry No. 127071); glycodialdehyde inosine (NSC 118994; CAS Registry No. 23590990); macbecin II (NSC 330500; CAS Registry No. 73341738); pyrazoloimidazole (NSC 51143; CAS Registry No. 6714290); thioguanine (NSC 752; CAS Registry No. 154427); thiopurine (NSC 755; CAS Registry No. 50442).

[00150] Cell cycle modulators: silybin (CAS Registry No. 22888-70-6); epigallocatechin gallate (EGCG; CAS Registry No. 989515); procyanidin derivatives (for example, procyanidin Al [CAS Registry No. 103883030], procyanidin B1 [CAS Registry No. 20315257], procyanidin B4

[CAS Registry No. 29106512], arecatanine B1 [CAS Registry No. 79763283]); isoflavones (eg, genistein [4% S, 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 Registry No. 2998574); nocodazole (CAS Registry No. 31430189); podophyllotoxin (CAS Registry No. 518285); vinorrelbine tartrate (NSC 608210; CAS Registry No. 125317397); cryptoficina (NSC 667642; CAS Registry No. 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); erlotinibo (NSC 718781; CAS Registry No. 183319699); everolimus (NSC 733504; CAS Registry No. 159351696); fostamatinib (NSC 745942; CAS Registry No. 901119355); gefitinib (NSC 715055; CAS Registry No. 184475352); ibrutinib (CAS Registry No. 936563961); imatinib (NSC 716051; CAS Registry No. 220127571); lapatinib (CAS Registry No. 388082788); lenvatinib (CAS Registry No. 857890392); mubritinib (CAS 366017096); nilotinib (CAS Registry No. 923288953); nintedanib (CAS Registry No. 656247175); palbocyclib (CAS Registry No. 571190302); pazopanib (NSC 737754; CAS Registry 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); INCBO018424 (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. 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); XLO019 (CAS Registry No. 945755566); PD-325901 (CAS Registry No. 391210109); PD-98059 (CAS Registry No. 167869218); ATP competitive TORCI1 / 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 Registry No. 1245537681), BEZ235 (CAS Registry No. 915019657), XL-765 (CAS Registry No. 934493762).

[00152] Protein synthesis inhibitors: acriflavin (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 Registry No. 1181540); cycloheximide (CAS Registry No. 66819); dactinomycin (NSC 3053; CAS Registry No. 50760); dalfopristin (CAS Registry No. 112362502); demeclocycline (CAS Registry No. 127333); dibecacin (CAS Registry No. 34493986); dihydroestreptomycin (CAS Registry No. 128461); dirithromycin (CAS Registry No. 62013041); doxycycline (CAS Registry No. 17086281); emetin (NSC 33669; CAS Registry No. 483181);

erythromycin (NSC 55929; CAS Registry No. 114078); flurithromycin (CAS Registry No. 83664208); framicetin (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 Registry No. 8063078); ketolides such as telithromycin (CAS Registry No. 191114484), ketromycin (CAS Registry No. 205110481) and solithromycin (CAS Registry No. 760981837); lincomycin (CAS Registry No. 154212); limecycline (CAS Registry No. 992212); meclocycline (NSC 78502; CAS Registry No. 2013583); metacycline (rondomycin; NSC 356463; CAS Registry No. 914001); midecamycin (CAS Registry No. 35457808); minocycline (NSC 141993; CAS Registry No. 10118908); myocamycin (CAS Registry No. 55881077); neomycin (CAS Registry No. 119040); netilmicina (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 azidamphenicol (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); pirlimycin (CAS Registry No. 79548735); puromycin (NSC 3055; CAS Registry No. 53792); quinupristine (CAS Registry No. 120138503); ribostamycin (CAS Registry No. 53797356);

roquitamicina (CAS Registry No. 74014510); rolithetracycline (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 Registry No. 4478937); thioureidobutyronitrile (Kevetrinº; CAS Registry No. 6659890); valproic acid (NSC 93819; CAS Registry No. 99661); vorinostat (NSC 701852; CAS Registry No. 149647789); ACY-1215 (rocilinostat; CAS Registry No. 1316214524); CUDC-101 (CAS Registry No. 1012054599); CHR-2845 (tefinostat; CAS Registry No. 914382608); CHR-3996 (CAS Registry No. 1235859138); 45C-202 (CAS Registry 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 113 (Registration

CAS No. 865070377); aspirin (NSC 406186; CAS Registry No. 50782); ellipticin (CAS Registry No. 519233); berberine (CAS Registry No. 633658); cerulenin (CAS Registry No. 17397896), GX015-070 (Obatoclax &O; 1H- Indole, 2- (2 - (((3,5-dimethyl-1 H-pyrrol-2-yl) methylene) -3-methoxy-2H-pyrrole -5-ila) -; NSC 729280; CAS Registry No. 803712676); celastrol (tripterin; CAS Registry No. 34157830); metformin (NSC 91485; CAS Registry No. 1115704); Bright green (NSC 5011; CAS Registry No. 633034); ME-344 (CAS Registry No. 1374524556).

[00155] Agents - Antimitotics: alocolchicine (NSC 406042); auristatins, such as MMAE (monomethyl auristatin E; CAS Registry No. 474645-27-7) and MMAF (monomethyl auristatin F; CAS Registry No. 745017- 94-1; halichondrin B (NSC 609395); colchicine (NSC 757; CAS Registry No. 64868); colchicine derivative (N-benzoyl-deacetyl benzamide; 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 Registry No. 33069624); taxol derivative ((2-N- [3- (dimethylamino) propyl Jglutaramate taxol; NSC 608832); tiocolchicine (3- demethyltiocolchicine; NSC 361792); trityl cysteine (NSC 49842; CAS Registry No. 2799077); vinblastine sulfate (NSC 49842; CAS Registry No. 143679); vincristine sulfate (NSC 67574; CAS Registry No. 2068782).

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

[00157] In a 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 (“MMAFE”) or monomethyl auristatin F (“MMAF”).

5.3.2. Connectors

[00159] In the anti-glyco-MUCI ADCs of the description, 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 can be short, long, hydrophobic, hydrophilic, flexible or rigid or it can be composed of segments that each independently has one or more of the properties mentioned above such that the linker can include segments having different properties. The linkers can be polyvalent such that they covalently bind more than one agent to a single site on the antibody or monovalent such that they covalently bind a single agent to a single site on the antibody.

[00160] As will be evaluated by the skilled professionals, the linkers bind cytotoxic and / or cytostatic agents to the antibody by forming a covalent bond to the cytotoxic and / or cytostatic agent in one location and a covalent bond to the antibody in another. 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 (1) unconjugated forms of the linker that include a functional group capable of covalently attaching the linker to a cytotoxic and / or cytostatic agent and a functional group capable of covalently attaching the linker an antibody; (11) partially conjugated forms of the linker which include a functional group capable of covalently attaching the linker to an antibody and which is covalently linked to a cytotoxic and / or cytostatic agent or vice versa and (iii) fully conjugated forms of the linker which are covalently linked to both a cytotoxic and / or cytostatic agent and an antibody. In some specific embodiments of anti-glyco-MUC1 linkers and ADCs of the description, as well as syntones used to conjugate binding agents to antibodies, portions comprising the functional groups on the linker and covalent bonds formed between the linker and antibody are specifically illustrated as R, and XY, respectively.

[00161] The linkers are preferably, but need not be, chemically stable to conditions outside the cell and can 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 can 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. Agents that are selective or targeted and have lower toxicity than normal cells can use, 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 link the cytotoxic and / or cytostatic agents to the anti-glyco-MUCI ADC antibody of the description.

Exemplary polyvalent linkers that can be used to bind 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 connector technology developed by Mersana et al. has the potential to allow ADCs with high DAR with good physicochemical properties. As shown below, Mersana technology is based on the incorporation of drug molecules into a solubilizing polyacetal backbone via an ester binding sequence. The methodology makes ADCs highly loaded (DAR up to 20) while maintaining good physical and chemical properties.

[00163] Additional examples of dendritic linkers can be found in US 2006/116422; US 2005/27 1615; 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 linkers 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 er al., 2011, J. Med. Chem. 54: 3606-3623; U.S. Pat. No. 7,223,837; U.S. Pat. No. 8,568,728; U.S. Pat. No. 8,535,678; and WO2004010957, each of which is incorporated herein by reference.

[00165] By way of example and not by way of limitation, some cleavable and non-cleavable linkers that can be included in the anti-glyco-MUC ADCs of the description are described below.

5.3.3. Cleavable Connectors

[00166] In certain embodiments, the selected linker is cleavable in vivo. Cleavable linkers can include chemically or enzymatically unstable or degradable bonds. 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 bonds that are chemically or enzymatically cleavable while the rest of the linker is not cleavable. In certain embodiments, a linker comprises a chemically unstable group such as hydrazone and / or disulfide groups. Linkers - comprising “chemically unstable groups exploit differential properties between plasma and some cytoplasmic compartments. Intracellular conditions to facilitate drug release for hydrazone-containing linkers are the acid 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] The acid-unstable groups, such as hydrazone, remain intact during systemic circulation in the neutral blood pH environment (pH 7.3 to 7.5) and undergo hydrolysis and release the drug once the ADC is internalized within the mildly acid 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 non-specific drug release. To increase the stability of the hydrazone group on the linker, the linker can be varied by chemical modification, for example, substitution, allowing tuning to achieve more efficient release in the lysosome with a minimized loss in circulation.

Hydrazone-containing linkers may contain additional cleavage sites, such as additional acid unstable cleavage sites and / or enzymatically unstable cleavage sites. ADCs including exemplary hydrazone-containing linkers include the following structures:

L o eg)

N Y AN

Á X N Ss NAT Ab) 'D º n O (Ih)

N THE

N EO Granddaughter

The Don

N SS (1)

DÓ À

H H3C NAHAb TIA n

The one where D and Ab represent the cytotoxic and / or cytostatic agent (drug) and Ab, respectively and n represents the number of drug-linkers bound to the antibody. In certain linkers such as linker (Ig), the linker comprises two cleavable groups - a disulfide moiety and a hydrazone moiety. For such linkers, effective release of the unmodified free drug requires acidic pH or reduction of disulfide and acidic pH. Linkers such as (Ih) and (Ii) 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 the acidic cellular compartments include carbonates. Such linkers can be useful in cases where the cytotoxic and / or cytostatic agent can be covalently attached via oxygen.

[00170] Other acid-unstable groups that can be included in the linkers include linkers containing cis-aconityl. The cis-aconityl chemistry uses a carboxylic acid juxtaposed to an amide bond to accelerate the amide hydrolysis under acidic conditions.

[00171] Cleavable linkers can also include a disulfide group. Disulfides are thermodynamically stable at physiological pH and are designed to release the drug on internalization within cells, where the cytosol provides a significantly more reducing environment compared to the extracellular environment. Fission of the disulfide bond generally requires the presence of a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH), such that the disulfide-containing linkers are reasonably stable in the circulation, selectively releasing the drug in the cytosol. The intracellular enzyme protein disulfide isomerase or similar enzymes capable of cleaving the disulfide bond can also contribute to the preferential disulfide bond cleavage 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, circulating in approximately 5 tumor cells, where the irregular blood flow leads to a hypoxic state, results in enhanced activity of reducing 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 linkage.

[00172] ADCs including exemplary disulfide-containing linkers include the following structures:

1)

R R D Ss N Ab AX A: R R O n (1) PN so n a)

R R p PAD so n where D and Ab represent the drug and antibody, respectively, n represents the number of drug-linkers bound to the antibody and R is independently selected for each occurrence of hydrogen or alkyl, for example. In certain embodiments, increasing the steric impedance adjacent to the disulfide bond increases the stability of the bond. Structures such as (Ij) and (II) show increased in vivo stability when one or more R groups are selected from 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. Linker-based peptides tend to be more stable in the plasma and extracellular medium than chemically unstable linkers. Peptide bonds generally have good serum stability, since lysosomal proteolytic enzymes have very low activity in the blood due to endogenous inhibitors and the unfavorably high pH value of the blood compared to lysosomes. The release of a drug from an antibody occurs specifically due to the action of lysosomal proteases, for example, cathepsin and plasmin. These proteases can be present at high 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-Leu- Ala-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- (DJ) Asp , Ala- (DJAsp 5, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, FenilGIy- (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 they are preferred over the longer polypeptides due to the hydrophobicity of the longer peptides.

[00175] A variety of dipeptide-based cleavable linkers useful for binding drugs such as doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepine, talisomycin 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 by reference). All of these dipeptide linkers, or modified versions of these dipeptide linkers, can be used in the anti-glyco-MUCI1 ADCs of the description. Other dipeptide linkers that can be used include those found in ADCs such as Brentuximab Vendotin SGN-35 from Seattle Genetics (Adcetris8 &), SGN-75 from Seattle Genetics (anti-CD-70, Val-Cit-monormethyl auristatin FMMAF), SGN- Seattle Genetics CD33A (anti-CD-33, Val-Ala- (SGD-1882)), Celldex Therapeutics glembatumumab (CDX-011) (anti-NMB, Val-Cit-monomethyl auristatin E (MMAE) and PSMA-ADC gives

Citogen (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).

[00176] Enzymatically cleavable linkers may include an auto-immolating spacer to spatially separate the drug from the enzymatic cleavage site. Direct attachment 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 an auto-immolating spacer allows the elimination of the fully active drug, chemically unmodified in the hydrolysis of the amide bond.

[00177] An auto-immolating spacer is the group of bifunctional para-aminobenzyl alcohol, which is linked to the peptide through the amino group, forming an amide bond, while amine-containing drugs can be attached through carbamate functionalities to the benzyl hydroxyl group of the connector (PABC). The resulting prodrugs are activated in protease-mediated cleavage, leading to an elimination reaction in 1.6 releasing the unmodified drug, carbon dioxide and remnants of the linker group. The following scheme represents the fragmentation of p-amidobenzyl ether and the release of the drug:

o o Oo Do protease: eptide À,

PEP H o ad —P disposal 1.61 HoN Xx — D LL * ez

HN where X-D represents the unmodified drug.

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

[00179] In some embodiments, the enzymatically cleavable linker is a linker based on B-glucuronic acid. Easy drug release can be achieved by cleaving the glycosidic bond of B-glucuronide by the lysosomal enzyme B-glucuronidase. This enzyme is abundantly present within lysosomes and is overexpressed in some types of tumor, although enzyme activity outside cells is low. B-glucuronic acid based linkers can be used to circumvent an ADC's tendency to aggregate due to the hydrophilic nature of B-glucuronides. In some embodiments, B-glycuronic acid based linkers are preferred as linkers for ADCs linked to hydrophobic drugs. The following scheme represents the release of the drug from a

T1 / 172 linker based on B-glucuronic acid and containing ADC:

HO Oo Ho O À B-glucuronidase

HO O D Y

HO o Ho o

HN O

The DNA Ô ne bu OH o O Sb elimination 1,6 (OW Yi

HO D HN NO »

O o + CO,

HN AT THE O

[00180] A variety of B-glucuronic acid-based cleavable linkers useful for binding drugs such as auristatins, camptothecin and doxorubicin analogs, CBI minor groove binders and psimberin to antibodies have 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,

T2 / 172 each of which is incorporated by reference). All of these B-glucuronic acid based linkers can be used in the anti-glyco-MUCI ADCs of the description.

[00181] Additionally, cytotoxic and / or cytostatic agents containing a phenol group can be covalently linked to a linker through phenolic oxygen. Such a linker, described in WO 2007/089149, relies on a methodology in which a diamino-ethane “SpaceLink” is used in conjunction with traditional “PABO” based autoimolative groups to release phenols. The cleavage of the linker is schematically represented below, where D represents a cytotoxic and / or cytostatic agent having a phenolic hydroxyl group. HO connector. Oo representative with PABO HO unit », 9" SpaceLink "DA 5 enzyme e: o o | lysosomal OH À N O. - - 0 OUR CT "| ô Lo for mAb ACD O. HO — D

MONS | 3 V / the final destination of d = SpaceLink is a cyclic urea

THE

[00182] Cleavable connectors may include portions or segments not

Cleavable T3 / 172 and / or cleavable segments or portions may be included in an otherwise non-cleavable linker to make it cleavable. Just by way of example, polyethylene glycol (PEG) and related polymers can include cleavable groups in the polymer chain. For example, a polyethylene glycol or polymeric linker can include one or more cleavable groups such as a disulfide, hydrazone or dipeptide.

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

[00184] In certain embodiments, the linker comprises an enzymatically cleavable peptide moiety, for example, a linker comprising the structural formulas (IVa) or (TVb):

7T4 / 172 o (IVa) to X AO 5a y R: nPpeptide Tt x NHO yo IVb XP o (1Vb) aa OHE) or a salt thereof, where: peptide represents a peptide (illustrated CN and not showing the “terminals ”Carboxy and amino) cleavable by a lysosomal enzyme; T represents a polymer comprising one or more ethylene glycol units or an alkylene chain or combinations thereof; Rº is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; p is an interior number ranging from 0 to 5; géOoul; xédoul; yéloul; * 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.

[00185] In certain embodiments, the peptide is selected from a tripeptide or a dipeptide. In particular modalities, the dipeptide is selected from: Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; 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; Ile-Cit; Phe-Arg and Trp-Cit. In certain embodiments, the dipeptide is selected from: Cit-Val and Ala-Val.

[00186] Exemplary specific modalities of linkers according to structural formula (IVa) that can be included in the anti-glyco-MUCI ADCs of the description include the linkers illustrated below (as illustrated,

the linkers include a group suitable for covalently attaching the linker to an antibody): o (Val) o o o DA t H QDO AAA "o HN 'o o (IVa2) o o z o O i H

CODOPOOSODOÉS o o (IVa.3) o o Di o

N À AN CON Tr Yo O 'sOs o (Vad)

III: H [e] N.; Ao o o (IVaS5) j A AX j ss and À AN É NH; > o (IVa6) ME Ron Ú

OX N Oo Oo

NH io o (Va7) j A XL j e. 'F .. Z NH? AND.

[00187] Exemplary specific embodiments of linkers according to structural formula (IVb) that can be included in the anti-glyco-MUCI ADCs of the description include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the binding to an antibody): o (Vb1) oooo

H N H JS ê H o q

NH Au o (1Vb2) K o ea

A-N-A

N N) Y Ss HN 'to 1 (IVb.3) C o o Oo It is FRO NA à) Ras o

TIAT2 o (DV)

LAO

N H Í N As ns (VS) o o o o and N H 3 ê H Di ma o (IVP6)

LA N H Í à H SM o í “(VP?) N H Í ê H Di

A o (VS)? To CI IA o ”N v N.:"

o (IVb9) / o o qi o o LILA, 8 H Í ê H

L

PS No (IVb.10) o RA o OA A, RÁ 8 H J Ê H

LL

THE ; (IVb.11) / o Ace o om

H Ho — S = 0 o ú

Í DJ NH

THE ! (IVb.12) / fo

H Ho — S = 0 o ú

Í DJ

NH Pv

(IVb.13) OH o o o o E (À RA 8 1 o o ã

LL

 (IVb.14) Lao

N FS HN

MS (IVb.15) o 7 q bh a "IA and

ANTA the

NH

A (IVb.16) o o o Du o o H i H

AEE o o OSs

NH ÂÁu

(IVb.17) o / o o oOO

H ço, RA, 8 H Ê H

LL Pv NH? (IVb.18) o N ne o o '

COPA o O.

Y (IVb.19) o / o o Oo

H WA E, O o ê

[00188] In certain embodiments, the linker comprises an enzymatically cleavable peptide moiety, for example, a linker comprising the structural formula (IVc) or (IVd): (IVc) o 9 É H, / peptide T x NO yoo (IVd ) Cm NA Rê or a salt thereof, where: peptide represents a peptide (shown CN 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; Rº is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; p is an integer ranging from 0 to 5; géOloul; xéboul; yéOoul; 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] Exemplary specific embodiments of linkers according to structural formula (IVc) that can be included in the anti-glyco-MUCI ADCs of the description include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the binding to an antibody): (IVc.1) oooo

H

LA A TA o 'J

HN io (IVc.2) o o o o

H CUP YEAR! o º 'o VS) (IVc.4) o o o v 6' "do o so; o E: 2 (ves) (IVc.6) TIE A AA of º C NHo o o W> NH Ace

(IVc.7) o o o

H is C NH> Es

[00190] Exemplary specific embodiments of linkers according to structural formula (IVd) that can be included in the anti-glyco-MUCI 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): oo (IVd.1) o (IVd.2) o "/ oo AA Ae RNA, Re Y o 8 Pd o À D

NH HN Pu to o (IVd.3) (IVd.4) / o "o [o Ad u o Õ o ê o  o q

NH Âm

NH (IVd.5) (IVd.6) o o o o o o

H H A Re A A Ae X o XY o is q o

NH Au

KEN Oo (IVd.7) o (IVd.8) <C À the N í o o º o a

H And oH XY. 3 Y

NH Av NH> / º o om (Vd9) o (IVd.10) H / o o ió ca do bh X x Ace o (IVd.11) P IVd.12 Á / (IVd.12) NIX! SN UAXA "õ Y y Rr Õ WI PÃO º q popo o õ no |. And Au o INd.13 INd.14”, oo / FX gta AAA A Ô i Ras

Ô X

NH HN Âm Ms

(IVd.15) (IVd.16) ESA oo o o o o so; Nº Nº e x and o (IVd.17) / o o o o ã

LL

[00191] In certain embodiments, the linker comprising structural formulas (IVa), (IVb), (IVc) or (IVd) further comprises a portion of carbonate cleavable by exposure to an acidic medium. In particular embodiments, the linker is attached via oxygen to a cytotoxic and / or cytostatic agent.

5.3.4. Non-cleavable connectors

[00192] Although cleavable linkers may provide certain advantages, the linkers comprising the anti-glyco-MUCI1 ADC of the description need not be cleavable. For non-cleavable linkers, drug release does not depend on the differential properties between plasma and some cytoplasmic compartments. The release of the drug is postulated to occur after the internalization of the ADC through antigen-mediated endocytosis and delivery to the lysosomal compartment, where the antibody is degraded to the level of amino acids 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 has been covalently attached. The amino acid drug metabolites of conjugates with non-cleavable linkers are more hydrophilic and generally less permeable to the membrane, which leads to less spectator effects and less non-specific toxicities compared to those conjugated to a cleavable linker. In general, ADCs with non-cleavable linkers have greater stability in circulation than ADCs with cleavable linkers. The 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 can include segments of alkylene, 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 er al., 2005, J. Am. Chem. Soc. 127: 11254-11255, each of which is incorporated herein by reference. All of these linkers can be included in the anti-glyco-MUCI ADCs of the description.

[00194] In certain embodiments, the linker is non-cleavable in vivo, for example a linker according to structural formulas (VIa), (VIb), (VIC) or (VId) (as illustrated, the linkers include a suitable group to covalently bind the linker to an antibody: oo (Vla)

ANNAN o (Vlb) o o (Vo) q (VId)

AE EN H Ra

[00195] or salts thereof, where: Rº is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; R * is a moiety including a functional group capable of covalently attaching the linker to an antibody and * represents the attachment point of the linker to a cytotoxic and / or cytostatic agent.

[00196] Exemplary specific modalities of linkers according to structural formula (VIa) - (VId) that can be included in the anti-glyco-MUCI ADCs of the description include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently attaching the linker to an antibody and * represents the attachment point to a cytotoxic and / or cytostatic agent): (Vla) oo O. R * NOS À, PAPAS ys: ooo (Via) o. o À EA YA NADO O) OD o o (Vlc.1) o (Vlc.2) Ú do V

N N do x OS o o O. o N (VId.1) o o. (VId.2) NV v

DONA P SO3H o (VId.3)? O.

AS SS NX O

5.3.5. Groups Used to Attach Binders to Antibodies

[00197] A variety of groups can be used to attach linker-drug synons to antibodies to produce ADCs. Fixation groups can be electrophilic in nature and include: maleimide groups, activated disulfides, activated esters such as NHS esters and HOBt esters, haloformiatides, acid halides, alkyl and benzyl halides such as haloacetamides. As discussed below, there are also emerging technologies related to “self-stabilizing” maleimides and “bridging disulfides” that can be used according to the description. 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 shown in the schematic below. See US20130309256 A1; also Lyon et al., Nature Biotech published online, doi: 10.1038 / nbt.2968. Normal system: O. a

NH mAb a | the S.

NH mao. ? N o S plasma easy protein a F—— fe) = - N —— O. a, NH o

O o o. Here,

NH mAb | The Ss? N

O o. The,

NH

The Pro.

N

O Leads to “loss of DAR” over time SGN MalDPR system (dipropylamido maleimido): o O o mAb NH | o Spontaneous Po | W mAb-SH S NH at pH 7.4 ——õãõõ — D "A" —— $ N

S HN

HN US20130309256A1 mAb | o o ro

S NH h stable in plasma (retro reaction HN hetero-Michael shown above O. slow) OH H2oN

[00199] Politherics has described a method to bridge a

89/172 pair of sulfhydryl groups derived from the reduction of a native hinge disulfide bond. See, Badescu et al., 2014, Bioconjugated Chem. 25: 1124-

1136. The reaction is represented in the diagram below. An advantage of this methodology is the ability to synthesize DARA4 ADCs enriched by the total reduction of IgGs (to give 4 pairs of sulfhydryl) followed by the reaction with 4 equivalents of the alkylating agent. ADCs containing "bridged disulfides" are also claimed to have increased stability. q 025 A H elimination in situ ——>

SO O

The disulfide C o o o reduced er O Q "Q / o o O (C $ E ArO, S. N o Q o í N; H ro" bridged disulfide "

[00200] Similarly, as shown below, a maleimide derivative (1, below) that is capable of bridging a pair of sulfhydryl groups has been developed. See WO2013 / 085925.

FR

N, | Ds + o Ss o Ss b o

Õ FT =.

5.3.6. Connector Selection Considerations

[00201] As is known to the skilled professionals, the linker selected for a particular ADC can be influenced by a variety of factors, including, but not limited to the antibody attachment site (eg, lys, cys or other amino acid residues) , structural restrictions of the drug's pharmacophore and the lipophilicity of the drug. The specific linker selected for an ADC should 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 exterminate antigen-negative cells present in the vicinity of antigen-positive tumor cells. The mechanism of extermination of the expectant cell by ADCs has indicated that the metabolic products formed during the intracellular processing of ADCs may play a role. The neutral cytotoxic metabolites generated by the metabolism of ADCSs in antigen-positive cells appear to play a role in the extermination of the expectant cell whereas the charged metabolites can be prevented from spreading through the membrane into the medium and therefore cannot affect the extermination of expectant cells. . In certain embodiments, the linker is selected to attenuate the exterminating effect of spectators caused by ADC cell metabolites. In certain embodiments, the connector is selected to increase the extermination effect of the spectators.

[00203] The properties of the linker can also impact the aggregation of the ADC under conditions of use and / or storage. Typically, ADCSs reported in the literature do not contain more than 3 to 4 molecules of drug per antibody molecule (see, for example, Chari, 2008, Acc Chem Res 41: 98-107). Attempts to obtain higher drug to antibody ratios (“DAR”) have often 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 er 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 potency. 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 portions that reduce the aggregation of ADCs during storage and / or use. A linker can incorporate polar or hydrophilic groups such as charged groups or groups that become charged under physiological pH to reduce the aggregation of ADCs. For example, a linker can 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 bind numerous cytotoxic and / or cytostatic agents to an antibody are described 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 contents of which are incorporated herein by reference in their entirety.

[00205] In particular modalities, 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. METHODS OF MANUFACTURING ADCS ANTI-GLICO-MUCI

[00206] The anti-glyco-MUCI ADCs of the description can be synthesized using chemicals that are well known. The chemistries selected will depend, among other things, on the identity of the cytotoxic (s) and / or cytostatic agent (s), the linker and the groups used to attach the linker to the antibody. Generally, ADCs according to formula (1) can be prepared according to the following scheme: DLR * + Ab-R *> [DL-XY], - Ab (D) where D, L, Ab, XY and n are as previously defined and R * and R represent complementary groups capable of forming a covalent bond with each other, as discussed above.

[00207] The identities of the R * and Rº groups will depend on the chemistry used to bind the D-L-R * tunon 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 resembled those of the unconjugated antibody. A variety of chemicals and techniques for conjugating molecules to biological molecules such as antibodies are known in the art and in particular to 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: À Review,” in: Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera er 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; PCT publication WO 89/12624. Any of these chemicals can be used to bind syntones to an antibody.

[00208] Various functional R * and chemical groups useful for binding syntones to accessible lysine residues are known and include by way of example and not limitation NHS esters and isothiocyanates.

[00209] Various functional R * and chemical groups useful for linking synthes to the free sulfhydryl groups accessible from cysteine residues are known and include by way of example and not limitation haloacetyl 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 binding sites available in the antibody by conjugating small, multifunctional molecules to the side chains of accessible amino acid residues of the antibody. Functional groups R * suitable for covalently linking the tunons to these "converted" functional groups are then included in the tunons.

[00211] The antibody can also be engineered to include amino acid residues for conjugation. An approach for 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 group useful for binding syntones to non-coded amino acids.

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

[00213] For bonds where R * is a sulfhydryl group (for example, when R * is a maleimide), the antibody is usually first totally or partially reduced to break the disulfide bonds interchain between cysteine residues.

[00214] Cysteine residues that do not participate in the disulfide bridges can be engineered into an antibody by mutating one or more codons. The reduction of these unpaired cysteines produces a sulfhydryl group suitable for conjugation. Preferred positions for incorporating engineered cysteines include, by way of example and without limitation, positions S112C, S113C, AL14C, SI115C, AI176C, 5180C, S252C, V286C, V292C, S357C, A359C, S398C, S428C (Kabat numbering) human IgG heavy chain and positions VI110C, S114C, SI21C, S127C, S168C, V205C (Kabat numbering) on the human Ig cap light chain (see, for example, US Pat. No. 7,521,541, US Pat. No. 7,855 .275 and US Pat.

8,455,622).

[00215] As it will be evaluated by the skilled professionals, the number of cytotoxic and / or cytostatic agents linked to an antibody molecule can vary, such that a collection of ADC can be heterogeneous in nature,

where some antibodies contain a bound agent, some two, some three, etc. (and some none). The degree of heterogeneity will depend, among other things, on the chemicals used to bind cytotoxic and / or cytostatic agents. For example, where antibodies are reduced to produce sulfhydryl groups for attachment, heterogeneous mixtures of antibodies having zero, 2, 4, 6 or 8 linked agents per molecule are often produced. In addition, by limiting the molar ratio of fixing compound, antibodies having zero, 1, 2, 3, 4, 5, 6, 7, or 8 linked agents per molecule are often produced. Thus, it will be understood that depending on the context, established DARs can be averages for a collection of antibodies. For example, “DAR4” may refer to an ADC preparation that has not undergone purification to isolate specific DAR peaks and may comprise a heterogeneous mixture of ADC molecules having different numbers of antibody-fixed cytostatic and / or cytotoxic agents ( eg, 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 linked 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 using HPLC or other chromatography and purity assessed by analyzing areas under the resulting peak curves.

5.4. Chimeric Antigen Receptors

[00218] The present description provides chimeric antigen receptors

(CARs) comprising the anti-glyco-MUCI1 antibodies or antigen binding fragments described herein.

[00219] The CARSs of the description typically comprise an extracellular domain operably linked to a transmembrane domain which is in turn operably linked to an intracellular domain for signaling.

The extracellular domains of the CARs of the description comprise the sequence of an anti-glyco-MUCI1 antibody or antigen-binding fragment (for example, as described in Section O or modalities 1 ao).

[00221] The transmembrane domain sequence and exemplary intracellular domain sequences are described in Sections O and O, respectively.

[00222] Several fusion proteins described herein (for example, modalities O and O to 0) are CARs and the CAR related descriptions 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 can be derived from a natural or a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane regions of particular use in this description can be derived (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, CDI54. In some cases, a variety of human hinges can also be used including the human Ig (immunoglobulin) hinge.

[00225] In one embodiment, the transmembrane domain is synthetic (ie, non-naturally occurring). Examples of synthetic transmembrane domains are peptides predominantly comprising hydrophobic residues such as leucine and valine. Preferably a phenylalanine, tryptophan and valine triplet 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 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 transmembrane domain CD8. 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 transmembrane domain CD28. In one embodiment, the transmembrane domain CD28 comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWYV.

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

5.4.2. Intracellular Domain

[00229] The CAR intracellular signaling domain of the description is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell, for example, can be cytolytic activity or auxiliary activity including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces the signal of the effector function and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can usually 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 signal from the effector function. The term intracellular signaling domain is thus intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the signal of the effector function.

[00230] Examples of preferred intracellular signaling domains for use in the CAR of the description include the T cell receptor (TCR) cytoplasmic sequences and co-receptors 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 capacity.

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

[00232] Primary cytoplasmic signal sequences regulate the primary activation of the TCR complex in a stimulator mode or in an inhibitor mode. Primary cytoplasmic signaling sequences that act in a stimulatory manner 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 of the description include those derived from zeta TCR, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79% a, CD79b and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR of the description comprises a CD3-zeta cytoplasmic signaling sequence.

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

[00235] The co-stimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory 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, CDA40, PD-1, ICOS, antigen-1 associated with lymphocyte function (LFA-1), CD2, CD7, LIGHT, NKG2C , B7-H3 and a linker that specifically binds with CD83 and the like.

[00236] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the description can be linked together in a random or specified order. Optionally, an oligomeric connector

or short polypeptide, preferably between 2 and 10 amino acids in length, can form the bond. 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 encompasses nucleic acid molecules encoding immunoglobulin light and heavy chain genes for anti-glyco-MUCI antibodies, vectors comprising such nucleic acids and host cells capable of producing the anti-glyco-MUCI1 antibodies of the description. In certain respects, nucleic acid molecules encode and host cells are able to express, anti-glyco-MUCI1 antibodies and antibody binding fragments of the description (for example, as described in Section O and modalities O to O) as well as fusion proteins (for example, as described in modalities O to O) and chimeric antigen receptors (for example, as described in Section O and modalities O to O) containing them. The exemplary vectors of the description are described in the O to O modalities and the exemplary host cells are described in the O to O modalities.

[00239] An anti-glyco-MUCI1I antibody of the description can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express a recombinantly antibody, a host cell is transfected with one or more recombinant expression vectors carrying fragments of DNA encoding the antibody's immunoglobulin light and heavy chains such that the light and heavy chains are expressed in the host cell and, optionally, secreted. in the medium in which host cells are grown, medium from which antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into 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 Pat . U.S. No.

4,816,397.

[00240] To generate nucleic acids encoding such anti-glyco-MUCI antibodies, fragments of DNA 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 heavy and light chain sequences, for example using the polymerase chain reaction (PCR). Germline DNA sequences for 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 er al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, UJS. 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 Vu and Vr segments related to the DNA fragments encoding anti-glyco-MUCI1 antibody are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the genes of the region variable for full-length antibody chain genes, for Fab fragment genes, or for an scFv gene. In these manipulations, a DNA fragment encoding Vy or Vr is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably 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 Vu region can be converted to a full-length heavy chain gene by operably linking the DNA encoding Vu to another DNA molecule encoding the constant regions of the heavy chain (CH ,, CH, CH; and, optionally, CH,). The sequences of genes in the human heavy chain constant region 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 covering these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG ,, IgG, I8G3, I8Ga, IgA, IgE, IZGM or IgD constant region, but in certain embodiments it is an IgG, or IgG constant region ... For a heavy chain gene of Fab fragment, the DNA encoding Vu can be operably linked to another DNA molecule encoding only the CH1 constant region of the heavy chain.

[00243] Isolated DNA encoding the Vr region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding Vr to another DNA molecule encoding the constant region light chain, CL. The gene sequences of the human light chain constant region 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 covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a cap or lambda constant region, but in certain embodiments it is a cap constant region.

[00244] To create a scFv gene, the DNA fragments encoding Vu and VL can be operably linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4-Ser) 3, such that the Vu sequences and V ”can be expressed as a contiguous single chain protein, with the Vu and V1 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 of the description, DNAs encoding light and heavy chains of partial or full size, obtained as described above, are inserted into the expression vectors such that the genes are operably linked to the control sequences transcriptional and translational. In this context, the term “operably linked” is intended to mean that an antibody gene is linked in a vector such that the transcriptional and translational control sequences within the vector serve its intended function to regulate the transcription and translation of the gene. antibody. The expression vector and 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 can 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 (for example, binding of complementary restriction sites on the antibody gene fragment and vector or abrupt end binding if none of the restriction sites are present). Before the insertion of the light or heavy chain sequences related to the

104/172 anti-glyco-MUCI antibody, the expression vector can already carry the sequences of the antibody constant region. For example, an approach to convert the Vu and Vr sequences related to the anti-glyco-MUCI monoclonal antibody to full-length antibody genes is to insert them into expression vectors already encoding the heavy chain constant and chain constant regions. take, respectively, such that the segment Vu is operatively linked to the segment (s) CH within the vector and the segment V is operably linked to the segment CL within the vector. In addition or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is attached in the frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a protein other than immunoglobulin).

[00247] In addition to the antibody chain genes, the recombinant expression vectors of the description carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other elements of expression control (for example, 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 evaluated by those skilled in the art that design the expression vector, including selection regulatory sequences may depend on factors such as the choice of host cell to be transformed, the level of protein expression desired, etc. Regulatory sequences suitable for mammalian host cell expression include viral elements that target 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 major adenovirus late promoter (AGMLP)) and polyoma. For further description of viral regulatory elements and their sequences, see, for example, Pat. No. 5,168,062 by Stinski, Pat. No. 4,510,245 by Bell et al. and Pat. No. 4,968,615 by Schaffner et al.

[00248] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the description can 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. Pat. No. 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 'host cells with methotrexate selection / amplification) 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 by standard techniques. The various forms of the term “transfection” are intended to cover a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, for example, electroporation, lipofection, precipitation with calcium phosphate, DEAE transfection -dextran and

106/172 similar.

[00249] It is possible to express the antibodies of the description in prokaryotic or eukaryotic host cells. In certain embodiments, antibody expression is carried out on 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 of the description include Chinese Hamster Ovary cells (CHO cells) (including CHO DHFR 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 expression of the antibody in the host cells or secretion of the antibody within the medium. culture in which 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 portions of intact antibodies, such as Fab fragments or scFv molecules. It is understood that variations in the above procedure are within the scope of this 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-MUCI1 antibody of this description.

[00250] For the expression of a CAR of the description, for example as described in Section O and in modalities O and O, it is preferable that the host cell is a T cell, preferably a human T cell. In some embodiments, the host cell exhibits anti-tumor immunity

107/172 when the cell is cross-linked with MUC1 in a tumor cell. The detailed methods for producing the T cells in the description are described in Section O

[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 necessary for binding to glyco-MUCI. Molecules expressed from such truncated DNA molecules are also covered by the antibodies of the description.

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

[00253] Since a nucleic acid encoding one or more portions of an anti-glyco-MUCI antibody, additional changes 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-MUCI1 antibodies of the description can also be produced by chemical synthesis (for example, by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Il1.). Variant antibodies can also be generated using a cell-free platform (See, for example, Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology, 17: 420-426).

[00255] Since an anti-glyco-MUCI1 antibody of the description has been

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 (for example, ion exchange, affinity and column chromatography by size classification) , centrifugation, differential solubility or any other standard technique for protein purification. In addition, the anti-glyco-MUC] 1 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-MUCI1 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 SuperdexO 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-MUCI1 CARSs of the description are released into the cells using a retroviral or lentiviral vector. Retroviral and lentiviral vectors expressing CAR can be released in different types of eukaryotic cells as well as within entire tissues and organisms using cells transduced as carriers or cell-free local or systemic delivery of encapsulated, linked or naked vectors. The method used can be for any purpose where stable expression is required or sufficient.

[00258] In other embodiments, the CAR sequences are delivered within the cells using mRNA transcribed in vitro. The mRNA CAR transcribed in vitro can be delivered within different types of eukaryotic cells as well as within entire tissues and organisms using transfected cells as carriers or free local or systemic delivery.

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 transponsons.

[00260] An advantage of the RNA transfection methods of the description is that the RNA transfection is essentially transient and a vector free: an RNA transgene can be delivered to a lymphocyte and expressed there after a brief activation of the in vitro cell. in vitro, 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. Cloning of cells is not necessary because of the efficiency of RNA transfection and its ability to uniformly modify the entire lymphocyte population.

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

[00262] Some IVT vectors are known in the literature that are used in a standardized way as a standard for in vitro transcription and that have been genetically modified in such a way that stabilized RNA transcripts are produced. Currently protocols used in the technique are based on a plasmid vector with the following structure: a 5th RNA polymerase promoter allowing RNA transcription, followed by a gene of interest that is flanked 3rd and / or 5th by untranslated regions (RTU) and a 3rd polyadenyl cassette containing 50-70 A nucleotides. Before in vitro transcription, the circular plasmid is linearized 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 3rd end. It is not clear whether this non-physiological projection affects the amount of protein produced intracellularly from such a construction.

[00263] RNA has several advantages over more traditional plasmid or viral approaches. The gene expression of an RNA source does not require transcription and the protein product is produced quickly after transfection. Furthermore, since RNA has gained access only to the cytoplasm, rather than the nucleus, and therefore, typical transfection methods result in an extremely high transfection rate. In addition, 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, the construction of RNA can be delivered inside the cells by electroporation. See, for example, formulations and methodology for electroporation of nucleic acid constructs within mammalian cells as described in US 2004/0014645, US 2005 / 0052630A1l, US 2005 / 0070841Al, US 2004 / 0059285Al, US 2004 / 0092907A1. The various parameters including the strength of the electric field required for the 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, Pat. U.S. No.

6,678,556, Pat. No. 7,171,264 and Pat. No. 7,173,116. Apparatus for the therapeutic application of electroporation are commercially available, for example, the MedPulsere6 DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.) And are described in patents such as

Pat. No. 6,567,694, Pat. No. 6,516,223, Pat. No. 5,993,434, Pat. No. 6,181,964, Pat. US. No. 6,241,701 and Pat. US. No. 6,233,482; electroporation can also be used for transfecting cells in vitro as described for example in US20070128708A1. Electroporation can also be used to release nucleic acids into cells in vitro. Consequently, electroporation-mediated administration within nucleic acid cells including expression constructs using any of the many available devices and electroporation systems known to those skilled in the art presents an exciting new way to deliver 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 (for example, mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subjects are human beings.

[00266] T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments of the present description, any number of T cell lines 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 Ficollº separation. In a preferred embodiment, an individual's circulating blood cells are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, 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 an appropriate buffer or medium 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, bivalent cations. Again, surprisingly, initial activation steps in the absence of calcium lead to increased activation. How those of ordinary skill in the art would easily assess a washing step can be performed by methods known to those in the art, such as using a semi-automated “direct flow” centrifuge (eg, Cobe 2991 cell processor, Baxter CitoMate or 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, PlasmaLyte A or other saline with or without buffer. Alternatively, the 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 depleting monocytes, for example, by centrifugation through a PERCOLLº gradient or by centrifugal elutriation in reverse flow. 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 granules conjugated to anti-CD3 / anti-CD28 (i.e., 3 x 28), such as DYNABEADSO M-450 CD3 / CD28 T, for a period of time sufficient for the positive selection of the desired T cells.

In one mode, the time period is about 30 minutes.

In an additional modality, the time period ranges from 30 minutes to 36 hours or longer and all values in between.

In an additional embodiment, the time period is at least 1, 2, 3,4,5 or 6 hours.

In another preferred mode, 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, can increase cell production.

Longer incubation times can be used to isolate T cells in any situation where there are few T cells when compared to other cell types, such as in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or immunocompromised individuals.

In addition, the use of longer incubation times can increase the capture efficiency of CD8 + T cells. Thus, simply by shortening or, prolonging the time, T cells are allowed to bind to CD3 / CD28 granules and / or by increasing or decreasing the granule to T cell ratio (as further described here), subpopulations of T cells can preferably be selected for or against the initiation of culture or at other time points during the process.

In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies in the granules or other surface, subpopulations of T cells can be preferably selected for or against at the beginning of the 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 modalities, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process.

The “unselected” cells can also be subjected to additional selection rounds.

[00268] The enrichment of a T cell population by negative selection can be performed with a combination of antibodies directed to unique surface markers for the negatively selected cells. One method is cell classification and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present in negatively selected cells. For example, to enrich for CD4 * cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CDIA4, CD20, CD11b, CDI6, HLA-DR and CD8. In certain embodiments, it may be desirable to enrich how much or positively select for regulatory T cells that typically express CD4 *, CD25 *, CD62L ", GITR * and FoxP3 *. Alternatively, in certain embodiments, regulated T cells are depleted by anti -C25 conjugates or other similar selection method.

[00269] For the isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (for example, particles such as granules) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume at which granules and cells are mixed together (that is, they increase the concentration of cells), to ensure maximum contact of cells and granules. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In an additional embodiment, more than 100 million cells / ml are used. In an additional embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells / ml is used. In another embodiment, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells / ml is used. In additional embodiments, concentrations of 125 or 150 million cells / ml can be used. The use of high concentrations can result in increased cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28 negative T cells or samples where there are many tumor cells present (ie, leukemic blood, tissue tumor, etc.). Such cell populations can be of therapeutic value and would be desirable to obtain. For example, using a high concentration of cells allows more efficient selection of CD8 + T cells that normally have weaker CD28 expression.

[00270] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the T-cell and surface mixture (for example, particles such as granules), interactions between the particles and the cells are minimized. This selects 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 in diluted concentrations. In one embodiment, the cell concentration used is 5 x 10º / ml. In other modalities, the concentration used can be from about 1 x 10º / ml to 1 x 10º / ml and any integer value between them.

[00271] In other modalities, cells can be incubated on a rotator for varying lengths of time at varying speeds from 2 to 10º 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 freezing and thawing step provides a more uniform product by removing granulocytes and to some degree monocytes in 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 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% 5% Dextrose, 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 -80º C. at a rate of 1 per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used as well as uncontrolled freezing immediately at -20º C. or in liquid nitrogen.

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

[00274] Also considered in the context of the description is the collection of blood samples or apheresis product from a subject in a period of time prior to when the expanded cells as described here were necessary. 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 benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis sample is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen and used at a later time. In certain embodiments, samples are collected from a patient shortly after the diagnosis of a particular disease as described here, but before any treatment. In an additional embodiment, cells are isolated from a blood sample or apheresis from a subject before any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, “agents — antiviral, chemotherapy , radiation, immunosuppressive agents, such as cyclosporine, azatioprinay methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATEH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, 28 steroids, and steroids, 28 and 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 embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (for example, before, simultaneously or after) a bone marrow or stem cell transplant or ablative T cell therapy using chemotherapy agents such as such as, fludarabine, external beam radiation therapy (XRT), cyclophosphamide.

[00275] In an additional embodiment of the present description, T cells are obtained from a patient directly after treatment. In this regard, it was noted that following certain cancer treatments, in particular treatments with drugs that damage the immune system, right after treatment during the period when patients would normally be recovering from treatment, the quality of the obtained T cells may be ideal or improved in its ability to expand ex vivo.

Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced graft and in vivo expansion. Thus, it is considered within the context of the present description to collect blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage, during this recovery phase. In addition, in certain embodiments, mobilization (for example, GM-CSF mobilization) and conditioning regimes can be used to create a condition in a subject in which the repopulation, recirculation, regeneration and / or expansion of particular cell types are favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells and other cells of the immune system.

5.5.1.2 T Cell Activation and Expansion

[00276] T cells are activated and expanded generally using methods as described, for example, in Pats. No. 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 in the description are expanded by contact with a surface having attached 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 T cells. 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 kinase C activator from protein (eg, briostatin) in conjunction with a calcium ionophor. For the co-stimulation of an accessory molecule on the surface of T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under appropriate conditions to stimulate the proliferation of T cells. To stimulate the proliferation of CD4 * T cells or CD8 + T cells, a anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, 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): 13191328, 1999; Garland et al., J. Imnmunol Meth. 227 (1-2): 53-63, 1999).

[00278] In certain embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell can be provided by different protocols. For example, the agents providing each signal can be in solution or attached to a surface. When coupled to a surface, the agents can be coupled to the same surface (that is, in the "cis" formation) or on separate surfaces (that is, in the "trans" formation). Alternatively, one agent can be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the co-stimulatory signal is attached 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 can 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, U.S. Patent Application Publications No. 20040101519 and 20060034810 for cells presenting artificial antigen (aAPCs) which 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, in the same granule, that is, "cis," or in separate granules, that is, "trans." For example, the agent providing the primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof and both agents are co-immobilized in the same granule in equivalent molecular amounts. In one embodiment, a 1: 1 ratio of each antibody bound to the granules for CDA4 * T cell expansion and T cell cultivation is used. In certain aspects of the present description, a ratio of anti CD3: CD28 antibodies bound to the granules is used such that an increase in T cell expansion is seen when compared to 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 to the expansion observed using a 1: 1 ratio. In one embodiment, the ratio of CD3: CD28 antibody bound to the granules ranges from 100: 1 to 1: 100 and all integers between them. In one aspect of the present description, more anti-CD28 antibody is bound to the particles than the anti-CD3 antibody, i.e., 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 a particular embodiment, a CD3: CD28 ratio of 1: 100 of antibody bound to the granules is used. In another embodiment, a CD75: CD28 ratio of 1:75 of antibody bound to the granules is used. In an additional 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 another embodiment, a CD3: CD28 ratio of 1: 3 of antibody bound to the granules is used. In another embodiment, a CD3: CD28 ratio of 3: 1 of antibody bound to the granules is used.

[00280] Particle to cell ratios from 1: 500 to 500: 1 and any integer values between these can be used to stimulate T cells or other target cells.

Since those of ordinary skill in the art can easily assess, the particle to cell ratio may depend on the particle size in relation to the target cell.

For example, small granules would only bind a few cells, while larger granules would bind many.

In certain embodiments the cell to particle ratio ranges from 1: 100 to 100: 1 and any integer values between them and in additional embodiments the ratio comprises 1: 9 to 9: 1 and any integer values between them, 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 may 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, 8:11, 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: cell ratio is 1: 5. In additional embodiments, the particle to cell ratio can be varied depending on the stimulation day.

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 other day or every other day thereafter for up to 10 days, in final ratios. 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, the particles are added on a daily basis or every other day at a final ratio of 1: 1 on the first day and 1: 5 on the third and fifth days of stimulation.

In another embodiment, the particle to cell ratio is 2: 1 on the first stimulation day and adjusted to 1:10 on the third and fifth stimulation days.

In another embodiment, the particles are added on a daily or every other day basis to a final ratio of

1: 1 on the first day and 1:10 on the third and fifth days of stimulation. A person skilled in the art will appreciate that a variety of other reasons may be suitable for use in the present description. In particular, the ratios will vary depending on the particle size and cell size and type.

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

[00282] By way of example, cell surface proteins can be bound by allowing paramagnetic granules to which anti-CD3 and anti-CD28 are attached (3 x 28 granules) to contact T cells. In one embodiment, cells ( for example, 10th to 10th T cells) and granules (for example, DYNABEADSG M-450 CD3 / CD28 T paramagnetic granules in a 1: 1 ratio) are combined in a buffer, preferably PBS (without bivalent cations such as calcium and magnesium). Again, those of ordinary skill in the art can easily assess any cell concentration that can be used. For example, the target cell can be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) can comprise the target cell of interest. Consequently, any cell number is within the context of the present description. In certain embodiments, it may be desirable to significantly decrease the volume at which the particles and cells are mixed together (i.e., to increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, more than 100 million cells / ml are used. In an additional embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells / ml is used. In another embodiment, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells / ml is used. In additional embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations allows more efficient capture of cells that can 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 embodiments. For example, the use of high concentration of cells allows for more efficient selection of CD8 + T cells that normally have weaker CD28 expression.

[00283] In an embodiment of the present description, the mixture can be grown for several hours (about 3 hours) to about 14 days or any whole hourly value in between. In another embodiment, the mixture can be grown for 21 days. In one embodiment of the description, granules and T cells are grown together for about eight days. In another embodiment, granules and T cells are grown together for 2 to 3 days. Various stimulation cycles can also be desired such that the T cell culture times can be 60 days or more. Suitable conditions for T cell culture include an appropriate medium (for example, Minimum Essential Medium or RPMI 1640 Medium, or X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (for example , fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFB and TNF -o or any other cell growth additives known to the skilled practitioner. Other additives for cell growth include, but are not limited to, surfactant, plasmanate and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Means can include RPMI 1640, AIM-V, DMEM, MEM, oa-MEM, F-12, X-Vivo 15 and X-Vivo 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 an amount of cytokine (s) sufficient for the growth and expansion of T cells. Antibiotics, for example, penicillin and streptomycin, are included only in cultures experimental, not in cell cultures that should be infused into a subject. Target cells are maintained under conditions necessary to support growth, for example, at an appropriate temperature (for example, 37º C.) and atmosphere (for example, air plus 5% CO).

[00284] T cells that have been exposed to varying stimulation times may exhibit different characteristics. For example, peripheral blood mononuclear cell products typical of blood or undergoing apheresis have an auxiliary T cell population (Tn, CD4 *) that is larger than the cytotoxic or suppressive T cell population (Tc, CD8 *). The ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a population of T cells that before about 8 to 9 days consists predominantly of Tu cells, while after about 8 to 9 days, the population of T cells 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 Ty cells may be advantageous. Similarly, if a specific subset of Tc cell antigen has been isolated it may be beneficial to expand this subset to a greater degree.

[00285] Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers vary significantly, but in larger parts, reproducibly during the course of the cell expansion process. Like this,

such reproducibility enables the ability to adapt an activated T cell product for specific purposes.

5.6 COMPOSITIONS

[00286] The anti-glyco-MUCI1 antibodies and / or anti-glyco-MUC1 ADCs of the description may be in the form of compositions comprising the anti-glyco-MUCI1 antibody and / or ADC and one or more carriers, excipients and / or diluents . The compositions can be formulated for specific uses, such as for veterinary or pharmaceutical uses in humans. The form of the composition (eg, dry powder, liquid formulation, etc.) and 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 mode of administration.

[00287] For therapeutic uses, the compositions can be supplied as part of a pharmaceutical, sterile composition that includes a pharmaceutically acceptable carrier. This composition can be in any suitable form (depending on the desired method of administering it to a patient). The pharmaceutical composition can 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 for 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] The pharmaceutical compositions can conveniently be presented in unit dosage forms containing a predetermined amount of an anti-glyco-MUCI1 antibody and / or anti-glyco-MUCI ADC from the dose description. 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 can

126/172 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. Dry powder unit dosage forms can be packaged in a Kkit with a syringe, an appropriate amount of diluent and / or other components useful for administration. Unit dosages in liquid form can be conveniently supplied in the form of a syringe filled with an amount of antibody and / or ADC suitable for a single administration.

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

[00290] Pharmaceutical compositions can 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 referred to here as "carriers"), that is, buffering agents, stabilizing agents, preservatives, isotonifying agents, non-ionic detergents, antioxidants and other various additives. See, Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be non-toxic to the recipients at the dosages and concentrations used.

[00291] Buffering agents help maintain the pH in the range that approaches physiological conditions. They can be present in a wide variety of concentrations, but will typically 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 (for example, mixture of monosodium citrate-disodium citrate, mixture of citric acid-trisodium citrate, mixture citric acid-citrate

127/172 monosodium, etc.), succinate buffers (e.g., successinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (for example, mixture of tartaric acid-sodium tartrate, mixture of tartaric acid-potassium tartrate, mixture of tartaric acid-sodium hydroxide, etc.), fumarate buffers (for example, mixture of fumaric acid-monosodium fumarate , mixture of fumaric acid-disodium fumarate, mixture of monosodium fumarate-disodium fumarate, etc.), gluconate buffers (for example, mixture of glyconic acid-sodium gluconate, mixture of glyconic acid-sodium hydroxide, mixture glyconic acid-potassium gluconate, etc.), oxalate buffer (eg mixture of oxalic acid-sodium oxalate, mixture of oxalic acid-sodium hydroxide, mixture of oxalic acid-potassium oxalate, etc.), lactate buffers (eg mixture of lactic acid-sodium lactate, mixture of lactic acid-sodium hydroxide, mixture of lactic acid-potassium lactate, etc.) and acetate buffers (for example, mixture of acetic acid-sodium acetate, mixture of acetic acid- sodium hydroxide, etc.). In addition, phosphate buffers, histidine buffers and trimethylamine salts such as Tris can be used.

[00292] Preservatives can be added to slow microbial growth and can be added in amounts ranging from about 0.2% to 1% (w / v). Preservatives suitable for use with the present description include phenol, benzyl alcohol, metacresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide and iodide), hexamethonium chloride and such parabens alkyl such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonifiers sometimes known as "stabilizers" can be added to ensure the isotonicity of liquid compositions of the present description and include polyhydric sugar alcohols, for example three-alcohol 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 adherence to the container wall. Typical stabilizers can 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 (for example, 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 can be present in amounts ranging from 0.5 to 10% by weight by weight of ADC.

[00293] Surfactants or non-ionic detergents (also known as "wetting agents") can 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 shear from the stressed surface without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.) and pluronic polyols. Nonionic surfactants can be present in a range of about 0.05 mg / ml to about 1.0 mg / ml, for example about 0.07 mg / ml to about 0.2 mg / ml.

[00294] Additional varied 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-MUCI1 antibody or binding fragments described herein can be used in various diagnostic assays. For example, 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), activated cell classification fluorescence (FACS) and Western blots.

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

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

[00298] When using the description CARs for therapy, the therapeutic methods of the description comprising administering to a subject with a tumor expressing glyco-MUCI an amount of a genetically modified cell engineered to express a description CAR, for example as described in Section O or in mode O or mode O. Methods of modifying cells, particularly T cells, to express a CAR, are described in Section 0.

6. Examples

6.1 Example 1: Identification of Anti-Glyco-MUC1 Antibodies

6.1.1. General view

[00299] The chemoenzymatic synthesis of multiple repeat MUCI1 glycopeptides with different O-glycan density and Tn glycoforms (GalNAcal1-O-Ser / Thr) was developed using recombinant glycosyltransferases. Different polypeptide GalNAc-transferase isoforms have been used to target O-glycan occupation sites (Bennett et al., 1998). The ideal vaccine project was found to be Tn glycoforms with a high density of O-glycan and KLH-conjugated glycopeptides were found to overcome tolerance. In wild-type Balb / c mice, glycopeptides with full O-glycan occupation evoked the strongest antibody response by reacting with MUCI1 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 MUCI glycopeptides

[00300] 60-mer MUCI1 peptide (VTISAPDTRPAPGSTAPPAHG) 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 ) from MUC2 (PTTTPISTTTMVTPTPTPITC) (SEQ ID NO 51) and MUCA4 (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.

6,465,220. The glycosylation with GalNAc of the peptides was carried out in a reaction mixture (1 mg peptide / mL) containing 25 mM cacodylate buffer (pH 7.4), 10 mM MnCL ,, 0.25% Triton X-100 and 2 mM UDP-GalNA-rc.

Glycosylation of 1 mg of 60 mer peptide with two GalNAc per TR (MUCI60Tn6) was obtained using GalNAc-TIl.

The incorporation of three GalNAc by TR (MUCI60Tn9) was obtained using GalNAc-T2. The replacement of all five putative O-glycosylation sites on MUCI TR (MUCI60Tn15) was performed using MUC160Tn9 as a substrate in a reaction with GalNAc-T4. Glycosylation was monitored using nano-scale reverse phase columns (Poros R3, PerSeptive Biosystems, Framingham, MA) and MALDI-TOF mass spectrometry.

The glycopeptides were purified by high performance liquid chromatography (HPLC) on a Zorbax 300SB-C3 (9.4 mm x 25 in) column (Agilent Technologies, Palo Alto, CA) on a Hewlett Packard 1100 system (Avondale, PA) using 0.1% trifluoroacetic acid (TFA) and a gradient of O to 80% acetonitrile.

The quantification and estimation of yields of glycosylation reactions were performed by comparing HPLC peaks by UV 210 absorbance using 10 µg of heavy peptide as standard.

The glycosylation in GalNAc of peptides generally produced 80 to 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 time-consuming 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 / μL were prepared for analysis by placing 1 μL of sample solution in a probe tip followed by 1 μL of matrix.

All mass spectra were obtained in the linear mode.

Data processing was performed using the software

GRAMS / 386 (Galactic Industries, Salem, NH).

6.1.2.2 Immunization Protocol

[00301] The glycopeptides were coupled to KLH (Pierce, Rockford, IL) using glutaraldehyde. The conjugation efficiency was evaluated by analyzing the reaction by size exclusion chromatography on a PD-10 column using anti-MUCI1 ELISA of the fractions. Essentially all reactivity was found with the excluded fraction and negligible reactivity in the included fractions expected to contain peptides. Additional evaluation included comparative titration analysis of KLH conjugated to the corresponding glycopeptide in the ELISA. Both analyzes indicated that the conjugation was almost complete, which should result in a 1: 300 KLH to glycopeptide ratio. Female wild-type Balb / c mice were injected subcutaneously with 10 or 15 µg of (glyco) peptide in a total volume of 200 µL (1: 1 mixture with Freunds adjuvant, Sigma). The mice received four immunizations 14 days separately 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 MAb anti-Tn-MUC1 mice

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

6.1.2.4 ELISA

[00303] ELISA were performed using 96-well MaxiSorp plates (Nunc, Denmark). The plates were coated overnight at 4ºC with | ug / mL of glycopeptides in bicarbonate buffer — carbonate (pH 9.6),

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-immunoglobulins (DakoCytomation, Glostrup, Denmark) or peroxidase-conjugated goat isotype-specific antibodies IgM, IgG1l, IgG2a, IgG2b or IgG3 (Southern Biotechnology Associates, Birmingham, AL). The plates were developed with O-phenylenediamine tablets (DakoCytomation) and read at 492 nm. Control antibodies included anti-MUC1 antibodies HMFG2 and SM3 (Burchell et al., 1987) and anti-carbohydrate antibodies 5SF4 (Tn) and 3F1 (STn) (Mandel et al., 1991). Control sera included mice immunized with MUCA mucin peptide linked to KLH.

6.1.3 Results

[00304] glycopeptide-specific mAbs were produced for GalNAcCc-MUCI using 60-mer glycopeptide GalNAc-MUCI conjugated to KLH as an immunogen. Using an ELISA Assay, the generated mAb GO2 (5F7) reacted specifically with the MUCI tandem repeat (VTSAPDTRPAPGSTAPPAHG); (SEQ ID NO: 47) which was glycosylated in vitro using purified recombinant human glycosyltransferases GalNAc-T1, GalNAc-T2 and GalNAc-T4, without any reaction with the corresponding MUC peptide] without GalNAc residues or irrelevant glycopeptides with the same type of glycoform Tn. The results of the ELISA assay are shown in Figure 1.

6.2 Example 2: Characterization of Anti-Glyco-MUC1 Antibodies

6.2.1 Overview

[00305] GO2 monoclonal antibody (5F7) has been distinguished for the specificity of its binding to the MUCI1 Tn glycoforms associated with cancer cells.

6.2.2 Materials and Methods

6.2.2.1 Immunocytochemistry

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

6.2.2.2 Immunohistochemistry

[00307] Tissues fixed in formalin, embedded in paraffin wax from breast carcinoma were obtained. All cases were conventionally classified by histological type. The avidin-biotin peroxidase complex method was used for immunoting. The paraffin sections were dewaxed, rehydrated and treated with 0.5% H7O; in methanol for 30 min. The sections were rinsed in TBS and incubated for 20 min with non-immune rabbit serum. The sections were rinsed and incubated overnight at 5ºC with primary antibody. The sections were rinsed and incubated with biotin labeled 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% H7O. The sections were stained with hematoxylin, dehydrated and assembled.

6.2.3 Results

[00308] Immunohistochemistry of colorectal carcinoma, pancreatic carcinoma and invasive breast adenocarcinomas was performed with GO2. Staining of colorectal cancer tissue (Figure 2) demonstrated strong labeling of intracellular and superficial 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 has been 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 none or limited reactivity with intracellular structures in the surrounding healthy epithelia or connective tissue cells.

6.3 Example 3: Sequence Analysis of Anti-Glyco-MUC1 Antibodies

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

The nucleotide sequence encoding the variable regions of heavy and light chain with their signal sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The heavy and light chain variable regions encoded by SEQ ID NO: 11 and SEQ ID NO: 12 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The sequences of the mature predicted variable region (following truncation of the signal peptide) are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively and are encoded by SEQ ID NO: 13 and SEQ ID NO: 4, respectively. The predicted CDR heavy chain sequences (IMGT definition) are shown in SEQ ID NO: 5 to 7, respectively, and the predicted CDR light chain sequences (IMGT definition) are shown in SEQ ID NO: 8 to 10, respectively.

6.4 Example 4: Drug Delivery to Cancer Cells with

Anti-Glico-MUC1 Antibodies

6.4.1 Overview

[00311] GO2 monoclonal antibody (5F7) has been tested for its ability to release a cytotoxic agent to target cells.

6.4.2 Materials and Methods

[00312] OVCar human ovarian carcinoma cells were added to a 24-well cell culture plate at 1,000 cells / well. GO2 monoclonal antibody and a secondary antibody conjugated to the antitubulin agent monomethyl auristatin F (MMAF) (anti-mMFc-NC-MMAF) (Moradec catalog no. AM-101-AF) were added to the plate to give the following concentrations (in ug / ml) of GO2 and ADC: Eae 2 Ps' bh 20 jaDC20 | ADCI2ZO JADC: 20 —JADC: 20 —JADC: 2.0 POR fines faithful FES ADC file: 0.6 | ADC: 0.66 —JADC: 06 | ADC: 06 —JADC: 06 —ADC: 0.6 ADC : 0,2 - | ADC: 02 | ADC: 02 ADC: 02 —ADC: 02 —ADC: 02 poses fan ft MES elis ADC: O ADC: O ADC: O ADC: O ADC: O

[00313] The plate was incubated at 37ºC for 48 hours. After 48 hours of incubation, AlamarBlueG & (Invitrogen) was added to each well and the fluorescence measured at 600 nm.

6.4.3 Results

[00314] The results are shown in Figure 5. The results show that cell toxicity is dependent on the concentration of the primary antibody (GO2) and the presence of the antibody and concentration and presence of the antibody conjugated to secondary ADC. In other words, GO2 induces the cellular toxicity of its cancer cell line when coupled with a secondary antibody that carries an MMAF cytotoxic agent.

6.5 Example 5: Quantification of Circulating Tumor Cell with Anti-MUC1 Antibodies

6.5.1 Overview

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[00315] The monoclonal antibody GO2 (5F7) has been tested for its ability to be used to quantify circulating tumor cells.

6.5.2 Materials and Methods

[00316] GO? 2 was conjugated to a magnetic separation granule and allowed to interact with samples of different concentrations of tumor cells. Cells and granules were pulled out of the solution with a magnet and washed several times to remove unbound material. GO2 which was conjugated to horseradish peroxidase was then applied to the magnetic separation granules containing linked cancer cells, incubated and then the unbound conjugated GO2 was washed. 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 test results demonstrated the GO2 binding of tumor cells.

6.6 Example 6: Immunohistochemical staining of tumor tissue using anti-glyco-MUCI1 antibodies

6.6.1 Materials and Methods

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

[00319] The dyeing was carried out in a Discovery XT autotector (Ventana Medical Systems). Following antigen recovery with cell conditioning solution 1 (CC1) (Ventana Medical Systems), the

GO? it was applied at a concentration of 1 µg / mL in antibody diluent of green Dako medium and incubated for 60 min at 37ºC. 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 MUCI1 positive TMA tumor nuclei are shown in Figure 7. In BC and OVC TMAs, most spots (> 90%) showed moderate or strong binding of GO2 to the 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 the stains in TMA 1 revealed a moderate or strong dyeing intensity when applying GO-2. Dyeing patterns have always been cytoplasmic and in many cases attached to the membrane. A pattern of apical membrane dyeing was observed in a few cores.

6.7 Example 7: Production and purification of an anti-MUC1 antibody in bispecific T cell format (TCB)

6.7.1 Materials and Methods

6.7.1.1 Production of expression vector

[00321] The GO? 2 antibody was converted to the TCB format, including P329G / L234A / L235A (“PGLALA”) mutations in the Fc region and residues loaded in the MUC1 CHI (147E / 213E; “EE” regions) ) and CL (123R / 124K; “RK”) (see SEQ ID NO: 43 to 46). The TCB antibody is illustrated in Figure 8. In summary, the variable heavy and variable light chains of mAb GO? have been 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 À with 5'º RTU and a BGH polyadenylation site. In addition, plasmids contain the

139/172 oriP region of the Epstein Barr virus for stable maintenance in HEK293 cells harboring the nuclear EBV 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 the production of antibodies in a 500 ml shaken flask, 300 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all quantities have been adjusted accordingly). For transfection, the cells are centrifuged for 10 min at 210 x ge and the supernatant is replaced with 20 ml of preheated CD CHO medium. The 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 s and subsequently incubated for 10 min at room temperature. After that, the cells are mixed with the DNA / PELI solution, transferred to a 500 ml shaken flask and incubated for 3 hours at 37ºC in an incubator with a 5% CO 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 of glucose is 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 in a final concentration of 0.01% w / v is added. The solution is then stored at 4ºC.

6.7.1.3 Antibody purification

[00323] The secreted proteins were purified by affinity chromatography using Protein A, followed by

140/172 size. For affinity chromatography, the supernatant was loaded onto a Protein A MabSelect SuRe column (GE Healthcare) equilibrated with 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unbound protein was removed by washing with equilibration buffer. The bound protein was eluted using a stepwise 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 column (GE Healthcare) equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[00324] The aggregate content of the eluted fractions was analyzed by the analytical size exclusion chromatography. Therefore, 30 ul of each fraction was applied to a TSK G3000 SWXL column (TOSOH, 7.8 mm x cm) balanced with 200 mM Arginine, 25 mM K; PO ,, 125 mM sodium chloride, 0.02 % NaN; 3, 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 -80ºC.

6.7.2 Results

[00325] The yield and quality of TCB GO? 2 antibody production are shown in Table 5.

mcBeor bi baw bo ba bB6e |

6.8 Example 8: Jurkat-NFAT reporter assay to monitor ex vivo target expression in undigested patient-derived tumor samples

6.8.1 Overview

[00326] A Jurkat NFAT reporter assay was used to monitor target expression (glyco-MUCI) ex vivo in undigested primary human tumor samples using a GO? 2 TCB.

6.8.2 Materials and Methods

6.8.2.1 Materials * TCB GO? 2 (see Example 7) * DP47 TCB (unbleached, negative control) * Matrigel (Item No. 734-1101, Corning / VWR, Switzerland) * Corning8 & Costar & Ultra-low clamping multiple reservoir plates (Item No. CLS7007-24EA, Sigma) * Cell culture microplate, 96 wells (Item No. 655098, Greiner Bio-A, Switzerland) * RPMII640 medium (Item No. 42401-018, FisherScientific, Schweiz) * Jurkat medium : RPMI1640 medium with 2 g / 1 D-Glucose, 2 g / 1 NaHCO ;, 10% FCS, 25 mM HEPES, 2 mM L-Glutamine, 1 x NEAA, 1 x Sodium Pyruvate, 200 ug / ml Hygromycin B * Jurkat NFAT luciferase reporter cells (Promega) * Tumor samples were received from Indivumed GmbH, Germany. The samples were sent overnight in a means of transport. About 24 h 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 the tumor pieces were added (triplicates). 33 ul of cold matrigel was added per well and the plate was incubated again for 2 min at 37 ° C. 100 µl (50 nM or 5 nM) dilution of TCB antibody (diluted in Jurkat medium without Hygromycin but with 2X Penicillin / Streptomycin) was added per well. The reporter cells

Jurkat-NFAT were harvested and viability was assessed using ViCell. The cells were centrifuged at 350 x g for 7 min before they were resuspended in Jurkat medium without hygromycin. 50 µl of the cell suspension was added per well (50,000 cells / well). The plate was incubated for 4 to 5 h at 37ºC in a humidified incubator before it was collected for luminescence reading. 50 µl of the ONE-Glo solution was added to each well and incubated for 10 min at room temperature in the dark. Luminescence was detected using WALLAC Victor3 ELISA reader (PerkinElmer2030), 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 bronchial and lung neoplasm: 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 bronchial and lung neoplasm: 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 bronchial and lung neoplasm: 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 dotted line indicates luminescence for T Jurkat NFA cells incubated with tumor samples without any TCB. 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 than samples incubated with DP47 negative control TCB.

6.9 Example 9: In vitro characterization of TCB GO2

6.9.1 Overview

[00329] TCB GO? 2 (Example 7) that recognized the aberrantly tumor-specific glycosylated MUCI was functionally distinguished in the tumor cells expressing MUCI1.

6.9.2 Materials and Methods

6.9.2.1 Cell lines and PBMCs

The engineered tumor cell lines T3M4 pfzv and MCF7 cs were cultured in DMEM with 10% FCS and 2 mM Glutamine. MCFIOA is a human non-tumorigenic mammary epithelial cell line (ATCC & CRL-10317). HBEpiC are human bronchial epithelial cells (Sciencell 43210). 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 on 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. The 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, cells were washed twice with FACS buffer before the addition of the diluted secondary antibody (Goat anti-human IgG Fcy Specific Fragment F (ab ') 2 AffiniPure conjugated with PE, Jackson ImmunoResearch * 109 -116-170). After 30 min of incubation at 4 ° C the unbound secondary antibody was washed. Before measurement, cells were resuspended in 200 µl of FACS buffer and analyzed by flow cytometry using BD Fortessa. The tests

144/172 were performed in triplicates.

6.9.2.3 T-cell-mediated tumor killing cells and T-cell activation

[00332] The target cells were harvested with Trypsin / EDTA, counted and the viability was 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 was transferred into each well of a 96-well flat bottom plate. The plate was incubated overnight at 37ºC in the incubator to allow the cells to adhere to the plate. The next day the PBMCs were isolated from whole blood. The blood was diluted 2: 1 with PBS and covered with 15 ml of Histopaque-1077 (% 10771, Sigma-Aldrich) in Leucosep tubes and centrifuged for 30 min at 450g without brake. After centrifugation, the strip containing the cells was collected with a 10 ml pipette and transferred into 50 ml tubes. The tubes were filled with PBS up to 50 ml and centrifuged (400g, 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 combined and the washing step was repeated (this time centrifugation 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 of 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 determined after 24 h and 48 h using Cytotoxicity Detection Kit (1644793, Roche Applied Science). The overloading of activation marker on T cells after tumor cell extermination was measured by flow cytometry. In summary, PBMCs were harvested, transferred into a 96-well round bottom plate and stained with CD4 APC antibodies (300514, BioLegend), CD8 FITC (344704, BioLegend), CD25 BV421 (302630, BioLegend), CD69 PE ( 310906, BioLegend) 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 MU, 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] The cytokine / chemokine secretion in the supernatant was measured by flow cytometry, using the cytometric granule arrangement (CBA), according to the manufacturer's guidelines. Extermination assays mediated by T cell supernatants were collected and stored at -20ºC. The supernatants were subsequently thawed and tested according to the manufacturer's instructions. The following CBA kits (BD Biosciences) were used: human gamma interferon CBA (IFNy) Flex Set (E7), Granzyme B human CBA Flex Set (D7), human IL6 CBA Flex Set (A7), human IL8 CBA Flex Set (A9 ), Human ILIO CBA Flex Set (B7) and human tumor necrosis factor CBA (TNF) Flex Set (D9). The samples were measured using BD FACS Canto II and the analysis was performed using Software Diva (BD Biosciences). The tests were carried out in triplicates.

6.9.3 Results

[00334] The binding of TCB GO2 to the breast cancer cell line MCF7 and to the pancreatic cancer cell line T3MA4, both engineered to express aberrantly glycosylated MUCI1, was

146/172 confirmed (Figure 12). Subsequently, TCB GO? 2 activity was tested on both tumor cell lines, MCF7 and T3MA, using freshly isolated PBMCs (Figure 13 and Figure 14). The tumor cell extermination of both cell lines was detected after 24 h and even stronger after 48 h. This was accompanied by the strong activation of CD4 T cells and CD8 T cells determined by the overloading of the two activation markers CD25 and CD69 and the release of IL6, IL8, IL10, IFNY, TNFa and Granzima B in the supernatant. As a negative control, the respective non-targeted TCB was included.

[00335] To prove that TCB GO? 2 does not bind to MUC1 normally glycosylated in epithelial cells, binding to MCFIOA, which is a human non-tumorigenic mammary epithelial cell line and to HBEpiC, which are primary human bronchial epithelial cells, tested. As a positive control, the HMFG1 TCB, which does not discriminate between MUCI1 expressed in normal and tumor cells, was included. The HFMGI1 TCB was found to bind to both cells tested, confirming MUCI expression, but TCB GO? it was not stable to bind to these cells (Figure 15). Also, TCB GO? was tested to see if it would induce T cell extermination or activation in the presence of normal epithelial cells expressing MUCI. This was tested on MCFI0A cells and there was no detectable extermination or T cell activation with TCB GO? 2, whereas HMFG1 TCB induced extermination as well as T cell activation (Figure 16).

6.10 Example 10: Functional characterization of GO2 and TCB GO antibodies? by superficial plasmon resonance

6.10.1 Overview

[00336] GO2 and TCB GO2 (Example 7) were distinguished by superficial plasmon resonance.

6.10.2 Materials and Methods

6.10.2.1 Connection of GO2 and TCB GO? to immobilized glycopeptides

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[00337] The binding of GO2 and TCB GO antibodies? to human and cinomolgo glycopeptides (Table 6) was evaluated by superficial plasmon resonance (SPR). All SPR experiments were performed in a Biacore T200 at 25ºC with HBS-EP as conduction buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% P20 surfactant , Biacore, Freiburg / Germany).

ED NO: 49)

[00338] The biotinylated glycopeptides were dissolved in PBS and the final concentration was between 0.9 and 1.8 mg / ml (Table 6). The biotinylated glycopeptides were directly coupled to a flow cell of a streptavidin (SA) sensor chip. Immobilization levels up to 880 resonance units (UK) were used. The GO2 antibody or the TCB GO? 2 were injected with a flow of 30 µL / minute through the flow cells, in 240 seconds and at a concentration of 1000 nM (Figure 17). Dissociation was monitored for 500 s. The differences in the mass refractive index were corrected by subtracting the response obtained in a reference flow cell, where no protein was immobilized.

6.10.2.2 Greed for GO2 and TCB GO? to immobilized glycopeptides

[00339] The greed of GO2 and TCB GO? was assessed by superficial plasmon resonance (SPR). All SPR experiments were performed in a Biacore T200 at 25ºC with HBS-EP as conduction buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% P20 surfactant , Biacore, Freiburg / Germany). The biotinylated glycopeptides (Table 6) were directly coupled to a flow cell of a streptavidin (SA) sensor chip. Immobilization levels up to 200 resonance units (UK) were used.

[00340] The GO2 antibody or the TCB GO? 2 were injected with a

148/172 flow of 30 uL / minute through the flow cells in 120 seconds and in a concentration range of 3.9 to 1000 nM (dilution 1: 2). Dissociation was monitored for 400 s. The differences in the mass refractive index were corrected by subtracting the response obtained in a reference flow cell, where no protein was immobilized. The KDs were derived, in spite of the bivalence of the interaction, adjusting the curve to a 1: 1 Langmuir bond using the Biaeval software (GE Healtheare). The "apparent" KG can therefore only be used for comparison purposes.

6.10.3 Results

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

[00342] As can be seen in Figure 19, the binding of a divalent GO2 binder (IZG, TCB) to the human glycopeptide is in the three nanomolar digits (Figure 19A and 19C), while the connection to the cinomolgo glycopeptide is in both nanomolar digits (Figure 19B and 19D).

6.11 Example 11: Pharmacokinetic and tolerability study of single exploratory dose of TCB GO2 in cynomolgus monkeys

6.11.1 Overview

[00343] The objectives of this study are to determine the pharmacokinetics and tolerability of the TCB GO? 2 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 frozen TCB GO? 2 stock solution (2.12 mg / mL) and formulation buffer (20 mM Histidine, 140 mM

149/172 NaCl, 0.01% Tween 20; pH 6.0) is done overnight in a refrigerator to maintain 4ºC. The test item dosage formulations are prepared under sterile conditions in appropriate concentrations to meet the dose level requirements by diluting 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, shaken or shaken. Any mixing is done by gentle pipetting or gentle oscillation of the container.

6.11.2.2 Animals

[00346] Cynomolgus monkeys (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 test facility for at least 6 weeks prior to the start of dosing.

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

[00348] The animals selected for the study are randomly allocated in cages based on the group compatibility information provided and then allocated with individual study numbers. The animals are placed in 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 floors measuring 1.61 x 1.66 x 2.5 m on the lower floor and 1.61 x 1.66 x 2.03 m on the upper floor. The forage material is sawdust. There was no known contaminant in the forage that interfered with the objectives of the study.

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

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

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

[00353] The animal's domestic environment is enriched to promote social interaction, play and exploration. The animals have perches and materials such as plastic toys, balls, climbers and stainless steel mirrors. These are changed frequently to reduce familiarity. Before changing, all toys and climbers are thoroughly cleaned to avoid cross-contamination. Animals are also offered a range of other treatments such as forage mix, vegetables, nuts, cookies and fruits 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 ensured by clinical signs or other changes.

6.11.2.4 Experimental design

[00355] A male and a female animal are administered with TCB GO2 | Group number Dose level (ug / kg) (mL / kg) (ug / kg) [LU male and 1 female) - oo hoo [2 (1 male and 1 female) Boo Bo |

[00356] TCB GO? it is administered to the appropriate animals by a single slow intravenous bolus injection (1 to 2 min) into the saphenous vein or tail vein at least 8 days separately. It is staggered so that only one male and one female receive a new dose level on any single day. Based on observations of 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 fixed Vygon infusion needle.

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

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

6.11.3 Results

[00359] TCB GO? is tolerated at the doses tested.

7. SPECIFIC MODALITIES, CITATION OF REFERENCES

[00360] Although several specific modalities have been illustrated and described, it will be assessed that several changes can be made without diverging from the spirit and scope of the description (s). This description is exemplified by the numbered modalities presented below.

1. An anti-glyco-MUCI antibody or antigen binding fragment that: a. preferably binds to a glyco-MUCI epitope that is overexpressed in cancer cells when compared to normal and b cells. 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 cell line of breast cancer MCF7 or T47D.

2. An anti-glyco-MUCI antibody or antigen binding fragment that connects to the MUCI tandem repeat (VTSAPDTRPAPGSTAPPAHG); which has been glycosylated in vitro using purified GalNAc-TI, GalNAc-T2 and GalNAc-T4 e - human (recombinant) glycosyltransferases (hereinafter referred to below 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 cell line of breast cancer MCF7 or T47D.

3. The anti-glyco-MUCI1 antibody or antigen-binding fragment of mode 1 or mode 2 comprising a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-H2 comprising the sequence 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.

4. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality O, wherein CDR-H1l comprises the amino acid sequence of SEQ ID NO: 5.

5. The anti-glyco-MUCI1 antibody or antigen binding fragment of modality 0, wherein CDR-H1l comprises the amino acid sequence of SEQ ID NO: 23.

6. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality O, wherein CDR-HIL comprises the amino acid sequence of SEQ ID NO: 28.

7. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality 0, wherein CDR-H1l comprises the amino acid sequence of SEQ ID NO: 32.

8. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the O to O modalities, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6.

9. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the O to O modalities, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24.

10. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the O to O modalities, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7.

11. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the O to O modalities, wherein CDR-LI comprises the amino acid sequence of SEQ ID NO: 30.

12. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the O to O modalities, wherein CDR-LI comprises the amino acid sequence of SEQ ID NO: 26.

13. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the O to O modalities, wherein CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27.

14. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the O to O modalities, wherein CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10.

15. The anti-glyco-MUCI1 antibody or antigen-binding fragment of mode 1 or mode 2 where VH comprises the complementarity determining regions (CDRs) of SEQ ID NOS: 5a 7 and VL comprises the CDRs of SEQ ID NOS: 8 to 10.

16. The anti-glyco-MUCI1 antibody or antigen-binding fragment of mode 1 or mode 2 where VH comprises the complementarity determining regions (CDRs) of SEQ ID NOS: 23 a and VL comprises the CDRs of SEQ ID NOS: 26,27 and 10.

17. The anti-glyco-MUCI1 antibody or antigen-binding fragment of mode 1 or mode 2 where VH comprises the complementarity determining regions (CDRs) of SEQ ID NOS: 28, 29 and 25 and VL comprises CDRs SEQ ID NOS: 30, 9 and 31.

18. The anti-glyco-MUCI1 antibody or antigen binding fragment of any of modalities 1 to O which is a chimeric or humanized antibody.

19. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the modalities 1 to O where the VH comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3 and the VL comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4.

20. The anti-glyco-MUCI antibody or antigen-binding fragment of any of the modalities 1 to O where 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 to SEQ ID NO: 4.

21. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the modalities 1 to O where 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-MUCI antibody or antigen-binding fragment of mode 1 or mode 2 where 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-MUCI1 antibody or antigen-binding fragment of any of modalities 1 to O is multivalent.

24. The anti-glyco-MUCI antibody or antigen binding fragment of any of modalities 1 to O which is in the form of a single chain variable fragment (scFv).

25. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality O in which scFv comprises the N-terminal heavy chain variable fragment relative to the light chain variable fragment.

26. The anti-glyco-MUCI1 antibody or antigen binding fragment of modality O in which the heavy chain variable fragment and scFv light chain variable fragment are covalently linked to a 4 to 15 amino acid linker sequence.

27. The anti-glyco-MUCI antibody or antigen binding fragment of any of modalities 1 to O which is in the form of a multispecific antibody.

28. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality O 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-MUCI1 antibody or antigen-binding fragment of modality O, where the bispecific antibody is a CrossMab, a Fab arm exchange antibody, a bispecific T cell engender (BiTE) or a targeting molecule double affinity (DART).

30. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality O, where the bispecific antibody is a CrossMab.

31. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality 0, where the bispecific antibody is a CrossMab "ºE,

32. The anti-glyco-MUCI antibody or antigen-binding fragment of modality 0, where the bispecific antibody is a CrossMabY "vL

33. The anti-glyco-MUCI antibody or antigen-binding fragment of modality 0, where the bispecific antibody is a CrossMabC Her,

34. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality O, wherein the bispecific antibody is a Fab arm exchange antibody.

35. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality 0, wherein the bispecific antibody is a double affinity targeting molecule (DART).

36. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality O, in which the bispecific antibody is an

157/172 bispecific T cell (BiTE).

37. The anti-glyco-MUCI1 antibody or antigen-binding fragment of any of the O to O modalities, wherein the second epitope is an MUCI epitope.

38. The antigen-binding anti-glyco-MUCI1 antibody of any of the O to O modalities, wherein the second epitope is an MUCI1 epitope that is overexpressed in cancer cells when compared to normal cells.

39. The anti-glyco-MUCI1 antibody or antigen binding fragment of any of the O to O modalities, wherein the second epitope is a T cell epitope.

40. The anti-glyco-MUCI1 antibody or antigen-binding fragment of modality O, wherein the T cell epitope comprises a CD3 epitope, a CD8 epitope, a CDI16 epitope, a CD25 epitope, a CD28 epitope or an NKG2D epitope .

41. The anti-glyco-MUCI antibody or antigen-binding fragment of modality O, wherein the T cell epitope comprises a CD3 epitope, which is optionally an epitope present on human CD3.

42. The anti-glyco-MUCI1 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-MUCI1 antibody or antigen-binding fragment of any of modalities 1 to O that is conjugated to a detectable moiety.

44. The anti-glyco-MUCI antibody or antigen-binding fragment of modality O where the detectable marker is an enzyme, a radioisotope or a fluorescent label.

45. A bispecific antibody comprising a first antigen binding domain that binds to CD3 (optionally human CD3) and a second antigen binding domain that binds to glyco-MUCI, where the bispecific antibody competes with an antibody or fragment antigen binding sequence comprising a 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 heavy chain variable region comprising the heavy chain CDR-H1 of SEQ ID NO: 34, CDR-H2 of SEQID NO: 35e and CDR-H3 of SEQ ID NO: 36 and a light chain variable region comprising the light chain CDR-L1 of SEQ ID NO: 37, CDR-L2 of SEQ ID NO: 38 and CDR-L3 of SEQ ID NO: 39.

46. The bispecific O mode antibody, wherein the second antigen binding domain comprises (1) a heavy chain variable 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 light chain variable region comprising the CDR-L1 light chain of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9 and CDR-L3 of SEQ ID NO: 10.

47. The bispecific antibody of modality O or modality O, wherein the first antigen binding domain comprises a sequence of the heavy chain variable region 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 sequence of the light chain variable region that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of amino acid of SEQ ID NO: 41.

48. The bispecific antibody of modality O, the first antigen binding domain comprises the sequence of the heavy chain variable region of SEQ ID NO: 40 and the sequence of the light chain variable region of SEQ ID NO: 41.

49. The bispecific antibody of any of the O to O modalities, wherein the second antigen binding domain comprises a sequence of the heavy chain variable region 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 sequence of the light chain variable region that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid sequence of SEQ ID NO: 4.

50. The bispecific antibody of modality 0, wherein the second antigen binding domain comprises the sequence of the heavy chain variable region of SEQ ID NO: 3 and the sequence of the light chain variable region of SEQ ID NO: 4.

51. The bispecific antibody of any of the O to O modalities, wherein the first and / or the second antigen binding domain is a Fab molecule.

52. The bispecific antibody of modality 0, where the first antigen binding domain is a crossed Fab molecule, in which the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged.

53. The bispecific antibody of mode 0, where the first and the second antigen binding domain of the bispecific antibody are both Fab molecules and in one of the antigen binding domains (particularly the first antigen binding domain) the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, where a. in the CL constant domain 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 CHI constant domain 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 (numbered according to the EU Kabat index); or b. in the CL constant domain 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 CHI constant domain 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 (numbered according to the EU Kabat index), where the constant domains CL and CHI of the antigen binding domain having the VH / VL exchange are not replaced by one other.

54. The bispecific antibody of modality O, where a. in the CL constant domain of the first antigen binding domain the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbered according to Kabat) and in the CH1 constant domain of the first domain antigen binding the amino acid at position 147 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbered according to the EU Kabat index); or b. in the CL constant domain of the second antigen-binding domain the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbered according to Kabat) and in the CH1 constant domain of the second domain antigen binding the amino acid at position 147 or the amino acid at position 213 is replaced independently by glutamic acid (E) or aspartic acid (D) (numbered according to the EU Kabat index).

55. The bispecific antibody of modality O, where 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) (numbered according to Kabat) and in the CH1 constant domain 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) (numbered according to the EU index of Kabat ).

56. The bispecific antibody of modality O, where 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) (numbered according to Kabat) and in the constant domain CH1 of the second antigen binding domain the amino acid at position 147 is replaced independently by glutamic acid (E) or aspartic acid (D) (numbered according to the EU index of Kabat).

57. The bispecific antibody of modality O, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is replaced independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is replaced independently by lysine (K), arginine (R) or histidine (H) (numbered according to Kabat) and in the constant domain CH1 of the second antigen binding domain the amino acid at position 147 is replaced independently by glutamic acid (E) or aspartic acid (D) (numbered according to the EU Kabat index) and the amino acid at position 213 is replaced independently by glutamic acid (E) or aspartic acid (D) (numbered according to the EU Kabat index).

58. The bispecific antibody of modality 0, where in the constant domain CL of the second antigen binding domain the amino acid at position 124 is replaced by lysine (K) (numbered according to Kabat) and the amino acid at position 123 is replaced by lysine (K) (numbered according to Kabat) and in the CH1 constant domain of the second antigen binding domain the amino acid at position 147 is replaced by glutamic acid (E) (numbered according to the EU Kabat index) and the amino acid in position 213 it is replaced by glutamic acid (E) (numbered according to the EU Kabat index).

59. The bispecific antibody of modality O, where in the constant domain CL of the second antigen binding domain the amino acid at position 124 is replaced by lysine (K) (numbered according to Kabat) and the amino acid at position 123 is replaced by arginine (R) (numbered 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) (numbered according to the EU Kabat index) and the amino acid in position 213 it is replaced by glutamic acid (E) (numbered according to the EU Kabat index).

60. The bispecific antibody of any of the modalities O to O, wherein the constant domain CL of the second antigen binding domain is of the kappa isotype.

61. The bispecific antibody of any of the modalities O to O, wherein the first and second antigen binding domains are fused to each other, optionally via a peptide linker.

62. The bispecific antibody of modality 0, where the first and second antigen binding domain are each Fab molecule and (1) 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 (li) the first antigen binding domain is fused at the C terminal of the Fab heavy chain to the N terminal of the Fab heavy chain of the second antigen binding domain.

63. The bispecific antibody of any of the modalities O to O, where the bispecific antibody provides monovalent binding to CD3.

64. The bispecific antibody of modality O, which comprises two antigen binding domains that specifically bind to glyco-MUCI.

65. The bispecific antibody of modality O, wherein the two antigen binding domains that specifically bind to glyco-MUCI1 comprise the same amino acid sequence.

66. The bispecific antibody of any of the modalities O to O, wherein the bispecific antibody further comprises an Fc domain composed of a first and a second subunit.

67. The bispecific antibody of mode 0, where the Fc domain is an IgG Fc domain.

68. The bispecific antibody of mode 0, wherein the Fc domain is an IgG Fc domain.

69. The bispecific antibody of mode 0, wherein the Fc domain is an IgG Fc domain.,.

70. The bispecific antibody of modality 0, wherein the IgG Fc domain, comprises an amino acid substitution at position S228 (numbering the EU index of Kabat), preferably the substitution of amino acid S228P.

71. The bispecific O mode antibody, where the Fc domain is a human Fc domain.

72. The bispecific antibody of modality O, wherein the Fc domain is a human IgGl Fc domain, which optionally comprises SEQ ID NO: 42.

73. The bispecific antibody of any of the modalities O to O, where the first, the second and, where present, the third antigen binding domain are each Fab molecule and (a) (1) the second binding domain antigen is fused at the C terminal of the Fab heavy chain to the N terminal of the Fab heavy chain of the first antigen binding domain and the first antigen binding domain is fused at the C terminal

164/172 of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain or (11) 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 domain of antigen binding and the second antigen binding domain is fused at the C terminal of the Fab heavy chain to the N terminal of the first subunit of the Fc domain and (b) the third antigen binding domain, where present, is fused at the C terminal from the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

74. The bispecific antibody of any of the modalities O to O, in which the Fc domain comprises a modification promoting the association of the first and second subunits of the Fc domain.

75. The bispecific antibody of any of the modalities O to O, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or the effector function.

76. The bispecific antibody of modality O, comprising a first antigen binding domain that specifically binds to CD3, wherein the first antigen binding domain is a crossed Fab molecule in which the variable or constant regions, preferably the variable regions, the Fab light chain and the Fab heavy chain are switched; B. a second and third antigen binding domains that specifically bind to glyco-MUCI1, comprising a heavy chain variable region comprising the heavy chain CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6 and CDR-H3 of SEQ ID NO: 7 and a light chain variable region comprising the light chain CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9 and CDR-L3 of SEQ ID NO: 10, where the second and third antigen binding domains are each Fab molecules; ç. 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 terminal of the Fab heavy chain to the N terminal of the Fab heavy chain of the first antigen binding domain and the first antigen binding domain is fused at the C terminal of the Fab heavy chain to the N terminal of the first subunit of the Fc domain and where the third antigen binding domain is fused at the C terminal of the Fab heavy chain to the N terminal the second subunit of the Fc domain.

77. The bispecific antibody of modality 0, wherein the first antigen binding domain comprises a heavy chain variable region comprising the heavy chain CDR-H1 of SEQ ID NO: 34, CDR-H2 of SEQ ID NO: 35 and the CDR-H3 of SEQ ID NO: 36 and a light chain variable region comprising the light chain CDR-L1 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 modality 0, wherein the first antigen binding domain comprises a sequence of the heavy chain variable region 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 sequence of the light chain variable region 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 O mode antibody, wherein the first antigen binding domain comprises the sequence of the heavy chain variable region of SEQ ID NO: 40 and the sequence of the light chain variable region of SEQ ID NO: 41.

80. The bispecific antibody of any of the modalities 0 to O, wherein 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 sequence of the light chain variable region 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 O mode antibody, wherein the second and third antigen binding domains comprise the heavy chain variable region of SEQ ID NO: 3 and the light chain variable region of SEQ ID NO: 4.

82. The bispecific antibody of any of the modalities O to O, in which the Fc domain incorporates, alone or in combination, all the resources described in Sections O and O in relation to the Fc domains.

83. The bispecific antibody of any of the O to O modalities, in which the antigen binding domains and the Fc region are fused to each other by the peptide linkers.

84. The bispecific antibody of modality O, wherein the peptide linkers comprise the peptide linkers as in SEQ ID NO: 45 and / or SEQ ID NO: 46.

85. The bispecific antibody of any of the modalities O to O, where in the constant domain CL of the second and third Fab molecules, the amino acid at position 124 is replaced by lysine (K) (numbered according to Kabat) and the amino acid at position 123 is replaced by lysine (K) or arginine (R), preferably by arginine (R) (numbered according to Kabat) and in the CH1 constant domain of the second and third Fab molecules under (11) the amino acid in position 147 is replaced by glutamic acid (E) (numbered according to the EU Kabat index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbered according to the EU Kabat index).

86. The bispecific antibody of any of the modalities O to O, which comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to

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 the 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 the sequence of SEQ ID NO: 45 and a polypeptide comprising a sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 46.

87. The bispecific antibody of mode 0, wherein the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 43, a polypeptide comprising the sequence of SEQ ID NO: 44, a polypeptide comprising the sequence of SEQ ID NO: 45 and a polypeptide comprising the sequence of SEQ ID NO: 46.

88. The bispecific antibody of modality O, which comprises two polypeptides comprising the sequence of SEQ ID NO: 43.

89. The bispecific antibody of any of the modalities O to O which is conjugated to a detectable portion.

90. The bispecific antibody of modality O where 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-MUCI1 antibody or antigen binding fragment of any of the modalities 1 to O or the bispecific antibody of any of the modalities O to O operably linked to at least one second amino acid sequence.

92. The fusion protein of modality O, in which the second amino acid sequence is that of 4-1BB, CD3-zeta or a fragment thereof.

93. The O-mode fusion protein, where the second amino acid sequence is that of a fusion peptide.

168/172

94. The mode 0 fusion protein, wherein the fusion peptide is a CD28-CD3-zeta or 4-IBB (CD137) -CD3-zeta fusion peptide.

95. The fusion protein of modality O, in which the second amino acid sequence is that of a modulator of T cell activation or a fragment thereof.

96. The O mode fusion protein, where the modulator of T cell activation is IL-15 or IL-15Ra.

97. A chimeric antigen (CAR) receptor comprising scFv of any of the O to O modalities.

98. The CAR of the O modality, comprising in order from the amino terminus to the 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-MUCI antibody or antigen-binding fragment of any of the modalities 1 to O or the bispecific antibody of any of the modalities O to O or the fusion protein of any of the modalities O to O conjugated to a cytotoxic agent.

100. The antibody-drug conjugate of modality O, in which the cytotoxic agent is an auristatin, a minor DNA slot binding agent, an alkylating agent, an enediine, a lexitropsin, a duocarmicin, a taxane, a dolastatin , a maytansinoid or a vinca alkaloid.

101. The O-mode antibody-drug conjugate, wherein the anti-glyco-MUCI1 antibody or antigen-binding fragment or bispecific antibody is conjugated to the cytotoxic agent via a linker.

102. The antibody-drug conjugate of modality O, where the linker is cleavable under intracellular conditions.

103. The O-mode antibody-drug conjugate, in

169/172 that the cleavable linker is cleavable by an intracellular protease.

104. The antibody-drug conjugate of modality O, wherein the linker comprises a dipeptide.

105. The O-mode antibody-drug conjugate, wherein the dipeptide is val-cit or phe-lys.

106. The O-mode antibody-drug conjugate, where the cleavable linker is hydrolyzable at a pH of less than 5.5.

107. The antibody-drug conjugate of modality O, wherein the hydrolyzable linker is a hydrazone linker.

108. The antibody-drug conjugate of modality O, wherein 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 modalities 1 to O or the bispecific antibody of any of modalities O to O, the fusion protein of any one of the modes O to O or the CAR of mode O or mode O.

110. The nucleic acid of modality O in which the coding region is optimized in the codon for expression in a human cell.

111. A vector comprising nucleic acid of modality O or modality O.

112. The modality vector What is a viral vector.

113. The mode vector O in which the viral vector is a lentiviral vector.

114. A host cell engineered to express nucleic acid of modality O or modality O.

115. The host cell of mode O, which is a human T cell engineered to express CAR of mode O or mode O.

116. A host cell comprising the vector of any of the O to O modalities.

117, The host cell of modality What is a T cell and where the vector encodes the CAR of modality O or modality 0.

118. A pharmaceutical composition comprising (a) the anti-glyco-MUCI1 antibody or antigen binding fragment of any of modalities 1 to O, the bispecific antibody of any of modalities O to O, the fusion protein of any of of the modalities O to 0, the CAR of the modality O or modality O, the antibody-drug conjugate of any of the modalities O to O, the nucleic acid of modality O or modality 0, the vector of any of the modalities O to O or the host cell of the modality of any of the modalities O to O and (b) a physiologically suitable buffer, adjuvant or diluent.

119. A method of treating cancer comprising administering to a subject in need thereof an effective amount of the anti-glyco-MUCI1 antibody or antigen binding fragment of any of modalities 1 to O, the bispecific antibody of any of the modalities O a O, the fusion protein of any of the O to 0 modalities, the CAR of the O modality or the O modality, the antibody-drug conjugate of any of the O to O modalities, the nucleic acid of the O modality or the 0 modality, the vector of any of the O to O modalities, the host cell of any of the O to O modalities or the pharmaceutical composition of the 0 modality.

120. The O 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 one of modalities 1 to O and detecting the binding of the anti-glyco- MUCI] or antigen-binding fragment.

122. The method of modality O, further comprises quantifying the binding of the anti-glyco-MUCI1 antibody or antigen binding fragment.

123. Method of mode O or mode O, where binding 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 individually indicated to be incorporated by reference for all purposes. In the event that there is an inconsistency between the descriptions of one or more of the references incorporated herein and this description, the descriptions of this specification are considered.

8. REFERENCES

1. Bennett, EP, Hassan, H., Mandel, U., Mirgorodskaya, E., Roepstorff, P., Burchell, J., Taylor-Papadimitriou, J., Hollingsworth, MA, Merkx, G., van Kessel, AG, and others. (1998) Cloning of a human UDP-N- acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl- transferase that complements other GalNAc-transferases in complete O- glycosylation of the MUCI1 tandem repeat. J. Biol. Chem., 273, 3047230481.

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313.

Claims (77)

CLAIMS: 1. Anti-glyco-MUCI1 antibody or antigen binding fragment, characterized by the fact that: a. preferably it binds to a glyco-MUCI1 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 cell line of breast cancer MCF7 or T47D. 2. Anti-glyco-MUCI1 antibody or antigen binding fragment, characterized by the fact that it is linked to the MUCI tandem repeat (VTSAPDTRPAPGSTAPPAHG); which has been glycosylated in vitro using purified GalNAc-TI, GalNAc-T2 and GalNAc-T4 e - human (recombinant) glycosyltransferases (hereinafter referred to below 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 cell line of breast cancer MCF7 or T47D. 3. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 1 or 2, characterized in that it comprises a complementarity determining region (CDR) HI 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. 4. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5. 5. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-HI1 comprises the amino acid sequence of SEQ ID NO: 23. 6. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-HI comprises the amino acid sequence of SEQ ID NO: 28. 7. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-HI comprises the amino acid sequence of SEQ ID NO: 32. 8. Anti-glyco-MUC1 antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6. 9. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24. 10. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7. 11. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30. 12. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26. 13. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27. 14. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 3, characterized by the fact that CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10. 15. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 1 or 2, characterized by the fact that VH comprises the complementarity determining regions (CDRs) of SEQ ID NOS: 5 to 7 and VL comprises the CDRs of SEQ ID NOS: 8 to 10. 16. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim | or 2, characterized by the fact that the VH comprises the complementarity determining regions (CDRs) of SEQ ID NOS: 23 to 25 and the VL comprises the CDRs of SEQ ID NOS: 26,27 and 10. 17. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 1 or 2, characterized by the fact that VH comprises the complementarity determining regions (CDRs) of SEQ ID NOS: 28, 29 and 25 and the VL comprises the CDRs of SEQ ID NOS: 30.9 and 31. 18. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 1 or 2, characterized by the fact that it is a chimeric or humanized antibody. 19. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim | or 2, characterized by the fact that 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: 3 SEQ ID NO: 4. 20. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 1 or 2, characterized in that the VH comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 3 and the VL comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 4. 21. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 1 or 2, characterized in that the VH comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 3 and the VL comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 4. 22. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim | or 2, characterized by the fact that VH comprises the amino acid sequence of SEQ ID NO: 3 and VL comprises the amino acid sequence of SEQ ID NO: 4. 23. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 1 or 2, characterized by the fact that it is multivalent. 24. Anti-glyco-MUCI1 antibody or antigen binding fragment according to claim 1 or 2, characterized in that it is in the form of a single chain variable fragment (scFv). 25. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 24, characterized in that the scFv comprises the N-terminal heavy chain variable fragment to the light chain variable fragment. 26. Anti-glyco-MUCI antibody or antigen binding fragment according to claim 24, characterized in that the heavy chain variable fragment and scFv light chain variable fragment are covalently linked to a 4 to 15 amino acids. 27. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 1 or 2, characterized in that it is in the form of a multispecific antibody. 28. Anti-glyco-MUCI1 antibody or antigen binding fragment according to claim 27, characterized in that the multispecific antibody is a bispecific antibody that binds to a second epitope that is different from the first epitope. 29. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 28, characterized by the fact that the bispecific antibody is a CrossMab, a Fab arm exchange antibody, a bispecific T cell engaging (BITE) or a double affinity targeting molecule (DART). 30. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 29, characterized in that the bispecific antibody is a CrossMab. 31. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 30, characterized by the fact that the bispecific antibody is a CrossMab "º" E, 32. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 30, characterized by the fact that the bispecific antibody is a CrossMabY "-Y, 33. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 30, characterized in that the bispecific antibody is a CrossMabC HH! L, 34. Anti-glyco-MUCI antibody or antigen binding fragment according to claim 29, characterized in that the bispecific antibody is a Fab arm exchange antibody. 35. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 29, characterized by the fact that the bispecific antibody is a double affinity enhancing molecule (DART). 36. Anti-glyco-MUCI1 antibody or antigen binding fragment according to claim 29, characterized by the fact that the bispecific antibody is a bispecific T cell engender (BiTE). 37. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 28, characterized in that the second epitope is a MUCI epitope. 38. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 28, characterized in that the second epitope is an MUCI1 epitope that is overexpressed in cancer cells when compared to normal cells. 39. Anti-glyco-MUCI antibody or antigen-binding fragment according to claim 28, characterized in that the second epitope is a T cell epitope. 40. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 39, characterized in that the T cell epitope comprises a CD3 epitope, a CD8 epitope, a CD 16 epitope, a CD25 epitope, a epitope CD28 or an NKG2D epitope. 41. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 40, characterized in that the T cell epitope comprises a CD3 epitope, which is optionally an epitope present in human CD3. 42. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 41, characterized in that the CD3 epitope comprises a CD3 gamma epitope, a CD3 delta epitope, a CD3 epsilon epitope or a CD3 zeta epitope. 43. Anti-glyco-MUCI1 antibody or antigen-binding fragment according to claim 1 or 2, characterized in that it is conjugated to a detectable portion. 44. Anti-glyco-MUCI1 antibody or antigen binding fragment according to claim 43, characterized in that the detectable marker is an enzyme, a radioisotope or a fluorescent label. 45. Fusion protein, characterized by the fact that it comprises the amino acid sequence of the anti-glyco-MUCI antibody] or antigen binding fragment as defined in claim 1 or 2 operably linked to at least a second amino acid sequence. 46. Fusion protein according to claim 45, characterized in that the second amino acid sequence is that of 4-1BB, CD3-zeta or a fragment thereof. 47. Fusion protein according to claim 45, characterized in that the second amino acid sequence is that of a fusion peptide. 48. Fusion protein according to claim 47, characterized in that the fusion peptide is a CD28-CD3-zeta or 4-IBB (CD137) -CD3-zeta fusion peptide. 49. Fusion protein according to claim 45, characterized in that the second amino acid sequence is that of a T cell activation modulator or a fragment thereof. 50. Fusion protein according to claim 49, characterized in that the T cell activation modulator is IL-15 or IL-15Ra. 51. Chimeric antigen (CAR) receptor, characterized by the fact that it comprises scFv as defined in claim 24. 52. The chimeric antigen receptor according to claim 51, characterized in that it comprises in the order of amino terminal for carboxy: a leading human CD8 peptide, scFv, a human CD8 hinge domain, a CD8 transmembrane domain human and a CD3-zeta signaling domain. 53. Antibody-drug conjugate, characterized in that it comprises the anti-glyco-MUCI antibody or antigen binding fragment as defined in claim 1 or 2 conjugated to a cytotoxic agent. 54. Antibody-drug conjugate according to claim 53, characterized by the fact that the cytotoxic agent is an auristatin, a minor DNA slot binding agent, an alkylating agent, an enedin, a lexitropsin, a duocarmycin, a taxane, dolastatin, maytansinoid or vinca alkaloid. 55. Antibody-drug conjugate according to claim 54, characterized in that the anti-glyco-MUCI] antibody or antigen binding fragment is conjugated to the cytotoxic agent via a linker. 56. Antibody-drug conjugate according to claim 55, characterized in that the linker is cleavable under intracellular conditions. 57. Antibody-drug conjugate according to claim 56, characterized in that the cleavable linker is cleavable by an intracellular protease. 58. Antibody-drug conjugate according to claim 57, characterized in that the linker comprises a dipeptide. 59. Antibody-drug conjugate according to claim 58, characterized in that the dipeptide is val-cit or phelys. 60. Antibody-drug conjugate according to claim 56, characterized in that the cleavable linker is hydrolyzable at a pH of less than 5.5. 61. Antibody-drug conjugate according to claim 60, characterized in that the hydrolyzable linker is a hydrazone linker. 62. Antibody-drug conjugate according to claim 56, characterized in that the cleavable linker is a disulfide linker. 63. Nucleic acid, characterized in that it comprises a coding region for an anti-glyco-MUC! 1 antibody or antigen binding fragment as defined in claim 1 or 2. 64. Nucleic acid according to claim 63, characterized by the fact that the coding region is optimized in the codon for expression in a human cell. 65. Vector, characterized by the fact that it comprises the nucleic acid as defined in claim 63. 66. Vector according to claim 65, characterized by the fact that it is a viral vector. 67. Vector according to claim 66, characterized by the fact that the viral vector is a lentiviral vector. 68. Host cell, characterized by the fact that it is engineered to express nucleic acid as defined in the claim 63. 69. Host cell, characterized by the fact that it is a human T cell engineered to express the CAR as defined in claim 51. 70. Host cell, characterized by the fact that it comprises the vector as defined in claim 65. 71. Host cell, characterized in that it is a T cell comprising a vector encoding the CAR as defined in claim 51. 72. Pharmaceutical composition, characterized in that it comprises (a) the anti-glyco-MUC1 antibody or antigen binding fragment as defined in claim 1 or 2 and (b) a physiologically suitable buffer, adjuvant or diluent. 73. Method of treating cancer, characterized in that it comprises administering to a subject in need thereof an effective amount of the anti-glyco-MUCI1 antibody or antigen binding fragment as defined in claim 1 or 2. 74. Method according to claim 73, characterized by the fact that the subject is suffering from breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer or colorectal cancer. 75. Method of detecting cancer in a biological sample, characterized by the fact that it comprises contacting a sample with an anti-glyco-MUCI1 antibody or antigen-binding fragment as defined in the claim | or 2 and detecting binding of the anti-glyco-MUC1 antibody or antigen binding fragment. 76. The method of claim 75, characterized in that it further comprises quantifying the binding of the anti-glyco-MUCI1 antibody or antigen binding fragment. 77. The method of claim 75, characterized in that the binding is compared to a normal tissue control as a negative / baseline control and / or to a cancerous tissue control as a positive control.