Antiglyco-CD44 antibody and its use

Abstract

The present disclosure relates to anti-glyco-CD44 antibodies and antigen-binding fragments thereof that specifically bind to cancer-specific glycosylation variants of CD44, as well as related fusion proteins and antibody-drug conjugates, as well as nucleic acids encoding such biomolecules. The present disclosure further relates to the use of the antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates, and nucleic acids for cancer therapy.

Description

[Technical Field]

[0001] 1. Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 986,083, filed on March 6, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application includes an electronically submitted sequence listing in ASCII format, which is incorporated in its entirety by reference. The ASCII copy was created on March 5, 2021, named GOT-003-WO_SL.txt, and is 202,651 bytes in size. [Background technology]

[0003] 3.Background Therapies that redirect T-cell responses using chimeric antigen receptors (CARs) have emerged as a powerful tool in cancer immunotherapy and have proven highly effective in hematological malignancies. This targets non-essential tissue-shared antigens such as CD19 in B-cell malignancies (Brentjens et al., 2013, Sci Transl Med. 5(177):177ra38-177ra38; Grupp et al., 2013, N Engl J Med. 368(16):1509-1518; Kalos et al., 2011, Sci Transl Med. 3(95):95ra73-95ra73; Kochenderfer et al., 2010, Blood. 116(20):4099-4102; Porter et al., 2011, N Engl J Med. 365(8):725-733). However, since most CAR targets are normal autoantigens overexpressed in solid tumors, employing CAR therapy in solid tumors has presented problems. Consequently, adverse effects due to cross-reactivity with essential healthy tissues have frequently been reported in studies targeting solid tumors with CAR T cells (Bin Hou et al., 2019, Dis Markers, Article ID 3425291). To overcome the problems of employing CAR therapy in solid tumors, novel cancer-specific antigens that enable selective targeting are needed.

[0004] Many cancers express abnormally glycosylated proteins that differ from those in healthy tissue. These abnormally glycosylated proteins contain glycopeptide epitopes that may be suitable for immunotherapy of solid tumors, although few such glycopeptide epitopes have been identified. CD44 is a transmembrane protein with a heavy chain glycosylated, involved in cell-cell interactions, cell adhesion, and migration, and has also been suggested to be a marker for cancer stem cells. In humans, there are 10 CD44 variants, including a standard variant. These 10 variants are differentially expressed in various tumors (see Chen, et al., 2018, J Hematol Oncol. 11(1):64). Within the CD44 variant region, there are 117 possible O-linked glycosylation sites, including 54 serine and 63 threonine sites.

[0005] Antibodies that target CD44, such as vivatuzumab, which recognizes the cancer-associated isoform CD44v6, are well known in the industry. However, vivatuzumab induces severe skin toxicity due to low expression of CD44v6 in healthy skin. Borjesson et al., 2003. Clin Cancer Res. 9(10 Pt 2):3961S-72S; Brentjens et al., 2013. Sci Transl Med. 5(177):177ra38- 177ra38; Goodison et al., 1999, Mol Pathol. 52(4):189-196; Grupp, et al. al. 2013, N Engl J Med. 368(16):1509-1518; Hou et al., 2019, Dis Markers. 2019:1-11; Julien et al., 2012, Biomolecules. 2(4):435-466; Kalos et al., 2011, Sci Transl Med. 3(95):95ra73-95ra73; King et al., 2017, Blood Adv. 1(7):429-442; Kochenderfer et al., 2010, Blood. 116(20):4099-4102; Porter et al., 2011, N Engl J Med. 365(8):725-733; Posey et al., 2016, Immunity. 44(6):1444- 1454; Prochazka et al., 2014, Cell Signal. 26(10):2234-2239; Radhakrishnan et al., 2014, Proc Natl Acad Sci. 111(39):E4066-E4075; Sneath et al., 1998, Mol Pathol. 51(4):191-200; Sorensen et al., 2006, Glycobiology. 16(2):96-107; Stanley, 2011, Cold Spring Harb Perspect Biol. 3(4); Steentoft et al.See also: EMBO J. 32(10):1478-1488, 2013; Steentoft et al., 2011, Nat Methods. 8(11):977-982; Stroomer et al., 2000, Clin Cancer Res. 6(8):3046-3055; Thapa et al., 2016, Stem Cells Int. 2016:1-15; Tijink et al., 2006, Clin Cancer Res. 12(20):6064-6072; Wandall et al., 2010, Cancer Res. 70(4):1306-1313.

[0006] Therefore, there is a need to identify glyco-CD44 epitopes that are overexpressed in cancer cells, and to develop new therapeutic approaches that utilize such glyco-CD44 epitopes, such as antibodies and CARs. [Overview of the project]

[0007] 4. Summary This disclosure provides therapeutic and diagnostic agents based on antibodies and antigen-binding fragments that are selective for cancer-specific epitopes of glycoCD44, thereby achieving tumor specificity for glycopeptide variants.

[0008] The present invention provides an anti-glyco-CD44 antibody that binds to a cancer-specific glycosylated variant of CD44 and its antigen-binding fragment. The present invention further provides fusion proteins and antibody-drug conjugates comprising the anti-glyco-CD44 antibody and antigen-binding fragment, as well as nucleic acids encoding the anti-glyco-CD44 antibody, antigen-binding fragment, and fusion protein.

[0009] The disclosures of this invention further provide methods for using anti-glyco-CD44 antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates, and nucleic acids for cancer therapy.

[0010] In certain embodiments, the disclosure provides anti-glyco-CD44 antibodies and antigen-binding fragments having bispecificity and other multispecificity, which bind to cancer-specific glycosylated variants of CD44 and to a second epitope. The second epitope may be CD44 itself, another protein co-expressed with CD44 on cancer cells, or another protein present on different cells such as activated T cells. Furthermore, nucleic acids encoding antibodies are also disclosed, such as nucleic acids containing codon-optimized coding regions and nucleic acids containing non-codon-optimized coding regions for expression in specific host cells.

[0011] The anti-glyco-CD44 antibody and binding fragment may be in the form of a fusion protein containing a fusion partner. The fusion partner may be useful for providing a second function, such as signaling function of the signaling domain of a T cell signaling protein, a peptide modulator of T cell activation, or an enzymatic element of a labeling system. Exemplary T cell signaling proteins include 4-1BB, CO3C, and fusion peptides, such as CD28-CD3-zeta and 4-1BB-CD3-zeta. 4-1BB, or CD137, is a T cell costimulatory receptor, and CD3-zeta is a signaling element of the T cell antigen receptor. The portion providing the second function may be a T cell activation modulator, such as IL-15, IL-15Ra, or an IL-15 / IL-15Ra fusion, an MHC class I chain-related (MIC) protein domain useful for MicAbody construction, or it may encode a label or enzymatic element of a labeling system useful for monitoring the degree and / or location of binding in vivo or in vitro. Constructs encoding these prophylactic and therapeutic biomolecules, placed under the conditions of T cells, such as autologous T cells, provide a robust platform for supplementing adoptive T cells for preventing or treating various cancers, in some embodiments of this disclosure.

[0012] In certain embodiments, the anti-glyco-CD44 antibodies or antigen-binding fragments of this disclosure include (or are encoded by nucleotide sequences) heavy-chain and / or light-chain variable sequences as described in Tables 1A-1E. For clarity, where the term “anti-glyco-CD44 antibody” is used in this document, it is intended to include monospecific and multispecific (including bispecific) anti-glyco-CD44 antibodies, antigen-binding fragments of monospecific and multispecific antibodies, and fusion proteins and conjugates containing antibodies and their antigen-binding fragments, unless the context indicates otherwise. Similarly, where the term “anti-glyco-CD44 antibody or antigen-binding fragment” is used, it is also intended to include monospecific and multispecific (including bispecific) anti-glyco-CD44 antibodies and their antigen-binding fragments, as well as fusion proteins and conjugates containing such antibodies and antigen-binding fragments, unless the context indicates otherwise.

[0013] In other embodiments, the anti-glyco-CD44 antibodies or antigen-binding fragments of this disclosure comprise (or are encoded by) the heavy and / or light chain CDR sequences listed in Tables 1-3. The CDR sequences listed in Tables 1A-1E comprise CDR sequences defined according to the schemes of IMGT (Lefranc et al., 2003, Dev Comparat Immunol 27:55-77), Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and Chothia (Al-Lazikani et al., 1997, J. Mol. Biol 273:927-948) for defining the CDR boundaries. The CDR sequences listed in Tables 1F, 1G, and 1H are consensus sequences derived from the CDR sequences listed in Tables 1A-1D according to the definitions of IMGT, Kabat, and Chothia, respectively. The CDR sequences listed in Tables 1I, 1J, and 1K are consensus sequences derived from the CDR sequences listed in Tables 1A to 1E, respectively, according to the definitions of IMGT, Kabat, and Chothia. The CDR sequences listed in Tables 2A to 2E are combined overlapping regions of the CDR sequences listed in Tables 1A to 1E, with IMGT, Kabat, and Chothia sequences indicated in underlined bold. The CDR sequence listed in Table 2F is a combined overlapping region of the consensus CDR sequences listed in Tables 1F to 1H. The CDR sequence listed in Table 2G is a combined overlapping region of the consensus CDR sequences listed in Tables 1I to 1K. The CDR sequences listed in Tables 3A to 3E are common overlapping regions of the CDR sequences shown in Tables 1A to 1E. The CDR sequence listed in Table 3F is a common overlapping region of the CDR sequences listed in Tables 1F to 1H. The CDR sequence listed in Table 3G is a common overlapping region of the CDR sequences listed in Tables 1I to 1K.Such anti-glycoCD44 antibody and antigen-binding fragment framework sequences may be natural mouse framework sequences of VH and VL sequences as listed in Tables 1A-1D, natural rabbit framework sequences of VH and VL sequences as listed in Table 1E, or non-natural (e.g., humanized or human) framework sequences.

[0014] [Table 1-1]

[0015] [Table 1-2]

[0016] [Table 2-1]

[0017] [Table 2-2]

[0018] [Table 3-1]

[0019] [Table 3-2]

[0020] [Table 4-1]

[0021] [Table 4-2]

[0022] [Table 5-1]

[0023] Table 5-2

[0024] Table 6

[0025] Table 7

[0026] Table 8

[0027] Table 9

[0028] Table 10

[0029] Table 11

[0030] Table 12

[0031] Table 13

[0032] Table 14

[0033] Table 15

[0034] Table 16

[0035] Table 17

[0036] Table 18

[0037] Table 19

[0038] Table 20

[0039] Table 21

[0040] Table 22

[0041] Table 23

[0042] Table 24

[0043] Table 25

[0044] In certain embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of this disclosure comprises a combination of CDRs selected from the CDR sequences listed in Tables 1-3. In some embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID NOs: 3, 9, 15, 25, 31, 37, 47, 53, 59, 69, 75, 81, 89, 93, 97, 101, 107, 113, 119, 125, 129, 135, 141, 147, 153, 208, 214, 220, 228, 232, 236, 240, 246, 250, or 256. In some embodiments, CDR-H2 includes the amino acid sequence of SEQ ID NOs: 4, 10, 16, 26, 32, 38, 48, 54, 60, 70, 76, 82, 90, 94, 98, 102, 108, 114, 120, 126, 130, 136, 142, 148, 154, 209, 215, 221, 229, 233, 237, 241, 247, 251, or 257. In some embodiments, CDR-H3 includes the amino acid sequence of SEQ ID NOs: 5, 11, 17, 27, 33, 39, 49, 55, 61, 71, 77, 83, 103, 109, 115, 121, 131, 137, 143, 149, 210, 216, 222, 242, or 252. In some embodiments, CDR-L1 includes the amino acid sequences of SEQ ID NOs. 6, 12, 18, 28, 34, 40, 50, 56, 62, 72, 78, 84, 104, 110, 116, 122, 132, 138, 144, 150, 211, 217, 223, 243, and 253. In some embodiments, CDR-L2 includes the amino acid sequences of SEQ ID NOs. 7, 13, 19, 29, 35, 41, 51, 57, 63, 73, 79, 85, 91, 95, 99, 105, 111, 117, 123, 127, 133, 139, 145, 151, 155, 212, 218, 224, 230, 234, 238, 244, 248, 254, and 258. In some embodiments, CDR-L3 contains the amino acid sequences of SEQ ID NOs: 8, 14, 20, 30, 36, 42, 52, 58, 64, 74, 80, 86, 92, 96, 100, 106, 112, 118, 124, 128, 134, 140, 146, 152, 156, 213, 219, 225, 231, 235, 239, 245, 249, 255, and 259.

[0045] In certain embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the present disclosure comprises a CDR containing the amino acid sequence of any of the CDR combinations described in numbered embodiments 13 to 275. Therefore, in certain embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the present disclosure comprises: CDR-H1 containing the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 125, SEQ ID NO: 153, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 246, or SEQ ID NO: 256; CDR-H2 containing the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO: 229, SEQ ID NO: 233, SEQ ID NO: 237; SEQ ID NO: 103, SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 131, Includes CDR-H3 containing the amino acid sequence of sequence number 137, SEQ ID NO: 143, SEQ ID NO: 149, SEQ ID NO: 242, or SEQ ID NO: 252; CDR-L1 containing the amino acid sequence of SEQ ID NO: 104, SEQ ID NO: 110, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 132, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 150, SEQ ID NO: 234, or SEQ ID NO: 253; CDR-L2 containing the amino acid sequence of SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 230, or SEQ ID NO: 234; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 231.

[0046] In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 3-5 and light chain CDRs of SEQ ID NOs. 6-8. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 9-11 and light chain CDRs of SEQ ID NOs. 12-14. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 15-17 and light chain CDRs of SEQ ID NOs. 18-20.

[0047] In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 25-27 and a light chain CDR of SEQ ID NOs. 28-30. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 31-33 and a light chain CDR of SEQ ID NOs. 34-36. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 37-39 and a light chain CDR of SEQ ID NOs. 40-42.

[0048] In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 47-49 and a light chain CDR of SEQ ID NOs. 50-52. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 53-55 and a light chain CDR of SEQ ID NOs. 56-58. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 59-61 and a light chain CDR of SEQ ID NOs. 62-64.

[0049] In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 69-71 and light chain CDRs of SEQ ID NOs. 72-74. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 75-77 and light chain CDRs of SEQ ID NOs. 78-80. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 81-83 and light chain CDRs of SEQ ID NOs. 84-86.

[0050] In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 208-210 and light chain CDRs of SEQ ID NOs. 211-213. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 214-216 and light chain CDRs of SEQ ID NOs. 217-219. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 220-222 and light chain CDRs of SEQ ID NOs. 223-225.

[0051] In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 101-103 and light chain CDRs of SEQ ID NOs. 104-106. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 107-109 and light chain CDRs of SEQ ID NOs. 110-112. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 113-115 and light chain CDRs of SEQ ID NOs. 116-118. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 119-121 and light chain CDRs of SEQ ID NOs. 122-124. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises heavy chain CDRs of SEQ ID NOs. 240-242 and light chain CDRs of SEQ ID NOs. 243-245.

[0052] In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 129-131 and a light chain CDR of SEQ ID NOs. 132-134. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 135-137 and a light chain CDR of SEQ ID NOs. 138-140. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 141-143 and a light chain CDR of SEQ ID NOs. 144-146. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 147-149 and a light chain CDR of SEQ ID NOs. 150-152. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure comprises a heavy chain CDR of SEQ ID NOs. 250-252 and a light chain CDR of SEQ ID NOs. 253-255.

[0053] The antibodies and antigen-binding fragments of this disclosure may be mouse, rabbit, chimeric, humanized, or human.

[0054] In a further embodiment, the anti-glyco-CD44 antibody or antigen-binding fragment of the present disclosure competes with the antibody or antigen-binding fragment containing the heavy chain and light chain variable regions of SEQ ID NOs: 1 and 2, respectively. In yet another embodiment, the present disclosure provides an anti-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity of SEQ ID NOs: 1 and 2, respectively.

[0055] In yet another embodiment, the anti-glyco-CD44 antibody or antigen-binding fragment of the present disclosure competes with the antibody or antigen-binding fragment containing the heavy chain and light chain variable regions of SEQ ID NOs. 23 and 24, respectively. In yet another embodiment, the present disclosure provides an anti-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NOs. 23 and 24, respectively.

[0056] In yet another embodiment, the anti-glyco-CD44 antibody or antigen-binding fragment of the present disclosure competes with the antibody or antigen-binding fragment containing the heavy chain and light chain variable regions of SEQ ID NOs. 45 and 46, respectively. In yet another embodiment, the present disclosure provides an anti-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NOs. 45 and 46, respectively.

[0057] In yet another embodiment, the anti-glyco-CD44 antibody or antigen-binding fragment of the present disclosure competes with the antibody or antigen-binding fragment containing the heavy chain and light chain variable regions of SEQ ID NOs. 67 and 68, respectively. In yet another embodiment, the present disclosure provides an anti-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NOs. 67 and 68, respectively.

[0058] In yet another embodiment, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure competes with the antibody or antigen-binding fragment containing the heavy chain and light chain variable regions of SEQ ID NOs. 206 and 207, respectively. In yet another embodiment, the Disclosure provides an anti-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NOs. 206 and 207, respectively.

[0059] In yet another embodiment, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure is a single-chain variable fragment (scFv). An exemplary scFv includes a heavy-chain variable fragment at the N-terminal end of a light-chain variable fragment. In some embodiments, the heavy-chain and light-chain variable fragments of the scFv are covalently bound to a linker sequence of 4-15 amino acids. The scFv may be in the form of a bispecific T-cell engager or may be within a chimeric antigen receptor (CAR).

[0060] The anti-glyco-CD44 antibody and antigen-binding fragment may be in the form of a single-chain variable fragment multimer, a bispecific single-chain variable fragment, and a bispecific single-chain variable fragment multimer. In some embodiments, the single-chain variable fragment multimer is selected from a bivalent single-chain variable fragment, a tribody, or a tetrabody. In some of these embodiments, the bispecific single-chain variable fragment multimer is a bispecific T cell engager.

[0061] Other aspects of this disclosure are of interest to nucleic acids encoding anti-glyco-CD44 antibodies and antibody-binding fragments of this disclosure. In some embodiments, the portion of the nucleic acid encoding the anti-glyco-CD44 antibody or antigen-binding fragment is codon-optimized for expression in human cells. In certain embodiments, this disclosure provides an anti-glyco-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions encoded by a heavy chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1, SEQ ID NO: 23, SEQ ID NO: 45, SEQ ID NO: 67, or SEQ ID NO: 206, and a light chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 46, SEQ ID NO: 68, or SEQ ID NO: 207. A vector containing the nucleic acid (e.g., a viral vector such as a lentiviral vector) and a host cell are also within the scope of this disclosure. The sequences encoding the heavy chain and light chain may be present in a single vector or in separate vectors.

[0062] A further aspect of the present disclosure is a pharmaceutical composition comprising an anti-glyco-CD44 antibody, an antigen-binding fragment, a nucleic acid (or a pair of nucleic acids), a vector (or a pair of vectors) or a host cell according to the present disclosure, and a physiologically suitable buffer, adjuvant, or diluent.

[0063] A further aspect of the present disclosure is a method for producing a chimeric antigen receptor, comprising incubating cells containing the nucleic acid or vector according to the present disclosure under conditions suitable for the expression of a coding region, and collecting the chimeric antigen receptor.

[0064] Another aspect of the present disclosure is a method for detecting cancer, comprising contacting a cell or tissue sample with an anti-glycoCD44 antibody or antigen-binding fragment of the present disclosure, and detecting whether the antibody is bound to the cell or tissue sample.

[0065] A further aspect of this disclosure is an anti-glyco-CD44 antibody or antigen-binding fragment according to this disclosure for use in detecting cancer.

[0066] A further aspect of the present disclosure is a method for treating cancer, comprising administering a prophylactic or therapeutically effective amount of an anti-glycoCD44 antibody, antigen-binding fragment, nucleic acid, vector, host cell, or pharmaceutical composition according to the present disclosure to a subject in need thereof.

[0067] A further aspect of the present disclosure is an anti-glyco-CD44 antibody, antigen-binding fragment, nucleic acid, vector, host cell, or pharmaceutical composition according to the present disclosure for use in the treatment of cancer.

[0068] A further aspect of the present disclosure is the use of anti-glyco-CD44 antibodies, antigen-binding fragments, nucleic acids, vectors, host cells, or pharmaceutical compositions according to the present disclosure for the manufacture of pharmaceuticals for the treatment of cancer.

[0069] CD44v6 peptides are also provided herein. The peptides may be 12 to 30 amino acids in length and contain amino acids 4 to 13 of SEQ ID NO: 165. CD44v6 peptides are described in Section 6.8 and numbered embodiments 534 to 539. The peptides may be included in compositions as described in Section 6.8.1 and numbered embodiments 540 to 541. CD44v6 peptides can be used in methods for producing antibodies in animals and / or for inducing an immune response in animals. Methods for using CD44v6 peptides are described in Section 6.8.2 and numbered embodiments 542 to 545. [Brief explanation of the drawing]

[0070] [Figure 1-1] This figure shows that antibody 4C8 specifically binds to Tn glycosylated CD44. Figure 1A: ELISA performed with 1 μg / mL of 4C8 mAb against various concentrations of unglycosylated and Tn glycosylated CD44 and MUC1. Figure 1B: Affinity of 4C8 mAb for CD44v6 glycopeptide determined using Biacore and Octet techniques. [Figure 1-2] This figure shows that antibody 4C8 specifically binds to Tn glycosylated CD44. Figure 1C: Staining of HaCaT WT and COSMC KO cells using α-Golgi, 4C8mAb supernatant, α-CD44v6, and various dilutions of mouse IgG isotype control. [Figure 1-3] This figure shows that antibody 4C8 specifically binds to Tn glycosylated CD44. Figure 1D: Immunofluorescence staining of HaCaT WT and COSMC KO cells using 4C8mAb, α-CD44v6, and α-Tn. Figure 1E: HaCaT WT and COSMC KO cells grown in an organ-type skin model on a collagen gel containing human fibroblasts, fixed, paraffin-embedded, and stained for immunofluorescence using 4C8mAb and α-CD44v6. Figure 1F: Biopsy from healthy human skin stained for immunofluorescence using 4C8mAb and α-CD44v6. [Figure 2-1] This figure shows that the antibody 4C8 selectively stains several types of primary cancer tissue. Figure 2A-1: Tissue microarrays of several carcinomas and adjacent healthy tissues stained for immunohistochemistry using 4C8mAb, α-CD44, and mouse IgG isotype controls. [Figure 2-2] This figure shows that the antibody 4C8 selectively stains several types of primary cancer tissue. Figure 2A-2: Tissue microarrays of several carcinomas and adjacent healthy tissues stained for immunohistochemistry using 4C8mAb, α-CD44, and mouse IgG isotype controls. [Figure 2-3] This figure shows that the antibody 4C8 selectively stains several types of primary cancer tissue. Figure 2A-3: Tissue microarrays of several carcinomas and adjacent healthy tissues stained for immunohistochemistry using 4C8mAb, α-CD44, and mouse IgG isotype controls. [Figure 2-4] Figure 2B: A table showing the distribution of strongly stained, weakly stained, and negatively stained tissue sections observed by immunohistochemistry as depicted in Figure 2A, divided into Grade 1, Grade 2, and Grade 3 carcinomas for each cancer type. [Figure 3] This figure shows that 4C8 CAR T cells selectively lethal Tn-positive cancer cells. Figure 3A: Results of cytotoxic assays performed using 4C8 CAR T cells (Construct 1) co-cultured with HaCaT WT and COSMC KO cells. Figure 3B: Concentration of IFN-γ in co-culture supernatant analyzed by ELISA. Figure 3C: Expression of (assedded) T cell activation markers evaluated using flow cytometry. [Figure 4] This figure shows the results of a cytotoxic assay performed using 4C8 CAR T cells co-cultured with HaCaT WT and COSMC KO cells in a 3:1 ratio. NV (no vector) represents T cells alone that do not express 4C8 CAR. The orientation of the N-terminal light chain (L) (construct 1) was found to be more effective than that of the N-terminal heavy chain (construct 4). [Figure 5-1] These are schematic diagrams of typical 4C8 CAR constructs 1-8. Figure 5A: Construct 1 (LH-4C8-CD8a-CART); Figure 5B: Construct 2 (LH-4C8-IgG4-CART). Figures 5A-5H disclose "(GGGGS)3" as sequence number 184 and "(GGGGS)1" as sequence number 183. [Figure 5-2] These are schematic diagrams of typical 4C8 CAR constructs 1-8. Figure 5C: Construct 3 (LH-4C8-IgG4-long-chain-CART); Figure 5D: Construct 4 (HL-4C8-CD8a-CART). Figures 5A-5H disclose "(GGGGS)3" as Sequence ID 184 and "(GGGGS)1" as Sequence ID 183. [Figure 5-3] These are schematic diagrams of typical 4C8 CAR constructs 1-8. Figure 5E: Construct 5 (HL-4C8-IgG4-CART); Figure 5F: Construct 6 (HL-4C8-IgG4-Long Chain-CART). Figures 5A-5H disclose "(GGGGS)3" as Sequence ID 184 and "(GGGGS)1" as Sequence ID 183. [Figure 5-4] These are schematic diagrams of typical 4C8 CAR constructs 1-8. Figure 5G: Construct 7 (LHx2-4C8-CD8-CART); Figure 5H: Construct 8 (HLx2-4C8-CD8-CART). Figures 5A-5H disclose "(GGGGS)3" as sequence number 184 and "(GGGGS)1" as sequence number 183. [Figure 6] This is a schematic diagram of a typical 10H4 CAR construct.

[0071] 6. Detailed explanation 6.1 Antibodies Each of the 117 possible O-linked glycosylation sites within the CD44 variant region has the potential to be a target for therapeutic antibodies. It is unclear which glycosylation sites can be effectively targeted. The CD44v6 domain alone contains 13 possible O-linked glycosylation sites, including four serine and nine threonine sites. Each of these sites could potentially be used as an antibody target. This disclosure provides novel antibodies directed at specific glycoforms of CD44v6 present on tumor cells. These are exemplified by antibodies 4C8, 2B2, 18G9, 1D12, and 10H4. To mimic the glycosylation pattern of CD44v6 present on tumor cells, specific glycoforms of CD44v6 glycosylated with GalNAc on serine and threonine residues, indicated in bold and underlined, are identified as GYRQ. T PKEDSH S Screening for mouse antibodies that bind to the glycosylated peptide ("CD44v6 glycopeptide") present in TTGTAAA (SEQ ID NO: 165) identified 4C8, 2B2, 18G9, and 1D12. Screening for rabbit antibodies that bind to the same CD44v6 glycopeptide identified 10H4.

[0072] The anti-glyco-CD44 antibodies of this disclosure, exemplified by antibodies 4C8, 2B2, 18G9, 1D12, and 10H4, are useful as tools in the diagnosis and therapy of cancer.

[0073] Accordingly, in certain embodiments, the Disclosure provides antibodies and antigen-binding fragments that preferably bind to a CD44v6 glycopeptide, which is a glycoform of CD44 (referred to herein as "glyco-CD44") presented on tumor cells.

[0074] The anti-glyco-CD44 antibodies of this disclosure may be polyclonal, monoclonal, genetically modified, and / or otherwise modified in nature, and include, but are not limited to, chimeric antibodies, humanized antibodies, human antibodies, primate-modified antibodies, single-chain antibodies, bispecific antibodies, and bivariable domain antibodies. In various embodiments, the antibody comprises all or part of the constant region of the antibody. In some embodiments, the constant region is an isotype selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), and IgM. In a specific embodiment, the anti-glyco-CD44 antibody of this disclosure comprises an isotype of the constant region of IgG1.

[0075] The term “monoclonal antibody,” as used herein, is not limited to antibodies produced via hybridoma technology. Monoclonal antibodies include any eukaryotic, prokaryotic, or phage clone, which are derived from a single clone by any means available or known in the art. Monoclonal antibodies useful in the disclosure of this invention can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombination, and phage display technologies, or combinations thereof. Chimeric, primate-modified, humanized, or human antibodies can be suitably used in many uses of the disclosure of this invention, including the in vivo use of anti-glycoCD44 antibodies in humans.

[0076] As used herein, the term “chimeric” antibody refers to an antibody having a variable sequence derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a constant region of a human immunoglobulin, typically selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent No. 5,807,715; No. 4,816,567; and No. 4,816,397, which are incorporated herein by this reference in their entirety.

[0077] The “humanized” form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin containing a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody is expected to contain at least one, typically two, substantially all variable domains, where all or substantially all of the CDR region corresponds to the CDR region of the non-human immunoglobulin, and all or substantially all of the FR region corresponds to the FR region of the human immunoglobulin sequence. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin consensus sequence. Methods for antibody humanization are publicly known in the art. For example, all of the following are incorporated herein by reference: Riechmann et al., 1988, Nature 332:323-7; U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 by Queen et al.; European Patent No. 239400; PCT International Publication No. 91 / 09967; U.S. Patent No. 5,225,539; European Patent No. 592106; European Patent No. 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., See Proc. Natl. Acad. Sci. 91:969-973, 1994, and U.S. Patent No. 5,565,332.

[0078] "Human antibodies" include antibodies having the amino acid sequence of human immunoglobulins, and further include antibodies isolated from human immunoglobulin libraries, or antibodies isolated from animals that are transgenic with respect to one or more human immunoglobulins and do not express endogenous immunoglobulins. Human antibodies can be prepared by various methods known in the art, such as phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111, as well as PCT International Publication Nos. 98 / 46645, 98 / 50433, 98 / 24893, 98 / 16654, 96 / 34096, 96 / 33735, and 91 / 10741, each of which is incorporated herein by reference in whole. Human antibodies can also be produced by using transgenic mice that can express human immunoglobulin genes but cannot express functional endogenous immunoglobulins. See, for example, PCT International Publication No. 98 / 24893; International Publication No. 92 / 01047; International Publication No. 96 / 34096; International Publication No. 96 / 33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, for example, which are incorporated herein by reference in their entirety. Fully human antibodies that recognize selected epitopes can be produced using a technique called "guided selection." In this approach, selected non-human monoclonal antibodies, such as mouse antibodies, are used to guide the selection of fully human antibodies that recognize the same epitope (see Jespers et al., 1988, Biotechnology 12:899-903).

[0079] A "primate-like antibody" includes a monkey variable region and a human constant region. Methods for producing primate-like antibodies are known in the art. See, for example, U.S. Patent Nos. 5,658,570; 5,681,722; and 5,693,780, which are incorporated herein by reference in their entirety.

[0080] The anti-glycoCD44 antibody of this disclosure comprises both a full-length (intact) antibody molecule and an antigen-binding fragment capable of binding to glycoCD44. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv fragments, and single-domain fragments.

[0081] The Fab fragment contains the constant domain of the light chain (CL) and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the hinge region of the antibody. The F(ab') fragment is produced by cleavage of the disulfide bond at the hinge cysteine ​​of the F(ab')2 pepsin digestion product. Additional chemical coupling of antibody fragments is known to those skilled in the art. The Fab and F(ab')1 fragments lack the Fc fragment of the intact antibody, are eliminated from the animal circulation more rapidly than the intact antibody, and may have less nonspecific tissue binding than the intact antibody (see, e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

[0082] The "Fv" fragment is the smallest fragment of an antibody that contains the complete target recognition and binding site. This region is a dimer of one heavy chain and one light chain variable domain in a tight non-covalent association (V H -V L It consists of a dimer. In this configuration, the three CDRs of each variable domain interact, V H -VL Target binding sites on the surface of the dimer are defined. Often, six CDRs confer target binding specificity to an antibody. However, in some cases, even a single variable domain (or half of the Fv containing only three CDRs specific for the target), although with an affinity lower than that of the entire binding site, may have the ability to recognize and bind to the target.

[0083] A "single-chain Fv" or "scFv" antigen-binding fragment contains the V H and V L domains, in which case these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further contains a polypeptide linker between the V H domain and the V L domain, which enables the scFv to form a structure desirable for target binding.

[0084] A "single-domain antibody" is composed of a single V H or V L domain that exhibits sufficient affinity for glycoCD44. In a specific embodiment, the single-domain antibody is a camelized antibody (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0085] Furthermore, the anti-glyco-CD44 antibodies of this disclosure may be bispecific and other multispecific antibodies. A bispecific antibody is a monoclonal antibody, often a human antibody or humanized antibody, that has binding specificity to two different epitopes on the same or different antigen. In the disclosure of this invention, one of the binding specificities may be directed to glyco-CD44, and the other may be directed to any other antigen, such as cell surface proteins, receptors, receptor subunits, tissue-specific antigens, virus-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins, or bacterial surface proteins. In certain embodiments, anti-glyco-CD44 antibodies and antigen-binding fragments having bispecific and other multispecificity may specifically bind to a second CD44 epitope, an epitope on another protein co-expressed with CD44 on cancer cells, or an epitope on another protein present on different cells such as activated T cells. The bispecific antibodies of this disclosure include IgG-type bispecific antibodies and single-chain-based bispecific antibodies.

[0086] The IgG-type bispecific antibodies of this disclosure may be any of the various types of IgG-type bispecific antibodies known in the art, such as quadroma bispecific antibodies, knob-in-hole bispecific antibodies, CrossMab bispecific antibodies, charge-paired bispecific antibodies, general light-chain bispecific antibodies, one-arm single-chain Fab-immunoglobulin gamma bispecific antibodies, disulfide-stabilized Fv bispecific antibodies, DuetMab, controlled Fab-arm exchange bispecific antibodies, chain-exchanged domain-body bispecific antibodies, two-arm leucine zipper heterodimer monoclonal bispecific antibodies, κλ-body bispecific antibodies, bivariable domain bispecific antibodies, and cross-bivariable domain bispecific antibodies.For example, by this reference, the entirety of these references is incorporated herein: Kohler and Milstein, 1975, Nature 256:495-497; Milstein and Cuello, 1983, Nature 305:537-40; Ridgway et al., 1996, Protein Eng. 9:617-621; Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92; Gunasekaran et al., 2010, J Biol Chem 285:19637-46; Fischer et al., 2015 Nature Commun 6:6113; Schanzer et al., 2014, J Biol Chem 289:18693-706; Metz et al., 2012 Protein Eng Des Sel 25:571-80;Mazor et al., 2015 MAbs 7:377-89;Labrijn et al., 2013 Proc Natl Acad Sci USA 110:5145-50;Davis et al., 2010 Protein Eng Des 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. See 8:38 and Yang et al., 2017, Int. J. Mol. Sci. 18:48.

[0087] In some embodiments, the bispecific antibody of this disclosure is CrossMab. CrossMab technology is described in detail in International Publication Nos. 2009 / 080251, 2009 / 080252, 2009 / 080253, 2009 / 080254, 2013 / 026833, 2016 / 020309, and Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92, which are incorporated herein by this reference in their entirety. Briefly, CrossMab technology is based on the crossing of domains between the heavy and light chains within one Fab arm of bispecific IgG to facilitate correct chain association. The CrossMab bispecific antibody of this disclosure is a bispecific IgG antibody in which the heavy and light chains of the Fab portion of one arm are swapped. FAB It may also be an antibody. In other embodiments, the CrossMab bispecific antibody of this disclosure is a "CrossMab" antibody in which only the variable domains of the heavy and light chains of the Fab portion of one arm of a bispecific IgG antibody are exchanged. VH-VL It may also be an antibody. In further embodiments, the CrossMab bispecific antibody of this disclosure is a "CrossMab" antibody in which only the constant domains of the heavy and light chains of the Fab portion of one arm of a bispecific IgG antibody are exchanged. CH1-CL "Antibodies may also be used. CrossMab CH1-CL The antibody is CrossMab FAB and ClawsMab VH-VL In contrast, it does not have the expected byproducts, and therefore, in some embodiments, CrossMab CH1-CL Bispecific antibodies are preferred. See Klein et al., 2016, mAbs, 8(6):1010-1020.

[0088] In some embodiments, the bispecific antibodies of this disclosure are controlled Fab-arm exchange bispecific antibodies. Methods for producing Fab-arm exchange bispecific antibodies are described in PCT International Publication No. 2011 / 131746 and Labrijn et al., 2014 Nat Protoc. 9(10):2450-63, which are incorporated herein by reference in their entirety. Briefly, controlled Fab-arm exchange bispecific antibodies can be produced by separately expressing two parental IgG1 molecules containing a single matching point mutation in the CH3 domain, mixing the parental IgG1 molecules in vitro under redox conditions to allow recombination of half the molecules, and removing the reduced form to reoxidize the interchain disulfide bond, thereby forming a bispecific antibody.

[0089] In some embodiments, the bispecific antibodies of this disclosure are bispecific antibodies in the form of “bottle opener,” “mAb-Fv,” “mAb-scFv,” “central scFv,” “central Fv,” “one-armed central-scFv,” or “dual scFv.” The contents of these forms of bispecific antibodies are described in PCT International Publication No. 2016 / 182751, which is incorporated herein by reference in its entirety. All of these forms rely on the self-assembly properties of the Fc domain of the antibody heavy chain, in which a “monomer” containing two Fc subunits assembles into a “dimer” containing an Fc domain.

[0090] In the bottle-opener configuration, the first monomer optionally comprises an scFv covalently linked to the N-terminus of an Fc subunit via a linker, and the second monomer comprises a heavy chain (containing VH, CH1, and a second Fc subunit). The bottle-opener configuration bispecific antibody further comprises a light chain capable of pairing with the second monomer to form Fab.

[0091] The mAb-Fv bispecific antibody pattern relies on an "extra" VH domain attached to the C-terminus of one heavy chain monomer and an "extra" VL domain attached to the other heavy chain monomer forming a third antigen-binding domain. In some embodiments, the mAb-Fv bispecific antibody comprises a first monomer containing a first VH domain, a CH1 domain and a first Fc subunit, with the VL domain covalently attached to the C-terminus. The second monomer contains a VH domain, a CH1 domain, a second Fc subunit and VH covalently attached to the C-terminus of the second monomer. The two C-terminus-attached variable domains constitute the Fv. The mAb-Fv further contains two light chains, which, when associated with the first and second monomers, form the Fab.

[0092] The mAb-scFv bispecificity mechanism relies on the attachment of the scFv to one of the mAb monomers at its C-terminus, thus forming a third antigen-binding domain. Thus, the first monomer contains a first heavy chain (containing VH, CH1, and a first Fc subunit) along with the scFv covalently attached to its C-terminus. The mAb-scFv bispecific antibody further contains a second monomer (containing VH, CH1, and a first Fc subunit), as well as two light chains that, upon association with the first and second monomers, form Fab.

[0093] The central scFv bispecificity mechanism relies on the use of an scFv domain inserted into the mAb, and thus the formation of a third antigen-binding domain. The scFv domain is inserted between one Fc subunit and the CH1 domain of the monomer, thus providing a third antigen-binding domain. Thus, the first monomer may contain a VH domain, a CH1 domain (and an optional hinge), and a first Fc subunit, with the scFv covalently attached between the C-terminus of the CH1 domain and the N-terminus of the first Fc subunit using an optional domain linker. The other monomer may be a standard Fab-side monomer. The central scFv bispecific antibody further contains two light chains, which, when associated with the first and second monomers, form a Fab.

[0094] The central Fv bispecificity mechanism relies on the use of an inserted Fv domain, and thus the formation of a third antigen-binding domain. Each monomer may contain components of Fv (for example, one monomer may contain a variable heavy chain domain and the other a variable light chain domain). Thus, one monomer may contain a VH domain, a CH1 domain, a first Fc subunit, and optionally a VL domain covalently attached between the C-terminus of the CH1 domain and the N-terminus of the first Fc subunit using a domain linker. The other monomer may contain a VH domain, a CH1 domain, a second Fc subunit, and optionally an additional VH domain covalently attached between the C-terminus of the CH1 domain and the N-terminus of the second Fc domain using a domain linker. The central Fv bispecific antibody further comprises two light chains that, upon association with the first and second monomers, form Fab.

[0095] The one-arm central scFv bispecificity configuration comprises one monomer containing only the Fc subunit, while the other monomer contains an inserted scFv domain, thus forming a second antigen-binding domain. Thus, one monomer may contain a VH domain, a CH1 domain, and a first Fc subunit, and the scFv is covalently attached between the C-terminus of the CH1 domain and the N-terminus of the first Fc subunit using a domain linker, optionally. The second monomer may contain an Fc domain. This embodiment further utilizes a light chain containing a variable light chain domain and a constant light chain domain that associate with the first monomer to form a Fab.

[0096] The dual scFv bispecificity form comprises a first monomer containing an scFv covalently attached to the N-terminus of a first Fc subunit via a linker (optional), and a second monomer containing an scFv covalently attached to the N-terminus of a second Fc subunit via a linker (optional).

[0097] The bispecific antibodies of this disclosure may include 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 IgG1Fc domain. In another embodiment, the Fc domain is an IgG4Fc domain. In a more specific embodiment, the Fc domain is an IgG4Fc domain containing an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU index numbering). Unless otherwise specified herein, the numbering of amino acid residues in the Fc domain or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. This amino acid substitution reduces Fab arm exchange of the IgG4 antibody in vivo (see Stubenrauch et al., 2010, Drug Metabolism and Disposition 38:84-91). In a further specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG1Fc domain. An exemplary sequence of the human IgG1Fc region is shown in SEQ ID NO: 166.

[0098] In certain embodiments, the Fc domain includes modifications that facilitate the association of the first and second subunits of the Fc domain. The site of the largest protein-protein interaction between the two subunits of the human IgG Fc domain is located in the CH3 domain. Therefore, in one embodiment, the modifications are located in the CH3 domain of the Fc domain.

[0099] In specific embodiments, modifications that facilitate the association of the first and second subunits of the Fc domain are so-called “knob-into-hole” modifications, which include a “knob” modification on one of the two subunits of the Fc domain and a “hole” modification on the other of the two subunits of the Fc domain. Knob-into-hole techniques are described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 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 projection ("knob") at the boundary of a first polypeptide and a corresponding cavity ("hole") at the boundary of a second polypeptide so that the projection can enter a cavity, thereby promoting heterodimerization and inhibiting homodimerization. The protrusions are constructed by replacing smaller amino acid side chains (e.g., tyrosine or tryptophan) from the boundary of the first polypeptide. By replacing the larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a corresponding cavity is created at the boundary of the second polypeptide, having the same or similar size as the protrusions.

[0100] Therefore, in some embodiments, by replacing an amino acid residue in the CH3 domain of the first subunit of the Fc domain with an amino acid residue having a larger side chain volume, a projection is created within the CH3 domain of the first subunit that can enter a cavity in the CH3 domain of the second subunit, and by replacing an amino acid residue in the CH3 domain of the second subunit of the Fc domain with an amino acid residue having a smaller side chain volume, a cavity is created within the CH3 domain of the second subunit that can accommodate the projection in the CH3 domain of the first subunit. Preferably, the 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, the 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 projections and cavities can be produced, for example, by modifying the nucleic acid encoding the polypeptide by site-directed mutagenesis, or by peptide synthesis.

[0101] In certain such embodiments, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), 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 the Kabat EU index). In further embodiments, in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced with a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is additionally replaced with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine ​​residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is additionally replaced with a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). In certain embodiments, the first subunit of the Fc domain includes amino acid substitutions S354C and T366W, and the second subunit of the Fc domain includes amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the Kabat EU index).

[0102] In some embodiments, fixed steering (as described, for example, in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to facilitate the association of the first and second subunits of the Fc domain.

[0103] In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.

[0104] In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In specific embodiments, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group of complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and cytokine secretion. In certain embodiments, the effector function is ADCC.

[0105] Typically, the same one or more amino acid substitutions are present in each of the two subunits of the Fc domain. In one embodiment, one or more amino acid substitutions reduce the binding affinity of the Fc domain to the Fc receptor. In one embodiment, one or more amino acid substitutions reduce the binding affinity of the Fc domain to the Fc receptor by at least half, at least one-fifth, or at least one-tenth.

[0106] In one embodiment, the Fc domain contains an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331, and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain contains an amino acid substitution at a position selected from the group L234, L235, and P329 (numbered according to the Kabat EU index). In some embodiments, the Fc domain contains amino acid substitutions L234A and L235A (numbered according to the Kabat EU index). In one such embodiment, the Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. In one embodiment, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbered according to the Kabat EU index). In one embodiment, the Fc domain includes an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (numbered according to the Kabat EU index). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain includes amino acid substitutions at positions P329, L234, and L235 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain includes amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA", or "LALAPG").Specifically, in certain embodiments, each subunit of the Fc domain includes amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), namely, in each of the first and second subunits of the Fc domain, the leucine residue at 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) (Kabat EU index numbering). In one such embodiment, the Fc domain is an IgG1Fc domain, in particular a human IgG1Fc domain.

[0107] The single-chain-based bispecific antibodies of this disclosure may be any of the various types of single-chain-based bispecific antibodies known in the industry, such as bispecific T cell engagers (BiTEs), diabodies, tandem diabodies (tandabs), dual-affinity retargeting molecules (DARTs), and bispecific killer cell engagers. For example, see Loffler et al., 2000, Blood 95:2098-103; Holliger et al., 1993, Proc Natl Acad Sci USA, 90:6444-8; Kipriyanov et al., 1999, Mol Biol 293:41-56; Johnson et al., 2010, Mol Biol 399:436-49; Wiernik et al., 2013, Clin Cancer Res 19:3844-55; Liu et al., 2017, Front. Immunol. 8:38; and Yang et al., 2017, Int. J. Mol. Sci. 18:48, which are incorporated herein by reference in their entirety.

[0108] In some embodiments, the bispecific antibodies of this disclosure are bispecific T cell engagers (BiTEs). A BiTE is a single polypeptide chain molecule having two antigen-binding domains, one of which binds to a T cell antigen and the second antigen-binding domain binds to an antigen presented on the surface of a target (see PCT International Publication No. 05 / 061547, which are incorporated herein by reference; Baeuerle et al., 2008, Drugs of the Future 33: 137-147; Bargou, et al., 2008, Science 321: 974-977). Thus, the BiTEs of this disclosure have an antigen-binding domain that binds to a T cell antigen and a second antigen-binding domain directed to glyco-CD44.

[0109] In some embodiments, the bispecific antibodies of this disclosure are biaffinity retargeting molecules (DARTs). A DART comprises at least two polypeptide chains that associate (particularly via covalent interactions) to form at least two epitope-binding sites, which may recognize the same epitope or different epitopes. Each polypeptide chain of a DART contains 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. Rather, the immunoglobulin heavy chain variable region of one (e.g., the first) DART polypeptide chain interacts with the immunoglobulin light chain variable region of a different (e.g., the second) DART® polypeptide chain to form an epitope-binding site. Similarly, the immunoglobulin light chain variable region of one (e.g., the first) DART polypeptide chain interacts with the different (e.g., the second) immunoglobulin heavy chain variable region of the DART polypeptide chain to form an epitope-binding site. DARTs may be monospecific, bispecific, triplicate, etc., and therefore can bind simultaneously to one, two, three or more different epitopes (which may be of the same or different antigens). In addition, DARTs may be monovalent, divalent, trivalent, tetravalent, pentavalent, hexavalent, etc., and therefore can bind simultaneously to one, two, three, four, five, six or more molecules. Combining these two properties of DARTs (i.e., degree of specificity and valency) can produce, for example, a tetravalent (i.e., capable of binding to four sets of epitopes) bispecific antibody (i.e., capable of binding to two epitopes), etc. DART molecules are disclosed in PCT International Publication No. 2006 / 113665, International Publication No. 2008 / 157379, and International Publication No. 2010 / 080538, whose entirety is incorporated herein by reference.

[0110] In some embodiments of the bispecific antibodies of this disclosure, one binding specificity is directed to glyco-CD44, and the other is directed to an antigen expressed on an immune effector cell. The term “immune effector cell” or “effector cell,” as used herein, refers to a cell in the natural repertoire of cells in the mammalian immune system that can be activated and affect the viability of target cells. Examples of immune effector cells include lymphoid cells, such as natural killer (NK) cells, T cells including cytotoxic T cells, or B cells, as well as myeloid cells such as monocytes or macrophages, dendritic cells, and neutrophils. Thus, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophils. The replacement of abnormal cells with effector cells means that the immune effector cells are taken up in close proximity to the abnormal target cells so that the effector cells can directly lethal the abnormal cells to which they are replaced, or indirectly initiate their lethality. To avoid nonspecific interactions, the bispecific antibodies of this disclosure preferably specifically recognize antigens on the immune effector cells that are at least overexpressed by these immune effector cells compared to other cells in the body. Examples of target antigens presented on immune effector cells include CD3, CD8, CD16, CD25, CD28, CD64, CD89, NKG2D, and NKp46. Preferably, the antigen on the immune effector cells is CD3 expressed on T cells.

[0111] As used herein, “CD3” refers to all natural CD3 derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses all forms of CD3 resulting from processing in cells, in addition to “full-length” unprocessed CD3. The term also encompasses naturally occurring variants of CD3, such as splice variants or allele variants. The most preferred antigen on immunoeffector cells is the CD3 epsilon chain. This antigen has been shown to be highly effective in T cell replacement for abnormal cells. Therefore, the bispecific antibodies of this disclosure preferably specifically recognize CD3 epsilon. The amino acid sequence of human CD3 epsilon is shown in UniProt (www.uniprot.org) accession number P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3 epsilon is shown in NCBI GenBank number BAB71849.1. For therapeutic use in humans, a bispecific antibody is used in which the CD3 binding domain specifically binds to human CD3 (e.g., the human CD3 epsilon chain). For preclinical trials in non-human animals or cell lines, a bispecific antibody can be used in which the CD3 binding domain specifically binds to CD3 in the species used in the preclinical trial (e.g., cynomolgus monkey CD3 in the case of primate trials).

[0112] As used herein, a binding domain that "specifically binds" to or "specifically recognizes" a target antigen of a particular species does not exclude binding to or recognition of antigens of other species, and therefore, one or more binding domains may contain antibodies that exhibit cross-reactivity between species. For example, a CD3 binding domain that "specifically binds" to or "specifically recognizes" human CD3 may also bind to or recognize cynomolgus monkey CD3, and vice versa.

[0113] In some embodiments, the bispecific antibody of this disclosure can compete with the monoclonal antibody H2C (described in PCT International Publication No. 2008 / 119567) for binding to the CD3 epitope. In other embodiments, the bispecific antibody of this disclosure can compete with the monoclonal antibody V9 (described in Rodrigues et al., 1992, Int J Cancer Suppl 7:45-50 and U.S. Patent No. 6,054,297) for binding to the CD3 epitope. In yet another embodiment, the bispecific antibody of this disclosure can compete with the monoclonal antibody FN18 (described in Nooij et al., 1986, Eur J Immunol 19:981-984) for binding to the CD3 epitope. In further embodiments, the bispecific antibodies of this disclosure can compete with the monoclonal antibody SP34 (described in Pessano et al., 1985, EMBO J 4:337-340) for binding to the CD3 epitope.

[0114] The anti-glycoCD44 antibodies of this disclosure include derivatized antibodies. For example, but not limited to, derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, cleavage by proteolysis, or linkage to cellular ligands or other proteins. Any of the various chemical modifications, but not limited to these, can be carried out by known techniques such as specific chemical cleavage, acetylation, formylation, or metabolic synthesis of tunicamycin. In addition, derivatives may contain one or more non-natural amino acids, for example, using the ambrx technique (see, e.g., Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0115] An antiglyco-CD44 antibody or binding fragment may be an antibody or fragment whose sequence has been modified to alter the biological effector function mediated by at least one constant region. For example, in some embodiments, an antiglyco-CD44 antibody may be modified to reduce the biological effector function mediated by at least one constant region compared to an unmodified antibody, for example, to reduce binding to the Fc receptor (FcγR). Binding to FcγR can be reduced by mutating the immunoglobulin constant region segment of the antibody in a specific region required for FcγR interaction (see, e.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; and Lund et al., 1991, J. Immunol. 147:2657-2662). Reducing the FcγR binding ability of an antibody can also reduce other effector functions that rely on FcγR interactions, such as opsonization, phagocytosis, and antigen-dependent cell-mediated cytotoxicity ("ADCC").

[0116] The anti-glyco-CD44 antibodies or binding fragments described herein include antibodies and / or binding fragments that have been modified to acquire or improve upon biological effector function mediated by at least one constant region compared to an unmodified antibody, for example, to enhance FcγR interaction (see, for example, U.S. Patent Application Publication No. 2006 / 0134709). For example, the anti-glyco-CD44 antibodies of this disclosure may have a constant region that binds to FcγRIIA, FcγRIIB, and / or FcγRIIIA with greater affinity than the corresponding wild-type constant region.

[0117] Accordingly, the antibodies of this disclosure may have modifications in biological activity that cause opsonization, phagocytosis, or an increase or decrease in ADCC. Such modifications are known in the art. For example, modifications in antibodies that reduce ADCC activity are described in U.S. Patent No. 5,834,597. An exemplary ADCC-reducing variant corresponds to “Mutant 3” in which residue 236 is deleted and residues 234, 235 and 237 (using EU numbering) are substituted with alanine (shown in Figure 4 of U.S. Patent No. 5,834,597). Other exemplary ADCC-reducing variants include amino acid mutations L234A, L235A and P329G ("P329G LALA"). The amino acid substitution combination "P329G LALA" almost completely disrupts the Fcγ receptor binding (and complement binding) of the human IgG1 Fc domain, as described in PCT Publication WO2012 / 130831, which is incorporated herein in its entirety by reference. WO2012 / 130831 also describes methods for preparing such mutant Fc domains and for determining their properties, such as Fc receptor binding or effector function.

[0118] In some embodiments, the anti-glycoCD44 antibodies of this disclosure have low levels of fucose or are fucose-free. Fucose-free antibodies, particularly at low doses, have been shown to correlate with enhanced ADCC activity. See Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. One method for preparing low-fucose antibodies is proliferation in rat myeloma YB2 / 0 cells (ATCC CRL1662). YB2 / 0 cells express low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase, an enzyme necessary for polypeptide fucosylation.

[0119] In some embodiments, the anti-glycoCD44 antibody or binding fragment includes a bisected oligosaccharide, for example, a bibranched oligosaccharide bound to the Fc domain that is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., 1999, Nat Biotechnol 17:176-180; Ferrara et al., 2006, Biotechn Bioeng 93: 851-861; WO99 / 54342; WO2004 / 065540; and WO2003 / 011878.

[0120] In yet another embodiment, the anti-glyco-CD44 antibody or binding fragment includes modifications that increase or decrease its binding affinity to the fetal Fc receptor, FcRn, for example, by mutating an immunoglobulin constant region segment in a specific region involved in FcRn interaction (see, for example, International Publication No. 2005 / 123780). In a particular embodiment, the IgG class anti-glyco-CD44 antibody is mutated such that at least one of the amino acid residues 250, 314, and 428 in the heavy chain constant region is replaced, for example, at positions 250 and 428, or 250 and 314, or 314 and 428, or 250, 314, and 428, either alone or in any combination thereof, the specific combination being positions 250 and 428. In the case of position 250, the substituted amino acid residue may be any amino acid residue other than threonine, and may include, but are 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. In the case of position 314, the substituted amino acid residue may be any amino acid residue other than leucine, and may include, but are 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. In the case of position 428, the substituted amino acid residue may be any amino acid residue other than methionine, and may include, for example, 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 preferred amino acid substitutions are identified in Table 1 of U.S. Patent No. 7,217,797, which is incorporated herein by reference.Such mutations increase binding to FcRn, thereby protecting the antibody from degradation and increasing its half-life.

[0121] In yet other embodiments, the anti-glycoCD44 antibodies of the antigen-binding fragments of the present disclosure have one or more amino acids inserted into one or more of their hypervariable regions, as described, for example, in Jung and Pluckthun, 1997, Protein Engineering 10:9, 959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17; and U.S. Patent Application Publication No. 2007 / 0280931.

[0122] In yet other embodiments particularly useful for diagnostic applications, the anti-glycoCD44 antibodies of the antigen-binding fragments of the present disclosure are attached to a detectable moiety. Detectable moieties include radioactive moieties, colorimetric molecules, fluorescent moieties, chemiluminescent moieties, antigens, enzymes, detectable beads (e.g., magnetic or high electron density (e.g., gold) beads), or molecules that bind to another molecule (e.g., biotin or streptavidin).

[0123] Radioisotopes or radionuclides include 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0124] Fluorescent labels include rhodamine, lanthanide fluorescence, fluorescein and its derivatives, fluorochromes, GFP (GFP stands for "Green Fluorescent Protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0125] Examples of enzyme labels include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase.

[0126] Chemiluminescent labels or chemiluminescers, such as isoluminol, luminol, and dioxetane.

[0127] Other detectable moieties include molecules such as biotin, digoxigenin, or 5-bromodeoxyuridine.

[0128] In certain embodiments, the anti-glycoCD44 antibodies or antigen-binding fragments of the present disclosure compete with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of 4C8 or 4C8 (SEQ ID NOs: 1-2, respectively).

[0129] In other embodiments, the anti-glycoCD44 antibodies or antigen-binding fragments of the present disclosure compete with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of 2B2 or 2B2 (SEQ ID NOs: 23-24, respectively).

[0130] In other embodiments, the anti-glycoCD44 antibodies or antigen-binding fragments of the present disclosure compete with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of 18G9 or 18G9 (SEQ ID NOs: 45-46, respectively).

[0131] In other embodiments, the anti-glycoCD44 antibodies or antigen-binding fragments of the present disclosure compete with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of 1D12 or 1D12 (SEQ ID NOs: 67-68, respectively).

[0132] In other embodiments, the anti-glycoCD44 antibody or antigen-binding fragment of this disclosure competes with antibodies or antigen-binding fragments containing 10H4 or the heavy and light chain variable regions of 10H4 (SEQ ID NOs. 206-207, respectively).

[0133] The competition can be assayed using cells expressing a glyco-CD44 epitope that binds to 4C8, 2B2, 18G9, 1D12, or 10H4, or a glycosylated CD44 peptide containing an epitope that binds to 4C8, 2B2, 18G9, 1D12, or 10H4, such as the CD44v6 glycopeptide. As a control, cells that do not express the epitope or the non-glycosylated peptide can be used.

[0134] Cells that can be used in competitive assays include, but are not limited to, COSMC knockout HaCaT cells and recombinant cells (e.g., COSMC knockout HEK293 cells) that have been engineered to express the glyco-CD44 epitope. In one non-limiting example, HEK293 cells, which are inherently Tn-negative, may be induced to express the Tn antigen by knockout of the COSMC chaperone, and have been engineered to express CD44, resulting in cells expressing the Tn glycoform of CD44 bound to 4C8, 2B2, 18G9, 1D12, and 10H4. Cells expressing the unglycosylated form of CD44 can be used as a negative control. Cells expressing the Tn antigen can also be generated, for example, by treating CD44-expressing cells with a glycosylation inhibitor, core 1 synthase or ZIP9 knockout, or by cleaving existing glycans.

[0135] Assays for competition include, but are not limited to, radioactive material-labeled immunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), sandwich ELISAs, fluorescence-activated cell sorting (FACS) assays, surface plasmon resonance (e.g., Biacore) assays, and biolayer interferometry (BLI) assays. In some embodiments, antibody competition assays may be performed using BLI (e.g., using the Octet-HTX system (Molecular Devices)). Antibody competition or epitope binning of monoclonal antibodies may be evaluated in tandem against their specific antigens using BLI. In a BLI assay, the antigen may be immobilized on a biosensor in a series of steps and presented to two competing antibodies. Binding to non-overlapping epitopes occurs when saturation by the first antibody does not block binding by the second antibody. In some embodiments, antibody competition assays may be performed using surface plasmon resonance (e.g., using the Biacore system (Cytiva)). In a surface plasmon resonance assay, one or more antibodies may be immobilized on a biosensor and presented together with an analyte (e.g., the glyco-CD44v6 peptide of SEQ ID NO: 165, or a negative control analyte, e.g., the glyco-MUC1 peptide of SEQ ID NO: 205 or SEQ ID NO: 260, or the unglycosylated CD44v6 peptide of SEQ ID NO: 165). In some embodiments, the antibody is contacted with the analyte at a saturation concentration, e.g., at least about 0.5 μM. In some embodiments, the saturation concentration is about 1 μM, about 1.5 μM, or about 2 μM. When comparing the binding affinities of two antibodies, the affinities of both antibodies are preferably measured using the same concentration of both antibodies, e.g., using a concentration of 1 μM of each antibody.

[0136] In performing an antibody competition assay (regardless of species or isotype) between a reference antibody and a test antibody, the reference antibody may first be labeled with a detectable label, such as a fluorophore, biotin, or enzyme label (or even radioactive label), to allow for subsequent identification. In this case, cells expressing glycoCD44 are incubated with an unlabeled test antibody, the labeled reference antibody is added, and the intensity of the bound label is measured. If the test antibody competes with the labeled reference antibody by binding to overlapping epitopes, the intensity is expected to be reduced compared to a control reaction performed without the test antibody.

[0137] In a specific embodiment of this assay, the concentration of the labeled reference antibody that yields 80% of the maximum binding under assay conditions (e.g., specified cell density) is "conc 80% First, the ) is determined, and the competitive assay is performed using 10 × concrete unlabeled test antibodies. 80% and the conc of the labeled reference antibody 80% It will continue.

[0138] Inhibition is the inhibition constant, or K i It can be expressed as follows: K i =IC 50 / (1+[reference Ab concentration] / K D ) It is calculated according to, In the formula, IC 50 This is the concentration of the test antibody that results in a 50% reduction in the binding of the reference antibody, and K d is the dissociation constant of the reference antibody, which is a measure of its affinity for glyco-CD44. Antibodies that compete with the anti-glyco-CD44 antibodies disclosed herein are 10 pM to 10 nM K under the assay conditions described herein. i It may have.

[0139] In various embodiments, a test antibody is considered to compete with the reference antibody if, at a reference antibody concentration that is 80% of the maximum binding under the specific assay conditions used, and at a test antibody concentration 10 times higher than the reference antibody concentration, the test antibody reduces the binding of the reference antibody by at least about 20% or more, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, or in a percentage range between any of the aforementioned values.

[0140] In one example of a competitive assay, the CD44v6 glycopeptide is adhered to a solid surface, e.g., a microwell plate, by contacting the plate with a solution of the peptide (e.g., 1 μg / mL in PBS overnight at 4°C). The plate is washed (e.g., 0.1% Tween 20 in PBS) and blocked (e.g., with Superblock, Thermo Scientific, Rockford, IL). A mixture of unlabeled antibodies ("reference" antibody) or competing anti-glycoCD44 antibodies ("test" antibody) in subsaturated amounts (e.g., at a concentration of 80 ng / mL) and serially diluted (e.g., at concentrations of 2.8 μg / mL, 8.3 μg / mL, or 25 μg / mL) in ELISA buffer (e.g., 1% BSA and 0.1% Tween 20 in PBS) is added to the wells, and the plate is incubated for 1 hour with gentle shaking. Wash the plate, add streptavidin conjugated with 1 μg / mL HRP diluted in ELISA buffer to each well, and incubate the plate for 1 hour. Wash the plate and detect the bound antibody by adding the substrate (e.g., TMB, Biofx Laboratories Inc., Owings Mills, MD). Stop the reaction by adding the stop buffer (e.g., Bio FX Stop reagent, Biofx Laboratories Inc., Owings Mills, MD), and measure the absorbance at 650 nm using a microplate reader (e.g., VERSAmax, Molecular Devices, Sunnyvale, CA).

[0141] Variations of this competition assay can also be used to test the competition between 4C8, 2B2, 18G9, 1D12, 10H4 and other anti-glyco-CD44 antibodies. For example, in certain embodiments, the anti-glyco-CD44 antibody is used as a reference antibody, and 4C8, 2B2, 18G9, 1D12, and 10H4 are used as test antibodies. In addition, membrane-bound glyco-CD44 expressed on the cell surface during culture (e.g., on the surface of one of the cell types mentioned above) may be used instead of the glycosylated CD44 peptide of SEQ ID NO: 165. Generally, about 10 4 from 10 6 individual transformants, for example, about 10 5 One transformant is used. Other forms of the competing assay are known in the industry and can be employed.

[0142] In various embodiments, the anti-glyco-CD44 antibody of this disclosure reduces the binding of labeled 4C8, 2B2, 18G9, 1D12, and 10H4 by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or a percentage in the range of any of the aforementioned values ​​when the anti-glyco-CD44 antibody is used at concentrations of 0.08 μg / mL, 0.4 μg / mL, 2 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, or in a range of concentrations between any of the aforementioned values ​​(for example, in a range of concentrations from 2 μg / mL to 10 μg / mL).

[0143] In other embodiments, when 4C8, 2B2, 18G9, 1D12, and 10H4 are used at concentrations of 0.4 μg / mL, 2 μg / mL, 10 μg / mL, 50 μg / mL, 250 μg / mL, or at concentrations within the range between any of the foregoing values (e.g., at a concentration within the range from 2 μg / mL to 10 μg / mL), 4C8, 2B2, 18G9, 1D12, and 10H4 reduce the binding of the labeled anti-glycoCD44 antibody of the present disclosure by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at a percentage within the range between any of the foregoing values (e.g., 4C8, 2B2, 18G9, 1D12, and 10H4 reduce the binding of the labeled anti-glycoCD44 antibody of the present disclosure by 50% to 70%).

[0144] In the aforementioned assay, the 4C8, 2B2, 18G9, 1D12, and 10H4 antibodies can be replaced with any antibody or antigen-binding fragment that includes the CDRs or the variable regions of the heavy and light chains of 4C8, 2B2, 18G9, 1D12, and 10H4, such as humanized or chimeric counterparts of 4C8, 2B2, 18G9, 1D12, and 10H4.

[0145] In certain embodiments, the anti-glycoCD44 antibody or antigen-binding fragment of the present disclosure has an epitope that is the same as or similar to the epitope of 4C8, 2B2, 18G9, 1D12, or 10H4. The epitope of the anti-glycoCD44 antibody or antigen-binding fragment of the present disclosure can be characterized by performing alanine scanning. Each of the libraries of glycopeptides is changed from the CD44v6 glycopeptide by an alanine point mutation at one position of SEQ ID NO: 165 (or the CD44 peptide has alanine by a point mutation of glycine). By measuring the binding of the antibody or antigen-binding fragment to each of the peptides by ELISA, the epitope of the antibody or antigen-binding fragment can be mapped.

[0146] In certain embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of this disclosure comprises heavy-chain and / or light-chain variable sequences (or sequences encoded by nucleotides) as described in Tables 1A-1E. In other embodiments, the anti-glyco-CD44 antibody or antigen-binding fragment of this disclosure comprises heavy-chain and / or light-chain CDR sequences (or sequences encoded by nucleotides) as described in Tables 1-3. The framework sequences of such anti-glyco-CD44 antibodies and antigen-binding fragments may be natural mouse framework sequences of VH and VL sequences as described in Tables 1A-1D, natural rabbit framework sequences of VH and VL sequences as described in Table 1E, or non-natural (e.g., humanized or human) framework sequences.

[0147] In yet another embodiment, the disclosure provides an anti-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NOs: 1-2, respectively.

[0148] In yet another embodiment, the disclosure provides an anti-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NOs. 23-24, respectively.

[0149] In yet another embodiment, the disclosure provides an anti-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NOs. 45-46, respectively.

[0150] In yet another embodiment, the disclosure provides an anti-CD44 antibody or antigen-binding fragment having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NOs. 67-68, respectively.

[0151] In yet another embodiment, the disclosure provides anti-CD44 antibodies or antigen-binding fragments having heavy chain and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NOs. 206-207, respectively.

[0152] In yet another embodiment, the anti-glyco-CD44 antibody or antigen-binding fragment of the Disclosure is a single-chain variable fragment (scFv). An exemplary scFv includes a heavy-chain variable fragment from the N-terminus to the light-chain variable fragment. Another exemplary scFv includes a light-chain variable fragment on the N-terminal side of the heavy-chain variable fragment. In some embodiments, the scFv heavy-chain and light-chain variable fragments are covalently bound to a linker sequence of 4-15 amino acids. The scFv may be in the form of a bispecific T-cell engager or may be within a chimeric antigen receptor (CAR).

[0153] 6.2 Antibody-drug conjugates Another aspect of this disclosure relates to antibody-drug conjugates (ADCs) comprising the anti-glyco-CD44 antibody and / or antigen-binding fragment of this disclosure. An ADC generally comprises the anti-glyco-CD44 antibody and / or binding fragment described herein, to which one or more cytotoxic and / or cell proliferation inhibitory substances are linked via one or more linkers. In a specific embodiment, the ADC is structural formula (I): [DL-XY] n -Ab A compound or salt thereof, wherein each "D" independently represents a cytotoxic substance and / or a cell proliferation inhibitory substance ("drug"); each "L" independently represents a linker; "Ab" represents an antiglyco-CD44 antigen-binding domain, such as an antiglyco-CD44 antibody or binding fragment as described herein; and each "XY" represents a functional group R on the linker. x and the "complementary" functional group R on the antibody y This represents the linkage formed between the ADC and the ADC, where n represents the number of drugs linked to the ADC, or the drug-to-antibody ratio (DAR) of the ADC.

[0154] Specific embodiments of various antibodies (Ab) that may include ADC include various embodiments of the anti-glycoCD44 antibody and / or binding fragment described above.

[0155] In some specific embodiments of the ADC and / or salts of structural formula (I), each D is the same, and / or each L is the same.

[0156] In addition to the cytotoxic substances and / or cell proliferation inhibitors (D) and linkers (L) that may constitute the antiglyco-CD44 ADCs of this disclosure, specific embodiments of a number of cytotoxic substances and / or cell proliferation inhibitors linked to the ADCs are described in more detail below. do.

[0157] 6.2.1. Cytotoxic substances and / or cell proliferation inhibitors Cytotoxic and / or inhibitory substances can be any agent known to inhibit the growth and / or replication of cells, particularly cancer and / or tumor cells, and / or cause death of such cells. Numerous agents with cytotoxic and / or inhibitory properties are publicly known in the literature. Non-limiting examples of the class of cytotoxic and / or inhibitory substances include, but are not limited to, radionuclides, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA insertion agents (e.g., sub-groove binders), RNA / DNA antimetabolites, cell cycle modulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase inhibitors, mitochondrial inhibitors, and antimitotic agents.

[0158] The following are specific, non-limiting examples of drugs within a particular range of these various classes.

[0159] Alkylating agents: asaley ((L-leucine, N-[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-ethyl ester; NSC167780; CAS registry number 3577897)); AZQ ((1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-diethyl ester; NSC182986; CAS registry number 57998682)); BCNU ((N,N'-bis(2-chloroethyl)-N-nitrosourea; NSC409962; CAS Registry No. 154938); Busulfan (1,4-butanediol dimethanesulfonate; NSC750; CAS Registry No. 55981); (carboxyphthalate)platinum (NSC27164; CAS Registry No. 65296813); CBDCA ((cis-(1,1-cyclobutanedicarboxylate)diammineplatinum(II)); NSC241240; CAS Registry No. 41575944); CCNU ((N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea; NSC79037; CAS Registry No. 13010474)); CHI P (iproplatin; NSC256927); chlorambucil (NSC3088; CAS registry number 305033); chlorozotosin ((2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose; NSC178248; CAS registry number 54749905)); cisplatin (cisplatin; NSC119875; CAS registry number 15663271); clomesone (NSC338947; CAS registry number 88343720); cyanomorpholinodoxorubicin (NCS35 7704; CAS Registry No. 88254073); cyclodisone (NSC348948; CAS Registry No. 99591738); dianhydrogalactitol (5,6-diepoxydulcitol; NSC132313; CAS Registry No. 23261203); fluorodopan ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyluracil; NSC73754; CAS Registry No. 834913); hepsulfame (NSC329680; CAS Registry No. 96892578);Hicanton (NSC142982; CAS Registry No. 23255938); Melphalan (NSC8806; CAS Registry No. 3223072); Methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea; NSC95441; 13909096); Mitomycin C (NSC26980; CAS Registry No. 50077); Mitozolomide (NSC353451; CAS Registry No. 85622953); Nitrogen Mustard ((Bis(2-chloroethyl)methylamine hydrochloride; NSC762; CAS registry number 55867); PCNU ((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea; NSC95466; CAS registry number 13909029)); Piperazine alkylating agent ((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride; NSC344007)); Piperazinedione (NSC135758; CAS registry number 41109802); Pibob Roman ((N,N-bis(3-bromopropionyl)piperazine; NSC25154; CAS Registry No. 54911)); Porphyromycin (N-methylmitomycin C; NSC56410; CAS Registry No. 801525); Spirohydantoin mustard (NSC172112; CAS Registry No. 56605164); Teroxyrone (triglycidyl isocyanurate; NSC296934; CAS Registry No. 2451629); Tetraplatin (NSC363812; CAS Registry No. 6 2816982); Thiotepa (N,N',N''-tri-1,2-ethanediylthiophosphoramide; NSC6396; CAS Registry No. 52244); Triethylenemelamine (NSC9706; CAS Registry No. 51183); Uracil Nitrogen Mustard (desmethyldopan; NSC34462; CAS Registry No. 66751); Yoshi-864 ((Bis(3-methyloxypropyl)amine hydrochloride; NSC102627; CAS Registry No. 3458228).

[0160] Topoisomerase I inhibitors: Camptothecin (NSC94600; CAS Registry No. 7689-03-4); various camptothecin derivatives and analogs (e.g., NSC100880, NSC603071, NSC107124, NSC643833, NSC629971, NSC295500, NSC249910, NSC606985, NSC74028, NSC176323, NSC295501, NSC606172 SC606173, NSC610458, NSC618939, NSC610457, NSC610459, NSC606499, NSC610456, NSC364830, and NSC606497); Morpholine isoxorubicin (NSC354646; CAS registry number 89196043); SN-38 (NSC673596; CAS registry number 86639-52-3).

[0161] Topoisomerase II inhibitors: Doxorubicin (NSC123127; CAS registry number 25316409); Amonafide (Benzisoquinoline dione; NSC308847; CAS registry number 69408817); m-AMSA ((4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide; NSC249992; CAS registry number 51264143)); Anthrapyrazole Calcium derivatives ((NSC355644); Etoposide (VP-16; NSC141540; CAS registry number 33419420); Pyrazoloacridine ((Pyrazolo[3,4,5-kl]acridine-2(6H)-propanamine, 9-methoxy-N,N-dimethyl-5-nitro-, monomethanesulfonate; NSC366140; CAS registry number 99009219); Bisanthren hydrochloride Do (NSC337766; CAS Registry No. 71439684); Daunorubicin (NSC821151; CAS Registry No. 23541506); Deoxydoxorubicin (NSC267469; CAS Registry No. 63950061); Mitoxantrone (NSC301739; CAS Registry No. 70476823); Menogalyl (NSC269148; CAS Registry No. 71628961); N,N- Dibenzyl daunomycin (NSC268242; CAS registry number 70878512); oxanthrazole (NSC349174; CAS registry number 105118125); rubidazone (NSC164011; CAS registry number 36508711); teniposide (VM-26; NSC122819; CAS registry number 29767202).

[0162] DNA insertion agents: Anthramycin (CAS registry number 4803274); Thicamycin A (CAS registry number 89675376); Tomimycin (CAS registry number 35050556); DC-81 (CAS registry number 81307246); Sibiromycin (CAS registry number 12684332); Pyrrolobenzodiazepine derivatives (CAS registry number 945490095); SGD-1882((S)-2-(4-aminophenyl)-7-methoxy-8-(3-4(S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-di Hydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5(11aH)-one);SG2000(SJG-136;(11aS,11a'S)-8,8'-(propan-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5(11aH)-one);NSC694501;CAS Registry No. 232931576).

[0163] RNA / DNA metabolites: L-alanosine (NSC153353; CAS registry number 59163416); 5-azacitidine (NSC102816; CAS registry number 320672); 5-fluorouracil (NSC19893; CAS registry number 51218); asibicin (NSC163501; CAS registry number 42228922); aminopterin derivative N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino ]benzoyl-]L-aspartic acid (NSC132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC184692); aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acid monohydrate (NSC134033); folic acid antimetabolite (antifo) ((N α -(4-amino-4-deoxypterol)-N 7-Hemiphthaloyl-L-ornithine; NSC623017); Baker's soluble folate antagonist (antifol) (NSC139105; CAS registry number 41191042); Dichloroallyllawsone (lawsone) ((2-(3,3-dichloroaryl)-3-hydroxy-1,4-naphthoquinone; NSC126771; CAS registry number 36417160); Brequinal (NSC368390; CAS registry number 96201886); Futraful ((Prodrug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil; NSC148958; CAS registry number 37076689); 5,6-dihydro Ro-5-azacitidine (NSC264880; CAS Registry No. 62402317); methotrexate (NSC740; CAS Registry No. 59052); methotrexate derivative (N-[[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]L-glutamic acid; NSC174121); PALA ((N-(phosphonoacetyl)-L-aspartate; NSC224131; CAS Registry No. 603425565); pyrazofrine (NSC143095; CAS Registry No. 30868305); trimethrexate (NSC352122; CAS Registry No. 82952645).

[0164] DNA antimetabolites: 3-HP (NSC95678; CAS registry number 3814797); 2'-deoxy-5-fluorouridine (NSC27640; CAS registry number 50919); 5-HP (NSC107392; CAS registry number 19494894); α-TGDR (α-2'-deoxy-6-thioguanosine; NSC71851; CAS registry number 2133815); aphydicolinglycinate (NSC303812; CAS registry number 92802822); ara C (Cytosine Arabinoside; NSC63878; CAS Registry No. 69749); 5-Aza-2'-Deoxycytidine (NSC127716; CAS Registry No. 2353335); β-TGDR (β-2'-Deoxy-6-Thioguanosine; NSC71261; CAS Registry No. 789617); Cyclocitidine (NSC145668; CAS Registry No. 10212256); Guanazole (NSC1895; CAS Registry No. 1455772); Hid Roxyurea (NSC32065; CAS registry number 127071); Inosingricodial aldehyde (NSC118994; CAS registry number 23590990); Macbesyn II (NSC330500; CAS registry number 73341738); Pyrazolomidazole (NSC51143; CAS registry number 6714290); Thioguanine (NSC752; CAS registry number 154427); Thioprine (NSC755; CAS registry number 50442).

[0165] Cell cycle modulators: silibinin (CAS registry number 22888-70-6); epigallocatechin gallate (EGCG; CAS registry number 989515); procyanidin derivatives (e.g., procyanidin A1 [CAS registry number 103883030], procyanidin B1 [CAS registry number 20315257], procyanidin B4 [CAS registry number 29106512], arecatannin B1 [CAS registry number 79763283]); isoflavones (e.g., genistein [4% 5,7-trihydroxyisoflavones; CAS registry number 446720]) ], daidzein [4',7-dihydroxyisoflavon, CAS registry number 486668]; indole-3-carbinol (CAS registry number 700061); quercetin (NSC9219; CAS registry number 117395); estramustine (NSC89201; CAS registry number 2998574); nocodazole (CAS registry number 31430189); podophyllotoxin (CAS registry number 518285); vinorelbine tartrate (NSC608210; CAS registry number 125317397); cryptophycin (NSC667642; CAS registry number 124689652).

[0166] 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 (NSC732517; CAS Registry No. 302 962498); Erlotinib (NSC718781; CAS Registry No. 183319699); Everolimus (NSC733504; CAS Registry No. 159351696); Fostamatinib (NSC745942; CAS Registry No. 901119355); Gefitinib (NSC715055; CAS Registry No. 184475352); Ibrutinib (CAS Registry No. 936563961); Imatinib (NSC716051; CAS Registry No. 220127571); Lapatinib (CAS Registry No. 388082788); Lenvatinib (CAS Registry No. 857 890392); Mbritinib (CAS 366017096); Nilotinib (CAS Registry No. 923288953); Nintedanib (CAS Registry No. 656247175); Palbociclib (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 (Lidaforolimus) (CAS Registry No. 572924540); INCB018424 (Ruxolitinib) (CAS Registry No. 1092939177); ARRY-142886 (Selumetinib) (NSC741078; CAS Registry No. 606143-52-6); Sirolimus (NSC226080; CAS Registry No. 53123889); Sorafenib (NSC724772; CAS Registry No. 475207591); Sunitinib (NSC736511; CAS Registry No. 341031547); Tofacitinib (CAS Registry No. 477600752);Temsirolimus (NSC683864; 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. 11135888) 4); XL019 (CAS Registry No. 945755566); PD-325901 (CAS Registry No. 391210109); PD-98059 (CAS Registry No. 167869218); ATP-competitive TORC1 / TORC2 inhibitors, e.g., PI-103 (CAS Registry No. 371935749), PP242 (CAS Registry No. 1092351671), PP30 (CAS Registry No. 1092788094), Torin Examples include 1 (CAS Registry Number 1222998368), LY294002 (CAS Registry Number 154447366), XL-147 (CAS Registry Number 934526893), CAL-120 (CAS Registry Number 870281348), ETP-45658 (CAS Registry Number 1198357797), PX866 (CAS Registry Number 502632668), GDC-0941 (CAS Registry Number 957054307), BGT226 (CAS Registry Number 1245537681), BEZ235 (CAS Registry Number 915019657), XL-765 (CAS Registry Number 934493762), etc.

[0167] Protein synthesis inhibitors: Acriflavin (CAS registry number 65589700); Amikacin (NSC177001; CAS registry number 39831555); Arbekacin (CAS registry number 51025855); Astromycin (CAS registry number 55779061); Azithromycin (NSC643732; CAS registry number 83905015); Bekanamycin (CAS registry number 4696768); Chlortetracycline (NSC13252; CAS registry number 64722); Clarithromycin (NSC643733; CAS registry number 81103) 119); Clindamycin (CAS Registry No. 18323449); Chromocycline (CAS Registry No. 1181540); Cycloheximide (CAS Registry No. 66819); Dactinomycin (NSC3053; CAS Registry No. 50760); Dalfopristin (CAS Registry No. 112362502); Demeclocycline (CAS Registry No. 127333); Dibekacin (CAS Registry No. 34493986); Dihydrostreptomycin (CAS Registry No. 128461); Dilithromycin (CAS Registry No. 62013041); Doxycycline (CAS Registry No. 17086281); Emetine (NSC33669; CAS Registry No. 483181); Erythromycin (NSC55929; CAS Registry No. 114078); Flurithromycin (CAS Registry No. 83664208); Furamycetin (Neomycin B; CAS Registry No. 119040); Gentamicin (NSC82261; CAS Registry No. 1403663); Glycylcycline, e.g., tigecycline (CAS Registry No. 220620097); Hygromycin B (CAS Registry No. 31282049); Isepamycin (C AS Registry No. 67814760; Josamycin (NSC 122223; CAS Registry No. 16846245); Kanamycin (CAS Registry No. 8063078); Ketolides, e.g., telithromycin (CAS Registry No. 191114484), cesromycin (CAS Registry No. 205110481), and sorisromycin (CAS Registry No. 760981837); Lincomycin (CAS Registry No. 154212); Limecycline (CAS Registry No. 992212); Meclocycline (NSC 78502; CAS Registry No. 2013583);Metacyclines (rondomycin; NSC356463; CAS registry number 914001); midecamycin (CAS registry number 35457808); minocycline (NSC141993; CAS registry number 10118908); myokamycin (CAS registry number 55881077); neomycin (CAS registry number 119040); netylmycin (CAS registry number 56391561); oleandomycin (CAS registry number 3922905); oxazolidinones, e.g., epelezoli (CAS registry number 165800044), Nezolid (CAS Registry No. 165800033), posizolid (CAS Registry No. 252260029), ladezolid (CAS Registry No. 869884786), lambezolid (CAS Registry No. 392659380), stezolid (CAS Registry No. 168828588), tedizolid (CAS Registry No. 856867555); oxytetracycline (NSC9169; CAS Registry No. 2058460); paromomycin (CAS Registry No. 7542372); penimepicycline (CAS Registry No. 459960) 4); peptidyltransferase inhibitors, such as chloramphenicol (NSC3069; CAS registry number 56757) and derivatives such as azidamphenicol (CAS registry number 13838089), florfenicol (CAS registry number 73231342), and thiamphenicol (CAS registry number 15318453), and proiromucilines, such as letapamulin (CAS registry number 224452668), thiamuciline (CAS registry number 55297955), and barnemuciline (CAS registry number 101312929); pirurimycin (CAS registry number 56757) CAS Registry No. 79548735); Puromycin (NSC3055; CAS Registry No. 53792); Quinupristin (CAS Registry No. 120138503); Ribostamycin (CAS Registry No. 53797356); Rokitamycin (CAS Registry No. 74014510); Lolitetracycline (CAS Registry No. 751973); Roxithromycin (CAS Registry No. 80214831); Shisomycin (CAS Registry No. 32385118); Spectinomycin (CAS Registry No. 1695778); Spiramycin (CAS Registry No. 8025818);Streptomycin, for example, pristinamycin (CAS registry number 270076603), quinupristin / dalfopristin (CAS registry number 126602899), and virginiamycin (CAS registry number 11006761); streptomycin (CAS registry number 57921); tetracycline (NSC108579; CAS registry number 60548); tobramycin (CAS registry number 32986564); troreandomycin (CAS registry number 2751099); tylosin (CAS registry number 1401690); verdamicin (CAS registry number 49863481).

[0168] Histone deacetylase inhibitors: avexinostat (CAS registry number 783355602); bellinostat (NSC726630; CAS registry number 414864009); chidamide (CAS registry number 743420022); entinostat (CAS registry number 209783802); givinostat (CAS registry number 732302997); mosetinostat (CAS registry number 726169739); panobinostat (CAS registry number 404950807); xinostat (CAS registry number 875320299); resminostat (CAS registry number 864814880); romidepsin (CAS registry number 128517077); sulforaphane (CAS registry number 4478937); thioureidobacteria Tyronitrile (Kevetrin® trademark; CAS Registry No. 6659890); Valproic acid (NSC93819; CAS Registry No. 99661); Vorinostat (NSC701852; CAS Registry No. 149647789); ACY-1215 (Rocilinostat; CAS Registry No. 1316214524); CUDC-101 (CAS Registry No. 101 2054599); CHR-2845 (Tefinostat; CAS Registry Number 914382608); CHR-3996 (CAS Registry Number 1235859138); 4SC-202 (CAS Registry Number 910462430); CG200745 (CAS Registry Number 936221339); SB939 (Prasinostat; CAS Registry Number 929016966).

[0169] Mitochondrial inhibitors: Pancratistatin (NSC349156; CAS registry number 96281311); Rhodamine-123 (CAS registry number 63669709); Edelfosine (NSC324368; CAS registry number 70641519); d-Alpha-succinate tocopherol (NSC173849; CAS registry number 4345033); Compound 11β (CAS registry number 865070377); Aspirin (NSC406186; CAS registry number 50782); Ellipticin (CAS registry number 519233); Berberine (CAS registry number 633658); Se Lurenine (CAS Registry No. 17397896); GX015-070 (Obatoclax (registered trademark); 1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrole-2-yl)methylene)-3-methoxy-2H-pyrrole-5-yl)-; NSC729280; CAS Registry No. 803712676); Cerastrol (Tripterin; CAS Registry No. 34157830); Metformin (NSC91485; CAS Registry No. 1115704); Brilliant Green (NSC5011; CAS Registry No. 633034); ME-344 (CAS Registry No. 1374524556).

[0170] Antimitotic agents: allocolchicine (NSC406042); auristatins, e.g., MMAE (monomethyl auristatin E; CAS registry number 474645-27-7) and MMAF (monomethyl auristatin F; CAS registry number 745017-94-1); halichondrin B (NSC609395); colchicine (NSC757; CAS registry number 64868); colchicine derivative (N-benzoyl deacetylbenzamide; NSC33410; CAS registry number 63989753); dorastatin 10 (NSC376128; CAS registry number 110417-88-4); meitansine (NSC15385) 8; CAS Registry No. 35846-53-8); Rhozoxin (NSC332598; CAS Registry No. 90996546); Taxol (NSC125973; CAS Registry No. 33069624); Taxol derivative ((2'-N-[3-(dimethylamino)propyl]glutaramate)Taxol; NSC608832); Thiocorchicine (3-demethylthiocolchicine; NSC361792); Tritylcysteine ​​(NSC49842; CAS Registry No. 2799077); Vinblastine sulfate (NSC49842; CAS Registry No. 143679); Vincristine sulfate (NSC67574; CAS Registry No. 2068782).

[0171] Any of these drugs that include an attachment site for an antibody, or that can be modified to include an attachment site for an antibody, may be included in the ADCs disclosed herein.

[0172] In specific embodiments, the cytotoxic substance and / or cell proliferation inhibitor is an antimitotic agent.

[0173] In another specific embodiment, the cytotoxic substance and / or cell proliferation inhibitory substance is an auristatin, for example, monomethyl auristatin E ("MMAE") or monomethyl auristatin F ("MMAF").

[0174] 6.2.2. Linker In the antiglyco-CD44 ADCs of this disclosure, a cytotoxic substance and / or a cell growth inhibitor is linked to the antibody by a linker. The linker linking the cytotoxic substance and / or cell growth inhibitor to the antibody in the ADC may be short, long, hydrophobic, hydrophilic, flexible, or rigid, or may consist of segments each independently having one or more of the above properties, so that the linker may contain segments having different properties. The linkers may be polyvalent so that they covalently link more than one drug to a single site on the antibody, or they may be monovalent so that they covalently link a single drug to a single site on the antibody.

[0175] As those skilled in the art will expect to understand, the linker conjugates the cytotoxic substance and / or cell growth inhibitor to the antibody by forming a covalent linkage to the cytotoxic substance and / or cell growth inhibitor at one position and a covalent linkage to the antibody at the other position. The covalent linkage is formed by the reaction of a functional group on the linker with a functional group on the drug and / or antibody. As used herein, the expression “linker” is intended to include (i) an unconjugated form of the linker, comprising a functional group capable of covalently linking the linker to a cytotoxic substance and / or cell growth inhibitor, and a functional group capable of covalently linking the linker to the antibody; (ii) a partially conjugated form of the linker, comprising a functional group capable of covalently linking the linker to the antibody and / or cell growth inhibitor, or vice versa; and (iii) a fully conjugated form of the linker, covalently linked to both the cytotoxic substance and / or cell growth inhibitor and the antibody. In addition to the linker and antiglyco-CD44 ADC of this disclosure, in some specific embodiments of synthons used to conjugate the linker-drug to an antibody, the portion including the functional group on the linker and the covalent linkage formed between the linker and the antibody is, respectively, Rx And this is specifically exemplified as XY.

[0176] The linker may preferably, but not necessarily, be chemically stable to extracellular conditions and may be designed to be cleaved, destroyed, and / or otherwise specifically degraded inside the cell. Alternatively, a linker not designed to be specifically cleaved or degraded inside the cell may be used. The choice between a stable and an unstable linker may depend on the toxicity of the cytotoxic and / or cell growth inhibitor. For drugs toxic to normal cells, a stable linker is preferred. Drugs that are selective to or target normal cells to reduce toxicity may be used, and the chemical stability of the linker to the extracellular environment may be less critical. Various linkers useful for linking drugs to antibodies in the form of ADCs are known in the art. Any of these linkers, and other linkers, can be used to link cytotoxic and / or cell growth inhibitors to the antibodies of the antiglycoCD44 ADCs of this disclosure.

[0177] Exemplary multivalent linkers that can be used to link many cytotoxic and / or cell proliferation inhibitory substances to a single antibody molecule are described, for example, in International Publication No. 2009 / 073445; International Publication No. 2010 / 068795; International Publication No. 2010 / 138719; International Publication No. 2011 / 120053; International Publication No. 2011 / 171020; International Publication No. 2013 / 096901; International Publication No. 2014 / 008375; International Publication No. 2014 / 093379; International Publication No. 2014 / 093394; and International Publication No. 2014 / 093640, whose contents are incorporated herein by reference in their entirety. For example, the Fleximer linker technology developed by Mersana et al. has the ability to enable high-DAR ADCs with excellent physicochemical properties. As shown below, the Mersana technology is based on incorporating drug molecules into a soluble polyacetal backbone via an ester bond sequence. This approach results in highly loaded ADCs (up to 20 DARs) while maintaining excellent physicochemical properties.

[0178] Further examples of dendritic linkers are incorporated herein by reference: U.S. Patent Application Publication No. 2006 / 116422; U.S. Patent Application Publication No. 2005 / 271615; de Groot et al. (2003) Angew. Chem. Int. Ed. 42:4490-4494; Amir et al. (2003) Angew. Chem. Int. Ed. 42:4494-4499; Shamis et al. (2004) J. Am. Chem. Soc. 126:1726-1731; Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King et al. This can be found in al. (2002) Tetrahedron Letters 43:1987-1990.

[0179] Examples of monovalent linkers that can be used are, for example, listed in Nolting, 2013, Antibody-Drug Conjugates, Methods in Molecular Biology 1045:71-100; Kitson et al., 2013, CROs / CMOs--Chemica Oggi--Chemistry Today 31(4):30-38; Ducry et al., 2010, Bioconjugate Chem. 21:5-13; Zhao et al., 2011, J. Med. Chem. 54:3606-3623; U.S. Patent No. 7,223,837; U.S. Patent No. 8,568,728; U.S. Patent No. 8,535,678; and International Publication No. 2004010957, each of which is incorporated herein by reference.

[0180] As an example, but not limited to, some cleavable and non-cleavable linkers that may be included in the antiglyco-CD44 ADCs of this disclosure are described below.

[0181] 6.2.3. Cuttable Linker In certain embodiments, the selected linker is cleavable in vivo. The cleavable linker may contain chemically or enzymatically unstable or degradable linkages. The cleavable linker generally relies on intracellular processes to release the drug, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes, or cleavage by specific intracellular proteases or other enzymes. The cleavable linker generally incorporates one or more chemical bonds that are cleavable either chemically or enzymatically, while the remainder of the linker is incleavable. In certain embodiments, the linker contains a chemically unstable group, such as a hydrazone and / or disulfide group. Linkers containing a chemically unstable group take advantage of the different properties between plasma and certain intracellular compartments. In the case of a hydrazone-containing linker, the intracellular conditions that facilitate drug release are the acidic environments of endosomes and lysosomes, while a disulfide-containing linker is reduced in a cytosol with high thiol concentrations, such as glutathione. In certain embodiments, the plasma stability of a linker containing a chemically unstable group can be increased by introducing steric hindrance using substituents near the chemically unstable group.

[0182] Acid-unstable groups, such as hydrazones, remain intact during systemic circulation in the neutral pH environment of the blood (pH 7.3–7.5). However, when ADCs are internalized in the weakly acidic compartments of cells—endosomes (pH 5.0–6.5) and lysosomes (pH 4.5–5.0)—they undergo hydrolysis, releasing the drug. This pH-dependent release mechanism is associated with nonspecific drug release. To increase the stability of the hydrazone group in the linker, the linker may be modified, for example, by chemical modification, such as substitution, which can be adjusted to achieve more efficient release in lysosomes while minimizing loss in circulation.

[0183] A linker containing a hydrazone may contain additional cleavage sites, such as additional acid-instability cleavage sites and / or enzyme-instability cleavage sites. An example ADC containing a hydrazone-containing linker has the following structure:

[0184] [ka] In the formula, D and Ab represent a cytotoxic substance and / or a cell proliferation inhibitor (drug) and Ab, respectively, and n represents the number of drug-linkers linked to the antibody. In a particular linker, e.g., linker (Ig), the linker contains two cleavable groups, namely a disulfide moiety and a hydrazone moiety. For such linkers, effective release of the unmodified free drug requires an acidic pH or disulfide reduction and an acidic pH. Linkers such as (Ih) and (Ii) have been shown to be effective at a single hydrazone cleavage site.

[0185] Carbonates are examples of additional linkers that remain intact during systemic circulation but undergo hydrolysis to release drugs once ADCs are internalized within acidic cellular compartments. Such linkers may be useful if cytotoxic and / or cell proliferation inhibitors can be covalently attached via oxygen.

[0186] Other acid-unstable groups that may be present in linkers include those containing cis-aconityl. The cis-aconityl chemistry uses a carboxylic acid juxtaposed with the amide bond to facilitate amide hydrolysis under acidic conditions.

[0187] Cleavable linkers may also contain disulfide groups. Disulfides are thermodynamically stable at physiological pH and are designed to release drugs when internalized within the cell, in which case the cytosol provides a significantly more reducing environment compared to the extracellular environment. Separation of disulfide bonds generally requires the presence of cytoplasmic thiol cofactors, such as (reduced) glutathione (GSH), so that the disulfide-containing linkers are moderately stable in circulation and selectively release drugs in the cytosol. Intracellular enzyme proteins such as disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, may also contribute to the preferential cleavage of disulfide bonds within the cell. In approximately five tumor cells, GSH has been reported to be present in cells at concentrations ranging from 0.5 to 10 mM, compared to significantly lower concentrations of GSH or cysteine ​​(which is the most abundant low molecular weight thiol) in circulation. In this case, irregular blood flow causes hypoxia, enhancing the activity of reducing enzymes and thus leading to even higher glutathione concentrations. In certain embodiments, the in vivo stability of disulfide-containing linkers can be enhanced by chemical modification of the linker, for example, by the use of steric hindrance adjacent to the disulfide bond.

[0188] An example ADC containing a disulfide-containing linker has the following structure:

[0189] [ka] In the formula, D and Ab represent the drug and antibody, respectively, n represents the number of drug-linkers linked to the antibody, and R is independently selected from, for example, hydrogen or alkyl for each occurrence. In certain embodiments, increasing the steric hindrance adjacent to the disulfide bond increases the stability of the linker. Structures such as (Ij) and (Il) exhibit increased stability in vivo when one or more R groups are selected from lower alkyl groups, for example, methyl.

[0190] Another type of cleavable linker that can be used is one that is specifically cleaved by an enzyme. Such linkers are typically peptide-based or contain a peptide region that acts as a substrate for the enzyme. Peptide-based linkers tend to be more stable in plasma and the extracellular environment than chemically unstable linkers. Peptide bonds generally have superior stability in serum because lysosomal proteases have very low activity in blood due to endogenous inhibitors and the unfavorably high pH value of blood compared to lysosomes. Drug release from antibodies occurs specifically due to the action of lysosomal proteases, such as cathepsin and plasmin. These proteases may be present at high levels in certain tumor cells.

[0191] In exemplary embodiments, the cleavable peptides are tetrapeptides such as Gly-Phe-Leu-Gly (SEQ ID NO: 181), Ala-Leu-Ala-Leu (SEQ ID NO: 182), or Val-Cit, Val-Ala, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D)Asp5, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Asn-(D)Lys, AM Met-(D)Lys, Asn-(D)Lys, AW Dipeptides such as Met-(D)Lys and Asn-(D)Lys are selected. In certain embodiments, dipeptides are preferred over longer polypeptides because longer peptides are hydrophobic.

[0192] Various dipeptide-based cleavable linkers useful for linking drugs such as doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepines, talysomycin, and auristatin / auristatin family members to antibodies are described (each incorporated herein by reference: 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 See 122: 1455-1463; and Francisco et al., 2003, Blood 102:1458-1465). All of these dipeptide linkers, or modified versions thereof, may be used in the antiglyco-CD44 ADCs of this disclosure. Other dipeptide linkers that can be used include those found in ADCs such as Seattle Genetics' brentuximab vedotin SGN-35 (Adcetris®), Seattle Genetics' SGN-75 (anti-CD-70, Val-Cit-monomethyl auristatin F (MMAF)), Seattle Genetics' SGN-CD33A (anti-CD-33, Val-Ala-(SGD-1882)), Celldex Therapeutics' glenbatumumab (CDX-011) (anti-NMB, Val-Cit-monomethyl auristatin E (MMAE)), and Cytogen's PSMA-ADC (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).

[0193] As an enzymatically cleavable linker, a self-destructing spacer can be used to spatially separate the drug from the enzymatic cleavage site. Direct attachment of a drug to a peptide linker can lead to the release of amino acid adducts of the drug by proteolysis, thereby impairing its activity. The use of a self-destructing spacer allows for the detachment of a sufficiently active, chemically unmodified drug after hydrolysis of the amide bond.

[0194] One self-destructing spacer is a bifunctional para-aminobenzyl alcohol group, which is linked to the peptide via the amino group to form an amide bond, although the amine-containing drug may be attached to the hydroxyl group of the benzyl of the linker (PABC) via carbamic acid functionality. The resulting prodrug is activated by protease-mediated cleavage, causing a 1,6-elimination reaction that releases the unmodified drug, carbon dioxide, and the remainder of the linker group. The following scheme depicts the fragmentation of the p-amide benzyl ether and the release of the drug.

[0195] [ka] In the formula, XD represents the unmodified drug.

[0196] A heterocyclic variant of this self-destructing group is also described. See, for example, U.S. Patent No. 7,989,434, which is incorporated herein by reference.

[0197] In some embodiments, the enzymatically cleavable linker is a β-glucuronide-based linker. Facilitated drug release can be achieved via cleavage of the β-glucuronide glycosidic bond by the lysosomal enzyme β-glucuronidase. This enzyme is abundant in lysosomes and overexpressed in some tumor types, but its enzymatic activity is low outside the cell. β-glucuronide-based linkers can be used to avoid the tendency of ADCs to aggregate due to the hydrophilic nature of β-glucuronide. In some embodiments, β-glucuronide-based linkers are preferred as linkers for ADCs linked to hydrophobic drugs. The following scheme depicts drug release from an ADC containing a β-glucuronide-based linker.

[0198] [ka]

[0199] Various cleavable β-glucuronide-based linkers useful for conjugating drugs such as auristatin, camptothecin and doxorubicin analogs, CBI subgroove binders, and psymberin to antibodies are described (each incorporated herein by reference: 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., (See 2005, J. Am. Chem. Soc. 127:11254-11255). All of these β-glucuronide-based linkers can be used in the anti-glycoCD44 ADCs of this disclosure.

[0200] In addition, cytotoxic and / or cell proliferation inhibitory substances containing a phenol group can be covalently bonded to a linker via the oxygen of the phenol. One such linker is described in International Publication No. 2007 / 089149, which relies on a method that uses diaminoethane "SpaceLink" together with a conventional "PABO"-based self-destructing group to deliver phenol. The cleavage of the linker is schematically depicted below, where D represents a cytotoxic and / or cell proliferation inhibitory substance having a hydroxyl group of phenol.

[0201] [ka]

[0202] A cleavable linker may contain incleavable portions or segments, and / or a cleavable segment or portion may be contained within the other incleavable linker to make it cleavable. As just one example, polyethylene glycol (PEG) and related polymers may contain cleavable groups in the polymer backbone. For example, polyethylene glycol or polymer linkers may contain one or more cleavable groups, such as disulfides, hydrazones, or dipeptides.

[0203] Other degradable linkages that may be present in the linker include ester linkages formed by the reaction of carboxylic acids of PEG or activated carboxylic acids of PEG with alcohol groups on bioactive agents, in which case such ester groups generally hydrolyze under physiological conditions to release bioactive agents. Hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulting from the reaction of amines and aldehydes; phosphate ester linkages formed by the reaction of alcohols with phosphate groups; acetal linkages, which are reaction products of aldehydes and alcohols; orthoester linkages, which are reaction products of formates and alcohols; and, but are not limited to, oligonucleotide linkages formed by phosphoamidite groups at the ends of polymers and 5' hydroxyl groups of oligonucleotides.

[0204] In certain embodiments, the linker comprises an enzymatically cleavable peptide moiety, for example, the linker having structural formula (IVa) or (IVb):

[0205] [ka] or a salt thereof, wherein peptide represents a peptide that can be cleaved by lysosomal enzymes (illustrated from C to N, with carboxy and amino "terminuses" not shown); T represents a polymer comprising one or more ethylene glycol units or alkylene chains, or a combination thereof; R ap is selected from hydrogen, alkyl, sulfonate ions, and methylsulfonate ions; p is an integer in the range of 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

[0206] [ka] * represents the attachment site of the linker to the cytotoxic and / or cell proliferation inhibitory agent; * represents the attachment site of the rest of the linker.

[0207] In certain embodiments, the peptide is selected from tripeptides or dipeptides. In certain embodiments, 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.

[0208] Specific exemplary embodiments of the linker by structural formula (IVa) that may be included in the antiglyco-CD44 ADC of this disclosure include the linker illustrated below (as illustrated, the linker includes a group suitable for covalently linking the linker to the antibody):

[0209] [ka]

[0210] [ka]

[0211] Specific exemplary embodiments of the linker by structural formula (IVb) that may be included in the antiglyco-CD44 ADC of this disclosure include the linker illustrated below (as illustrated, the linker includes a group suitable for covalently linking the linker to the antibody):

[0212] [ka]

[0213] [ka]

[0214] [ka]

[0215] [ka]

[0216] [ka]

[0217] In certain embodiments, the linker comprises an enzymatically cleavable peptide moiety, for example, the linker having structural formula (IVc) or (IVd):

[0218] [ka] or a salt thereof, wherein peptide represents a peptide that can be cleaved by lysosomal enzymes (illustrated from C to N, with carboxy and amino "terminuses" not shown); T represents a polymer comprising one or more ethylene glycol units or alkylene chains, or a combination thereof; R ax is selected from hydrogen, alkyl, sulfonate ions and methylsulfonate ions; p is an integer in the range of 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1; x

[0219] [ka] * represents the attachment site of the linker to the cytotoxic and / or cell proliferation inhibitory agent; * represents the attachment site of the rest of the linker.

[0220] Specific exemplary embodiments of linkers by structural formula (IVc) that may be included in the antiglyco-CD44 ADC of this disclosure include the linker illustrated below (as illustrated, the linker includes a group suitable for covalently linking the linker to an antibody):

[0221] [ka]

[0222] Specific exemplary embodiments of the linker by structural formula (IVd) that may be included in the antiglyco-CD44ADC of this disclosure include the linker exemplified below (as exemplified, the linker includes groups suitable for covalently linking the linker to the antibody):

[0223] [ka]

[0224] [ka]

[0225] In certain embodiments, a linker comprising structural formula (IVa), (IVb), (IVc), or (IVd) further comprises a carbonate moiety that can be cleaved by exposure to an acidic medium. In certain embodiments, the linker is attached to a cytotoxic substance and / or a cell growth inhibitor via oxygen.

[0226] 6.2.4. Linkers that cannot be cut While cleavable linkers may offer certain advantages, the linkers containing the antiglyco-CD44 ADCs of this disclosure do not necessarily have to be cleavable. In the case of non-cleavable linkers, drug release does not depend on the different properties between plasma and certain intracytoplasmic compartments. Drug release occurs after the internalization of the ADC via antigen-mediated endocytosis and delivery to the lysosomal compartment, where the antibody is assumed to be degraded to the amino acid level via intracellular proteolysis. This process releases the drug, the linker, and the drug derivative formed by the amino acid residues to which the linker is covalently attached. Amino acid drug metabolites from conjugates with non-cleavable linkers are more hydrophilic and generally have lower membrane permeability compared to conjugates with cleavable linkers, thereby resulting in less bystander action and less nonspecific toxicity. Generally, ADCs with non-cleavable linkers have greater stability in circulation than ADCs with cleavable linkers. The uncleavable linker may be an alkylene chain, or a natural polymer, such as a polyalkylene glycol polymer, an amide polymer-based polymer, or it may contain segments of an alkylene chain, polyalkylene glycol (glocol), and / or an amide polymer.

[0227] Various non-cleavable linkers used to conjugate drugs to antibodies are described. See Jeffrey et al., 2006, Bioconjug. Chem. 17:831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280; and Jiang et al., 2005, J. Am. Chem. Soc. 127:11254-11255, each incorporated herein by reference. Any of these linkers may be included in the antiglyco-CD44 ADCs of this disclosure.

[0228] In certain embodiments, the linker is inviolable in vivo and is, for example, a linker with structural formula (VIa), (VIb), (VIc), or (VId) (as illustrated), and the linker has a suitable base for covalently linking the linker to the antibody:

[0229] [ka] or a salt thereof, in the formula, R a R is selected from hydrogen, alkyl, sulfonate, and methylsulfonate; x This is a portion containing a functional group that allows the linker to be covalently attached to the antibody;

[0230] [ka] This represents the linker's attachment point to cytotoxic and / or cell proliferation inhibitory substances.

[0231] Specific exemplary embodiments of linkers by structural formulas (VIa) to (VId) that may be included in the antiglyco-CD44 ADC of this disclosure include the linkers illustrated below (as illustrated, the linker comprises a group suitable for covalently linking the linker to the antibody,

[0232] [ka] This indicates an attachment point to a cytotoxic substance and / or a cell proliferation inhibitor.

[0233] [ka]

[0234] 6.2.5. Bases used to attach the linker to the antibody Various groups can be used to attach a linker-drug synth to an antibody to obtain an ADC. The attachment group may be electrophilic in nature, and examples include maleimide groups, activated disulfides, activated esters, e.g., NHS esters and HOBt esters, formic acid halides, acid halides, alkyl halides and benzyl halides, e.g., haloacetamides. There are also emerging technologies relating to “self-stabilizing” maleimides and “crosslinking disulfides” that can be used in accordance with this disclosure, as will be discussed below. The specific group used is expected to depend in part on the attachment site on the antibody.

[0235] An example of a "self-stabilizing" maleimide group that spontaneously hydrolyzes under antibody conjugation conditions to produce an ADC species with improved stability is illustrated in the following schematic diagram. See also U.S. Patent Application Publication No. 20130309256; and Lyon et al., Nature Biotech published online, doi:10.1038 / nbt.2968.

[0236] Normal system:

[0237] [ka]

[0238] SGN MalDPR (maleimidodipropylamino) system:

[0239] [ka]

[0240] Polytherics has disclosed a method for crosslinking pairs of sulfhydryl groups derived from the reduction of natural hinged disulfide bonds. See Badescu et al., 2014, Bioconjugate Chem. 25:1124-1136. This reaction is illustrated in the following schematic diagram. The advantage of this method is its ability to synthesize DAR4 ADCs enriched by the complete reduction of IgG (producing four pairs of sulfhydryls) followed by the reaction with four equivalents of an alkylating agent. ADCs containing "crosslinked disulfides" have also been reported to have increased stability.

[0241] [ka]

[0242] Similarly, maleimide derivatives (1, hereafter) capable of crosslinking sulfhydryl group pairs have been developed, as described below. See International Publication No. 2013 / 085925.

[0243] [ka]

[0244] 6.2.6. Considerations for Linker Selection As is known to those skilled in the art, the selected linker for a particular ADC may be influenced by various factors, including, but are not limited to, the attachment site to the antibody (e.g., lys, cys, or other amino acid residues), the structural constraints of the drug's pharmacological group, and the drug's lipophilicity. The selected linker for a specific ADC will need to balance these various factors for the specific antibody / conjugate. For a general overview of the factors influenced by linker selection 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.

[0245] For example, ADCs have been observed to kill bystander antigen-negative cells located near antigen-positive tumor cells. The mechanism of bystander cell lethality by ADCs has shown that metabolites formed during the intracellular processing of ADCs may play a role. Neutral cytotoxic metabolites produced by the metabolism of ADCs in antigen-positive cells appear to play a role in bystander cell lethality, while charged metabolites may have their diffusion into membrane-bound media inhibited and therefore cannot influence bystander lethality. In certain embodiments, linkers are selected to attenuate the bystander lethality caused by the cellular metabolites of ADCs. In certain embodiments, linkers are selected to increase the bystander lethality.

[0246] The properties of the linker can also affect the aggregation of ADCs under use and / or storage conditions. Typically, ADCs reported in the literature contain 3-4 or fewer drug molecules per antibody molecule (see, e.g., Chari, 2008, Acc Chem Res 41:98-107). Attempts to obtain a higher drug-to-antibody ratio ("DAR") often fail because the ADC aggregates, especially when both the drug and linker are hydrophobic (King et al., 2002, J Med Chem 45:4336-4343; Holander et al., 2008, Bioconjugate Chem 19:358-361; Burke et al., 2009 Bioconjugate Chem 20:1242-1250). In many cases, a DAR higher than 3-4 may be beneficial as a means of increasing potency. In cases where cytotoxic and / or cell proliferation inhibitory substances are inherently hydrophobic, particularly in cases where a DAR greater than 3-4 is desired, it may be desirable to select a relatively hydrophilic linker as a means of reducing ADC aggregation. Therefore, in certain embodiments, the linker incorporates chemical components that reduce ADC aggregation during storage and / or use. The linker may incorporate polar or hydrophilic groups, such as charged groups or groups that become charged at physiological pH, in order to reduce ADC aggregation. For example, the linker may incorporate charged groups, such as salts or groups, that, at physiological pH, for example, deprotonate carboxylic acids or protonate amines.

[0247] Exemplary polyvalent linkers that can be used to link numerous cytotoxic and / or cell proliferation inhibitory substances to antibodies, which have been reported to yield high DARs of up to 20, are described in International Publication Nos. 2009 / 073445; International Publication Nos. 2010 / 068795; International Publication Nos. 2010 / 138719; International Publication Nos. 2011 / 120053; International Publication Nos. 2011 / 171020; International Publication Nos. 2013 / 096901; International Publication Nos. 2014 / 008375; International Publication Nos. 2014 / 093379; International Publication Nos. 2014 / 093394; and International Publication Nos. 2014 / 093640, whose contents are incorporated herein by reference in their entirety.

[0248] In certain embodiments, the aggregation of ADCs during storage or use is less than about 10%, as determined by size exclusion chromatography (SEC).

[0249] 6.2.7. Method for producing antiglyco-CD44 ADCs The antiglyco-CD44 ADCs of this disclosure can be synthesized using well-known chemical methods. The selected chemical method is expected to depend, among many others, on the properties of the cytotoxic and / or cell proliferation inhibitory substance, the linker and the group used to attach the linker to the antibody. Generally, ADCs according to formula (I) can be prepared according to the following scheme: DLR x +Ab-R y →[DL-XY] n -Ab (I) In the formula, D, L, Ab, XY, and n are as previously defined, and R x and R yAs discussed above, these represent complementary groups that can form covalent bonds with each other.

[0250] R x and R y The properties of the base are, Synton DLR xThe binding properties are expected to depend on the chemical method used to conjugate the molecule to the antibody. Generally, the chemical method used should not alter the integrity of the antibody, for example, its ability to bind to its target. Preferably, the binding properties of the conjugated antibody are expected to be very similar to those of the unconjugated antibody. Various chemical methods and techniques for conjugating molecules to biomolecules such as antibodies are well known in the art, and those for conjugating to antibodies are particularly well known.For example, Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. Eds., Alan R. Liss, Inc., 1985;Hellstrom et al., “Antibodies For Drug Delivery,” in: Controlled Drug Delivery, Robinson et al. Eds., Marcel Dekker, Inc., 2nd Ed. 1987;Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in: Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., Eds., 1985;“Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in: Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. al., See Eds., Academic Press, 1985; Thorpe et al., 1982, Immunol. Rev. 62:119-58; and PCT International Publication No. 89 / 12624. Any of these chemical methods can be used to link synthons to antibodies.

[0251] Functional group R is useful for linking synthons to accessible lysine residues. x The number and chemical methods of these are well known, and examples include, but are not limited to, NHS esters and isothiocyanates.

[0252] Functional group R is useful for linking synthons to accessible free sulfhydryl groups of cysteine ​​residues. x The number and chemical methods of these compounds are well known, and examples, though not limited to these, include haloacetyl and maleimide.

[0253] However, the chemical properties of the conjugation are not limited to the available side chain groups. Side chains such as amines can be converted to other useful groups, such as hydroxyl, by linking a suitable small molecule to the amine. This strategy can be used to increase the number of available linkage sites on an antibody by conjugating polyfunctional small molecules to the side chains of accessible amino acid residues of the antibody. A suitable functional group R is then used to covalently link synthons to these "converted" functional groups. x However, it is included in Shinton.

[0254] Antibodies can also be modified to contain amino acid residues for conjugation. Approaches for modifying antibodies to contain non-gene-coding amino acid residues useful for conjugating drugs in the ADC state, along with useful chemical methods and functional groups for linking synthons to non-coding amino acids, are described by Axup et al., 2012, Proc Natl Acad Sci USA. 109(40):16101-16106.

[0255] Typically, synthons are linked to the side chains of amino acid residues in antibodies, for example, to the primary amino group of an accessible lysine residue or the sulfhydryl group of an accessible cysteine ​​residue. Free sulfhydryl groups can be obtained by reducing the interchain disulfide bond.

[0256] R y If R is a sulfhydryl group (for example, x In the ligation of (when maleimide is used), the antibody generally first completely or partially reduces the cysteine ​​residues, breaking down the disulfide crosslinks between the chains.

[0257] Mutations in one or more codons can modify the antibody so that cysteine ​​residues that do not participate in disulfide crosslinking can be incorporated. Reduction of these unpaired cysteines generates sulfhydryl groups suitable for conjugation. Preferred locations for incorporating the modified cysteine ​​include, but are not limited to, the S112C, S113C, A114C, S115C, A176C, 5180C, S252C, V286C, V292C, S357C, A359C, S398C, and S428C (Kabat numbering) on ​​the human IgG monohelic acid chain, and the V110C, S114C, S121C, S127C, S168C, and V205C (Kabat numbering) on ​​the human Ig kappa light chain (see, for example, U.S. Patents No. 7,521,541, 7,855,275, and 8,455,622).

[0258] As expected to be understood by those skilled in the art, the multiple cytotoxic and / or inhibitory agents linked to an antibody molecule can be modified so that the ADC assembly can be inherently heterogeneous, with some antibodies containing one linked agent, some containing two, some containing three, and so on (some antibodies containing no linked agents). The degree of heterogeneity is expected to depend, among many, on the chemical method used to link the cytotoxic and / or inhibitory agents. For example, if the antibody is reduced to produce sulfhydryl groups for attachment, heterogeneous mixtures of antibodies with 0, 2, 4, 6, or 8 linked agents per molecule are often produced. Furthermore, by limiting the molar ratio of the attachment compounds, antibodies with 0, 1, 2, 3, 4, 5, 6, 7, or 8 linked agents per molecule are often produced. Therefore, depending on the context, it is expected to be understood that the stated DAR may be the average of the antibody assembly. For example, "DAR4" may refer to an ADC preparation that has not been purified to isolate a specific DAR peak and may contain a heterogeneous mixture of ADC molecules with varying numbers of attached cell growth inhibitors and / or cytotoxic substances per antibody (e.g., 0, 2, 4, 6, or 8 drugs per antibody), but with an average drug-to-antibody ratio of 4. Similarly, in some embodiments, "DAR2" refers to a heterogeneous ADC preparation with an average drug-to-antibody ratio of 2.

[0259] If an enriched preparation is desired, antibodies having a predetermined number of linked cytotoxic and / or cell proliferation inhibitory substances can be obtained through the purification of heterogeneous mixtures, for example, by column chromatography, for example, by hydrophobic interaction chromatography.

[0260] Purity can be evaluated by various methods, as is well known in the industry. For example, ADC preparations may be analyzed via HPLC or other chromatography, and their purity may be evaluated by analyzing the area under the curve of the resulting peaks.

[0261] 6.3 Chimeric Antigen Receptors The disclosure of the present invention provides a chimeric antigen receptor (CAR) comprising an antiglyco-CD44 antibody or antigen-binding fragment as described herein. In some embodiments, the CAR comprises one or more scFvs (e.g., one or two) as described herein. For example, the CAR may comprise two scFvs covalently linked by a linker sequence (e.g., 4 to 15 amino acids). Exemplary linkers include GGGGS (SEQ ID NO: 183) and (GGGGS)3 (SEQ ID NO: 184).

[0262] The CARs of this disclosure typically comprise an extracellular domain operably linked to a transmembrane domain, which in turn operably links to an intracellular domain for signal transduction. The CAR may further comprise a signal peptide at the N-terminus of the extracellular domain (e.g., a human CD8 signal peptide). In some embodiments, the CARs of this disclosure comprise a human CD8 signal peptide comprising the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 175).

[0263] The extracellular domain of the CAR of this disclosure comprises a sequence of an anti-glycoCD44 antibody or antigen-binding fragment (for example, as described in Section 6.1 or in numbered embodiments 1 to 359).

[0264] Exemplary transmembrane domain sequences and intracellular domain sequences are described in sections 6.3.1 and 6.3.2, respectively.

[0265] The various fusion proteins described herein (e.g., numbered embodiments 395 to 429) are CARs, and the disclosures relating to CARs apply to such fusion proteins.

[0266] 6.3.1. Transmembrane domains With respect to the transmembrane domain, CARs can be designed to include a transmembrane domain that is operably linked (e.g., fused) to the extracellular domain of the CAR.

[0267] The transmembrane domain may be derived from either a natural or synthetic source. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain for a particular application in this disclosure may be derived from the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154 (i.e., may include at least their transmembrane domains). In some cases, various human hinges, such as human Ig (immunoglobulin) hinges, may also be employed.

[0268] In one embodiment, the transmembrane domain is synthetic (i.e., not naturally occurring). An example of a synthetic transmembrane domain is a peptide containing primarily hydrophobic residues such as leucine and valine. Preferably, a triplicate of phenylalanine, tryptophan, and valine is expected to be found at each terminus of the synthetic transmembrane domain. Optionally, a short oligo or polypeptide linker, preferably 2 to 10 amino acids in length, can form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine duo provides a particularly suitable linker.

[0269] In one embodiment, the transmembrane domain in the CAR of the present disclosure is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence YLHLGALGRDLWGPSPVTGYHPLL (SEQ ID NO: 185).

[0270] In one embodiment, the transmembrane domain in the CAR of the present disclosure is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 186).

[0271] In some cases, the transmembrane domain of the CAR of this disclosure is linked to the extracellular domain by a CD8a hinge domain. In one embodiment, the CD8a hinge domain comprises the amino acid sequence TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC (SEQ ID NO: 187). In another embodiment, the CD8a hinge domain comprises the amino acid sequence TTTAPRPPTPAPTIASPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 176).

[0272] In some cases, the transmembrane domain of the CAR of this disclosure is linked to the extracellular domain by a human IgG4-short chain hinge. In one embodiment, the human IgG4-short chain hinge comprises the amino acid sequence ESKYGPPCPSCP (SEQ ID NO: 177).

[0273] In some cases, the transmembrane domain of the CAR of this disclosure is linked to the extracellular domain by a human IgG4-long hinge. In one embodiment, the human IgG4-long hinge is an amino acid sequence

[0274] [ka] Includes.

[0275] 6.3.2. Intracellular Domains The intracellular signaling domains of the CARs described herein are involved in the activation of at least one normal effector function of the immune cells on which the CARs are expressed. The term “effector function” refers to a specialized function of a cell. For example, the effector function of a T cell may be helper activity, including cytolytic activity or cytokine secretion. Thus, the term “intracellular signaling domain” refers to a portion of a protein that translates the effector function signal and directs the cell to perform its specialized function. While the entire intracellular signaling domain can usually be employed, in many cases it is not necessarily required to use the entire chain. As long as a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, provided that it translates the effector function signal. Thus, the term intracellular signaling domain means that it includes any truncated portion of an intracellular signaling domain that is sufficient to translate the effector function signal.

[0276] Preferred examples of intracellular signaling domains for use in CARs of this disclosure include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act cooperatively with antigen receptors to initiate signaling, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0277] Signals generated by the TCR alone may be insufficient for complete T cell activation, and secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0278] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex either by stimulation or inhibition. Primary cytoplasmic signaling sequences that act by stimulation may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0279] Examples of primary intracytoplasmic signaling sequences containing ITAMs with specific applications in the CARs of this disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. The intracytoplasmic signaling molecules in the CARs of this disclosure are particularly preferably those that include a cytoplasmic signaling sequence derived from CD3-zeta.

[0280] In a preferred embodiment, the cytoplasmic domain of the CAR is designed to include the primary cytoplasmic signaling sequence domain containing the ITAM (e.g., that of CD3-zeta) itself, or in combination with any other desirable cytoplasmic domain useful in the context of the CAR of this disclosure. For example, the cytoplasmic domain of the CAR may include the CD3-zeta chain portion and the co-stimulatory signaling region.

[0281] The co-stimulatory signaling region refers to the CAR portion of a co-stimulatory molecule that includes the intracellular domain. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for the efficient response of lymphocytes to antigens. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, as well as DAP10 and GITR.

[0282] The intracellular signaling sequences within the intracellular signaling portion of the CARs of this disclosure may be linked to one another in a random or specified order. Optionally, short oligo or polypeptide linkers, preferably 2 to 10 amino acids in length, can form the linkages. Glycine-serine pairs provide particularly suitable linkers.

[0283] In one embodiment, the cytoplasmic domain includes a CD3-zeta signaling domain and a CD28 signaling domain. In some embodiments, the CD3-zeta signaling domain includes the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 180). In some embodiments, the CD28 signaling domain includes the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 179).

[0284] In another embodiment, the cytoplasmic domain includes a CD3-zeta signaling domain and a 4-1BB signaling domain.

[0285] 6.4 MicAbody The present invention provides a MicAbody comprising an anti-glyco-CD44 antibody and an antigen-binding fragment. A MicAbody is a fusion protein comprising an antibody or antigen-binding fragment and a modified MHC-class I chain-related (MIC) protein domain. The MIC protein is a native ligand for the human NKG2D receptor expressed on the surface of NK cells, and the α1-α2 domain of the MIC protein provides a binding site for the NKG2D receptor. By fusing a modified MIC protein domain (e.g., a modified α1-α2 domain) to a cancer-targeting antibody or antigen-binding fragment, T cells expressing a modified NKG2D receptor capable of binding to the modified MIC protein domain can be targeted to cancer cells. Modified MIC protein domains which may be included in the MicAbody of this Disclosure, as well as NKG2D receptors capable of binding to the modified MIC protein domains, CARs and CAR T cells containing the NKG2D receptor, are described in U.S. Patent Application Publications No. 2011 / 0183893, 2011 / 0311561, 2015 / 0165065, and 2016 / 0304578, and in PCT Publications International Publications No. 2016 / 090278, International Publications

[0286] In some embodiments, the MicAbody of this disclosure includes an α1-α2 domain that is at least 80% identical or homologous to the α1-α2 domain of an NKG2D ligand (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or OMCP). Exemplary amino acid sequences of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and OMCP are as described in Sequence IDs 1-9 of International Publication No. 2019 / 191243, respectively, and these sequences are incorporated herein by reference. In other embodiments, the α1-α2 domain is 85% identical to the native or natural α1-α2 domain of the NKG2D ligand. In yet another embodiment, the α1-α2 domain is 90% identical to the native or natural α1-α2 domain of a natural NKG2D ligand protein and binds to non-natural NKG2D.

[0287] In some embodiments, the MicAbody of this disclosure includes an α1-α2 domain that is at least 80% identical or homologous to the native or natural α1-α2 domain of a human MICA or MICB protein and binds to NKG2D. In some embodiments, the α1-α2 domain is 85% identical to the native or natural α1-α2 domain of a human MICA or MICB protein and binds to NKG2D. In other embodiments, the α1-α2 domain is 90%, 95%, 96%, 97%, 98%, or 99% identical to the native or natural α1-α2 platform domain of a human MICA or MICB protein and binds to NKG2D.

[0288] In some embodiments, specific mutations in the α1-α2 domain of the NKG2D ligand can be created to produce a non-natural α1-α2 domain that binds to a non-natural NKG2D receptor that has been modified to have reduced affinity for the natural NKG2D ligand. This may be done, for example, via genetic engineering. Using such a modified non-natural NKG2D receptor, it is possible to create NKG2D-based CARs that can preferentially bind to molecules composed of non-natural α1-α2 domains on the surface of NK or T cells of the immune system, thereby enabling activation. Such pairs of non-natural NKG2D receptors and their cognitive non-natural NKG2D ligands can offer significant safety, efficacy, and manufacturing advantages for the treatment of cancer and viral infections compared to conventional CAR-T cells and CAR-NK cells. Activation of CAR-T cells and CAR-NK cells with NKG2D-based CARs can be controlled by administration of MicAbody. In events where adverse events occur, the MicAbody dose regimen may be modified to destroy the injected CAR cells rather than to utilize the induced suicide mechanism.

[0289] MicAbody can be generated by attaching an antibody or antigen-binding fragment to an α1-α2 domain modified via a linker, for example, APTSSSGGGGS (SEQ ID NO: 188) or GGGS (SEQ ID NO: 189). For example, the α1-α2 domain may be fused to the C-terminus of an IgG heavy or light chain, as described, for example, in International Publication No. 2019 / 191243.

[0290] In some embodiments, the MicAbody of this disclosure is an amino acid sequence

[0291] [ka] Includes modified α1-α2 domains.

[0292] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0293] [ka] Includes modified α1-α2 domains.

[0294] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0295] [ka] Includes modified α1-α2 domains.

[0296] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0297] [ka] Includes modified α1-α2 domains.

[0298] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0299] [ka] Includes modified α1-α2 domains.

[0300] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0301] [ka] Includes modified α1-α2 domains.

[0302] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0303] [ka] Includes modified α1-α2 domains.

[0304] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0305] [ka] Includes modified α1-α2 domains.

[0306] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0307] [ka] Includes modified α1-α2 domains.

[0308] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0309] [ka] Includes modified α1-α2 domains.

[0310] In other embodiments, the MicAbody of this disclosure is an amino acid sequence

[0311] [ka] Includes modified α1-α2 domains.

[0312] An example of a modified NKG2D receptor is the amino acid sequence

[0313] [ka] It includes tyrosine at position 73, which is replaced by another amino acid, such as alanine.

[0314] Another exemplary modified NKG2D receptor has an amino acid sequence

[0315] [ka] This includes a derivative in which the tyrosine at positions 75 and 122 is replaced by another amino acid, for example, alanine at position 75 and phenylalanine at position 122.

[0316] 6.5 Nucleic acids, recombinant vectors, and host cells The disclosure of the present invention encompasses nucleic acid molecules encoding immunoglobulin light chain and heavy chain genes of anti-glyco-CD44 antibodies, vectors containing such nucleic acids, and host cells capable of producing the anti-glyco-CD44 antibodies of the present disclosure. In certain embodiments, the nucleic acid molecules encode the anti-glyco-CD44 antibodies and antibody-binding fragments of the present disclosure (e.g., as described in Section 6.1 and numbered embodiments 1 to 384), in addition to fusion proteins containing them (e.g., as described in numbered embodiments 385 to 394) and chimeric antigen receptors (e.g., as described in Section 6.3 and numbered embodiments 395 to 429), and host cells capable of expressing them. Exemplary vectors of the present disclosure are described in numbered embodiments 442 to 444, and exemplary host cells are described in numbered embodiments 445 to 448.

[0317] The anti-glyco-CD44 antibodies of this disclosure can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in host cells. To recombinantly express the antibody, host cells are transfected with one or more recombinant expression vectors having DNA fragments encoding the antibody's immunoglobulin light and heavy chains, thereby causing the light and heavy chains to be expressed in the host cells, and optionally they are secreted into the culture medium in which the host cells are cultured, from which the antibody can be recovered. Standard recombinant DNA techniques are used to obtain antibody heavy and light chain genes, incorporate these genes into a recombinant expression vector, and introduce the vector into host cells as described, for example, 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 U.S. Patent No. 4,816,397.

[0318] To generate nucleic acids encoding such anti-glycoCD44 antibodies, first, DNA fragments encoding the light and heavy chain variable regions are obtained. These DNAs can be obtained, for example, by amplifying and modifying germline DNA or cDNA encoding the light and heavy chain variable sequences using polymerase chain reaction (PCR). Germline DNA sequences of human heavy and light chain variable region genes are publicly known in the art (see, for example, the "VBASE" human germline sequence database; see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J. Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J. Immunol. 24:827-836; the contents of each of these are incorporated herein by reference).

[0319] V related to anti-glyco-CD44 antibodies H and V L Once DNA fragments encoding segments are obtained, these DNA fragments can be further modified using standard recombinant DNA techniques to convert, for example, variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these modifications, V H or V L A DNA fragment encoding a protein is operably linked to another DNA fragment encoding a protein, such as an antibody constant region or a flexible linker. The term "operably linked," when used in this context, is intended to mean that the two DNA fragments fuse together such that the amino acid sequences encoded by both fragments remain within the frame.

[0320] VH The isolated DNA encoding the region is V H The DNA encoding the heavy chain can be converted into a full-length heavy chain gene by operably ligating it to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3, and optionally CH4). The sequences of human heavy chain constant region genes are publicly known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, but in certain embodiments it is the constant region of IgG1 or IgG4. In the case of the Fab fragment heavy chain gene, V H The DNA encoding this may be operably ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0321] V L The isolated DNA encoding the region is V L The DNA encoding CL can be converted into a full-length light chain gene (and Fab light chain gene) by operably ligating it to the light chain constant region, another DNA molecule encoding CL. The sequences of human light chain constant region genes are publicly known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but in certain embodiments, it is a kappa constant region.

[0322] In order to create the scFv gene, V H and V L The DNA fragment that encodes V H and V L The array is V H and V L The region may be operably ligated to another fragment encoding a flexible linker, such as another fragment encoding the amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 184), so that it can be expressed as a continuous single-chain protein linked by a 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).

[0323] To express the anti-glycoCD44 antibody of this disclosure, the partial or full-length light and heavy chain encoding DNA obtained as described above is inserted into an expression vector such that the genes are operably ligated to transcriptional and translational regulatory sequences. In this context, the term “operably ligated” is intended to mean that the antibody genes are ligated to the vector such that the transcriptional and translational regulatory sequences within the vector exert their intended functions of regulating the transcription and translation of the antibody genes. The expression vector and expression regulatory sequences are selected to be suitable for the expression host cells in which they are used. The antibody light chain gene and antibody heavy chain gene may be inserted into separate vectors, or more typically, both genes may be inserted into the same expression vector.

[0324] The antibody gene is inserted into the expression vector by a standard method (e.g., complementary restriction site ligation on the antibody gene fragment and vector, or blunt-end ligation if no restriction site exists). Prior to the insertion of the antiglyco-CD44 antibody-related light chain or heavy chain sequence, the expression vector may already contain the antibody constant region sequence. For example, the V H and V L One approach to converting a sequence into a full-length antibody gene is V H The segment is operably linked to the CH segment in the vector, V L The process involves inserting the heavy chain constant and light chain constant regions into an expression vector that pre-encodes them, respectively, so that the segments are operably linked to the CL segments within the vector. In addition, or alternatively, the recombinant expression vector may encode a signal peptide that facilitates the secretion of the antibody chain from host cells. The antibody chain gene can be cloned into the vector so that the signal peptide is linked to the amino terminus of the antibody chain gene within the frame. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0325] In addition to the antibody chain gene, the recombinant expression vectors of this disclosure have regulatory sequences that control the expression of the antibody chain gene in host cells. The term “regulatory sequence” is intended to include promoters, enhancers, and other expression regulatory elements that control the transcription or translation of the antibody chain gene (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990. Those skilled in the art will expect to understand that the design of an expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of host cells to be transformed and the desired level of protein expression. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), promoters and / or enhancers derived from Simianvirus 40 (SV40) (e.g., SV40 promoter / enhancer), promoters and / or enhancers derived from adenovirus (e.g., adenovirus major late promoter (AdMLP)), and promoters and / or enhancers derived from polyoma. For further descriptions of viral regulatory elements and their sequences, see, for example, U.S. Patent No. 5,168,062 by Stinski, U.S. Patent No. 4,510,245 by Bell et al., and U.S. Patent No. 4,968,615 by Schaffner et al.

[0326] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of this disclosure may have additional sequences such as sequences that regulate vector replication in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents No. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). Typically, for example, a selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. A preferred selectable marker gene is the dihydrofolate reductase (DHFR) gene (DHFR - Examples include the use of methotrexate selection / amplification in host cells and neo genes (for G418 selection). For light and heavy chain expression, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term “transfection” are intended to encompass a variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, and DEAE-dextran transfection.

[0327] The antibodies of this disclosure may be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, antibody expression, or optimal secretion of properly folded and immunologically active antibodies, is carried out in eukaryotic cells, e.g., mammalian host cells. An exemplary mammalian host cell for expressing the recombinant antibodies of this disclosure is Chinese hamster ovary (CHO cells) (e.g., DHFR cells as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220). -Examples include CHO cells (used with a DHFR-selectable marker, as described, for example, Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient to allow antibody expression in the host cells or secretion of the antibody into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods. The host cells can also be used to produce a portion of the intact antibody, such as a Fab fragment or scFv molecule. Variations of the above procedure are understood to be within the scope of the disclosure of this invention. For example, it may be desirable to transfect host cells with DNA encoding either (but not both) the light chain or heavy chain of the anti-glycoCD44 antibody of this disclosure.

[0328] With respect to the expression of CARs in this disclosure, the host cell is preferably a T cell, preferably a human T cell, as described in, for example, Section 6.3, and further in numbered embodiments 395 to 429. In some embodiments, the host cell exhibits anti-tumor immunity when the cell is crosslinked with CD44 on tumor cells. Detailed methods for producing the T cells of this disclosure are described in Section 6.5.1.

[0329] Recombinant DNA technology can also be used to remove part or all of the DNA encoding either or both of the light and heavy chains that are not necessarily required for binding to glycoCD44. Molecules expressed from such shortened DNA molecules are also included in the antibodies of this disclosure.

[0330] For recombinant expression of the anti-glycoCD44 antibody of this disclosure, host cells may be co-transfected with two expression vectors of this disclosure, namely, a first vector encoding a heavy-chain polypeptide and a second vector encoding a light-chain polypeptide. The two vectors may contain the same selectable marker, or they may each contain different selectable markers. Alternatively, a single vector encoding both heavy-chain and light-chain polypeptides may be used.

[0331] Once a nucleic acid encodes one or more portions of an anti-glycoCD44 antibody, further modifications or mutations can be introduced into the coding sequence to produce nucleic acids encoding antibodies with different CDR sequences, antibodies with reduced affinity to the Fc receptor, or antibodies of a different subclass.

[0332] The anti-glycoCD44 antibody of this disclosure can also be produced by chemical synthesis (e.g., by the method described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Variant antibodies can also be generated using a cell-free platform (see, 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).

[0333] Once the anti-glyco-CD44 antibody of this disclosure is produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, fractional lysis, or any other standard protein purification technique. Furthermore, the anti-glyco-CD44 antibody and / or conjugated fragment of this disclosure can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0334] Once isolated, the anti-glycoCD44 antibody may be further purified, if necessary, by, for example, high-performance liquid chromatography (see Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980) or by gel filtration chromatography on a Superdex® 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0335] 6.5.1. Recombination Production of CARs in T Cells In some embodiments, the nucleic acids encoding the anti-glycoCD44 CAR of this disclosure are delivered to cells using retroviral or lentiviral vectors. Retroviral and lentiviral vectors expressing the CAR can be delivered to different types of eukaryotic cells, and even to tissues and whole organisms, using transduced cells as carriers, or using cell-free local or systemic delivery of encapsulated, conjugated, or naked vectors. The methods used may be those where stable expression is required or sufficient for any purpose.

[0336] In other embodiments, the CAR sequence is delivered to cells using in vitro transcribed mRNA. In vitro transcribed mRNA CARs can be delivered to different types of eukaryotic cells, and even to tissues and whole organisms, using transfected cells as carriers, or using cell-free local or systemic delivery of encapsulated, bound, or naked mRNA. The methods used may be those requiring transient expression or sufficient for any purpose.

[0337] In another embodiment, the desired CAR can be expressed in cells by a transposon.

[0338] One advantage of the RNA transfection method disclosed herein is that RNA transfection is inherently transient and vector-free; that is, the RNA transgene is delivered to lymphocytes and, after a short period of in vitro cell activation, is expressed there as a minimal expression cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely to occur. Cell cloning is not necessarily due to the efficiency of RNA transfection or its ability to uniformly modify an entire lymphocyte population.

[0339] Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two distinct strategies, both of which have been continuously tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. Preferably, the IVT-RNA is stabilized using various modifications to achieve sustained expression of the transfected IVT-RNA.

[0340] It is known from the literature that some IVT vectors are used in a standardized manner as templates for in vitro transcription and are genetically modified to produce stabilized RNA transcripts. Currently, the protocols used in the industry are based on plasmid vectors having the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by the gene of interest flanked by an untranslated region (UTR) at either the 3' or 5' end, and a 3' polyadenylic cassette (SEQ ID NO: 204) containing 50–70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylic cassette with a type II restriction enzyme (recognition sequence corresponding to the cleavage site). Thus, the polyadenylic cassette corresponds to the late poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzymatic cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced intracellularly from such constructs.

[0341] RNA offers numerous advantages over more traditional plasmid or viral approaches. Gene expression from RNA sources does not require transcription, and protein products are produced immediately after transfection. Furthermore, because RNA only needs to enter the cytoplasm rather than the nucleus, typical transfection methods result in extremely high transfection rates. In addition, plasmid-based approaches require activating a promoter that drives the expression of the target gene in the cells under study.

[0342] In another embodiment, RNA constructs can be delivered to cells by electroporation. See, for example, formulations and methods for the electroporation of nucleic acid constructs to mammalian cells, as taught in U.S. Patent Publication No. 2004 / 0014645, U.S. Patent Publication No. 2005 / 0052630, U.S. Patent Publication No. 2005 / 0070841, U.S. Patent Publication No. 2004 / 0059285, and U.S. Patent Publication No. 2004 / 0092907. Various parameters, such as the electric field strength required for electroporation of any known cell type, are generally publicly known in numerous patents and applications, in addition to relevant research literature in this field. See, for example, U.S. Patent No. 6,678,556, U.S. Patent No. 7,171,264, and U.S. Patent No. 7,173,116. Devices for the therapeutic application of electroporation, such as the MedPulser® DNA electroporation therapeutic system (Inovio / Genetronics, San Diego, Calif.), are commercially available and described in U.S. patents such as U.S. Patent No. 6,567,694; U.S. Patent No. 6,516,223; U.S. Patent No. 5,993,434; U.S. Patent No. 6,181,964; U.S. Patent No. 6,241,701; and U.S. Patent No. 6,233,482. Electroporation can also be used for in vitro cell transfection, as described, for example, in U.S. Patent Application Publication No. 20070128708. Electroporation can also be used for in vitro delivery of nucleic acids to cells. Therefore, electroporation-mediated delivery of nucleic acids, including expression constructs, to cells using any of the many available devices and electroporation systems known to those skilled in the art provides an exciting new means for delivering target RNA to target cells.

[0343] 6.5.1.1 T cell sources The T cell source is obtained from a subject before it is increased and genetically modified. The term "subject" is intended to include organisms (e.g., mammals) capable of eliciting an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is human.

[0344] T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural exudate, splenic tissue, and tumors. In certain embodiments of the disclosure of this invention, various T cell lines available in the art can be used. In certain embodiments of the disclosure of this invention, T cells can be obtained from blood units collected from a subject using various techniques known to the art, such as Ficoll® isolation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets, including T cells. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in a suitable buffer or medium for subsequent processing steps. In one embodiment of this disclosure, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or partially or entirely deficient in many divalent cations. Here again, surprisingly, the initial activation step in the absence of calcium results in further activation. As those skilled in the art will expect to readily understand, the washing step may be achieved by methods known to those skilled in the art, for example, by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other salt solutions with or without buffers. Alternatively, unwanted components of the apheresis sample may be removed, and the cells may be resuspended directly in the culture medium.

[0345] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLL® gradient or countercurrent centrifugation. A specific subpopulation of T cells, for example, CD3 + CD28', CD4 + CD8 + CD45RA + and CD45RO +T cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3 × 28) conjugate beads, e.g., DYNABEADS® M-450 CD3 / CD28T, for a period sufficient to positively select the desired T cells. In one embodiment, the period is about 30 minutes. In a further embodiment, the period is in the range of 30 minutes to 36 hours or longer, within any integer range in between. In a further embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours. In a preferred embodiment, the incubation period is 24 hours. In the case of isolating T cells from patients with leukemia, using a longer incubation time, e.g., 24 hours, can increase the cell yield. Longer incubation times can be used to isolate T cells in any situation where T cells are extremely scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunodeficient individuals. Furthermore, the use of longer incubation times can increase the capture efficiency of CD8+ T cells. Thus, a subpopulation of T cells, whether present or absent, can be preferentially selected at the start of culture or at other time points during the process, simply by shortening or lengthening the time that T cells are bound to CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as further described herein). In addition, a subpopulation of T cells, whether present or absent, can be preferentially selected at the start of culture or at other desired time points, either by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces. Those skilled in the art will expect to recognize that multiple selections can also be used in the context of this disclosure. In certain embodiments, it may be desirable to perform a selection procedure and use cells that were "not selected" in the activation and augmentation process."Unselected" cells can also be subjected to additional rounds of selection.

[0346] Enrichment of a T cell population by negative selection can be achieved by using in combination an antibody against a surface marker specific to the negatively selected cells. One method is negative magnetic immuno - adhesion or cell sorting and / or selection via flow cytometry using a cocktail of monoclonal antibodies against cell surface markers presented on the negatively selected cells. For example, to enrich CD4 + cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA - DR, and CD8. In certain embodiments, typically CD4 + , CD25 + , CD62L hi , GITR + , and FoxP3 + expressing regulatory T cells may be desirable to enrich or to positively select them. Alternatively, in certain embodiments, regulatory T cells are depleted by anti - C25 conjugated beads or other similar selection methods.

[0347] For the isolation of desired cell populations by positive or negative selection, the concentrations of cells and surfaces (e.g., particles such as beads) may vary. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and the beads. For example, in one embodiment, a concentration of 2 billion cells per ml is used. In one embodiment, a concentration of 1 billion cells per ml is used. In further embodiments, more than 100 million cells per ml are used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per ml are used. In yet another embodiment, cell concentrations from 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per ml are used. In further embodiments, concentrations of 125 million or 150 million cells per ml may be used. The use of high concentrations can result in increased cell yield, cell activation, and cell proliferation. Further, the use of high cell concentrations enables more efficient capture of cells in which the expression of the target antigen of interest may be weak, such as CD28-negative T cells, or from samples in which many tumor cells are present (i.e., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are expected to be desirable to obtain. For example, the use of high concentrations of cells enables more efficient selection of CD8 + T cells, which typically have relatively weak CD28 expression.

[0348] In related embodiments, it may be desirable to use lower cell concentrations. By significantly diluting the mixture of T cells and surfaces (e.g., particles such as beads), the interaction between the particles and the cells is minimized. This selects for cells that express large amounts of the desired antigen to be bound to the particles. For example, CD4 + T cells express higher levels of CD28 and at dilute concentrations CD8 +They are captured more efficiently than T cells. In one embodiment, the cell concentration used is 5 × 10 6 The concentration is / ml. In other embodiments, the concentration used is approximately 1 × 10⁻⁶ / ml. 5 From / ml to 1 x 10 6 / ml, or any integer range in between.

[0349] In other embodiments, the cells may be incubated in a rotating vessel at varying speeds for varying lengths of time at either 2°C to 10°C or room temperature.

[0350] The T cells for stimulation may also be frozen after the washing step. While not intended to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and some monocytes from the cell population. After the washing step to remove plasma and platelets, the cells may be suspended in the freezing solution. Many freezing solutions and parameters are known in the art and are expected to be useful in this situation, but one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or a 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 media containing, for example, Hespan and PlasmaLyte A, the cells then being frozen to -80°C at a rate of 1° / min and stored in the gas phase of a liquid nitrogen storage tank. Other controlled freezing methods can also be used, as can immediate freezing at -20°C or uncontrolled freezing in liquid nitrogen.

[0351] In certain embodiments, before activation using the method disclosed in the present invention, cryogenically preserved cells are thawed, washed as described herein, and left to stand at room temperature for 1 hour.

[0352] Furthermore, in the context of this disclosure, it is also anticipated that blood samples or apheresis products be collected from subjects at a time prior to when the increased cells described herein are deemed necessary. Thus, the source of the cells to be increased can be collected at any necessary time point, and the desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for various diseases or conditions that are expected to benefit from T cell therapy, such as those described herein. In one embodiment, the blood sample or apheresis is taken from an overall healthy subject. In a particular embodiment, the blood sample or apheresis is taken from an overall healthy subject who is at risk of developing the disease but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In a particular embodiment, the T cells can be increased, frozen, and used at a later time. In a particular embodiment, the sample is collected from a patient immediately after diagnosis of a particular disease described herein, but before any treatment. In further embodiments, cells are isolated from a blood sample or apheresis from the subject before treatment with various related treatment methods, for example, but not limited to, natalizumab, efalizumab, antiviral agents, chemotherapeutic agents, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunosuppressants such as CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate, steroids, FR901228, and radiation. These drugs either inhibit calcineurin, a calcium-dependent 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 a further embodiment, cells are isolated and frozen on a patient-by-patient basis for subsequent use (e.g., before, concurrently with, or after) bone marrow or stem cell transplantation or T-cell depletion therapy using any of the chemotherapeutic agents, such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide.

[0353] In a further embodiment of the disclosure of the present invention, T cells are obtained from the patient immediately after treatment. In this regard, it has been observed that the quality of T cells obtained immediately after treatment, particularly after treatment with drugs that damage the immune system, during the period when the patient is normally expected to recover from the treatment, may be optimal or improved with respect to their ability to proliferate ex vivo. Similarly, after ex vivo modification using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo proliferation. Therefore, in the context of the disclosure of the present invention, it is expected that blood cells, including T cells, dendritic cells, or other hematopoietic lineage cells, will be collected during this recovery period. Furthermore, in certain embodiments, recruitment (e.g., recruitment with GM-CSF) and conditioning regimens can be used to produce conditions in the subject that are favorable for the re-engraftment, recirculation, regeneration, and / or proliferation of specific cell types, particularly within a defined time frame after therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

[0354] 6.5.1.2 Activation and Increase of T Cells T cells are generally activated and increased using methods such as those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Publication No. 20060121005.

[0355] Generally, the T cells of this disclosure are increased by contacting them with a surface to which a drug that stimulates CD3 / TCR complex-related signaling and a ligand that stimulates a co-stimulatory molecule on the T cell surface are attached. In particular, a T cell population can be stimulated as described herein, for example, by contacting it with an anti-CD3 antibody or its antigen-binding fragment, or with an anti-CD2 antibody immobilized on the surface, or by contacting it with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For co-stimulation of accessory molecules on the T cell surface, ligands that bind to the accessory molecules are used. For example, a T cell population can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. CD4 + T cells or CD8 +Anti-CD3 antibodies and anti-CD28 antibodies are used to stimulate the proliferation of either T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), which can generally be used in the same way as other methods 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. Immunol Meth. 227(1-2):53-63, 1999).

[0356] In certain embodiments, primary stimulatory and co-stimulatory signals for T cells can be provided by a variety of protocols. For example, the agents providing each signal may be in solution form or may be surface-coupled. If surface-coupled, the agents may be coupled to the same surface (i.e., in a "cis" configuration) or to a different surface (i.e., in a "trans" configuration). Alternatively, one agent may be surface-coupled and the other agent in solution form. In one embodiment, the agent providing the co-stimulatory signal is bound to the cell surface, and the agent providing the primary activation signal is in solution form or surface-coupled. In certain embodiments, both agents may be in solution form. In another embodiment, the agents may be in soluble form and then crosslinked to the surface of cells expressing Fc receptors, or to antibodies or other binders expected to bind to the agent. In this regard, see, for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 with respect to artificial antigen-presenting cells (aAPCs) expected to be used in the T cell activation and proliferation disclosed in the present invention.

[0357] In one embodiment, the two agents are immobilized on beads either on the same bead, i.e., "cis," or on different beads, i.e., "trans." For example, the agent providing the primary activation signal is an anti-CD3 antibody or its antigen-binding fragment, and the agent providing the co-stimulatory signal is an anti-CD28 antibody or its antigen-binding fragment, and both agents are immobilized together with the same bead in equivalent molecular amounts. In one embodiment, CD4 + Each antibody conjugated to beads in a 1:1 ratio is used for T cell increase and T cell proliferation. In certain embodiments of the disclosure of the present invention, a ratio of anti-CD3:CD28 antibodies conjugated to beads is used such that an increase in T cell increase is observed compared to the increase observed using a 1:1 ratio. In one particular embodiment, an increase of about 1 to about 3 times is observed compared to the increase observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies conjugated to beads is in the range of 100:1 to 1:100 and all integer values ​​in between. In one embodiment of the disclosure of the present invention, more anti-CD28 antibodies are conjugated to the particles than anti-CD3 antibodies, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of the disclosure, the ratio of anti-CD28 antibodies conjugated to beads to anti-CD3 antibodies is greater than 2:1. In one particular embodiment, antibodies conjugated to beads in a CD3:CD28 ratio of 1:100 are used. In another embodiment, antibodies conjugated to beads with a CD3:CD28 ratio of 1:75 are used. In yet another embodiment, antibodies conjugated to beads with a CD3:CD28 ratio of 1:50 are used. In yet another embodiment, antibodies conjugated to beads with a CD3:CD28 ratio of 1:30 are used. In a preferred embodiment, antibodies conjugated to beads with a CD3:CD28 ratio of 1:10 are used. In yet another embodiment, antibodies conjugated to beads with a CD3:CD28 ratio of 1:3 are used. In yet another embodiment, antibodies conjugated to beads with a CD3:CD28 ratio of 3:1 are used.

[0358] Ratios of particles to cells ranging from 1:500 to 500:1, and any integer values ​​in between, can be used to stimulate T cells or other target cells. As will be readily apparent to those skilled in the art, the ratio of particles to cells may depend on the particle size relative to the target cells. For example, small beads can bind to only a few cells, while larger beads can bind to many cells. In certain embodiments, the ratio of cells to particles is in the range of 1:100 to 100:1, and any integer value in between, and in further embodiments, the ratio includes the range of 1:9 to 9:1, and any integer value in between, and these can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 coupled particles to T cells that induces T cell stimulation may vary as described above, but specific preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least 1:1 for particles to T cells. In one embodiment, a particle-to-cell ratio of 1:1 or lower is used. In one particular embodiment, a preferred particle-to-cell ratio is 1:5. In further embodiments, the particle-to-cell ratio may be changed depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is 1:1 to 10:1 on day 1, and then additional particles are added to the cells daily or every other day (based on the number of cells on the day of addition) in a final ratio of 1:1 to 1:10 until day 10. In one particular embodiment, the ratio of particles to cells is 1:1 on day 1 of stimulation and adjusted to 1:5 on days 3 and 5 of stimulation. In another embodiment, particles are added daily or every other day, reaching a final ratio of 1:1 on day 1 and 1:5 on days 3 and 5 of stimulation. In yet another embodiment, the ratio of particles to cells is 2:1 on day 1 of stimulation and adjusted to 1:10 on days 3 and 5 of stimulation.In another embodiment, particles are added daily or every other day, reaching a final ratio of 1:1 on day 1, and 1:10 on days 3 and 5 of stimulation. Those skilled in the art will understand that various other ratios may be suitable for use in the disclosure of the present invention. In particular, the ratios are expected to vary depending on particle size and cell size and type.

[0359] In a further embodiment of the disclosure of the present invention, cells, such as T cells, are combined with drug-coated beads, then the beads and cells are separated, and the cells are then cultured. In an alternative embodiment, the drug-coated beads and cells are not separated before culturing, but are cultured together. In a further embodiment, the beads and cells are first enriched by the application of a force such as magnetism, thereby increasing the ligation of cell surface markers and thus inducing cell stimulation.

[0360] As an example, cell surface proteins can be ligated by bringing paramagnetic beads (3 × 28 beads) to which anti-CD3 and anti-CD28 proteins are attached into contact with T cells. In one embodiment, cells (e.g., 10 4 from 10 9The T cells and beads (e.g., DYNABEADS® M-450CD3 / CD28T paramagnetic beads in a 1:1 ratio) are combined in a buffer, preferably in PBS (which does not contain divalent cations such as calcium and magnesium). Again, those skilled in the art will readily understand that any cell concentration can be used. For example, the target cells may be very sparse in the sample, constitute only 0.01% of the sample, or the entire sample (i.e., 100%) may consist of the target cells of interest. Thus, any number of cells is included in the disclosure of this invention. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and particles. For example, in one embodiment, a concentration of about 2 billion cells per ml is used. In another embodiment, more than 100 million cells per ml is used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per ml are used. In yet another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per ml are used. In yet another embodiment, concentrations of 125 million or 150 million cells per ml may be used. The use of high concentrations can result in increased cell yield, cell activation, and cell growth. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may have weak expression of the target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and are expected to be desirable to obtain in certain embodiments. For example, the use of high concentrations of cells allows for more efficient selection of CD8+ T cells, which typically have relatively weak CD28 expression.

[0361] In one embodiment of the disclosure of the present invention, the mixture can be cultured for several hours (about 3 hours) to about 14 days, or any integer value in between. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the disclosure, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may be desirable to allow the T cell culture time to be 60 days or longer. Suitable conditions for T cell culture include a suitable medium (e.g., minimal essential medium or RPMI medium 1640 or X-vivo15 (Lonza)) which may contain factors necessary for proliferation and survival such as serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for cell proliferation known to those skilled in the art. Other additives for cell proliferation include, but are not limited to, surfactants, plasmamenates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Examples of culture media include RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a predetermined set of hormones and / or sufficient amounts of cytokines for T cell proliferation and increase, supplemented with amino acids, sodium pyruvate, and vitamins. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in the culture of cells intended for injection into the target. Target cells are maintained under conditions necessary to support proliferation, for example, at an appropriate temperature (e.g., 37°C) and atmosphere (e.g., 5% CO2 in addition to air).

[0362] T cells exposed to various stimulation times may exhibit different characteristics. For example, typical blood or apheresis-treated peripheral blood mononuclear cell products may exhibit cytotoxic or suppressor T cell populations (TC CD8 + A larger population of helper T cells (T H CD4 + ) has. The increase in T cells by stimulating CD3 and CD28 receptors ex vivo occurs mainly before day 8-9. H It produces a population of T cells, but after about 8-9 days, the population of T cells becomes even larger. C It contains a population of cells. Therefore, depending on the purpose of the treatment, primarily T H In some cases, it may be advantageous to inject T cell populations that include T cells. C If an antigen-specific subset of cells is isolated, it may be beneficial to increase the concentration of this subset to a higher degree.

[0363] Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers change significantly, but mostly reproducibly, during the cell proliferation process. This reproducibility, therefore, allows for the ability to tailor activated T cell products to specific purposes.

[0364] 6.6 Composition The anti-glyco-CD44 antibody, fusion protein, and / or anti-glyco-CD44 ADC of this disclosure may be in the form of a composition comprising the anti-glyco-CD44 antibody, fusion protein, and / or ADC, and one or more carriers, excipients, and / or diluents. The composition may be formulated for a specific use, for example, for veterinary use or for pharmaceutically use in humans. The form of the composition used (e.g., dry powder, liquid formulation, etc.) and the form of the excipients, diluents, and / or carriers are expected to be determined by the intended use of the antibody, fusion protein, and / or ADC, and further by therapeutic use and mode of administration.

[0365] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition may be in any preferred form (depending on the preferred method of administration to the patient). The pharmaceutical composition can be administered to the patient by various routes, including orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, subarachnoidally, topically, or topically. The most preferred route of administration in any given case is expected to depend on the specific antibody and / or ADC, the subject, and the nature and severity of the subject's disease and physical condition. Typically, the pharmaceutical composition is expected to be administered intravenously or subcutaneously.

[0366] The pharmaceutical composition can be conveniently provided in unit dosage forms containing a predetermined amount of the anti-glyco-CD44 antibody and / or anti-glyco-CD44 ADC of this disclosure per dose. The amount of antibody and / or ADC contained in the unit dose is expected to be determined by the disease being treated, in addition to other factors well known in the art. Such unit dosings may be in the form of a lyophilized dry powder containing an amount of antibody and / or ADC suitable for a single dose, or in liquid form. The dry powder unit dosage form may be packaged in a kit together with a syringe, a suitable amount of diluent, and / or other components useful for administration. The unit dosings in liquid form can be conveniently supplied in the form of a syringe pre-filled with an amount of antibody and / or ADC suitable for a single dose.

[0367] The pharmaceutical composition may also be supplied in bulk form containing an amount of antibody and / or ADC suitable for multiple doses.

[0368] Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing antibodies, fusion proteins, and / or ADCs of desired purity with any choice of pharmaceutically acceptable carriers, excipients, or stabilizers (all referred to herein as “carriers”) typically employed in the industry, namely buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and other compound additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be non-toxic to the recipient at the adopted dosage and concentration.

[0369] Buffers help maintain the pH within a range close to physiological conditions. While buffers may be present at various concentrations, they are typically expected to be in the range of approximately 2 mM to 50 mM. Suitable buffers for use in the disclosure of the present invention include, for example, citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citrate-trisodium citrate mixture, citrate-monosodium citrate mixture, etc.), succinate buffers (e.g., succinate-monosodium succinate mixture, succinate-sodium hydroxide mixture, succinate-disodium succinate mixture, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumarate-monosodium fumarate mixture, fumarate-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconate-sodium gluconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium gluconate mixture, etc.), and gluconate buffers (e.g., gluconate-sodium gluconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium gluconate mixture, etc.). Examples include both organic and inorganic acids, as well as their salts, such as gluconate (gluconate) mixtures, oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can be used.

[0370] Preservatives may be added to inhibit microbial growth, and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the disclosure of the present invention include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes known as “stabilizers,” can be added to ensure the isotonicity of the liquid compositions of the disclosure of the present invention, and examples include polyhydric sugar alcohols, such as sugar alcohols having three or more hydroxyl groups, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients that, in terms of function, can range from volume extenders to additives that solubilize therapeutic agents or help prevent denaturation or adhesion to container walls.Typical stabilizers include polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol, and cyclitol such as inositol; polyethylene glycol; amino acid polymers such as urea, glutathione, thioctic acid, and thioglycol. The stabilizers may include sulfur-containing reducing agents such as sodium thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides with 10 or fewer residues); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone monosaccharides, e.g., xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, sucrose, and trehalose; trisaccharides such as raffinose (trisaccacharide); and polysaccharides such as dextran. The stabilizers may be present in an amount ranging from 0.5 to 10 wt% relative to the weight of the ADC.

[0371] Nonionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize glycoproteins and to protect them from aggregation induced by stirring, thereby allowing the formulation to be exposed to stressed shear surfaces without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and pluronic polyols. The nonionic surfactant may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL, for example, in a range of about 0.07 mg / mL to about 0.2 mg / mL.

[0372] Additional excipients for the compound include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0373] 6.7 How to use The anti-glycoCD44 antibodies or binding fragments described herein can be used in a variety of diagnostic assays. For example, the antibodies and binding fragments can be used in immunoassays, such as competitive binding assays, direct and indirect sandwich assays, as well as immunoprecipitation assays, such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and Western blotting.

[0374] The anti-glyco-CD44 antibodies or conjugated fragments described herein are also useful for in vivo radiographic imaging, in which an antibody labeled with a detectable portion such as a radiopaque agent or radioisotope is administered to a subject, preferably into the bloodstream, and the presence and location of the labeled antibody in the host are assayed. This imaging technique is useful in the staging and treatment of malignant tumors.

[0375] The anti-glycoCD44 antibodies or binding fragments, fusion proteins, ADCs and CARs described herein are useful for treating cancers that express glycoCD44, such as breast cancer, lung cancer, pancreatic cancer, colorectal cancer, ovarian cancer, gastric cancer, or head and neck cancer, skin cancer, malignant melanoma, liver cancer, glioma, thyroid cancer, kidney cancer, prostate cancer and other genitourinary cancers, cervical cancer, and endometrial cancer.

[0376] Accordingly, this disclosure provides anti-glyco-CD44 antibodies, binding fragments, fusion proteins, ADCs, and CARs described herein for use as pharmaceuticals for use in the treatment of cancer, e.g., any of the cancers identified in the preceding paragraphs, for use in diagnostic assays, and for use in in vivo imaging of radiographs. This disclosure further provides the use of anti-glyco-CD44 antibodies, binding fragments, fusion proteins, ADCs, and CARs described herein in the manufacture of pharmaceuticals for the treatment of cancer, e.g., any of the cancers identified in the preceding paragraphs.

[0377] When using the CARs of the present disclosure in therapy, the therapeutic method of the present disclosure comprises administering to a subject having a tumor expressing glycoCD44 an effective amount of genetically modified cells modified to express the CARs of the present disclosure, for example, the CARs described in Section 6.3 or the numbered embodiments 395 to 429. Methods for modifying cells, in particular T cells, to express the CARs are described in Section 6.5.1.

[0378] When using the MicAbody of the present disclosure in therapy, the therapeutic method of the present disclosure involves targeting a tumor expressing glycoCD44 with a therapeutically effective dose of the MicAbody of the present disclosure, for example, the MicAbody described in Section 6.4 or the numbered embodiments 391-394, and genetically modified T cells modified to express a CAR comprising an NKG2D receptor capable of specifically binding to the MicAbody.

[0379] 6.8 CD44v6 peptide Isolated CD44v6 glycopeptides containing the amino acid GYRQTPKEDSHSTTGTAAA (SEQ ID NO: 165), or fragments thereof, are also provided herein. In some embodiments, the CD44v6 glycopeptide is glycosylated with GalNAc at serine and threonine residues indicated in bold and underlined letters (i.e., threonine at amino acid position 5 of SEQ ID NO: 165 and / or serine at amino acid position 12 of SEQ ID NO: 165), or fragments thereof. Exemplary isolated CD44v6 glycopeptides are described in numbered embodiments 534–539.

[0380] The disclosure of the present invention includes the synthetic synthesis of isolated CD44v6 glycoprotein and recombinant methods for producing isolated CD44v6 glycoprotein.

[0381] In certain embodiments, the isolated CD44v6 peptide is synthesized using a solid-phase peptide synthesis (SPPS) strategy. The SPPS method is known in the art. SPPS results in rapid assembly of a polypeptide via a series of reactions of amino acid derivatives on a solid support. The successive amino acid derivatives are added to the polypeptide via a repeating cycle of alternating N-terminal deprotection and coupling reactions. In other embodiments, the isolated CD44v6 peptide is synthesized using a liquid-phase peptide synthesis strategy. The liquid-phase peptide synthesis method is known in the art.

[0382] To ensure proper O-linked glycosylation at GalNAc in threonine at amino acid position 5 of SEQ ID NO: 165 and serine at amino acid position 12 of SEQ ID NO: 165, pre-synthesized glycosylated amino acids may be used in the extension reaction, as described in Section 7.1.2.1.

[0383] Provided are nucleic acid molecules encoding isolated CD44v6 glycopeptides, vectors containing such nucleic acids, and host cells capable of producing the isolated CD44v6 glycopeptides of the present disclosure. In a particular embodiment, the nucleic acid molecule encodes a fusion protein containing isolated CD44v6 glycopeptides, in addition to isolated CD44v6 glycoproteins, and the host cell is capable of expressing it.

[0384] The isolated CD44v6 glycopeptide of this disclosure can be prepared by recombinant expression in host cells. To recombinantly express the isolated CD44v6 glycopeptide, a recombinant expression vector containing the DNA encoding the glycopeptide can be transfected into host cells so that the glycopeptide is expressed in the host cells and, optionally, secreted into the culture medium in which the host cells are cultured, and the glycoprotein can be recovered from the medium. The CD44v6 glycoprotein gene can be obtained using standard recombinant DNA techniques, the gene can be incorporated into a recombinant expression vector, and the vector can be introduced into host cells, for example, as 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 U.S. Patent No. 4,816,397.

[0385] The antibodies of this disclosure can be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, antibody expression is performed in eukaryotic cells, for example, in mammalian host cells for optimal secretion of properly folded immunologically active antibodies. To produce the isolated CD44v6 glycoprotein of this disclosure, host cells are selected based on their ability to glycosylate threonine at amino acid position 5 of SEQ ID NO: 165 and serine at amino acid position 12 of SEQ ID NO: 165. An exemplary host cell is COSMC KO HEK293 cells.

[0386] 6.8.1. CD44v6 Peptide Composition The CD44v6 peptide of this disclosure may be in the form of a composition comprising the CD44v6 peptide and one or more carriers, excipients, diluents and / or adjuvants. The composition may be formulated for specific use, for example, veterinary use or pharmaceutical use in humans. The form of the composition used (e.g., dry powder, liquid formulation, etc.) as well as the excipients, diluents and / or carriers are expected to depend on the intended use of the antibody, fusion protein and / or ADC, as well as its therapeutic use and mode of administration.

[0387] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant. The composition may be in any preferred form (depending on the desired method of administration to the patient). The pharmaceutical composition can be administered to the patient by various routes, e.g., orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, subarachnoidally, topically, or stimulally. The most preferred route of administration in any given case is expected to depend on the specific CD44v6 peptide, the subject, the nature and severity of the disease, and the physical condition of the subject. Typically, the pharmaceutical composition is expected to be administered intravenously or subcutaneously.

[0388] The pharmaceutical composition may, in a convenient form, exist as a unit dosage form containing a predetermined amount of the CD44v6 peptide of this disclosure per dose. The amount of CD44v6 peptide contained in the unit dosage is expected to depend on the disease being treated, in addition to other factors well known in the art. Such unit dosages may be in the form of a lyophilized dry powder containing an amount of CD44v6 peptide suitable for single administration, or in liquid form. The dry powder unit dosage form may be packaged in a kit with a syringe, a suitable amount of diluent and / or other components useful for administration. The unit dosage in liquid form may, in a convenient form, be supplied in the form of a syringe pre-filled with an amount of CD44v6 peptide suitable for single administration.

[0389] The pharmaceutical composition may also be supplied in bulk form containing a large amount of CD44V6 peptide suitable for multiple doses.

[0390] Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing CD44v6 peptide of desired purity with any optional pharmaceutically acceptable carriers, excipients, adjuvants, or stabilizers (all referred to herein as “carriers”) typically employed in the industry, namely buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and other compound additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to the recipient at the dosage and concentration used.

[0391] In some embodiments, the composition comprises one or more pharmaceutically acceptable adjuvants in addition to the fusion protein and / or nanoparticles. Examples of adjuvants include aluminum salts (e.g., amorphous aluminum hydroxyphosphate sulfate; AAHS, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (Alam)), dsRNA analogs, lipid A analogs, flagellin, imidazoquinoline, CpG ODN, saponins (e.g., QS21), C-type lectin ligands (e.g., TDB), CD1d ligands (α-galactosylceramide), MF59, AS01, AS02, AS03, AS04, AS15, AF03, GLA-SE, IC31, CAF01, and virosomes. Other adjuvants known in the art, such as chemical adjuvants, genetic adjuvants, protein adjuvants, and lipid adjuvants, may also be included in the composition.

[0392] Buffers help maintain the pH within a range close to physiological conditions. While buffers may be present at various concentrations, they are typically expected to be in the range of approximately 2 mM to 50 mM. Suitable buffers for use with the disclosures of the present invention include, for example, citrate buffers (e.g., monosodium citrate-dinatotrium citrate mixture, citrate-trisodium citrate mixture, citrate-monosodium citrate mixture, etc.), succinate buffers (e.g., succinate-monosodium succinate mixture, succinate-sodium hydroxide mixture, succinate-disodium succinate mixture, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumarate-monosodium fumarate mixture, fumarate-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconate-sodium gluconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium gluconate mixture, etc.), and gluconate buffers (e.g., gluconate-sodium gluconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium gluconate). Examples include both organic and inorganic acids, as well as their salts, such as gluconate (gluconate) mixtures, oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can be used.

[0393] Preservatives may be added to inhibit microbial growth, and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the disclosure of the present invention include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes known as “stabilizers,” can be added to ensure the isotonicity of the liquid compositions of the disclosure of the present invention, and examples include polyhydric sugar alcohols, such as sugar alcohols having three or more hydroxyl groups, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients, which can vary in function from volume extenders to additives that solubilize therapeutic agents or help prevent denaturation or adhesion to container walls.Typical stabilizers include polyhydric sugar alcohols (as listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, etc., and cyclitol such as inositol; polyethylene glycol; amino acid polymers such as urea, glutathione, thioctic acid, and thioglycol. The stabilizers may include sulfur-containing reducing agents such as sodium acid, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides with 10 or fewer residues); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, sucrose, and trehalose; trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizers may be present in an amount ranging from 0.5 to 10 wt% relative to the weight of the CD44 peptide.

[0394] Nonionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize glycoproteins and to protect them from aggregation induced by stirring, thereby allowing the formulation to be exposed to stressed shear surfaces without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and pluronic polyols. The nonionic surfactant may be present in a concentration of about 0.05 mg / mL to about 1.0 mg / mL, for example, in the range of about 0.07 mg / mL to about 0.2 mg / mL.

[0395] Additional excipients for the compound include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0396] Exemplary CD44v6 peptide compositions of this disclosure are described in numbered embodiments 540-541.

[0397] 6.8.2. How to use CD44v6 peptide The CD44v6 peptide described herein can be used in the production of antibodies against tumor-associated forms of CD44v6. The CD44v6 peptide can be administered to animals. The amount of peptide administered may be effective in causing the animals to produce antibodies against the peptide. "Animals," as used herein, refer to multicellular eukaryotes from the biological animal kingdom. In some embodiments, the animals are mammals. In some embodiments, the animals are mice or rabbits. The antibodies obtained thereafter can be collected from the animals. The CD44v6 peptide can be administered as a purified peptide or as part of the compositions provided herein.

[0398] The CD44v6 peptide described herein can be used to induce an immune response to tumor-associated forms of CD44v6. The CD44v6 peptide can be administered to animals in an amount effective enough to initiate an immune response to the peptide (e.g., antibody production).

[0399] Exemplary methods for using the CD44v6 peptide of this disclosure are described in numbered embodiments 542–545. [Examples]

[0400] 7. Examples 7.1 Example 1: Identification and Characterization of Anti-GlycoCD44 Antibodies 7.1.1. Overview Glycans are essential membrane components, and neoplastic transformation of human cells is almost always associated with abnormal glycosylation of proteins and lipids. There are several types of protein glycosylation, including N-glycosylation and many types of O-glycosylation, but one of the most diverse types is mucin-type GalNAc-type O-glycosylation (hereinafter referred to as O-glycosylation). Cancer-related changes in O-glycans are of particular interest, and the most frequently observed abnormal glycophenotypes are the expression of the most immature, shortened O-glycan structures, named Tn(GalNAcα1-O-Ser / Thr), STn(NeuAcα2-6GalNAcα1-O-Ser / Thr), and T(Galβ1-3GalNAcα1-O-Ser / Thr) antigens. Shortened O-glycans are observed on almost all epithelial cancer cells and are strongly correlated with poor prognosis. In addition, glycans also play an important role in cancer development, and it is becoming clear that in cells prone to cancer, shortened O-glycans affect differentiation, intercellular and cell-matrix interactions, and directly induce tumorigenic characteristics.

[0401] The inventors identified the CD44 glycopeptide epitope in human cancer cells and used the defined glycopeptide to develop cancer-specific anti-glycoCD44 monoclonal antibodies.

[0402] 7.1.2. Materials and Methods 7.1.2.1 Synthesis of CD44v6 glycopeptide The CD44v6 glycopeptide, GYRQ, has O-linked GalNAc at the serine and threonine residues shown in bold and underlined text. T PKEDSH STTGTAAA (SEQ ID NO: 165) was synthesized using a standard FMOC peptide synthesis strategy. Pre-synthesized glycosylated amino acids were coupled stepwise to peptides elongated at specific positions using solid-phase or liquid-phase peptide chemistry. After completing the sequence and removing all protecting groups, the resulting glycopeptides were purified by high-performance liquid chromatography (HPLC) and characterized by mass spectrometry (electrospray ionization in positive mode).

[0403] 7.1.2.2 Synthesis of recombinant Tn glycosylated CD44 1 × 10 in 30 mL of Opti-MEM 6 COSMC KO HEK293 cells were transfected with 30 μg of a plasmid encoding his-tagged human CD44 and 60 μL of 293fectin® transfection reagent (Gibco). After 48 hours of culture, the cells were centrifuged, and the his-tagged recombinant CD44 protein was purified from the supernatant by eluting with 250 mM imidazole using a 50% Ni-NTA agarose slurry column (Invitrogen). This purification step was repeated to increase purity. The recombinant SC-CD44 protein was concentrated in PBS using an Amicon Ultra centrifuge filter.

[0404] 7.1.2.3 Immunization Protocol Female Balb / c mice were subcutaneously immunized with Tn glycosylated CD44v6 glycopeptide conjugated to KLH (keyhole limpet hemosinian) via a glutaraldehyde linker, or recombinant Tn glycosylated CD44. Mice were immunized with 50 μg, 45 μg, and 45 μg of KLH-glycopeptide on days 0, 14, and 35, respectively. The first immunization was performed using Freund's complete adjuvant. All subsequent immunizations were performed using Freund's incomplete adjuvant. On day 45, tail blood samples were evaluated for polyclonal response. On day 56 or thereafter, mice attempting to fuse were boosted with 15 μg of KLH-glycopeptide in Freund's incomplete adjuvant 3–5 days prior to hybridoma fusion. Splenocytes from mice were fused with SP2 / 0-Ag14 (ATCC, catalog number CRL-1581) myeloma cells using an electrocell manipulator (ECM2001) from BTX Harvard Apparatus. Hybridomas were inoculated into 96-well plates, cultured, scraped, and evaluated and selected for their specificity to CD44-Tn using ELISA, flow cytometry, and immunofluorescence to obtain monoclonal antibodies specific to CD44-Tn.

[0405] New Zealand white rabbits were subcutaneously immunized with Tn glycosylated CD44v6 glycopeptide conjugated to KLH (keyhole limpet hemosinian) via a glutaraldehyde linker, or with recombinant Tn glycosylated CD44. Mice were immunized with 200 μg, 100 μg, and 100 μg of KLH-glycopeptide on days 0, 28, and 47, respectively. The first immunization used Freund's complete adjuvant. All subsequent immunizations used Freund's incomplete adjuvant. Test blood collection was evaluated on day 58 for polyclonal response. On day 66 or thereafter, mice attempting to fuse were boosted with 50 ug of KLH-glycopeptide in Freund's incomplete adjuvant 3–5 days prior to hybridoma fusion. Rabbit splenocytes were fused with SP2 / 0-Ag14 (ATCC, catalog number CRL-1581) myeloma cells using an electrocell manipulator (ECM2001) from BTX Harvard Apparatus. Hybridomas were inoculated into 96-well plates, cultured, scraped, and evaluated and selected for specificity to CD44-Tn using ELISA, flow cytometry, and immunofluorescence to obtain monoclonal antibodies specific to CD44-Tn.

[0406] 7.1.2.4 ELISA A 96-well Corning high-binding microplate (Fisher) was coated overnight at 4°C with varying concentrations of protein, peptide, or glycopeptide in 0.2 M bicarbonate-carbonate buffer (pH 9.4). The plate was then blocked at room temperature for 1 hour in phosphate-buffered saline (PBS) (pH 7.4) containing 2.5% BSA. The plate contents were discarded, and purified antibodies, hybridoma supernatant, or serum for polyclonal response were added at varying concentrations and incubated at room temperature for 2 hours. The plate was washed with Tris-buffered saline and 0.05% Tween-20, and then incubated at room temperature for 1 hour with a 1:3000 dilution of HRP-conjugated goat anti-mouse IgGFcγ (Sigma). The plate was washed again and developed with TMB chromogen substrate. After adequate development (approximately 2-3 minutes), the reaction was stopped with 0.2 N H2SO4, and the absorbance was read at 450 nm. The data was analyzed using GraphPad Prism software.

[0407] 7.1.2.5 Flow Cytometry Adherent cells were dissociated using TrypLE Select (Gibco) and washed off the flask surface with cell culture medium (RPMI w / L-glutamine, 1% PenStrep, and 10% FBS). The cells were centrifuged at 4°C and 300×g for 5 minutes, and then washed several times by resuspending in PBS containing 1% BSA (PBS / 1% BSA). The cells were divided into 5×10⁶ cells. 5 cells / ml ~ cells 2×10 6The cells were resuspended at nucleotides / ml and then distributed into 96-well U-bottom plates. Diluted commercial antibodies (0.25–2 ug / ml), hybridoma supernatant, or serum for polyclonal response were added to the cells, and the cells were incubated on ice for 1 hour. After several washes with PBS / 1% BSA, the cells were incubated on ice for 30 minutes with a 1:1600 dilution of AlexaFluor647 conjugate F(ab)2 goat anti-mouse IgG Fcγ (JacksonImmunoResearch). The cells were washed again with PBS / 1% BSA and then fixed in 1% formaldehyde in PBS / 1% BSA. The cells were analyzed using either a 2 or 4-laser Attune NXT flow cytometer. Data were processed using FlowJo software.

[0408] 7.1.2.6 Immunofluorescence Cells were planted at 50% density on glass chamber slides (nunc) and incubated at 5% CO2, 37°C for 12–18 hours. After overnight growth, the medium was removed from the slides and the cells were fixed in 4% formaldehyde in PBS (pH 7.4) at room temperature for 10 minutes. The slides were washed in PBS. Diluted commercial antibodies (1–4 ug / ml), hybridoma supernatant, or serum for polyclonal response were added to the slides and incubated overnight at 4°C. The slides were washed in PBS and stained with a 1:800 dilution of AlexaFluor488 conjugate F(ab)2 rabbit anti-mouse IgG(H+L) (Invitrogen) at room temperature for 45 minutes. The slides were washed in PBS, mounted using a Prolong Gold Antifade Mountant (thermofisher) with DAPI, and examined using an Olympus FV3000 confocal microscope.

[0409] 7.1.2.7 Immunohistochemistry The paraffin was removed from paraffin-embedded tissue microarrays (TMAs) or tissue sections using xylene and ethanol, followed by antigen recovery with citrate buffer (pH 6.0) and heating in a microwave for 18 minutes. The TMAs were stained with the Ultra Vision Quanto detection system HRP DAB. Briefly, the TMAs were washed in TBS and incubated with mAb supernatant for 2 hours. After washing with TBS twice, the TMAs were incubated with the primary antibody amplifier Quanto for 10 minutes. After washing in TBS, the TMAs were incubated with HRP polymer Quanto (10 minutes), followed by incubation with DAB chromogen. Slides were counterstained with hematoxylin, dehydrated, and mounted. 7.1.3.Results 7.1.3.1 Glycopeptide-specific antibodies against Tn-CD44 Glycopeptide-reactive antibodies were generated using both Tn-glycosylated CD44v6 glycopeptide and recombinant Tn-glycosylated CD44. However, antibodies generated using CD44v6 glycopeptides, including 4C8, 2B2, 18G9, 1D12, and 10H4, were found to exhibit superior selectivity. For further characterization, antibody 4C8 was selected.

[0410] 7.1.3.2 Characterization of mAb 4C8 binding specificity To characterize the binding specificity of 4C8, ELISA was performed against unglycosylated and Tn-glycosylated CD44. ELISA was also performed against Tn-glycosylated MUC1 to evaluate cross-reactivity with Tn antigens. Regarding the ELISA, it was found that 4C8 reacted only with Tn-glycosylated CD44 and not with its unglycosylated counterpart or MUC1 (Figure 1A). The affinity of 4C8 for the CD44v6 glycopeptide was determined to be 128 nM as measured by Biacore. Using the Octet system, the apparent affinity of 4C8 for the CD44v6 glycopeptide (taking binding strength into account) was measured to be 7.9 nM (Figure 1B). Table 4 shows the dissociation constants (K) of 4C8 for different glycoforms of the CD44v6 peptide, as well as for unglycosylated CD44v6 and MUC1-Tn. D Summarize the following.

[0411] [Table 26]

[0412] To further evaluate the specificity of 4C8 in a more natural morphological context, 4C8 was used to stain HaCaT cells for flow cytometry and immunofluorescence. Keratinocyte-derived HaCaT cell lines are intrinsically Tn-negative but can be induced to express the Tn antigen by knockout of the COSMC chaperone. When stained for flow cytometry using 4C8, it was found that 4C8 selectively stained COSMC KO HaCaT cells but not their wild-type counterparts, even though both cell types stained positively for CD44v6 (Figure 1C). The reactivity of 4C8 could not be attributed to intracellular staining of Tn-positive intermediate CD44 protein localized in the secretory pathway, as the cells did not stain positively for Golgi markers. Consistent with these results, immunofluorescence stained CD44v6 + Tn + Only HaCaT COSMC KO cells were stained with 4C8, while CD44v6 stained them differently. + Tn- HaCaT WT cells were not stained (Figure 1D). In addition, when organ-type in vitro skin models were formed using HaCaT cells, 4C8 stained only COSMC KO skin but not wild-type skin (Figure 1E). Since clinical trials testing other CD44 antibodies have reported severe skin toxicity, 4C8 binding to healthy human skin was also evaluated. Human skin was positively stained for CD44v6, but 4C8 was found not to resonate with human skin (Figure 1F). Taken together, these results demonstrate that 4C8 selectively reacts with Tn glycosylated CD44 and does not cross-react with healthy human skin.

[0413] 7.1.3.3 Tissue expression of the Tn glycosylated CD44v6 epitope recognized by 4C8 When paraffin-embedded tissue sections fixed in formalin for immunofluorescence were stained, positive staining for 4C8 was observed in 7 / 10 lung sections, 7 / 24 ovarian sections, 7 / 14 HNSCC sections, 3 / 16 colon sections, 9 / 20 stomach sections, 3 / 8 kidney sections, and 3 / 10 breast cancer sections. In addition, immunohistochemistry of tissue microarrays using 4C8 showed strong staining of 22 / 89 and weak staining of 38 / 89 in colon cancer, strong staining of 4 / 24 and weak staining of 12 / 24 in pancreatic cancer, strong staining of 6 / 22 and weak staining of 8 / 22 in lung cancer, strong staining of 6 / 26 and weak staining of 7 / 26 in breast cancer, and weak staining of 2 / 24 in prostate cancer (Figures 2A-2B). Since this staining pattern correlated with staining of normal CD44 expression, it is suggested that CD44 expression in these cancers predicted responsiveness to 4C8. Importantly, no reactivity was observed when 4C8 was used to stain healthy adjacent tissue (Figure 2A). In conclusion, it was positively found that 4C8 reacts with several types of cancerous tissue sections but not with their healthy counterparts.

[0414] 7.2 Example 2: 4C8-based CAR We designed chimeric antigen receptors (CARs) possessing the VH and VL domains of 4C8. The selected CARs were then evaluated using target-specific cytotoxic assays.

[0415] 7.2.1. Materials and Methods 7.2.1.1 Vector Design We designed various CAR constructs containing scFv with 4C8 VH and VL domains. In some constructs, VH and VL were linked together by a single long linker (GGGGS)3 (SEQ ID NO: 184), while other constructs contained two scFv in a tandem configuration with a single short linker GGGGS (SEQ ID NO: 183) between VH and VL and a single long linker (GGGGS)3 (SEQ ID NO: 184) between each scFv (see Figures 5A-5H). VH and VL were linked in various directions to three different hinges (CD8a, IgG4-short chain, IgG4-long chain), followed by a second-generation CAR-T (CD28 intracellular signaling domain, and CD3-zeta intracellular chain). The N-terminus of the scFv was linked to the CD8a signaling sequence. 4C8 CAR-T was subcloned into the Virapower lentiviral vector pLENTI6.3-V5-DEST(Invitrogen).

[0416] Table 5A shows the nucleotide sequence encoding CAR. Table 5B shows the amino acid sequence of CAR.

[0417] [Table 27-1]

[0418] [Table 27-2]

[0419] [Table 27-3]

[0420] Table 27-4

[0421] Table 27-5

[0422] Table 27-6

[0423] Table 27-7

[0424] Table 27-8

[0425] Table 27-9

[0426] Table 27-10

[0427] Table 27-11

[0428] Table 28-1

[0429] Table 28-2

[0430] [Table 28-3]

[0431] 7.2.1.2 Transduction and Propagation Lentiviruses were produced in HEK293 T cells transfected with pGO-4C8, pVSVG, and pPAX2 using PEI overnight. Lentiviral supernatant was collected after 24 hours. To remove adherent cells, healthy donor PBMCs were isolated using Lymphoprep gravity centrifugation followed by plastic adhesion. Non-adherent PBMCs were cultured in RPMI-1640 Dutch modified containing 10% FBS, 50 μM 2-mercaptoethanol, and 20 ng / ml rIL-2 and activated with the human T-activator CD3 / CD28 Dynabead. After activation, T cells were transduced twice with viral supernatant for 24 hours each time. Transduced CAR T cells were stored in 0.5 × 10⁶ cells until ready for use in research. 6 cells / mL~cells 1×10 6 They expanded and grew in the culture medium at a density of cells / mL.

[0432] 7.2.1.3 Cytotoxic Assay HaCaT WT and COSMC KO cells were implanted in 96-well plates at a density of 20,000 cells per well and allowed to adhere overnight. Two days later, CAR T cells were added in an effector-target cell ratio of 5:1 or 3:1 and incubated for 6 hours. Cytotoxicity of target cells co-cultured with CAR T cells was assessed by lactate dehydrogenase cytotoxicity assay (abcam) according to the manufacturer's instructions. For a 100% cell death control, 1% tween in PBS was used for complete lysis of all cells. To assess IFN-γ production by CAR T cells, supernatant was collected from the co-culture and ELISA was performed according to the manufacturer's instructions (abcam).

[0433] 7.2.2.Results While approximately 45% cytotoxicity was observed in HaCaT WT co-cultures, 4C8 CAR T cells (Construct 1) exhibited approximately 2-3 times higher cytotoxicity against COSMC KO HaCaT cells than against their wild-type counterparts (Figure 3A). Consistent with this, even greater IFN-γ production was found in co-cultures with COSMC KO HaCaT cells compared to WT HaCaT cells, indicating stronger activation of CAR T cells by COSMC KO cells (Figure 3B).

[0434] Untransfected T cells did not show significant cytotoxicity against either HaCaT WT cells or COSMC KO HaCaT cells (Figure 4). In addition, CAR-T cells were observed to be more functional when the VL was positioned at the N-terminus of the VH (Figure 4).

[0435] 7.3 Example 3: 10H4-based CAR We designed a chimeric antigen receptor (CAR) possessing the VH and VL domains of 10H4.

[0436] 7.3.1. Materials and Methods 7.3.1.1 Vector Design A CAR construct was designed containing an scFv with 10H4 VH and VL domains. The VH and VL were joined together with a single long linker (GGGGS)3 (SEQ ID NO: 184) (see Figure 6). The VH and VL were ligated to a CD8a hinge, followed by a second-generation CAR-T (CD28 intracellular signaling domain and CD3-zeta intracellular chain). The N-terminus of the scFv was ligated to the CD8a signaling sequence. The 10H4 CAR-T was subcloned into the Virapower lentiviral vector pLENTI6.3-V5-DEST (Invitrogen).

[0437] Table 6A shows the nucleotide sequence encoding 10H4 CAR. Table 6B shows the amino acid sequence of 10H4 CAR.

[0438] [Table 29]

[0439] [Table 30]

[0440] 8. Specific embodiments and citation of references While various specific embodiments have been illustrated and described, it is expected that various modifications can be made without departing from the essence and scope of this disclosure. The disclosure of the present invention is illustrated by the numbered embodiments described below.

[0441] 1. CD44v6 peptide GYRQ, glycosylated by GalNAc at threonine at amino acid position 5 and serine at amino acid position 12 of SEQ ID NO: 165. T PKEDSH S An anti-glyco-CD44 antibody or antigen-binding fragment that specifically binds to TTGTAAA (SEQ ID NO: 165) ("CD44v6 glycopeptide").

[0442] 2. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 1, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 1 and the light chain variable (VL) sequence of SEQ ID NO: 2 for binding to the CD44v6 glycopeptide.

[0443] 3. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 1, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 23 and the light chain variable (VL) sequence of SEQ ID NO: 24 for binding to the CD44v6 glycopeptide.

[0444] 4. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 1, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 45 and the light chain variable (VL) sequence of SEQ ID NO: 46 for binding to the CD44v6 glycopeptide.

[0445] 5. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 1, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 67 and the light chain variable (VL) sequence of SEQ ID NO: 68 for binding to the CD44v6 glycopeptide.

[0446] 6. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 1, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 206 and the light chain variable (VL) sequence of SEQ ID NO: 207 for binding to the CD44v6 glycopeptide.

[0447] 7. An anti-glyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 1 to 6, which (i) specifically binds to COSMC knockout HaCaT cells and / or (ii) specifically binds to COSMC knockout HEK293 cells that recombinantly express CD44.

[0448] 8. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 7, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 1 and the light chain variable (VL) sequence of SEQ ID NO: 2 for binding to COSMC knockout HaCaT cells and / or COSMC knockout HEK293 cells that recombinantly express CD44.

[0449] 9. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 7, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 23 and the light chain variable (VL) sequence of SEQ ID NO: 24 for binding to COSMC knockout HaCaT cells and / or COSMC knockout HEK293 cells that recombinantly express CD44.

[0450] 10. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 7, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 45 and the light chain variable (VL) sequence of SEQ ID NO: 46 for binding to COSMC knockout HaCaT cells and / or COSMC knockout HEK293 cells that recombinantly express CD44.

[0451] 11. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 7, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 67 and the light chain variable (VL) sequence of SEQ ID NO: 68 for binding to COSMC knockout HaCaT cells and / or COSMC knockout HEK293 cells that recombinantly express CD44.

[0452] 12. The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 7, wherein the anti-glyco-CD44 antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment comprising the heavy chain variable (VH) sequence of SEQ ID NO: 206 and the light chain variable (VL) sequence of SEQ ID NO: 207 for binding to COSMC knockout HaCaT cells and / or COSMC knockout HEK293 cells that recombinantly express CD44.

[0453] 13. (a) Complementarity Determination Region (CDR) H1 containing the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 125, SEQ ID NO: 153, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 246, or SEQ ID NO: 256; (b) CDR-H2 containing the amino acid sequences of SEQ ID NOs. 90, 94, 98, 229, 233, and 237; (c) CDR-H3 containing the amino acid sequences of SEQ ID NO: 103, SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 137, SEQ ID NO: 143, SEQ ID NO: 149; SEQ ID NO: 242, or SEQ ID NO: 252; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 104, SEQ ID NO: 110, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 132, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 150, SEQ ID NO: 243, or SEQ ID NO: 253; (e) CDR-L2 containing the amino acid sequences of SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 230, and SEQ ID NO: 234; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 231 An anti-glycoCD44 antibody or antigen-binding fragment according to any one of embodiments 1 to 12, comprising:

[0454] 14. An antiglyco-CD44 antibody or antigen-binding fragment according to Embodiment 13, wherein the amino acid designated as X1 in SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 125 is Y.

[0455] 15. An antiglyco-CD44 antibody or antigen-binding fragment according to Embodiment 13, wherein the amino acid designated as X1 in SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 125 is F.

[0456] 16. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 15, wherein the amino acid designated as X2 in SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 125 is T.

[0457] 17. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 15, wherein the amino acid designated as X2 in SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 125 is S.

[0458] 18. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 17, wherein the amino acid designated as X3 in SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 125, and SEQ ID NO: 153 is Y.

[0459] 19. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 17, wherein the amino acid designated as X3 in SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 125, and SEQ ID NO: 153 is F.

[0460] 20. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 19, wherein the amino acid designated as X4 in SEQ ID NO: 89, SEQ ID NO: 93, and SEQ ID NO: 125 is W.

[0461] 21. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 19, wherein the amino acid designated as X4 in SEQ ID NO: 89, SEQ ID NO: 93, and SEQ ID NO: 125 is A.

[0462] 22. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 19, wherein the amino acid designated as X4 in SEQ ID NO: 89, SEQ ID NO: 93, and SEQ ID NO: 125 is G.

[0463] 23. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 22, wherein the amino acid designated as X5 in SEQ ID NO: 93 and SEQ ID NO: 125 is M.

[0464] 24. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 22, wherein the amino acid designated as X5 in SEQ ID NO: 93 and SEQ ID NO: 125 is I.

[0465] 25. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 24, wherein the amino acid designated as X6 in SEQ ID NO: 93 and SEQ ID NO: 125 is H.

[0466] 26. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 24, wherein the amino acid designated as X6 in SEQ ID NO: 93 and SEQ ID NO: 125 is S.

[0467] 27. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 26, wherein the amino acid designated as X7 in SEQ ID NO: 94 is N.

[0468] 28. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 26, wherein the amino acid designated as X7 in SEQ ID NO: 94 is E.

[0469] 29. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 26, wherein the amino acid designated as X7 in SEQ ID NO: 94 is Y.

[0470] 30. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 29, wherein the amino acid designated as X8 in SEQ ID NO: 90, SEQ ID NO: 94, and SEQ ID NO: 98 is Y.

[0471] 31. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 29, wherein the amino acid designated as X8 in SEQ ID NO: 90, SEQ ID NO: 94, and SEQ ID NO: 98 is S.

[0472] 32. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 31, wherein the amino acid designated as X9 in SEQ ID NO: 90, SEQ ID NO: 94, and SEQ ID NO: 98 is P.

[0473] 33. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 31, wherein the amino acid designated as X9 in SEQ ID NO: 90, SEQ ID NO: 94, and SEQ ID NO: 98 is S.

[0474] 34. X in SEQ ID NOs. 90, 94, and 98 10 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 33, wherein the amino acid specified is R.

[0475] 35. X in SEQ ID NOs. 90, 94, and 98 10 The anti-glyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 33, wherein the specified amino acid is G.

[0476] 36. X in SEQ ID NOs. 90, 94, and 98 11 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 35, wherein the amino acid specified is S.

[0477] 37. X in SEQ ID NOs. 90, 94, and 98 11 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 35, wherein the specified amino acid is G.

[0478] 38. X in SEQ ID NOs. 90, 94, and 98 12 An anti-glyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 37, wherein the specified amino acid is G.

[0479] 39. X in SEQ ID NOs. 90, 94, and 98 12 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 37, wherein the amino acid specified is S.

[0480] 40. X in SEQ ID NOs. 90, 94, and 98 13 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 39, wherein the specified amino acid is T.

[0481] 41. X in SEQ ID NOs. 90, 94, and 98 13 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 39, wherein the amino acid specified is Y.

[0482] 42. X in sequence numbers 90 and 94 14 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 41, wherein the specified amino acid is T.

[0483] 43. X in SEQ ID NOs. 90 and 94 14 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 41, wherein the amino acid specified is I.

[0484] 44. X in sequence number 94 15 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 43, wherein the amino acid specified is N.

[0485] 45. X in sequence number 94 15 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 43, wherein the amino acid specified is Y.

[0486] 46. ​​X in sequence number 94 16An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 45, wherein the amino acid specified is D.

[0487] 47. X in sequence number 94 16 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 45, wherein the amino acid specified is P.

[0488] 48. X in sequence number 94 16 The anti-glyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 45, wherein the amino acid specified is A.

[0489] 49. X in sequence number 94 17 An anti-glyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 48, wherein the specified amino acid is G.

[0490] 50. X in sequence number 94 17 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 48, wherein the amino acid specified is D.

[0491] 51. X in sequence number 94 18 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 50, wherein the amino acid specified is Y.

[0492] 52. X in sequence number 94 18 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 50, wherein the specified amino acid is T.

[0493] 53. X in sequence number 94 19 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 52, wherein the amino acid specified is F.

[0494] 54. X in sequence number 9419 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 52, wherein the amino acid specified is V.

[0495] 55. X in sequence number 94 20 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 54, wherein the designated amino acid is K.

[0496] 56. X in sequence number 94 20 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 54, wherein the specified amino acid is T.

[0497] 57. X in sequence number 94 21 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 56, wherein the amino acid specified is S.

[0498] 58. X in sequence number 94 21 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 56, wherein the specified amino acid is G.

[0499] 59. X in Sequence IDs 91 and 95 22 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 58, wherein the specified amino acid is G.

[0500] 60. X in Sequence ID No. 91 and Sequence ID No. 95 22 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 58, wherein the amino acid specified is S.

[0501] 61. X in Sequence ID No. 91 and Sequence ID No. 95 22 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 58, wherein the specified amino acid is L.

[0502] 62. X in Sequence IDs 91 and 95 23 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 61, wherein the specified amino acid is T.

[0503] 63. X in Sequence ID No. 91 and Sequence ID No. 95 23 The anti-glyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 61, wherein the amino acid specified is I.

[0504] 64. X in Sequence IDs 91 and 95 24 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 63, wherein the amino acid specified is N.

[0505] 65. X in SEQ ID NOs. 91 and 95 24 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 63, wherein the amino acid specified is S.

[0506] 66. X in sequence number 95 25 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 65, wherein the amino acid specified is N.

[0507] 67. X in sequence number 95 25 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 65, wherein the amino acid specified is R.

[0508] 68. X in sequence number 95 26 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 67, wherein the amino acid specified is R.

[0509] 69. X in sequence number 95 26An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 67, wherein the specified amino acid is L.

[0510] 70. X in sequence number 95 27 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 69, wherein the amino acid specified is A.

[0511] 71. X in sequence number 95 27 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 69, wherein the amino acid specified is H.

[0512] 72. X in sequence number 95 27 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 69, wherein the amino acid specified is F.

[0513] 73. X in sequence number 95 28 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 72, wherein the amino acid specified is P.

[0514] 74. X in sequence number 95 28 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 72, wherein the amino acid specified is S.

[0515] 75. X in sequence number 92 29 The anti-glyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 74, wherein the amino acid specified is A.

[0516] 76. X in sequence number 92 29 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 74, wherein the amino acid specified is Q.

[0517] 77. X in sequence number 9229 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 74, wherein the specified amino acid is L.

[0518] 78. X in sequence number 92 30 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 77, wherein the specified amino acid is L.

[0519] 79. X in sequence number 92 30 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 77, wherein the amino acid specified is Q.

[0520] 80. X in sequence number 92 31 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 79, wherein the specified amino acid is L.

[0521] 81. X in sequence number 92 31 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 79, wherein the specified amino acid is G.

[0522] 82. X in sequence number 92 31 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 79, wherein the specified amino acid is W.

[0523] 83. X in sequence number 92 32 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 82, wherein the amino acid specified is Y.

[0524] 84. X in sequence number 92 32 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 82, wherein the amino acid specified is S.

[0525] 85. X in sequence number 92 32 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 82, wherein the specified amino acid is T.

[0526] 86. X in sequence number 92 33 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 85, wherein the amino acid specified is S.

[0527] 87. X in sequence number 92 33 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 85, wherein the amino acid specified is T.

[0528] 88. X in sequence number 92 33 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 85, wherein the amino acid specified is H.

[0529] 89. X in sequence number 92 34 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 88, wherein the amino acid specified is N.

[0530] 90. X in sequence number 92 34 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 88, wherein the specified amino acid is L.

[0531] 91. X in sequence number 92 34 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 88, wherein the amino acid specified is Q.

[0532] 92. X in sequence number 92 35 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 91, wherein the amino acid specified is Y.

[0533] 93. X in sequence number 92 35 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 91, wherein the amino acid specified is P.

[0534] 94. X in sequence number 92 36 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 93, wherein the specified amino acid is W.

[0535] 95. X in sequence number 92 36 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 93, wherein the amino acid specified is F.

[0536] 96. X in sequence number 92 37 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 95, wherein the amino acid specified is Y.

[0537] 97. X in sequence number 92 37 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 to 95, wherein the amino acid specified is T.

[0538] 98. X in sequence numbers 228, 236, and 246 41 The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 13, wherein the specified amino acid is Y.

[0539] 99. X in sequence numbers 228, 232, and 246 41 The anti-glyco-CD44 antibody or antigen-binding fragment according to Embodiment 13, wherein the specified amino acid is F.

[0540] 100. X in sequence numbers 228, 236, and 246 42The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13, 98, and 99, wherein the amino acid specified is F.

[0541] 101. X in sequence numbers 228, 236, and 246 42 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13, 98, and 99, wherein the amino acid specified is I.

[0542] 102. X in sequence numbers 228, 236, and 246 43 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 101, wherein the amino acid specified is T.

[0543] 103. X in Sequence ID No. 228, Sequence ID No. 236, and Sequence ID No. 246 43 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 101, wherein the amino acid specified is S.

[0544] 104. X in Sequence ID No. 228, Sequence ID No. 236, and Sequence ID No. 246 43 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 101, wherein the amino acid specified is N.

[0545] 105. X in SEQ ID NOs. 228, 232, 236, 246, and 256 44 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 104, wherein the amino acid specified is S.

[0546] 106. X in SEQ ID NOs. 228, 232, 236, 246, and 256 44The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 104, wherein the amino acid specified is T.

[0547] 107. X in SEQ ID NOs. 228, 232, 236, 246, and 256 45 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 106, wherein the amino acid specified is Y.

[0548] 108. X in SEQ ID NOs. 228, 232, 236, 246, and 256 45 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 106, wherein the amino acid specified is F.

[0549] 109. X in Sequence ID No. 228, Sequence ID No. 232, and Sequence ID No. 246 46 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 108, wherein the amino acid specified is W.

[0550] 110. X in sequence numbers 228, 232, and 246 46 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 108, wherein the amino acid specified is A.

[0551] 111. X in Sequence ID No. 228, Sequence ID No. 232, and Sequence ID No. 246 46 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 108, wherein the specified amino acid is G.

[0552] 112. X in sequence numbers 228, 232, and 246 46An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 108, wherein the amino acid specified is H.

[0553] 113. X in Sequence ID No. 232 and Sequence ID No. 246 47 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 112, wherein the amino acid specified is M.

[0554] 114. X in Sequence IDs 232 and 246 47 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 112, wherein the amino acid specified is I.

[0555] 115. X in Sequence ID No. 232 and Sequence ID No. 246 48 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 114, wherein the amino acid specified is H.

[0556] 116. X in Sequence ID No. 232 and Sequence ID No. 246 48 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 114, wherein the amino acid specified is S.

[0557] 117. X in Sequence ID No. 232 and Sequence ID No. 246 48 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 114, wherein the amino acid specified is G.

[0558] 118. X in sequence number 233 49 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 117, wherein the amino acid specified is N.

[0559] 119. X in sequence number 233 49The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 117, wherein the amino acid specified is E.

[0560] 120. X in sequence number 233 49 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 117, wherein the amino acid specified is Y.

[0561] 121. X in sequence number 233 49 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 117, wherein the amino acid specified is I.

[0562] 122. X in sequence number 233 50 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 121, wherein the amino acid specified is I.

[0563] 123. X in sequence number 233 50 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 121, wherein the amino acid specified is V.

[0564] 124. X in sequence numbers 229, 233, and 237 51 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 123, wherein the amino acid specified is Y.

[0565] 125. X in Sequence ID No. 229, Sequence ID No. 233, and Sequence ID No. 237 51 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 123, wherein the amino acid specified is S.

[0566] 126. X in sequence numbers 229, 233, and 237 52An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 125, wherein the amino acid specified is P.

[0567] 127. X in sequence numbers 229, 233, and 237 52 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 125, wherein the amino acid specified is S.

[0568] 128. X in sequence numbers 229, 233, and 237 52 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 125, wherein the amino acid specified is H.

[0569] 129. X in sequence numbers 229, 233, and 237 53 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 128, wherein the amino acid specified is R.

[0570] 130. X in sequence numbers 229, 233, and 237 53 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 128, wherein the amino acid specified is G.

[0571] 131. X in sequence numbers 229, 233, and 237 53 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 128, wherein the amino acid specified is D.

[0572] 132. X in sequence numbers 229, 233, and 237 54 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 131, wherein the amino acid specified is S.

[0573] 133. X in SEQ ID NOs. 90, 94, and 98 54 The anti-glyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 131, wherein the amino acid specified is G.

[0574] 134. X in sequence numbers 229, 233, and 237 55 The anti-glyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 133, wherein the amino acid specified is G.

[0575] 135. X in sequence numbers 229, 233, and 237 55 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 133, wherein the amino acid specified is S.

[0576] 136. X in sequence numbers 229, 233, and 237 56 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 135, wherein the amino acid specified is T.

[0577] 137. X in sequence numbers 229, 233, and 237 56 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 135, wherein the amino acid specified is Y.

[0578] 138. X in sequence numbers 229 and 233 57 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 137, wherein the amino acid specified is T.

[0579] 139. X in sequence numbers 229 and 233 57The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 137, wherein the amino acid specified is I.

[0580] 140. X in sequence numbers 229 and 233 57 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 137, wherein the specified amino acid is absent.

[0581] 141. X in sequence number 233 58 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 140, wherein the amino acid specified is N.

[0582] 142. X in sequence number 233 58 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 140, wherein the amino acid specified is Y.

[0583] 143. X in sequence number 233 59 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 142, wherein the amino acid specified is D.

[0584] 144. X in sequence number 233 59 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 142, wherein the amino acid specified is P.

[0585] 145. X in sequence number 233 59 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 142, wherein the amino acid specified is A.

[0586] 146. X in sequence number 233 60The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 145, wherein the amino acid specified is G.

[0587] 147. X in sequence number 233 60 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 145, wherein the amino acid specified is D.

[0588] 148. X in sequence number 233 60 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 145, wherein the amino acid specified is T.

[0589] 149. X in sequence number 233 61 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 148, wherein the amino acid specified is Y.

[0590] 150. X in sequence number 233 61 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 148, wherein the amino acid specified is T.

[0591] 151. X in sequence number 233 61 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 148, wherein the amino acid specified is W.

[0592] 152. X in sequence number 233 62 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 151, wherein the amino acid specified is F.

[0593] 153. X in sequence number 233 62The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 151, wherein the amino acid specified is V.

[0594] 154. X in sequence number 233 62 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 151, wherein the amino acid specified is A.

[0595] 155. X in sequence number 233 63 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 154, wherein the amino acid specified is K.

[0596] 156. X in sequence number 233 63 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 154, wherein the amino acid specified is T.

[0597] 157. X in sequence number 233 64 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 156, wherein the amino acid specified is S.

[0598] 158. X in sequence number 233 64 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 156, wherein the specified amino acid is G.

[0599] 159. X in Sequence IDs 230 and 234 65 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 158, wherein the specified amino acid is G.

[0600] 160. X in sequence numbers 230 and 234 65An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 158, wherein the amino acid specified is S.

[0601] 161. X in sequence numbers 230 and 234 65 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 158, wherein the specified amino acid is L.

[0602] 162. X in Sequence IDs 230 and 234 65 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 158, wherein the designated amino acid is K.

[0603] 163. X in Sequence IDs 230 and 234 66 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 162, wherein the amino acid specified is T.

[0604] 164. X in sequence numbers 230 and 234 66 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 162, wherein the amino acid specified is I.

[0605] 165. X in Sequence IDs 230 and 234 66 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 162, wherein the amino acid specified is A.

[0606] 166. X in Sequence IDs 230 and 234 67 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 165, wherein the amino acid specified is N.

[0607] 167. X in Sequence IDs 230 and 23467 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 165, wherein the amino acid specified is S.

[0608] 168. X in sequence number 234 68 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 167, wherein the amino acid specified is N.

[0609] 169. X in sequence number 234 68 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 167, wherein the amino acid specified is R.

[0610] 170. X in sequence number 234 68 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 167, wherein the amino acid specified is T.

[0611] 171. X in sequence number 234 69 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 170, wherein the amino acid specified is R.

[0612] 172. X in sequence number 234 69 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 170, wherein the amino acid specified is L.

[0613] 173. X in sequence number 234 70 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 172, wherein the amino acid specified is A.

[0614] 174. X in sequence number 234 70An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 172, wherein the amino acid specified is H.

[0615] 175. X in sequence number 234 70 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 172, wherein the amino acid specified is F.

[0616] 176. X in sequence number 234 71 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 175, wherein the amino acid specified is P.

[0617] 177. X in sequence number 234 71 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 175, wherein the amino acid specified is S.

[0618] 178. X in sequence number 231 72 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 177, wherein the amino acid specified is A.

[0619] 179. X in sequence number 231 72 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 177, wherein the amino acid specified is Q.

[0620] 180. X in sequence number 231 72 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 177, wherein the amino acid specified is L.

[0621] 181. X in sequence number 231 73An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 180, wherein the amino acid specified is L.

[0622] 182. X in sequence number 231 73 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 180, wherein the amino acid specified is Q.

[0623] 183. X in sequence number 231 73 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 180, wherein the amino acid specified is G.

[0624] 184. X in sequence number 231 74 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 183, wherein the amino acid specified is L.

[0625] 185. X in sequence number 231 74 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 183, wherein the specified amino acid is G.

[0626] 186. X in sequence number 231 74 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 183, wherein the amino acid specified is W.

[0627] 187. X in sequence number 231 75 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 186, wherein the amino acid specified is Y.

[0628] 188. X in sequence number 231 75An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 186, wherein the amino acid specified is S.

[0629] 189. X in sequence number 231 75 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 186, wherein the amino acid specified is T.

[0630] 190. X in sequence number 231 76 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 189, wherein the amino acid specified is S.

[0631] 191. X in sequence number 231 76 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 189, wherein the amino acid specified is T.

[0632] 192. X in sequence number 231 76 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 189, wherein the amino acid specified is H.

[0633] 193. X in sequence number 231 76 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 189, wherein the amino acid specified is K.

[0634] 194. X in sequence number 231 77 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 193, wherein the amino acid specified is N.

[0635] 195. X in sequence number 231 77An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 193, wherein the amino acid specified is L.

[0636] 196. X in sequence number 231 77 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 193, wherein the amino acid specified is Q.

[0637] 197. X in sequence number 231 77 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 193, wherein the specified amino acid is G.

[0638] 198. X in sequence number 231 78 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 197, wherein the amino acid specified is Y.

[0639] 199. X in sequence number 231 78 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 197, wherein the amino acid specified is P.

[0640] 200. X in sequence number 231 78 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 197, wherein the amino acid specified is D.

[0641] 201. X in sequence number 231 79 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 200, wherein the amino acid specified is W.

[0642] 202. X in sequence number 231 79The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 200, wherein the amino acid specified is F.

[0643] 203. X in sequence number 231 79 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 200, wherein the amino acid specified is I.

[0644] 204. X in sequence number 231 80 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 203, wherein the amino acid specified is Y.

[0645] 205. X in sequence number 231 80 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 203, wherein the amino acid specified is T.

[0646] 206. X in sequence number 231 80 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 and 98 to 203, wherein the amino acid specified is H.

[0647] 207. X in sequence number 231 81 The antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 206, wherein the amino acid specified is P.

[0648] 208. X in sequence number 231 81 An antiglyco-CD44 antibody or antigen-binding fragment according to any one of embodiments 13 and 98 to 206, wherein the specified amino acid is absent.

[0649] 209. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 89.

[0650] 210. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 93.

[0651] 211. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 97.

[0652] 212. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 125.

[0653] 213. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 153.

[0654] 214. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 228.

[0655] 215. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 232.

[0656] 216. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 236.

[0657] 217. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 246.

[0658] 218. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 208, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 256.

[0659] 219. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 213, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 90.

[0660] 220. An anti-glyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 213, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 94.

[0661] 221. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 213, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 98.

[0662] 222. An anti-glyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 213, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 229.

[0663] 223. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 213, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 233.

[0664] 224. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 213, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 237.

[0665] 225. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 224, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 103.

[0666] 226. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 to 224, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 109.

[0667] 227. An antiglyco-CD44 antibody or antigen-binding fragment according to any one of Embodiments 13 t...

Claims

1. (a) heavy chain variable (VH) sequence of SEQ ID NO: 1, and light chain variable (VL) sequence of SEQ ID NO: 2; (b) heavy chain variable (VH) sequence of SEQ ID NO: 23, and light chain variable (VL) sequence of SEQ ID NO: 24; (c) heavy chain variable (VH) sequence of SEQ ID NO: 45, and light chain variable (VL) sequence of SEQ ID NO: 46; (d) heavy chain variable (VH) sequence of SEQ ID NO: 67, and light chain variable (VL) sequence of SEQ ID NO: 68; or (e) heavy chain variable (VH) sequence of SEQ ID NO: 206, and light chain variable (VL) sequence of SEQ ID NO: 207 An anti-glycoCD44 antibody or antigen-binding fragment thereof that comprises an antibody or antigen-binding fragment and competes with respect to binding to CD44v6 peptide GYRQTPKEDSHSTTGTAAA (SEQ ID NO: 165) glycosylated with GalNAc at threonine at amino acid position 5 of SEQ ID NO: 165 and serine at amino acid position 12 of SEQ ID NO:

165.

2. (a) heavy chain variable (VH) sequence of SEQ ID NO: 1, and light chain variable (VL) sequence of SEQ ID NO: 2; (b) heavy chain variable (VH) sequence of SEQ ID NO: 23, and light chain variable (VL) sequence of SEQ ID NO: 24; (c) heavy chain variable (VH) sequence of SEQ ID NO: 45, and light chain variable (VL) sequence of SEQ ID NO: 46; (d) heavy chain variable (VH) sequence of SEQ ID NO: 67, and light chain variable (VL) sequence of SEQ ID NO: 68; or (e) heavy chain variable (VH) sequence of SEQ ID NO: 206, and light chain variable (VL) sequence of SEQ ID NO: 207 An antibody or antigen-binding fragment containing the above competes with an anti-glyco-CD44 antibody or antigen-binding fragment for specific binding to COSMC knockout HaCaT cells and / or COSMC knockout HEK293 cells expressing the CD44v6 peptide GYRQTPKEDSHSTTGTAAAA (SEQ ID NO: 165), which is glycosylated by GalNAc at threonine at amino acid position 5 and serine at amino acid position 12 of SEQ ID NO:

165.

3. (a) VH, which includes complementarity-determining region (CDR)-H1 containing the amino acid sequence of SEQ ID NO: 3, CDR-H2 containing the amino acid sequence of SEQ ID NO: 4, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 5; and VL, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 8; (b) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 11, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; (c) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 15, CDR-H2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 18, CDR-L2 containing the amino acid sequence of SEQ ID NO: 19, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 20; (d) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; (e) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 31, CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36; (f) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 37, CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; (g) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 47, CDR-H2 containing the amino acid sequence of SEQ ID NO: 48, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 49, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 50, CDR-L2 containing the amino acid sequence of SEQ ID NO: 51, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 52; (h) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, CDR-H2 containing the amino acid sequence of SEQ ID NO: 54, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 55; and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 56, CDR-L2 containing the amino acid sequence of SEQ ID NO: 57, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 58; (i) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 61, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64; (j) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 69, CDR-H2 containing the amino acid sequence of SEQ ID NO: 70, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 71, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 74; (k) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80; (l) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 81, CDR-H2 containing the amino acid sequence of SEQ ID NO: 82, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 83, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 84, CDR-L2 containing the amino acid sequence of SEQ ID NO: 85, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 86; (m) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 208, CDR-H2 containing the amino acid sequence of SEQ ID NO: 209, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 210, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 211, CDR-L2 containing the amino acid sequence of SEQ ID NO: 212, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 213; (n) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 214, CDR-H2 containing the amino acid sequence of SEQ ID NO: 215, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 216, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 217, CDR-L2 containing the amino acid sequence of SEQ ID NO: 218, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 219; (o) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 223, CDR-L2 containing the amino acid sequence of SEQ ID NO: 224, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 225; An anti-glycoCD44 antibody or antigen-binding fragment having the properties of an anti-glycoCD44 antibody.

4. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 3, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 4, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 5, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

8.

5. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 11, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

14.

6. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 15, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 17, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 18, CDR-L2 containing the amino acid sequence of SEQ ID NO: 19, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

20.

7. (a) VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 2; (b) VH containing an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 1 and VL containing an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 2; (c) VH containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 1 and VL containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 2 The anti-glycoCD44 antibody or antigen-binding fragment according to claim 4, comprising:

8. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 4, comprising VH containing the amino acid sequence of SEQ ID NO: 1 and VL containing the amino acid sequence of SEQ ID NO:

2.

9. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and VH containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

30.

10. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 31, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

36.

11. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 37, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

42.

12. (a) VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 23, and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 24; (b) VH containing an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 23 and VL containing an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 24; or (c) VH containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 23 and VL containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 24 The anti-glycoCD44 antibody or antigen-binding fragment according to claim 9, comprising:

13. VH containing the amino acid sequence of SEQ ID NO: 23 and VL containing the amino acid sequence of SEQ ID NO: 24 The anti-glycoCD44 antibody or antigen-binding fragment according to claim 9, comprising:

14. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 47, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 48, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 49, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 50, CDR-L2 containing the amino acid sequence of SEQ ID NO: 51, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

52.

15. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 54, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 55, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 56, CDR-L2 containing the amino acid sequence of SEQ ID NO: 57, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

58.

16. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 61, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

64.

17. (a) VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 45, and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 46; (b) VH containing an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 45, and VL containing an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 46; or (c) VH containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 45, and VL containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

46. The anti-glycoCD44 antibody or antigen-binding fragment according to claim 14, comprising:

18. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 14, comprising VH containing the amino acid sequence of SEQ ID NO: 45 and VL containing the amino acid sequence of SEQ ID NO:

46.

19. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 69, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 70, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 71, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

74.

20. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

80.

21. An anti-glyco-CD44 antibody or antigen-binding fragment comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 81, VH containing CDR-H2 containing the amino acid sequence of SEQ ID NO: 82, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 83, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 84, CDR-L2 containing the amino acid sequence of SEQ ID NO: 85, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

86.

22. (a) VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 67, and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 68; (b) VH containing an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 67, and VL containing an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 68; or (c) VH containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 67, and VL containing an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

68. The anti-glycoCD44 antibody or antigen-binding fragment according to claim 19, comprising:

23. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 19, comprising VH containing the amino acid sequence of SEQ ID NO: 67 and VL containing the amino acid sequence of SEQ ID NO:

68.

24. An anti-glycoCD44 antibody or antigen-binding fragment having a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 208, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 209, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 210, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 211, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 212, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

213. **Claim 25** An anti-glycoCD44 antibody or antigen-binding fragment having a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 214, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 215, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 216, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 217, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 218, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

219. **Claim 26** An anti-glycoCD44 antibody or antigen-binding fragment having a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 223, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 224, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

225. **Claim 27** (a) a VH comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 206, and a VL comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 207; (b) a VH comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 206, and a VL comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 207; or (c) a VH comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 206, and a VL comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 207 The anti-glyco CD44 antibody or antigen-binding fragment according to claim 24, having

28. The anti-glyco CD44 antibody or antigen-binding fragment according to claim 24, having VH comprising the amino acid sequence of SEQ ID NO: 206 and VL comprising the amino acid sequence of SEQ ID NO:

207.

29. The anti-glyco CD44 antibody or antigen-binding fragment according to claim 3, which preferentially binds to a glyco CD44 epitope overexpressed in cancer cells as compared to normal cells.

30. To the CD44v6 peptide GYRQTPKEDSHSTTGTAAA (SEQ ID NO: 165) glycosylated with STn at threonine at amino acid position 5 of SEQ ID NO: 165 and serine at amino acid position 12 of SEQ ID NO: 165, as follows: (a) 1 nM to 200 nM when measured by surface plasmon resonance or biolayer interferometry; (b) 1 nM to 150 nM when measured by surface plasmon resonance or biolayer interferometry; (c) 1 nM to 100 nM when measured by surface plasmon resonance or biolayer interferometry; (d) 1 nM to 50 nM when measured by surface plasmon resonance or biolayer interferometry; (e) 5 nM to 200 nM when measured by surface plasmon resonance or biolayer interferometry; (f) 5 nM to 100 nM when measured by surface plasmon resonance or biolayer interferometry; (g) 5 nM to 50 nM when measured by surface plasmon resonance or biolayer interferometry; (h) 5 nM to 25 nM when measured by surface plasmon resonance or biolayer interferometry; (i) 5 nM to 10 nM when measured by surface plasmon resonance or biolayer interferometry; (j) When measured by surface plasmon resonance or biolayer interferometry, 10 nM to 200 nM; (k) When measured by surface plasmon resonance or biolayer interferometry, 10 nM to 150 nM; (l) When measured by surface plasmon resonance or biolayer interferometry, 10 nM to 100 nM; (m) When measured by surface plasmon resonance or biolayer interferometry, 10 nM to 50 nM; (n) When measured by surface plasmon resonance or biolayer interferometry, 10 nM to 25 nM; (o) When measured by surface plasmon resonance or biolayer interferometry, 50 nM to 200 nM; (p) When measured by surface plasmon resonance or biolayer interferometry, 50 nM to 150 nM; (q) When measured by surface plasmon resonance or biolayer interferometry, 50 nM to 100 nM; (r) When measured by surface plasmon resonance or biolayer interferometry, 100 nM to 200 nM; or (s) When measured by surface plasmon resonance or biolayer interferometry, 100 nM to 150 nM The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 3, which binds with a binding affinity (KD).

31. Sequences glycosylated in vitro using purified recombinant human glycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4 (VTSAPDTRPAPGSTAAPPAHG) ３ The anti-glycoCD44 antibody or antigen-binding fragment according to claim 3, which does not specifically bind to a first MUC1 glycopeptide having (SEQ ID NO: 205).

32. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 31, wherein the binding affinity to the CD44v6 glycopeptide is at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 200 times the binding affinity of the anti-glyco-CD44 antibody or antigen-binding fragment to the first MUC1 glycopeptide, and the CD44v6 glycopeptide is the CD44v6 peptide GYRQTPKEDSHSTTGTAAAA (SEQ ID NO: 165) glycosylated with GalNAc at threonine at amino acid position 5 of SEQ ID NO: 165 and serine at amino acid position 12 of SEQ ID NO:

165.

33. The anti-glycoCD44 antibody or antigen-binding fragment according to claim 32, wherein the binding affinity is measured by surface plasmon resonance.

34. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 3, which does not specifically bind to a second MUC1 glycopeptide having the sequence TAPPAHGVTSAPDTTRPAPGSTAAPPAHGVT (SEQ ID NO: 260), which is in vitro glycosylated with GalNAc at the serine and threonine residues indicated by the underlined letters.

35. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 34, wherein the binding affinity to the CD44v6 peptide is at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 200 times the binding affinity of the anti-glyco-CD44 antibody or antigen-binding fragment to the second MUC1 glycopeptide, and the CD44v6 glycopeptide is the CD44v6 peptide GYRQTPKEDSHSTTGTAAAA (SEQ ID NO: 165) glycosylated with GalNAc at threonine at amino acid position 5 of SEQ ID NO: 165 and serine at amino acid position 12 of SEQ ID NO:

165.

36. The anti-glycoCD44 antibody or antigen-binding fragment according to claim 35, wherein the binding affinity is measured by surface plasmon resonance.

37. A polyvalent anti-glycoCD44 antibody or antigen-binding fragment according to claim 3.

38. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 3, in the form of a single-chain variable fragment (scFv).

39. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 3, which is in the form of a multispecific antibody.

40. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 39, wherein the multispecific antibody is a bispecific antibody that binds to a second epitope different from a first epitope.

41. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 40, wherein the bispecific antibody is CrossMab, Fab-arm exchange antibody, bispecific T cell engager (BiTE), or biaffinity retargeting molecule (DART).

42. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 40, wherein the second epitope is a CD44 epitope.

43. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 42, wherein the second epitope is a CD44 epitope that is overexpressed in cancer cells compared to normal cells.

44. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 40, wherein the second epitope is a T cell epitope.

45. The anti-glyco-CD44 antibody or antigen-binding fragment according to claim 44, wherein the T cell epitope comprises a CD3 epitope, a CD8 epitope, a CD16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2D epitope.

46. A fusion protein comprising an amino acid sequence of an anti-glyco-CD44 antibody or antigen-binding fragment according to any one of claims 1 to 45, operably linked to at least a second amino acid sequence.

47. A chimeric antigen receptor (CAR) comprising scFv as described in claim 38.

48. The CAR according to claim 47, comprising the amino acid sequence of SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, or SEQ ID NO:

261.

49. An antibody-drug conjugate comprising an anti-glycoCD44 antibody or antigen-binding fragment according to any one of claims 1 to 45, conjugated to a cytotoxic substance.

50. A nucleic acid comprising the coding region of an antiglyco-CD44 antibody or antigen-binding fragment according to any one of claims 1 to 45.

51. A vector comprising the nucleic acid described in claim 50.

52. A host cell modified to express the nucleic acid described in claim 50.

53. A host cell comprising the vector according to claim 51.

54. (a) an anti-glycoCD44 antibody or antigen-binding fragment according to any one of claims 1 to 45, and (b) a pharmaceutical composition comprising a physiologically suitable buffer, adjuvant or diluent.

55. A pharmaceutical composition for treating cancer, comprising an effective amount of an anti-glyco-CD44 antibody or antigen-binding fragment according to any one of claims 1 to 45.

56. The pharmaceutical composition according to claim 55, wherein the cancer is breast cancer, lung cancer, genitourinary cancer, pancreatic cancer, colorectal cancer, ovarian cancer, stomach cancer, head and neck cancer, skin cancer, malignant melanoma, liver cancer, glioma, thyroid cancer, cervical cancer, or endometrial cancer.

57. A method for detecting cancer in a biological sample, comprising contacting the sample with an anti-glyco-CD44 antibody or antigen-binding fragment according to any one of claims 1 to 45, and detecting the binding of the anti-glyco-CD44 antibody or antigen-binding fragment.

58. The method according to claim 57, wherein the cancer is breast cancer, lung cancer, genitourinary cancer, pancreatic cancer, colorectal cancer, ovarian cancer, stomach cancer, head and neck cancer, skin cancer, malignant melanoma, liver cancer, glioma, thyroid cancer, cervical cancer, or endometrial cancer.

59. A peptide consisting of SEQ ID NO: 165, in which the threonine corresponding to position 5 of SEQ ID NO: 165 and the serine corresponding to position 12 of SEQ ID NO: 165 are O-glycosylated.

60. The peptide according to claim 59, wherein the O-glycosylation comprises or consists of GalNAc.

61. A composition comprising the peptide and adjuvant according to claim 60.

62. The composition according to claim 61, wherein the adjuvant comprises an aluminum salt.

63. A method for generating antibodies against tumor-associated forms of CD44v6, comprising administering the peptide described in claim 60 or the composition described in claim 61 or claim 62 to a non-human animal.

64. The method according to claim 63, further comprising collecting antibodies from the non-human animal.

65. A method for inducing an immune response to a tumor-associated form of CD44v6, comprising administering the peptide according to claim 60 or the composition according to claim 61 or claim 62 to a non-human animal.

66. The method according to any one of claims 63 to 65, wherein the non-human animal is a mouse or a rabbit.

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