Therapeutic antibodies that bind to biantennary Lewis B and Lewis Y antigens

Abstract

Disclosed herein are humanized antibodies, antigen-binding fragments thereof, and antibody conjugates that can specifically bind to certain biantennary Lewis antigens, which are expressed in a variety of cancers. The antibodies disclosed herein can be used to target cells expressing antigens to treat or detect diseases (including various cancers). Also provided are polynucleotides, vectors, and host cells for producing the disclosed antibodies and antigen-binding fragments thereof. Also provided are pharmaceutical compositions, treatment and detection methods, and uses of antibodies, antigen-binding fragments, antibody conjugates, and compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 678,890, filed May 31, 2018, pursuant to 35 U.S.C. §119(e), which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is 400100_401WO_SEQUENCE_LISTING.txt. The text file is 43.3KB, was created on May 30, 2019, and was submitted electronically through EFS-Web. Technical Field

[0005] Embodiments of the present disclosure generally relate to antibodies and antigen-binding fragments thereof that are capable of specifically binding to certain Lewis antigens, and methods of making and using the same. Compositions, polynucleotides, vectors, fusion proteins, and host cells related to the antibodies and antigen-binding fragments disclosed herein are also provided. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein are combined with biantennary Lewis antigens. B / Le B ,Le Y / Le Y ,Le B / Le Y and Le Y / Le B Antigens are capable of specific binding and can be used to treat or detect diseases characterized by expression of such antigens (eg, cancer).

[0006] background

[0007] Immunotherapy is an emerging modality for treating a variety of diseases, including various cancers. For example, since the late 1990s, the clinical efficacy of monoclonal antibodies (mAbs) with anti-tumor activity has been demonstrated (see, e.g., Topalian et al., J. Clin. Oncol. 29(36): 4828-4836 (2011)), and several best-selling cancer drugs are mAbs (e.g., rituximab, trastuzumab, and bevacizumab).

[0008] Antibody-based therapies can specifically target cells expressing antigens (such as tumor cells) and can, for example, produce cytotoxic activity in a variety of ways (including by eliciting antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and T-cell-mediated cytotoxicity). Other approaches involve the use of antibodies as carrier vehicles to selectively deliver, for example, cytotoxic agents or antiproliferative agents to kill target cells and / or inhibit the growth and metastatic spread of such cells.

[0009] The relevant criteria of monoclonal antibody therapy include, for example, the ability of antibody specific recognition and binding to the desired antigen, rather than indiscriminately binding to one or more other potential epitopes (e.g., proteins or polysaccharides expressed on healthy cells). Moreover, the antibody should not be immunogenic to the patient. In this regard, antibodies specific for human disease antigens are often produced in non-human hosts (such as mice and rabbits), and may have, for example, host species amino acid sequences and / or carbohydrate motifs that may be immunogenic in human patients. Therefore, the preferred antibodies for treatment lack or have minimized potential immunogenic properties. The characteristics of the target antigen must also be considered. Preferably, the antigen is selectively expressed or highly overexpressed in a specific disease state (e.g., cancer) so that the antibody-mediated therapy will not have an undesirable harmful "target, out of tumor" effect on healthy tissue.

[0010] Abnormal glycosylation (e.g., overexpression or misexpression of glycoproteins and glycolipids) is a common feature of many cancers (see, e.g., Blanas et al., Front. Oncol. 8: 39 (2018)). Without wishing to be bound by theory, abnormal glycosylation may affect many cellular processes that lead to cancer progression and metastasis (e.g., cell growth and metabolism, angiogenesis, cell-matrix interactions, and cell-cell adhesion). Exemplary carbohydrate antigens that are abnormally expressed in cancer include type I and type II Lewis antigens, which are terminal (at the non-reducing end) fucosylated carbohydrate epitopes of the Lewis antigen system. Type I and type II Lewis antigens include structurally related H1, H2, and Lewis A, B, X, and Y antigens, all of which share three monosaccharide units: (reducing) terminal acetylglucosamine (GlcNac); galactose (Gal); and fucose (Fuc). Type I Lewis antigens differ from type II Lewis antigens in the nature of the glycosidic bonds in their lactosamine core chains (Galβ1→3GlcNac: type I; Galβ1→4GlcNac: type II), and are present in a variety of structures (e.g., linear or branched, with one, two, or more antennae containing antigenic motifs). Lewis antigens have moderate expression levels in healthy adult tissues (e.g., digestive and reproductive epithelial cells), but are overexpressed on the cell surface in many solid cancers (including, for example, cancers of the lung, breast, liver, kidney, bladder, pancreas, and prostate), and are also associated with acute myeloid leukemia, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma.

[0011] Several therapies targeting Lewis antigens to treat cancer are known, including the discontinued Seattle Genetics / Bristol Meyers Squibb antibody drug conjugate "cBR96-Dox," which used the BR96 monoclonal antibody (Hellstrom et al., Cancer Res. 50(7):2183-2190 (1990)) as a carrier molecule to deliver doxorubicin to cells expressing Lewis antigens. Y Antigen tumor cells. Clearly, there is still a need in the art for additional therapies to treat cancers, such as cancers that express Lewis antigens as tumor-associated antigens or tumor-specific antigens. The presently disclosed embodiments address this need and provide other related advantages.

[0012] Overview

[0013] According to certain presently disclosed embodiments, an isolated antibody is provided, which comprises an immunoglobulin heavy chain having the amino acid sequence shown in SEQ ID NO:10 and an immunoglobulin light chain having the amino acid sequence shown in SEQ ID NO:11.

[0014] In certain embodiments, an isolated antibody or antigen-binding fragment thereof is provided, comprising: an immunoglobulin heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 35 and an immunoglobulin light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to:

[0015] Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen,

[0016] Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen,

[0017] Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen,

[0018] and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]

[0019] [VIII]'s Double Tentacles Y / Le B antigen,

[0020] and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] AAntigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV].

[0021] In certain embodiments, an isolated antibody or antigen-binding fragment thereof is provided, comprising: (a) an immunoglobulin heavy chain variable region, comprising a heavy chain complementary determining region 1 (VH CDR1), a heavy chain complementary determining region 2 (VH CDR2), and a heavy chain complementary determining region 3 (VH CDR3), wherein the heavy chain complementary determining region 1 (VH CDR1) comprises the amino acid sequence shown in SEQ ID NO: 2, the heavy chain complementary determining region 2 (VH CDR2) comprises the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain complementary determining region 3 (VH CDR3) comprises the amino acid sequence shown in SEQ ID NO: 4; and (b) an immunoglobulin light chain variable region, comprising a light chain complementary determining region 1 (VL CDR1), a light chain complementary determining region 2 (VL CDR2), and a light chain complementary determining region 3 (VLCDR3), wherein the light chain complementary determining region 1 (VL CDR1) comprises the amino acid sequence shown in SEQ ID NO: 6, and the light chain complementary determining region 2 (VL CDR2) comprises the amino acid sequence shown in SEQ ID NO: 7. NO:7, the light chain complementary determining region 3 (VLCDR3) comprises the amino acid sequence shown in SEQ ID NO:8; wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to:

[0022] Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen,

[0023] Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen,

[0024] Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI]B / Le Y antigen,

[0025] and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]

[0026] [VIII]'s Double Tentacles Y / Le B antigen,

[0027] and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV]

[0028] In certain embodiments described herein, the separated antibody can be a monoclonal antibody. In certain embodiments, the separated antibody or its antigen-binding fragment is a humanized antibody. In certain embodiments, the separated antibody or its antigen-binding fragment is selected from Fab fragment, F(ab')2 fragment, Fv fragment, single-chain Fv (scFv) antibody and bispecific antibody (adiabody).

[0029] Also provided herein are embodiments in which the isolated antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:35 and an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:5, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to:

[0030] Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen,

[0031] Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen,

[0032] Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen,

[0033] and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]

[0034] [VIII]'s Double Tentacles Y / Le B antigen,

[0035] and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A Antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV].

[0036] In certain embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising (a) an immunoglobulin heavy chain variable region comprising a heavy chain complementary determining region 1 (VH CDR1), a heavy chain complementary determining region 2 (VH CDR2), and a heavy chain complementary determining region 3 (VH CDR3), wherein the heavy chain complementary determining region 1 comprises the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:2; the heavy chain complementary determining region 2 (VH CDR2) comprises the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:3; the heavy chain complementary determining region 3 (VH CDR3) comprises the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4 NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:4; and (b) an immunoglobulin light chain variable region comprising a light chain complementary determining region 1 (VL CDR1), a light chain complementary determining region 2 (VL CDR2) and a light chain complementary determining region 3 (VL CDR3), wherein the light chain complementary determining region 1 (VL CDR1) comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:6; the light chain complementary determining region 2 (VL CDR2) comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:7. NO:7 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical; the light chain complementary determining region 3 (VL CDR3) comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:8.

[0037] In certain other embodiments, an isolated antibody or antigen-binding fragment thereof according to the present disclosure is provided, which comprises the following (i), (ii), (iii), (iv), (v) or (vi), or any combination thereof: (i) a VH CDR1 comprising a variant of the amino acid sequence shown in SEQ ID NO:2, wherein the variation consists of a Y→A substitution at position 33 according to Kabat numbering; (ii) a VH CDR3 comprising a variant of the amino acid sequence shown in SEQ ID NO:4, wherein the variation consists of a Y→A substitution at position 104 according to Kabat numbering; (iii) a VH CDR3 comprising a variant of the amino acid sequence shown in SEQ ID NO:4, wherein the variation consists of an H→A substitution at position 106 according to Kabat numbering; (iv) a VL CDR1 comprising a variant of the amino acid sequence shown in SEQ ID NO:6, wherein the variation consists of a Y→A substitution at position 30 according to Kabat numbering; (v) a VL CDR1 comprising a variant of the amino acid sequence shown in SEQ ID NO:7 CDR2, wherein the variant consists of a G→A substitution at position 50 according to Kabat numbering; or (vi) a VL CDR3 comprising a variant of the amino acid sequence shown in SEQ ID NO:8, wherein the variation consists of a T→S substitution at position 93 according to Kabat numbering.

[0038] In certain other embodiments, an isolated antibody or antigen-binding fragment is provided that comprises a complementarity determining region (CDR) as described herein (i.e., a CDR having at least 90% identity to SEQ ID NOs: 2, 3, 4, 6, 7, and 8, respectively, including those CDR variants having amino acid substitutions described herein), and comprises an immunoglobulin heavy chain variable region comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 35, and an immunoglobulin light chain variable region comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 35, ID NO:5 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:5, or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:5.

[0039] In any of the embodiments described herein, an isolated antibody or antigen-binding fragment thereof, or an antibody drug conjugate comprising an antibody or antigen-binding fragment of the present disclosure may exhibit reduced (e.g., reduced in a statistically significant manner as determined using an art-accepted method) binding (including, in certain embodiments, no binding) to a monoantennary Lewis B antigen or a monoantennary Lewis Y antigen as compared to a "BBC" antibody, as disclosed herein, comprising a VL domain having the amino acid sequence shown in SEQ ID NO: 27 and a VH domain having the amino acid sequence shown in SEQ ID NO: 28. Monovalent Lewis B is a blood group antigen expressed in normal human tissues; therefore, the antibodies, antigen-binding fragments, and antibody drug conjugates disclosed herein have improved specificity for cancer antigens and reduced binding to monoantennary Lewis B antigens expressed in normal human tissues as compared to BBC antibodies. In certain embodiments, the antibodies, antigen-binding fragments, or antibody drug conjugates disclosed herein have a 1-fold, 2-fold, 3-fold, 5-fold, 6-fold, 7-fold, 8-fold or greater increase in binding to a cell surface-expressed antigen according to any one or more of Formulae [I]-[VIII] herein compared to binding to a monoantennary Lewis B antigen.

[0040] In certain embodiments, the antibody, antigen-binding fragment, or antibody drug conjugate binds to the monoantennary Lewis B antigen with 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, or 1 / 2 the affinity of BR96 (e.g., as measured in an ELISA binding assay).

[0041] In any of the embodiments described herein, the isolated antibody or antigen-binding fragment thereof, or the antibody drug conjugate comprising the antibody or antigen-binding fragment of the disclosure binds to the monoantennary Le recognized by antibody BR96 as compared to BR96. Y The invention relates to an antigen that is different from an antigen and can allow safer and more specific targeting of cancer cells in therapeutic applications according to non-limiting theory. In any of the embodiments described herein, the isolated antibody or antigen-binding fragment thereof, or an antibody drug conjugate comprising an antibody or antigen-binding fragment of the present disclosure, binds to cancer cells expressing the antigen, is effectively and stably internalized into the lysosomes of such cells, and is safely tolerated at therapeutic doses in a non-human primate model.

[0042] On the other hand, the present disclosure provides isolated polynucleotides encoding antibodies or antigen-binding fragments thereof disclosed herein. In certain embodiments, the polynucleotides are codon-optimized for expression in host cells. In certain related embodiments, a recombinant vector comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein is provided. In certain embodiments, the recombinant vector comprises an expression control sequence operably connected to a polynucleotide encoding an antibody or antigen-binding fragment thereof. In a specific embodiment, the recombinant vector is an expression vector, wherein the expression control sequence comprises a promoter.

[0043] In another embodiment, a host cell comprising a recombinant and / or expression vector of the present disclosure is provided. In certain other embodiments, a related method for producing an antibody or antigen-binding fragment thereof disclosed herein is provided, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to:

[0044] Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen,

[0045] Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen,

[0046] Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen,

[0047] and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]

[0048] [VIII]'s Double Tentacles Y / Le B antigen,

[0049] and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV], wherein the method comprises: culturing the host cell under conditions and for a time sufficient for the host cell to express a polynucleotide encoding the antibody or its antigen-binding fragment, thereby obtaining a culture comprising the antibody or its antigen-binding fragment; and recovering the antibody or its antigen-binding fragment from the culture.

[0050] In another embodiment, an antibody conjugate comprising an antibody or antigen-binding fragment of the present invention and a payload molecule connected thereto are provided. In certain embodiments, the payload molecule is covalently linked to the antibody or antigen-binding fragment thereof through a linker. In certain embodiments, the linker is selected from a cleavable linker and a non-cleavable linker. In certain embodiments, the cleavable linker is a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. In certain embodiments, the cleavable linker is a protease-sensitive linker comprising a valine-citrulline dipeptide. In some embodiments, the linker comprises a maleimide group. In certain embodiments, the antibody or antigen-binding fragment thereof disclosed herein comprises a reduced disulfide bridge in the hinge region, and the reduced disulfide bridge is coupled to the maleimide group. The following embodiments are also provided herein: wherein the linker also comprises a self-disassembling group, such as, for example, p-aminobenzyl alcohol (PABC). In certain embodiments, the antibody conjugate comprises an antibody or antigen-binding fragment disclosed herein and a payload molecule, and the payload molecule is selected from a therapeutic agent and a detectable indicator. In certain embodiments, the payload molecule is a therapeutic agent selected from the group consisting of anti-mitotic agents targeting tubulin, peptide-based toxins, pyrrolobenzodiazepine (PBD) dimers, antibiotics, pyrimidine synthesis inhibitors, antimetabolites, DNA alkylating agents, and topoisomerase inhibitors. In certain embodiments, the payload molecule is selected from the group consisting of maytansinoids, an auristatin, doxorubicin, calicheamicin, PBD dimers, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF). In certain other embodiments, the payload molecule is a detectable indicator. In certain other embodiments, the detectable indicator is selected from the group consisting of radionuclides, dyes, radioactive metals, fluorescent moieties, MRI contrast agents, microbubbles, carbon nanotubes, gold particles, fluorodeoxyglucose, enzymes, chromophores, and radiopaque markers. In specific embodiments, the detectable indicator is a radionuclide selected from the following: 68 Ga, 64 Cu, 86 Y. 89 Zr, 124 I. 99m Tc, 123 I. 111 In, 177 Lu, 131 I. 76 Br, 78 Zr, 18 F and 124T. In certain embodiments, the antibody conjugate comprises the following radionuclide chelators: maleimide-labeled DOTA, N-hydroxysuccinimide-DOTA, and deferoxamine (DFO).

[0051] Also provided herein are pharmaceutical compositions, and in some embodiments, comprise an isolated antibody, antigen-binding fragment, or antibody conjugate disclosed herein and a pharmaceutical carrier.

[0052] In certain embodiments, the disclosure also provides a method of treatment or detection, which comprises using the antibodies, antigen-binding fragments and / or antibody conjugates disclosed in the present invention. In some embodiments, a method of treating or detecting cancer is provided, wherein the method comprises administering a pharmaceutical composition as disclosed herein to a subject in need thereof. In certain embodiments, the subject suffers from or is suspected of suffering from a cancer selected from the following: gastric cancer, colon cancer, breast cancer, lung cancer, lymphoma, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, uterine cancer and squamous cell carcinoma. In certain embodiments, the cancer is selected from: gastric adenocarcinoma, mucinous gastric adenocarcinoma, undifferentiated gastric adenocarcinoma, signet ring cell gastric cancer, colon adenocarcinoma, invasive mammary ductal carcinoma, hepatocellular carcinoma, lung adenocarcinoma, squamous cell carcinoma, metastatic lymph node adenocarcinoma, mucinous ovarian adenocarcinoma, pancreatic ductal adenocarcinoma, pancreatic papillary adenocarcinoma, prostate adenocarcinoma and endometrioid carcinoma. In certain embodiments, a method is provided, comprising administering to a subject a composition disclosed herein (e.g., comprising an antibody or antigen-binding fragment thereof disclosed herein) by a route selected from the following: intravenous, parenteral, intragastric, intrapleural, intrapulmonary, rectal, intradermal, intraperitoneal, intratumoral, subcutaneous, oral, topical, transdermal, intracisternal, intrathecal, intranasal, and intramuscular. In certain other embodiments of the methods disclosed herein for detecting or treating cancer, the subject is receiving or has previously received: (a) immunosuppressive therapy; (b) stimulatory immune checkpoint molecules; (c) radiation therapy; (d) chemotherapy; (e) cellular immunotherapy; or (f) any combination of (a)-(e).

[0053] These and other aspects and embodiments of the present disclosure will become apparent by reference to the following detailed description and accompanying drawings.All references disclosed herein are hereby incorporated by reference in their entirety as if each were individually incorporated.

[0054] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0055] Figure 1The polyamino acid sequence alignment of the heavy chain variable region of the "BBC" antibody disclosed in the present invention (SEQ ID NO: 28) and the selected candidate receptor amino acid sequences (1-8; corresponding to SEQ ID NO: 44-51, respectively) is shown. The third row (dashed box) shows the human receptor sequence selected for CDR transplantation. In the middle part of the figure, the single framed amino acid (V / I / M) is a rare methionine in the human receptor sequence; during the humanization process, this residue is replaced by the more common isoleucine.

[0056] Figure 2A Shown is an amino acid sequence alignment of the light chain (LC) variable region sequences of the IMH2 / BBC antibody (SEQ ID NO: 27) and humanized variants: hBBC.8 (SEQ ID NO: 31); hBBC.9 and hBBC.9.1 (SEQ ID NO: 33); and hBBC.10, hBBC.10.1 and hBBC.10.1FQ (SEQ ID NO: 5). The hBBC CDR sequences are represented by boxes (dashed lines), and the human receptor CDR sequences are underlined. At the bottom of the figure, two single boxed amino acid residues (R / G and Y / F) show the differences in the framework region sequences. Figure 2B The relative activities of BBC antibody, hBBC.8 antibody, hBBC.8 antibody with R66G substitution mutation, hBBC.9 antibody and hBBC.10 antibody in AGS cell binding assay are shown. hBBC-LC with F71Y mutation represents hBBC.9. hBBC-LC with F71Y and R66G mutations represents hBBC.10. Figure 2C-2E The binding specificity of other generated variant antibodies to AGS cells (2C) is shown, as well as to the synthetic monoantennary Le B The binding specificity of other generated variant antibodies against AGS cells (2D, 2E) was compared with that of the WT antigen.

[0057] Figure 3 A sequence alignment within the heavy chain framework 3 region of the hBBC.10.1 antibody (SEQ ID NO:38), the hBBC.10.1FQ antibody (SEQ ID NO:39), and two FDA-approved humanized therapeutic antibodies (reference 1 and reference 2) (SEQ ID NOs:40 and 41, respectively) is provided.

[0058] Figure 4A and Figure 4B MALDI-MSMS sequencing of Fuc4(LacNAc)3Lac at m / z 2521 of COLO205GSL cells in the unbound fraction (4A) and the BBC-bound (eluted) fraction (4B) is shown. Figures 4A-7BIn , glycans are depicted as follows: open circles = Gal; closed circles = Glc; closed squares = GlcNac; closed triangles = Fuc. Figure 8A-8B The same convention is used in , except that solid circle = Man.

[0059] Figure 5A and Figure 5B MALDI-MS spectra of BBC-bound (bottom) and non-bound (top) glycans of (A) NCI-N87 GSL cells and (B) SW1116 GSL cells are shown, respectively.

[0060] Fig. 6A and Figure 6B MALDI-MSMS sequencing of Fuc4(LacNAc)3Lac at m / z 2521 in the BBC-bound (eluted) fractions of NCI-N87 GSL cells (6A) and SW1116 GSL cells (6B) is shown.

[0061] Fig. 7A and Figure 7B MALDI-MSMS sequencing of Fuc4(LacNAc)3Lac and Fuc6(LacNAc)5Lac of NCI-N87 GSL cells in the BBC-bound (eluted) fraction at m / z 2970 (7A) and m / z 3767 (7B) is provided.

[0062] Fig. 8A and Figure 8B MALDI-Q / TOF MS / MS sequencing of BBC-enriched AGS N-glycans is shown.

[0063] Fig.9A and Fig. 9B Glycan antigen (A)Le Y -Pentose and (B)Le B - ITC titration graph of pentose with hBBC.10.1 antibody.

[0064] Figure 10A-10C ITC titration plots of additional glycan antigens with hBBC.10.1 antibody are shown: (A) Le Y / Le Y -ASGA; (B)Le Y / Le Y -I antigen; (C)Le Y / Le B -I antigen.

[0065] Figures 11A-11F ITC titration plots of other glycan antigens with hBBC.10.1 antibody are shown: (A) Le X-tetrasaccharide; (B)Le A -tetrasaccharide; (C) type I H antigen; (D) type II H antigen; (E) H-ASGP; (F) Le X / Le X -ASGP.

[0066] Figure 12A-12C ITC titration graphs of glycan antigen and reference antibody "BR96" are provided: (A) Le Y -pentose; (B)Le Y / Le Y -I antigen, and (C)Le Y / Le Y -ASGP.

[0067] Fig.13A and Fig. 13B Surface plasmon resonance (SPR) sensorgrams of hBBC.10.1 and BR96 against the indicated glycan antigens are shown: (A) Le Y -Gal and Le Y / Le Y -ASGA; (B)Le B -Gal and Le B / Le B -ASGA.

[0068] Fig.14A and Fig. 14B Results of indirect ELISA of hBBC.10.1 binding to coated Lewis antigen at the indicated concentrations are shown: (A) Le Y / Le Y -ASGA-biotin; (B)Le Y -Gal-Biotin.

[0069] Fig.15 The Lewis antigen (Lewis antigen) at the indicated concentrations is shown. B / Le B -ASGA relative to Le Y / Le Y Indirect ELISA results of hBBC.10.1 binding to LeB-ASGA and LeB-Gal).

[0070] Fig.16A The results of antigen binding ELISA of hBBC.10.1 to glycan antigens including Le Y -Gal-sp3-biotin (Le Y -Gal), Le B -Gal-LC-biotin (Le B -Gal), Le Y / Le Y -ASGA-Biotin(Le Y / Le Y -ASGA), Le B / Le B -ASGA-Biotin(Le B / Le B -ASGA)、3-Le Y / 6-Le B -ASGA-Biotin(Le Y / Le B -ASGA) and 3-Le B / 6-Le Y -ASGA-Biotin(Le B / Le Y -ASGA). All antigens were coated at the same amount (1.87 pmole). Fig. 16B and Fig. 16C Results from antigen binding ELISA experiments comparing the affinity and selectivity of (16B) BBC antibody and (16C) hBBC.10.1 antibody for the antigen are shown, respectively.

[0071] Fig.17 Fluorescence microscopy images are provided showing the endocytosis of hBBC.10.1 by AGS gastric cancer cells. Left: hBBC.10.1 stained by Alexa488-conjugated anti-human IgG (green channel). Right: Overlay of staining of F-actin labeled with phalloidin rhodamine (red channel). Areas where fluorescein and rhodamine are co-distributed appear yellow under the microscope and appear as co-localized fluorescent signals in the merged image.

[0072] Fig.18 Fluorescence microscopy images showing lysosomal localization of hBBC.10.1 in AGS gastric cancer cells are provided. Left: hBBC.10.1 stained by Alexa488-conjugated anti-human IgG (green channel). Middle: Lysosomes labeled with anti-Lamp-1 antibody and then by anti-rabbit IgG (red channel). Right: Merge.

[0073] Fig.19A and Fig.19B The antitumor activity of hBBC.10.1 is shown in in vivo xenograft experiments in which (A) human DLD-1 tumor cells or (B) COLO 205 tumor cells were administered to immunodeficient SCID mice, followed by antibody administration once tumors were established.

[0074] Fig. 20A and Fig. 20BAntitumor activity of various concentrations of (A) hBBC.10.1 and (B) BR96, respectively, in an in vivo xenograft experiment in which human AGS gastric adenocarcinoma cells were administered to immunodeficient SCID mice and then the antibodies were administered once tumors were established. Fig. 20C and Fig.20D The hBBC antibodies of the present disclosure were shown to have anti-tumor activity in an in vivo xenograft experiment.

[0075] Fig.21 Direct killing of AGS tumor cells by hBBC.10.1 ("hBBC") and BR96 at various antibody concentrations is shown. Killing was measured as the percentage of target cells stained with propidium iodide (PI).

[0076] Fig. 22 The antitumor activity of hBBC.10.1 ("hBBC") and BR96 was shown in an in vivo xenograft experiment in which human TSGH 9201 gastric cancer cells were administered to immunodeficient SCID mice, followed by administration of the antibodies once tumors were established.

[0077] Fig.23 The antitumor activity of hBBC.10.1 ("hBBC") at various concentrations was shown in an in vivo xenograft experiment in which human COLO 201 colorectal adenocarcinoma cells were administered to immunodeficient SCID mice, followed by administration of the antibody once tumors were established.

[0078] Fig.24A and Fig. 24B Shown are the antitumor activities of (A) hBBC.10.1 ("hBBC") and (B) BR96 in in vivo xenograft experiments in which human COLO 201 colorectal adenocarcinoma cells were administered to immunodeficient SCID mice, followed by administration of the antibodies once tumors were established.

[0079] Sequence Description

[0080] SEQ ID NO: 1 is the amino acid sequence of the heavy chain variable (VH) domain of antibody hBBC.10.1FQ:

[0081] QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWLG YIHYTGNTKYSPSLKSRLSISRDTSKNTFYLQMNSLTTEDTAVYYCGREALRGYD AGFWFTYWGQGTLVTV.

[0082] SEQ ID NO: 2 is the amino acid sequence of the heavy chain complementarity determining region 1 (VH CDR1) of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0083] SGYTWH.

[0084] SEQ ID NO:3 is the amino acid sequence of VH CDR2 of antibody hBBC.9.1, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0085] YIHYTGNTKYSPSLKS.

[0086] SEQ ID NO:4 is the amino acid sequence of VH CDR3 of antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0087] EALRGYDAGFWFTY.

[0088] SEQ ID NO:5 is the amino acid sequence of the light chain variable (VL) domain of antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0089] DIQMTQSPSSSLSASVGDRVTITTCTASEDIYNRLTWYQQKPGKVPRLLISGAT SLDTGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYWTTPWTFGQGTKLEI K.

[0090] SEQ ID NO:6 is the amino acid sequence of the light chain complementary determining region 1 (VL CDR1) of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0091] TASEDIYNRLT.

[0092] SEQ ID NO:7 is the amino acid sequence of VL CDR2 of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0093] GATSLDT.

[0094] SEQ ID NO:8 is the amino acid sequence of VL CDR3 of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0095] QQYWTTPWT.

[0096] SEQ ID NO: 9 is the amino acid sequence of a single chain variable region fragment (scFv) derived from antibody hBBC.10.1 in the [VL-VH] direction:

[0097] DIQMTQSPSSSLSASVGDRVTITTCTASEDIYNRLTWYQQKPGKVPRLLISGAT SLDTGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYWTTPWTFGQGTKLEI K(GGGGS) x QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGL EWLGYIHYTGNTKYSPSLKSRLSISRDTSKNQFFLKLSSVTTEDTAVYYCGREALRGYDAGFWFTYWGQGTLVTV, where "x" can be 1, or 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the sequence GGGGS shown in brackets.

[0098] SEQ ID NO: 10 is the amino acid sequence of the full-length heavy chain (HC) of antibody hBBC.10.1:

[0099] QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWLGYIHYTGNTKYSPSLKSRLSISRDTSKNQFFLKLSSVTTEDTAVYYCGREALRGYDAGFWFTYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0100] SEQ ID NO: 11 is the amino acid sequence of the full-length light chain (LC) of antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0101] DIQMTQSPSSSLSASVGDRVTITTCTASEDIYNRLTWYQQKPGKVPRLLISGATSLDTGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYWTTPWTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0102] SEQ ID NO: 12 is the nucleotide sequence encoding the heavy chain variable (VH) domain of antibody hBBC.10.1:

[0103] Caggtgcagctgcaggaaagcggcccgggcctggtgaaaccgagccagaccctgagcctgacctgcaccgtgagcggctatagcattaccagcggctatacctggcattggattcgccagcatccgggcaaaggcctggaatggctgggctatattcattataccggcaacaccaaatatag cccgagcctgaaaagccgcctgagcattagccgcgataccagcaaaaaccagttcttcctgaaactgagcagcgtgaccaccgaagataccgcggtgtattattgcggccgcgaagcgctgcgcggctatgatgctggcttctggtttacctattggggccaaggcaccctggtgaccgtg.

[0104] SEQ ID NO: 13 is the nucleotide sequence encoding the heavy chain complementary determining region 1 (VH CDR1) of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, hBBC.10.1 and antibody hBBC.10.1FQ:

[0105] agcggctatacctggcat.

[0106] SEQ ID NO: 14 is the nucleotide sequence encoding VH CDR2 of antibody hBBC.9.1, antibody hBBC.10 and antibody hBBC.10.1FQ:

[0107] tatattcattataccggcaacaccaaatatagcccgagcctgaaaagc.

[0108] SEQ ID NO: 15 is the nucleotide sequence encoding VH CDR3 of antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0109] gaagcgctgcgcggctatgatgctggcttctggtttacctat.

[0110] SEQ ID NO: 16 is the nucleotide sequence encoding the light chain variable (VL) domain of antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0111] gatattcagatgacccagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcaccgcgagcgaagatatttataaccgcctgacctggtatcagcagaaaccgggcaaagtgccgcgtctgctgatttctggcgcgaccagcct ggataccggcgtgccgagccgctttagcggcagcggcagcggcaccgattacaccctgaccattagcagcctgcagccggaagatgtggcgacctattattgccagcagtattggaccaccccgtggacctttggccagggcaccaaactggaaattaaa.

[0112] SEQ ID NO: 17 is the nucleotide sequence encoding the light chain complementary determining region 1 (VL CDR1) of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0113] accgcgagcgaagatattttataaccgcctgacc.

[0114] SEQ ID NO: 18 is the nucleotide sequence encoding the VL CDR2 of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0115] ggcgcgaccagcctggatacc.

[0116] SEQ ID NO: 19 is the nucleotide sequence encoding the VL CDR3 of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0117] cagcagtattggaccaccccgtggacc.

[0118] SEQ ID NO:20 is the nucleotide sequence encoding a single-chain variable region fragment (scFv) derived from antibody hBBC.10.1 in the [VL-(L)-VH] direction:

[0119] gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcaccgcgagcgaagatatttataaccgcctgacctggtatcagcagaaaccgggcaaagtgccgcgtctgctgatttctggcgcgaccagcctggataccggcgtgccgagccgctttagcggcagcggcagcggcaccgattacaccctgaccattagcagcctgcagccggaagatgtggcgacctattattgccagcagtattggaccaccccgtggacctttggccagggcaccaaactggaaattaaa(ggtggaggcggttct) x caggtgcagctgcaggaaagcggcccgg gcctggtgaaaccgagccagaccctgagcctgacctgcaccgtgagcggctatagcattaccagcggctatacctggcattggattcgccagcatccgggcaaaggcctggaatggctgggctatattcattataccggcaacaccaaatatagcccgagcctgaaaagccgcctgagcattagccgcgataccagcaaaaaccagttcttcctgaaactgagcagcgtgaccaccgaagataccgcggtgtattattgcggccgcgaagcgctgcgcggctatgatgctggcttctggtttacctattggggccaaggcaccctggtgaccgtg,

[0120] Wherein "x" may be 1, or 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the sequence ggtggaggcggttct shown in parentheses.

[0121] SEQ ID NO: 21 is the nucleotide sequence encoding the full-length heavy chain (HC) of the antibody hBBC.10.1:

[0122]

[0123] SEQ ID NO: 22 is the nucleotide sequence encoding the full-length light chain (LC) of antibody hBBC.10.1 and antibody hBBC.10.1FQ:

[0124] .

[0125] SEQ ID NO: 23 is an illustrative spacer amino acid sequence EGKSSGSGSESKVD.

[0126] SEQ ID NO: 24 is an illustrative spacer amino acid sequence KESGSVSSEQLAQFRSLD.

[0127] SEQ ID NO: 25 is the flexible multi-linker amino acid sequence GGGGS.

[0128] SEQ ID NO: 26 is the amino acid sequence of the flexible multilinker GGGGSGGGGSGGGGS. SEQ ID NO: 27 is the amino acid sequence of the VL domain of antibody IMH2 / BBC:

[0129] DIQMTQSSSSFSVSLGDRVTITTCTASEDIYNRLTWYQQKPGNVPRLLISGAT SLDTGVPSRFSGSRSGKDYALSITSLQTEDVATYYCQQYWTTPWTFGGGTRLEI K.

[0130] SEQ ID NO:28 is the amino acid sequence of the VH domain of antibody IMH2 / BBC:

[0131] DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYTWHWIRQFPGNTLEWLGY IHYSGNTKYSPSLKSRLSVTRDTSKNQFFLQLNSVTTEDTATYYCGREALRGYDH GFWFTYWGQGTLVTV.

[0132] SEQ ID NO:29 is the amino acid sequence of the VL domain of human acceptor framework AAS01771.1:

[0133] DIQMTQSPSSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPYTFGQGTKLEIK.

[0134] SEQ ID NO:30 is the amino acid sequence of the VH domain of the human acceptor framework CAD89404.1:

[0135] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGAYYWSWIRQHPGKGLEWIG YIYYSGTTYYNPSLKSRLSMSRDTSKNQFSLKLSSVTAADTAVYYCARGPYYDSP RPFDPWGQGTLVTV.

[0136] SEQ ID NO: 31 is the amino acid sequence of the VL domain of antibody hBBC.8:

[0137] DIQMTQSPSSSLSASVGDRVTITTCTASEDIYNRLTWYQQKPGKVPRLLISGAT SLDTGVPSRFSGSRSGTDFTLTISSLQPEDVATYYCQQYWTTPWTFGQGTKLEI K.

[0138] SEQ ID NO: 32 is the amino acid sequence of the VH domain of antibody hBBC.8, antibody hBBC.9 and antibody hBBC.10:

[0139] QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWLG YIHYSGNTKYSPSLKSRLSISRDTSKNQFFLKLSSVTTEDTAVYYCGREALRGYD HGFWFTYWGQGTLVTV.

[0140] SEQ ID NO: 33 is the amino acid sequence of the VL domain of antibody hBBC.9 and antibody hBBC.9.1:

[0141] DIQMTQSPSSSLSASVGDRVTITTCTASEDIYNRLTWYQQKPGKVPRLLISGAT SLDTGVPSRFSGSRSGTDYTLTISSLQPEDVATYYCQQYWTTPWTFGQGTKLEI K.

[0142] SEQ ID NO:34 is the amino acid sequence of the VH domain of antibody hBBC.9.1:

[0143] QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWLG YIHYTGNTKYSPSLKSRLSISRDTSKNQFFLKLSSVTTEDTAVYYCGREALRGAD HGFWFTYWGQGTLVTV.

[0144] SEQ ID NO: 35 is the amino acid sequence of the VH domain of antibody hBBC.10.1:

[0145] QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWLG YIHYTGNTKYSPSLKSRLSISRDTSKNQFFLKLSSVTTEDTAVYYCGREALRGYD AGFWFTYWGQGTLVTV.

[0146] SEQ ID NO: 36 is the amino acid sequence of a single chain variable region fragment (scFv) derived from antibody hBBC.10.1 in the [VH-VL] direction: QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWLGYIHY TGNTKYSPSLKSRLSISRDTSKNQFFLKLSSVTTEDTAVYYCGREALRGYDAGF WFTYWGQGTLVTV(GGGGS) x DIQMTQSPSSLSASVGDRVTITCTASEDIYNRLT WYQQKPGKVPRLLISGATSLDTGVPSRFSGSGSGTDYTLTISSLQPEDVATYYC QQYWTTPWTFGQGTKLEIK, where "x" can be 1 or 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the sequence GGGGS shown in brackets.

[0147] SEQ ID NO: 37 is the nucleotide sequence encoding a single-chain variable region fragment (scFv) derived from antibody hBBC.10.1 in the [VH-(L)-VL] direction:

[0148] caggtgcagctgcaggaaagcggcccgggcctggtgaaaccgagccagaccctgagcctgacctgcaccgtgagcggctatagcattaccagcggctatacctggcattggattcgccagcatccgggcaaaggcctggaatggctgggctatattcattataccggcaacaccaaatatagcccgagcc tgaaaagccgcctgagcattagccgcgataccagcaaaaaccagttcttcctgaaactgagcagcgtgaccaccgaagataccgcggtgtattat tgcggccgcgaagcgctgcgcggctatgatgctggcttctggtttacctattggggccaaggcaccctggtgaccgtg(ggtggaggcggttct) xgatattcagatgacccagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcaccgcgagcgaagatatttataaccgcctgacctggtatcagcagaaaccgggcaaagtgccgcgtctgctgatttctggcgcgaccagcct ggataccggcgtgccgagccgctttagcggcagcggcagcggcaccgattacaccctgaccattagcagcctgcagccggaagatgtggcgacctattattgccagcagtattggaccaccccgtggacctttggccagggcaccaaactggaaattaaa,

[0149] wherein "x" can be 1 or 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the sequence ggtggaggcggttct shown in brackets.

[0150] These and other sequences are provided in the accompanying Sequence Listing.

[0151] Details

[0152] The present disclosure relates to humanized antibodies and antigen-binding fragments thereof that are capable of specifically binding to certain Lewis antigens expressed on a variety of cancers. More specifically, as described for the first time herein and presented in more detail below, the chimeric humanized antibodies of the present invention unexpectedly bind with strong specificity to certain biantennary Lewis antigens expressed on cancer cells. B / Y Antigen binding, and have strong anti-tumor activity in vivo. In addition, compared to the BBC antibody comprising a VL domain having the amino acid sequence shown in SEQ ID NO: 27 and a VH domain having the amino acid sequence shown in SEQ ID NO: 28, the antibodies disclosed herein advantageously have surprisingly reduced (e.g., reduced in a statistically significant manner) binding to the monoantennary Lewis B antigen (which is expressed in healthy tissues). The antibodies disclosed herein bind to the monoantennary Lewis B antigen recognized by the antibody BR96. Y The antigens are different, and without wishing to be bound by theory, the antibodies of the present invention may allow safer and more specific targeting of cancer cells in therapeutic applications compared to BR96. In addition, the antibodies and antigen-binding fragments of the present disclosure also bind to cancer cells expressing the antigen, are effectively and stably internalized into the lysosomes of such cells, and are safely tolerated at therapeutic doses in non-human primate models.

[0153] According to certain preferred embodiments and further according to non-limiting theory, the beneficial uses of the antibodies and antigen-binding fragments thereof disclosed in the present invention relate to methods for diagnosing and / or treating cancer, such as, for example, various gastric cancers, ovarian cancers, lung cancers, prostate cancers, pancreatic cancers and other cancers. These embodiments and related embodiments are disclosed in more detail herein.

[0154] Peptides and proteins

[0155] The terms "polypeptide", "protein" and "peptide" and "glycoprotein" are used interchangeably and refer to polymers of amino acids that are not limited to any particular length. The term does not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The term "polypeptide" or "protein" may mean one or more amino acid chains, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein the polypeptide or protein may comprise multiple chains (which have the sequence of a native protein, i.e., a protein produced by a naturally occurring and specifically non-recombinant cell, or a genetically engineered or recombinant cell) linked together non-covalently and / or covalently by peptide bonds, and comprises molecules having the amino acid sequence of a native protein, or molecules having deletions, additions and / or substitutions of one or more amino acids of the native sequence. Thus, a "polypeptide" or "protein" may comprise one (referred to as a "monomer") or multiple (referred to as a "polymer") amino acid chains. The terms "peptide", "polypeptide" and "protein" specifically include the antibodies and antigen-binding fragments of the present disclosure, or sequences having deletions, additions and / or substitutions of one or more amino acids of the antibodies or antigen-binding fragments thereof.

[0156] As used herein, "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are subsequently modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bound to a hydrogen, carboxyl, amino, and R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0157] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule compared to a reference nucleic acid molecule or a reference polypeptide molecule, or compared to a wild-type nucleic acid molecule or a wild-type polypeptide molecule, respectively. A mutation can result in several different types of changes in a sequence, including substitutions, insertions or deletions of nucleotides or amino acids.

[0158] The term "polypeptide fragment" refers to a polypeptide (which may be monomeric or multimeric) having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally occurring or recombinantly produced polypeptide. As used herein, "contiguous amino acids" refers to covalently linked amino acids corresponding to an uninterrupted linear portion of a disclosed amino acid sequence. In certain embodiments, a polypeptide fragment may comprise an amino acid chain of at least 5 to about 500 amino acids in length. It will be appreciated that in certain embodiments, a fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length.

[0159] As used herein, the terms "isolated protein" and "isolated polypeptide" mean that the subject protein or polypeptide (1) is free of at least some other proteins or polypeptides that are normally found in nature, (2) is substantially free of other proteins or polypeptides from the same source (e.g., from the same species), (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or non-covalent interactions) with portions of the protein or polypeptide that the "isolated protein" or "isolated polypeptide" may be associated with in nature, (6) is operably associated (by covalent or non-covalent interactions) with a polypeptide with which the polypeptide is not associated in nature, or (7) does not exist in nature. Such isolated proteins or polypeptides may be encoded by genomic DNA, cDNA, mRNA or other RNA, may be of synthetic origin according to any of a number of well-known chemistries for the synthesis of artificial peptides and proteins, or any combination thereof. In certain embodiments, an isolated protein or polypeptide is substantially free of proteins or polypeptides or other contaminants found in its natural environment that interfere with its use (therapeutic, diagnostic, prophylactic, research, etc.).

[0160] The polypeptide may contain a signal (or leader) sequence at the N-terminus of the protein, which co-translates or post-translationally directs the transfer of the protein. The polypeptide may also be fused or conjugated in-frame with a linker or other sequence to facilitate the synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance the binding of the polypeptide to a solid support. As used herein, "fusion protein" or "fusion polypeptide" refers to a protein having at least two different domains in a single chain, wherein the domains are not naturally present together in the protein. Polynucleotides encoding fusion proteins may be constructed using PCR, recombinant engineering, etc., or such fusion proteins may be synthesized. The fusion protein may also contain other components, such as tags, linkers, or transduction markers. The fusion domain polypeptide may be connected to the polypeptide at the N-terminus and / or C-terminus, and may include, as non-limiting examples, immunoglobulin-derived sequences (e.g., Ig constant region sequences or portions thereof), affinity tags (e.g., His tags (e.g., hexahistidine or other polyhistidine)), FLAG TM Or myc or other peptide affinity tags, detectable polypeptide portions (such as green fluorescent protein (GFP) or variants thereof (e.g., yellow fluorescent protein (YFP), blue fluorescent protein (BFP), other aequorin or derivatives thereof, etc.) or other detectable polypeptide fusion domains, enzymes or portions thereof (such as glutathione-S-transferase (GST)) or other known enzymatic detection and / or reporter fusion domains, etc. Additional detectable portions are discussed herein.

[0161] Cysteine-containing peptides can be used as fusion peptides, which can be linked to the N-terminus and / or C-terminus of a polypeptide (such as an antibody or antigen-binding fragment thereof of the present disclosure) to allow such polypeptides to be easily assembled into disulfide-crosslinked dimers, trimers, tetramers or higher multimers according to established methods. For example, fusion polypeptides containing sequences derived from members of the immunoglobulin gene superfamily including cysteine ​​residues capable of forming interchain disulfide bridges are well known and are also other strategies for engineering SS-linked multimers (e.g., Reiter et al., 1994 Prot. Eng. 7: 697; Zhu et al., 1997 Prot. Sci. 6: 781; Mabry et al., 2010 Mabs 2: 20; Gao et al., 1999 Proc. Nat. Acad. Sci. USA 96: 6025; Lim et al., 2010 Biotechnol. Bioeng. 106: 27). Alternative approaches for grafting peptide sequences that promote multimeric assembly as fusion domains onto desired polypeptides (such as antibodies and antigen-binding fragments described herein) are also contemplated (eg, Fan et al., 2008, FASEB J. 22:3795).

[0162] Biosynthetic and / or chemical polypeptide modifications can be performed according to various well-known methods, and can also include conjugation to a carrier protein (e.g., keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), or other molecules), and covalent or non-covalent immobilization on a solid support. According to certain embodiments, chemical or biosynthetic conjugation to a carrier is contemplated for the production of a conjugate that is multivalent relative to the antibodies or antigen-binding fragments thereof described herein.

[0163] Also contemplated are detectable labeling with a detectable indicator moiety (sometimes referred to as a reporter moiety), such as a fluorophore (e.g., FITC, TRITC, Texas Red, etc.). Examples of a variety of detectable indicators (including colorimetric indicators) that can be selected for a particular purpose are described in Haugland, 2005 The Handbook: A Guide to Fluorescent Probes and Labeling Technologies-10th Edition, Invitrogen Corp. / Molecular Probes TM , Eugene, OR; Mohr, 1999 J. Mater. Chem., 9:2259-2264; Suslick et al., 2004 Tetrahedron 60:11133-11138; and U.S. Pat. No. 6,323,039. (See also, e.g., Fluka Laboratory Products Catalog, 2001 Fluka, Milwaukee, WI; and Sigma Life Sciences Research Catalog, 2000 Sigma, St. Louis, MO.). The detectable indicator can be a fluorescent indicator, a luminescent indicator, a phosphorescent indicator, a radiometric indicator, a dye, an enzyme, a substrate for an enzyme, an energy transfer molecule, or an affinity label.

[0164] Other detectable indicators for use in certain embodiments contemplated herein include affinity reagents such as antibodies, lectins, immunoglobulin Fc receptor proteins (e.g., Staphylococcus aureus protein A, protein G, or other Fc receptors), avidin, biotin, other ligands, receptors, or counter-receptors, or analogs or mimetics thereof, etc. For such affinity methods, reagents such as appropriately labeled antibodies or lectins can be prepared for immunoassay measurement, including, for example, with radionuclides (e.g., 76 Br, 78 Zr, 18Fl), those labeled with a fluorophore, with an affinity tag, with biotin or a biotin mimetic sequence, or prepared as an antibody-enzyme conjugate (see, e.g., Weir, DM, Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston; Scouten, WH, 1987 Methods in Enzymology 135:30-65; Harlow and Lane, Antibodies: A Laboratory Manual, 1988 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Haugland, Guide to Fluorescent Probes and Labeling Technologies-10th Edition, 2005 Invitrogen Corp. / Molecular Probes TM , Eugene, OR; Scopes, RK, Protein Purification: Principles and Practice, 1987, Springer-Verlag, NY; Hermanson, GT et al., Immobilized Affinity Lig and Techniques, 1992, Academic Press, Inc., NY; Luo et al., 1998 J. Biotechnol. 65:225 and references cited therein).

[0165] If desired, peptide linker / spacer sequences can also be used to separate the various polypeptide components by a sufficient distance to ensure that each polypeptide folds into its secondary and / or tertiary structure. Such peptide linker sequences can be incorporated into fusion polypeptides using standard techniques well known in the art.

[0166] Certain peptide spacer sequences can be selected, for example, based on: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that can interact with a functional epitope on the first polypeptide and the second polypeptide; and / or (3) lack of hydrophobic or charged residues that may react with a functional epitope of the polypeptide. In certain embodiments, the peptide spacer sequence contains, for example, Gly, Asn, and Ser residues. Other nearly neutral amino acids (such as Thr and Ala) can also be included in the spacer sequence. Other amino acid sequences that can be used as spacers include those described in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. Other illustrative and non-limiting examples of spacers can include, for example, Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO: 23) (Chaudhary et al., Proc. Natl. Acad. Sci. U.S.A. 87: 1066-1070 (1990)) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp (SEQ ID NO: 24) (Bird et al., Science 242: 423-426 (1988)).

[0167] In some embodiments, when the first polypeptide and the second polypeptide have a non-essential N-terminal amino acid region, no spacer sequence is required, and the non-essential N-terminal amino acid region can be used to separate functional domains and prevent spatial interference. Two coding sequences can be directly fused without any spacer, or by using a flexible multi-linker composed of, for example, a pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 25) when present in a single iteration or repeated 1 to 5 times or more times or more (see, e.g., SEQ ID NO: 26) directly fused. In some illustrative and non-limiting embodiments, the peptide spacer can be 1 to 5 amino acids, 5 to 10 amino acids, 5 to 25 amino acids, 5 to 50 amino acids, 10 to 25 amino acids, 10 to 50 amino acids, 10 to 100 amino acids, or any intermediate range of amino acids. In other illustrative embodiments, the peptide spacer comprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acid lengths.

[0168] According to certain embodiments, amino acid sequence modifications of the antibodies or antigen-binding fragments thereof described herein are also contemplated. Modifications include, for example, conservative and non-conservative amino acid substitutions. "Conservative substitutions" refer to amino acid substitutions that do not significantly affect or change a particular property (e.g., binding activity, such as specific binding activity) of a particular protein. Typically, conservative substitutions are substitutions in which the substituted amino acid residue is replaced by an amino acid residue with a similar side chain. Conservative substitutions include substitutions found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure or composition (e.g., acidic, basic, aliphatic, aromatic or sulfur-containing). For example, for substitution purposes, an aliphatic grouping may include Gly, Ala, Val, Leu, and Ile. Other conservative substitution groups include: sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0169] For example, it may be desirable to improve the binding affinity and / or other biological properties of an antibody or antigen-binding fragment thereof. The amino acid sequence variants may be prepared, for example, by introducing appropriate nucleotide changes into a polynucleotide encoding the amino acid sequence variant or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody or antigen-binding fragment thereof. Any combination of deletions, insertions, and substitutions may be performed to obtain the final antibody or antigen-binding fragment variant, provided that the final construct has the desired properties (e.g., maintaining specific binding to a biantennary Lewis antigen as described herein, while not being detectably bound to or showing binding to a monoantennary Lewis antigen as described elsewhere herein). X or Le A Amino acid changes may also alter post-translational processes of the antibody or antigen-binding fragment thereof, such as changing the number or position of glycosylation sites.

[0170] Determination of the three-dimensional structure of a representative antibody or antigen-binding fragment thereof can be performed by conventional methods, such that substitution, addition, deletion or insertion of one or more amino acids by selected natural or non-natural amino acids can be virtually simulated to determine whether the structural variants so derived retain the space-filling properties of the presently disclosed species. See, e.g., Donate et al., 1994 Prot. Sci. 3:2378; Bradley et al., Science 309:1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci., USA, 103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Raman et al., Science 327:1014-1018 (2010); Marcos et al., 2017 Science 355:201, and references cited therein. Some additional non-limiting examples of computer algorithms that can be used for these and related embodiments (e.g., for rational design of antibodies and antigen-binding fragments as provided herein) include VMD, which is a molecular visualization program that uses 3-D graphics and built-in scripts to display, animate, and analyze large biomolecular systems (see some other non-limiting examples of computer algorithms that can be used for these and related embodiments, e.g., for rational design of antibodies and antigen-binding fragments provided herein, including VMD, which is a program for displaying, animating, and analyzing large biomolecular systems using 3-D graphics and built-in scripts (see: the website of the Theoretical and Computational Biophysics Group, University of Illinois at Urbana-Champagne: ks.uiuc.edu / Research / vmd / ).

[0171] Many other computer programs are known in the art and available to the skilled person and allow the determination of atomic sizes from space filling models (van der Waals radii) of energy-minimized conformations. GRID (which aims to identify regions of high affinity for different chemical groups, thereby enhancing binding), Monte Carlo searches (which calculate mathematical alignments), and CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER (Weiner et al., (1981) J. Comput. Chem. 106:765), which evaluate the calculation and analysis of force fields (see also: Eisenfield et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ. Health Perspect. 61: 185-190; and Kini et al., (1991) J. Biomol. Struct. Dyn. 9: 475-488). Various suitable computer programs are also commercially available, such as from (Munich, Germany).

[0172] Antibody

[0173] Certain preferred embodiments of the present invention relate to antibodies or antigen-binding fragments thereof that specifically bind to:

[0174] Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] b / Le b antigen,

[0175] Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen,

[0176] Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen,

[0177] and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]

[0178] [VIII]'s Double Tentacles Y / Le B antigen,

[0179] and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV], and in certain other particularly preferred embodiments, the antibody or antigen-binding fragment thereof does not specifically bind to certain other biantennary or monoantennary Lewis antigens as described herein.

[0180] As used herein, the term "antibody" (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or trispecific antibodies), humanized antibodies, chimeric antibodies, heteroconjugate antibodies and antibody fragments, as long as they exhibit the desired biological activity, for example, retaining the ability to specifically bind to the biantennary Lewis antigen disclosed in the present invention. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0181] The basic antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each L chain is connected to the H chain by at least one (and usually one) covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (V H ), followed by three constant domains of each of the α and γ chains (C H ) and the four Cs of the μ and ε isoforms H Each L chain has a variable domain (V L ), followed by a constant domain at its other end (C L ). L With V H Aligned, and C L The first constant domain of the heavy chain (C H 1) Alignment. Specific amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. H and V L The pairs together form a single antigen binding site.

[0182] Based on their constant domain (C L ), L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda. H ) constant domains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, ε, γ, and μ, respectively. Based on the amino acid sequence of the C H Relatively minor differences in sequence and function further divide the gamma and alpha classes into subclasses, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgAl and IgA2. It will be appreciated that mammals encoding multiple Ig isotypes will be capable of isotype class switching.

[0183] IgM antibodies are composed of 5 basic heterotetramer units and another polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form a multivalent assembly comprising 2 to 5 basic four-chain units and J chains. In the case of IgG, the four-chain unit usually has a molecular weight of about 150,000 daltons. For the structure and properties of antibodies of different classes, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, p. 71, and Chapter 6.

[0184] The variable (V) domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. H The gene sequence of the domain has multiple copies of the variable (V) segment, the diversity (D) segment and the joining (J) segment. L The gene sequence of the domain contains multiple copies of V segments and J segments. H Area and V L The V domains undergo genetic rearrangement (ie, somatic recombination) to produce the various antigen specificities in antibodies. The term "variable" refers to the fact that certain segments of the V domains differ extensively in sequence between antibodies.

[0185] However, variability is not evenly distributed over the span of 110 amino acids in the variable domain. On the contrary, the V region is composed of a relatively constant segment of 15-30 amino acids called a framework region (FR), which is separated by a short region with extreme variability called a "hypervariable region". These hypervariable regions are the result of somatic hypermutation during affinity maturation, and they are usually 9-18 amino acids long each. However, it has been found that their length ranges from 4 to 28 amino acids, depending on the specific epitope. For example, CDR3 regions of at least 22 or 23 amino acids have been described. See, for example, Morea V et al., J Mol Biol. 275 (2): 269-94 (1998) and Kabat, EA et al., Sequences of Proteins of Immunological Interest, Fifth Edition. NIH published No. 91-3242 (1991). Antibody amino acid positions (eg, CDR sequences) can be identified according to known numbering schemes, such as the Kabat, Chothia, IMGT and / or EU numbering schemes.

[0186] The variable domains of native heavy and light chains each comprise four framework regions (FRs), which primarily adopt a β-sheet configuration connected by three hypervariable regions (also referred to as complementarity determining regions (CDRs), further defined below), which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs, and in some cases, together with the hypervariable regions from the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, Md. (1991)). The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) or other mechanisms that may involve the interaction of the constant region domains with cell surface Fc receptors (FcRs).

[0187] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. A hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., as determined according to Kabat numbering, between V L In the case of V H about residues 28-36 (H1), residues 50-65 (H2), and residues 95-102 (H3); see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, Md. (1991); and / or according to methods known in the art for identifying CDRs defined by Kabat, such as those described by Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains", In Antibody Engineering , R. Kontermann and S. Dubel, 2001, Springer-Verlag, Berlin, Germany, pp. 422-438), and / or those residues from the "hypervariable loops" (e.g., V LResidues 26-32 (L1), residues 50-52 (L2), and residues 91-96 (L3), as well as V H 26-32 (H1), residues 53-55 (H2), and residues 96-101 (H3); Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).

[0188] An "isolated antibody" is an antibody that has been separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the antibody is purified to: (1) greater than 95% by weight of the antibody as determined by the Bradford method, and most preferably greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, isolated antibodies are typically prepared by at least one purification step.

[0189] A "complete" antibody is one that contains both the antigen-binding site and the C L and at least the heavy chain constant domain (C H 1. C H 2 and C H 3). The constant domain may be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

[0190] An "antibody fragment" is a polypeptide that comprises or consists of a portion of an intact antibody, preferably the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0191] Papain digestion of antibodies produces two identical antigen-binding fragments (called "Fab" fragments) and a residual "Fc" fragment (a name reflecting the ability to crystallize readily). The Fab fragment consists of the entire L chain and the H chain (V H) and the first constant domain of a heavy chain (C H 1) Composition. Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with divalent antigen binding activity and is still able to cross-link antigen. Both Fab and F(ab')2 are examples of "antigen-binding fragments". Fab' fragments differ from Fab fragments in that the C H The carboxyl terminus of the constant domain has an additional small number of residues, which include one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains carry a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0192] The "Fc" fragment contains the carboxyl terminal portions of two H chains (i.e., the CH2 and CH3 domains of IgG) linked together by disulfide bonds. The effector functions of an antibody are determined by the sequences in the Fc region. The Fc domain is the portion of an antibody that is recognized by cellular receptors (e.g., FcRs), and to which the complement activating protein C1q binds. As discussed herein, the Fc domain can be modified (e.g., amino acid substitutions) in order to modify (e.g., improve, reduce, or eliminate) one or more functions of an Fc-containing polypeptide (e.g., an antibody of the present disclosure).

[0193] "Fv" is the smallest antibody fragment containing complete antigen recognition and antigen binding sites. The fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight non-covalent association. Six hypervariable loops (three loops each from the H chain and the L chain) are generated from the folding of these two domains, which contribute amino acid residues to antigen binding and give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is usually lower than that of the entire binding site.

[0194] "Single-chain Fv" is also abbreviated as "sFv" or "scFv" and is composed of V sequences linked into a single polypeptide chain. H and V L Preferably, the sFv polypeptide further comprises a V H and V LA polypeptide linker between domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, see below.

[0195] The term "diabody" refers to a H and V L Small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the V domains, thereby achieving interchain rather than intrachain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the V domains of the two antibodies are H Domain and V L Domains are present on different polypeptide chains. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Other antibody fragments and molecules comprising them include, for example, linear antibodies, tandem scFv, scFv-Fc, tandem scFv-Fc, scFv dimers, scFv-zipper, diabody-Fc, diabody-CH3, sc diabody, sc diabody-Fc, sc diabody-CH3, nanobodies, TandAb, minibodies, minibodies, triabodies, tetrabodies, scFab, Fab-scFv, Fab-scFv-Fc, scFv-CH-CL-scFv and F(ab')2-scFv2, all of which are also considered herein.

[0196] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are multispecific antibodies, such as bispecific antibodies or trispecific antibodies. The form of bispecific antibodies is disclosed in, for example, Spiess et al., Mol. Immunol. 67(2):95 (2015) and Brinkmann and Kontermann, mAbs 9(2):182-212 (2017), and these bispecific forms and methods of making them are incorporated herein by reference, and include, for example, bispecific T cell engagers. Engagers, BiTE), DART, Knobs-Into-Holes (KIH) assembly, scFv-CH3-KIH assembly, KIH common light chain antibody, TandAb, triabody, TriBi minibody, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCab, endosome, CrossMabs, dual-acting Fab (DAF) (two-in-one or four-in-one), DutaMab, DT-IgG, charge pair, Fab arm exchange, SEEDbodies, Triomab, LUZ-Y assembly, Fcab, κλ body, orthogonal Fab (orthogonal Fab), DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody and DVI-IgG (four in one).

[0197] As used herein, the term "polyclonal antibody" refers to an antibody obtained from a population of antigen-specific antibodies that recognize more than one epitope of a specific antigen. "Antigen" or "immunogen" refers to a peptide, lipid, polysaccharide or polynucleotide that is recognized by the adaptive immune system. Antigens can be self or non-self molecules. Examples of antigens include, but are not limited to, bacterial cell wall components, pollen and rh factors. The region of an antigen that is specifically recognized by a specific antibody is an "epitope" or "antigenic determinant". A single antigen can have multiple epitopes.

[0198] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous antibody population, that is, each antibody comprising the population is identical, except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. In addition, in contrast to polyclonal antibody preparations comprising different antibodies for different epitopes, each monoclonal antibody is directed to a single epitope of an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be interpreted as requiring the production of antibodies by any ad hoc method. For example, monoclonal antibodies that can be used in the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be prepared using recombinant DNA methods in bacteria, eukaryotic animals or plants (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0199] The monoclonal antibodies herein include "chimeric antibodies", in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the rest of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (see U.S. Pat. Nos. 4,816,567; 5,530,101 and 7,498,415; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). For example, a chimeric antibody may contain human and non-human residues. In addition, a chimeric antibody may contain residues not found in a recipient antibody or a donor antibody. These modifications are made to further improve antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Chimeric antibodies also include primate and humanized antibodies.

[0200] "Humanized antibodies" are generally considered to be human antibodies, which have one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are usually taken from the variable domain. Humanization is traditionally carried out by replacing non-human variable sequences with the corresponding sequences of human antibodies according to the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)). Therefore, such "humanized" antibodies are chimeric antibodies (U.S. Patent Nos. 4,816,567; 5,530,101 and 7,498,415), in which substantially less than the complete human variable domain has been replaced by the corresponding sequence from a non-human species. In some cases, "humanized" antibodies are produced by non-human cells or animals and contain human sequences (e.g., H C domain).

[0201] "Human antibodies" are antibodies that contain only sequences present in antibodies produced naturally by humans. However, as used herein, human antibodies may contain residues or modifications not found in naturally occurring human antibodies, including those modifications and variant sequences described herein. These are generally made to further improve or enhance antibody performance. In some cases, human antibodies are produced by transgenic animals. For example, see U.S. Patents Nos. 5,770,429; 6,596,541 and 7,049,426.

[0202] The "effector functions" of an antibody refer to those biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Amino acid modifications (e.g., substitutions) for modifying (e.g., improving, reducing, or eliminating) Fc function include, for example, T250Q / M428L, M252Y / S254T / T256E, H433K / N434F, M428L / N434S, E233P / L234V / L235A / G236+A327G / A330S / P331, E333A, S239D / A330L / I332E, P257 I / Q311, K326W / E333S, S239D / I332E / G236A, N297Q, K322A, S228P, L235E+E318A / K320A / K322A, L234A / L235A, and L234A / L235A / P329G mutations, which are summarized and annotated in "Engineered Fc Regions" published by InvivoGen (2011) and available online at: www.invivogen.com / PDF / review / review-Engineered-Fc-Regions-invivogen.pdf?utm_source=review&utm_medium=pdf&utm_campaign=review&utm_content=Engineered-Fc-Regions, which is incorporated herein by reference.

[0203] The phrase "functional fragment or analog" of an antibody or antigen-binding fragment thereof is a compound that shares qualitative biological activity with the full-length antibody or antigen-binding fragment thereof.

[0204] An antibody or antigen-binding fragment thereof having a "biological property" of a specified antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has one or more of the biological properties of the antibody or antigen-binding fragment thereof that distinguishes it from other antibodies or antibody-derived binding fragments. For example, in certain embodiments, an antibody or antigen-binding fragment thereof having a biological property of a specified antibody will bind to the same epitope as the epitope bound by the specified antibody and / or have the same effector function as the specified antibody.

[0205] As used herein, an antibody is considered to be expressed at a detectable level (preferably at a level greater than or equal to about 104 M -1 , or greater than or equal to about 10 5 M -1 , greater than or equal to about 10 6 M -1 , greater than or equal to about 10 7 M -1 , or greater than or equal to 10 8 M -1 The affinity of an antibody for its cognate antigen is also often expressed as the dissociation constant K. D , and in certain embodiments, if the antibody or antigen-binding fragment thereof is less than or equal to 10 -4 M, less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to 10 -7 M, or less than or equal to 10 -8 M of K D Combination, then it specifically binds to the Lewis antigen of the present disclosure. The affinity of antibodies and antigen-binding fragments can be easily determined using conventional techniques, such as those described by Scatchard et al. (Ann. NY Acad. Sci. USA, 51: 660 (1949)), or by surface plasmon resonance (SPR) (e.g., Hearty et al., 2012 Meths. Mol. Biol. 907: 411), by isothermal titration calorimetry (ITC) (e.g., Dam et al., 2008 J. Biol. Chem. 283: 31366), by enzyme-linked immunosorbent assay (ELISA) (e.g., Bobrovnik, 2003 J. Biochem. Biophys. Meths. 75 (3): 213), or by other methods familiar to those skilled in the art.

[0206] The binding properties of an antibody to its antigen, cell or tissue can generally be determined and assessed using immunoassays, including, for example, immunofluorescence-based assays such as immunohistochemistry (IHC) and / or fluorescence activated cell sorting (FACS). Other methods for determining antibody binding to an antigen include, for example, enzyme-linked immunosorbent assay (ELISA), isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) techniques.

[0207] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer that is nontoxic to cells or mammals exposed thereto at the dosages and concentrations used. Typically, a physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN TM ) Polyethylene glycol (PEG) and poloxamer (PLURONICS TM )wait.

[0208] Polynucleotides, vectors and host cells

[0209] In other aspects, the present disclosure provides in certain embodiments isolated polynucleotides encoding antibodies and antigen-binding fragments thereof as described herein, and also provides vectors comprising the polynucleotides. The nucleic acid comprising the polynucleotide may comprise DNA or RNA, and may be fully or partially synthetic.

[0210] As used herein, the term "polynucleotide" means a single-stranded or double-stranded nucleic acid polymer, and specifically includes DNA in single-stranded and double-stranded forms. Polynucleotides can be produced, for example, by polymerase chain reaction (PCR) or by in vitro translation, and fragments are produced by any connection, breakage, endonuclease action or exonuclease action. In certain embodiments, polynucleotides of the present disclosure are produced by PCR. Polynucleotides can be composed of monomers of naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (such as the α-enantiomeric form of naturally occurring nucleotides) or a combination of the two. In other embodiments, the nucleotides comprising polynucleotides can be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotides.

[0211] The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups, etc. (e.g., modified with bromouridine, arabinoside, or 2'3'-dideoxyribose). The term "oligonucleotide linkage" includes oligonucleotide linkages, such as phosphorothioate, phosphorodithioate, selenophosphate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoramidate, etc. See, e.g., LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, ed.), Oxford University Press, Oxford, England; Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are hereby incorporated by reference for any purpose. The oligonucleotide may include a detectable label to enable detection of the oligonucleotide or a hybrid thereof.

[0212] As used herein, the term "isolated polynucleotide" means a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, wherein, due to its origin, the isolated polynucleotide (1) is not associated with all or part of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur as part of a larger sequence in nature.

[0213] Unless the context requires otherwise, reference to a nucleotide sequence herein includes DNA molecules having the specified sequence, and includes RNA molecules having the specified sequence in which U is substituted for T.

[0214] The term "operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions under appropriate conditions. For example, a transcription control sequence "operably linked" to a protein coding sequence is linked thereto so as to achieve expression of the protein coding sequence under conditions compatible with the transcriptional activity of the control sequence.

[0215] As used herein, the term "control sequence" refers to a polynucleotide sequence that can affect the expression, processing or intracellular localization of a coding sequence connected or operably connected thereto. The nature of such control sequences may depend on the host organism. In specific embodiments, transcription control sequences for prokaryotes may include promoters, ribosome binding sites, and transcription termination sequences. In other specific embodiments, transcription control sequences for eukaryotes may include promoters, transcription enhancer sequences, transcription termination sequences, and polyadenylation sequences comprising one or more transcription factor recognition sites. In certain embodiments, "control sequences" may include leader sequences and / or fusion partner sequences. Expression control sequences may include appropriate transcription initiation sequences, termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals (such as splicing and polyadenylation signals); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that may enhance protein secretion. If the expression control sequence is adjacent to the target gene, they may be effectively connected, and the expression control sequence acts in trans or at a distance to control the target gene.

[0216] If introns are present, expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing.

[0217] As will be appreciated by those skilled in the art, polynucleotides may include genomic sequences, extragenomic and plasmid-encoded sequences, as well as smaller engineered gene fragments that express or can be adapted to express proteins, polypeptides, peptides, etc. Such fragments may be isolated from nature, or synthetically modified by the skilled artisan.

[0218] Those skilled in the art will also recognize that polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules can include hnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one manner, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences can, but need not be present in polynucleotides according to the present disclosure, and polynucleotides can, but need not be connected to other molecules and / or supporting materials. Polynucleotides can comprise native sequences or can comprise sequences encoding variants or derivatives of such sequences.

[0219] Thus, according to these and related embodiments, the disclosure also provides polynucleotides encoding the antibodies or antigen-binding fragments thereof as described herein.

[0220] In other related embodiments, polynucleotide variants can be substantially identical to the polynucleotide sequences encoding the antibodies or their antigen-binding fragments described herein. For example, using the methods described herein (e.g., BLAST analysis using standard parameters as described below), polynucleotides can be polynucleotides comprising at least 70% sequence identity (preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity) compared to reference polynucleotide sequences (such as sequences encoding antibodies or their antigen-binding fragments described herein). Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by considering codon degeneracy, amino acid similarity, reading frame positioning, etc.

[0221] Generally, polynucleotide variants will contain one or more substitutions, additions, deletions and / or insertions, preferably such that the binding affinity of the antibody or antigen-binding fragment thereof encoded by the variant polynucleotide is not substantially diminished relative to the antibody or antigen-binding fragment thereof encoded by the polynucleotide sequences specifically described herein (e.g., relative to the antibody referred to herein as hBBC.10.1, which comprises an immunoglobulin heavy chain having the amino acid sequence shown in SEQ ID NO:10 and an immunoglobulin light chain having the amino acid sequence shown in SEQ ID NO:11).

[0222] In certain other related embodiments, the polynucleotide fragments may comprise or consist essentially of continuous fragments of various lengths of sequences identical or complementary to the sequences encoding the antibodies or antigen-binding fragments thereof described herein. For example, polynucleotides comprising the following number of continuous nucleotides of sequences encoding the antibodies or antigen-binding fragments thereof disclosed herein and all intermediate lengths of sequences therebetween are provided, or polynucleotides consisting essentially of the following number of continuous nucleotides of sequences encoding the antibodies or antigen-binding fragments thereof disclosed herein and all intermediate lengths of sequences therebetween are provided. 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, or 1000 or more. It is readily understood that, in this article, "intermediate length" means any length between the cited values, such as 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers of 200-500, 500-1,000, etc. The polynucleotide sequences described herein may be extended at one or both ends by other nucleotides not found in the native sequence. The additional sequence may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides at either end of the polynucleotide encoding the antibody or antigen-binding fragment thereof described herein or at both ends of the polynucleotide encoding the antibody or antigen-binding fragment thereof described herein.

[0223] In another embodiment, polynucleotides are provided that can hybridize to polynucleotide sequences encoding antibodies or antigen-binding fragments thereof, or variants thereof, or fragments thereof, or complementary sequences thereof, provided herein, under moderate to high stringency conditions. Hybridization techniques are well known in the field of molecular biology. For illustrative purposes, suitable moderate stringency conditions for testing the hybridization of polynucleotides as provided herein with other polynucleotides include pre-washing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50° C.-60° C., 5×SSC; then washing twice with 2×, 0.5×, and 0.2×SSC containing 0.1% SDS at 65° C. for 20 minutes each. Those skilled in the art will appreciate that the stringency of hybridization can be easily manipulated, such as by changing the salt content of the hybridization solution and / or the temperature at which hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, except that the hybridization temperature is increased, for example, to 60° C.-65° C. or 65° C.-70° C.

[0224] In certain embodiments, the above-mentioned polynucleotides (e.g., polynucleotide variants, fragments, and hybridization sequences) encode antibodies or antigen-binding fragments thereof that bind to the biantennary Lewis antigens described herein. In other embodiments, such polynucleotides encode antibodies or antigen-binding fragments thereof, or variants thereof, that bind to the Lewis antigens disclosed herein by at least about 50%, at least about 70%, and in certain embodiments, at least about 90%, as well as antibodies or antigen-binding fragments thereof specifically described herein (e.g., antibody hBBC.10.1). In other embodiments, such polynucleotides encode antibodies or antigen-binding fragments thereof, or variants thereof, that bind to the Lewis antigens disclosed herein by greater affinity than the antibodies or antigen-binding fragments thereof specifically described herein (e.g., they quantitatively bind by at least about 105%, 106%, 107%, 108%, 109%, or 110%), as well as antibodies or antigen-binding fragments thereof specifically described herein.

[0225] As described elsewhere herein, the determination of the three-dimensional structure of the antibody or antigen-binding fragment thereof of the present disclosure can be carried out by conventional methods, so that the substitution, addition, deletion or insertion of the selected natural or non-natural amino acid of one or more amino acids can be virtually simulated to determine whether the structural variants so derived retain the space-filling properties of the species currently disclosed. A variety of computer programs are known to those skilled in the art for determining the appropriate amino acid substitution (or the appropriate polynucleotide encoding the amino acid sequence) within the antibody or antigen-binding fragment thereof, so as to, for example, maintain affinity or obtain better affinity. See, e.g., Donate et al., 1994 Prot. Sci. 3:2378; Bradley et al., Science 309:1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci. USA, 103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Raman et al., Science 327:1014-1018 (2010); Marcos et al., 2017 Science 355:201, and references cited therein.

[0226] Regardless of the length of the coding sequence itself, the polynucleotides described herein or fragments thereof can be combined with other DNA sequences (such as promoters, polyadenylation signals, other restriction enzyme sites, multiple cloning sites, other coding segments, etc.) so that their total length can vary significantly. Therefore, it is contemplated that nucleic acid fragments of almost any length can be used, wherein the total length is preferably limited by the ease of preparation and the use in the expected recombinant DNA protocol. For example, it is useful to consider illustrative polynucleotide fragments with the following total lengths: about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs, etc. (including all intermediate lengths).

[0227] As described below, when comparing polynucleotide sequences, if the nucleotide sequences in the two sequences are identical when the alignment is maximally corresponding, then the two sequences are considered to be "identical". The comparison between the two sequences is usually carried out by comparing the sequences on a comparison window to identify and compare the local regions of sequence similarity. As used herein, a "comparison window" refers to a section of at least about 20 continuous positions (usually 30 to about 75, 40 to about 50), wherein the sequence can be compared with the reference sequence of the same number of continuous positions after the two sequences are optimally aligned.

[0228] Optimal alignment of sequences for comparison can be performed using bioinformatics software (DNASTAR, Inc. Madison, WI). The Lasergene suite's Megalign program was used with default parameters. This program embodies several alignment schemes described in the following reference: Dayhoff, MO (1978) A model of evolutionary change in proteins–Matrices for detecting distant relationships. Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, Vol. 5, Supplement 3, pp. 345-358; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990); Methods in Enzymology Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, CABIOS 5:151-153 (1989); Myers, EW and Muller W., CABIOS 4:11-17 (1988); Robinson, ED, Comb. Theor 11:105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4:406-425 (1987); Sneath, PHA and Sokal, RR, Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA (1973); Wilbur, WJ and Lipman, DJ, Proc. Natl. Acad., Sci. USA, 80:726-730 (1983).

[0229] Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm of Smith and Waterman, Add. APL. Math 2:482 (1981), the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methods of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.

[0230] A preferred example of an algorithm suitable for determining sequence identity and sequence similarity percentages is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nucl. Acids Res. 25: 3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215: 403-410 (1990), respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine the sequence identity percentage between two or more polynucleotides. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. In an illustrative example, for nucleotide sequences, the cumulative score can be calculated using the parameters M (reward score for matching residue pairs; always> 0) and N (penalty score for mismatching residues; always< 0). When the cumulative alignment score drops by an amount X from its maximum achieved value; when the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored residue alignments; or when the end of either sequence is reached, the extension of word hits in each direction is stopped. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11 and an expectation value (E) of 10 as defaults, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89: 10915 (1989)) alignment, (B) is 50, the expected value (E) is 10, M = 5, N = -4, and compares two chains.

[0231] In certain embodiments, the "sequence identity percentage" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may include 20% or less (usually 5% to 15%, or 10% to 12%) additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the same nucleic acid base is present in the two sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to generate the percentage of sequence identity.

[0232] Those of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding antibodies or antigen-binding fragments thereof as described herein. Some such polynucleotides have minimal sequence identity to the nucleotide sequence of the native or original polynucleotide sequence encoding the antibodies or antigen-binding fragments thereof described herein. Nevertheless, polynucleotides that vary due to differences in codon usage are also explicitly contemplated by the present disclosure. In certain embodiments, sequences that have been codon-optimized for mammalian expression are specifically contemplated. Codon optimization can be performed using known techniques and tools, such as using OptimiumGene TM Tools. Codon-optimized sequences include partially codon-optimized sequences (ie, at least one codon is optimized for expression in a host cell) and those that are fully codon-optimized.

[0233] Therefore, in another embodiment, mutagenesis methods (such as site-specific mutagenesis) can be used to prepare variants and / or derivatives of the antibodies or antigen-binding fragments thereof described herein. In this way, specific modification of polypeptide sequences can be performed by mutagenizing the underlying polynucleotides encoding the polypeptide sequences. These techniques provide a straightforward method to prepare and test sequence variants, for example, by incorporating one or more of the aforementioned considerations by introducing one or more nucleotide sequence changes into the polynucleotides.

[0234] Site-specific mutagenesis allows the formation of mutations by using a specific oligonucleotide sequence encoding the desired mutated DNA sequence and a primer sequence of sufficient number of adjacent nucleotides to provide sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations can be used in selected polynucleotide sequences to improve, alter, reduce, modify or change the properties of the polynucleotide itself, and / or to alter the properties, activity, composition, stability or primary sequence of the encoded polypeptide.

[0235] In certain embodiments, the inventors of the present invention contemplate mutagenesis of a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein, or a variant thereof, to alter one or more properties of the encoded polypeptide (e.g., binding affinity to a Lewis antigen according to the present disclosure). Site-specific mutagenesis techniques are well known in the art and are widely used to generate variants of polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter specific portions of a DNA molecule. In such embodiments, a primer comprising a length of typically about 14 to about 25 nucleotides is used, wherein about 5 to about 10 residues on both sides of the sequence connection are altered.

[0236] As will be appreciated by those skilled in the art, site-specific mutagenesis techniques often use phage vectors in both single-stranded and double-stranded forms. Typical vectors for site-specific mutagenesis include vectors such as M13 phage. These phages are readily commercially available. Double-stranded plasmids that eliminate the step of transferring the target gene from a plasmid to a phage are also routinely used for site-specific mutagenesis.

[0237] Typically, the site-directed mutagenesis of the present invention is performed by first obtaining a single-stranded vector or unwinding the two strands of a double-stranded vector, which includes a DNA sequence encoding the desired peptide in its sequence. Oligonucleotide primers with the desired mutant sequence are typically prepared by synthesis. The primer is then annealed with the single-stranded vector and subjected to a DNA polymerase (such as E. coli polymerase I Klenow fragment) to complete the synthesis of the chain with the mutation. Thus, a heteroduplex is formed, in which one strand encodes the original non-mutated sequence and the second strand carries the desired mutation. The heteroduplex vector is then used to transform a suitable cell (such as an E. coli cell) and a clone of a recombinant vector comprising an arrangement of the mutant sequence is selected.

[0238] The use of site-directed mutagenesis to prepare sequence variants of a selected DNA fragment encoding a peptide provides a method for generating potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of peptides and the DNA sequences encoding them can be obtained. For example, a recombinant vector encoding a desired peptide sequence can be treated with a mutagen (such as hydroxylamine) to obtain sequence variants. Specific details about these methods and protocols are found in the teachings of the following references: Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each of which is incorporated herein by reference for this purpose.

[0239] As used herein, the term "oligonucleotide directed mutagenesis program" refers to a template-dependent process and vector-mediated proliferation, which results in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or results in an increase in the concentration of a detectable signal (such as amplification). As used herein, the term "oligonucleotide directed mutagenesis program" is intended to refer to a process involving template-dependent extension of a primer molecule. The term template-dependent process refers to nucleic acid synthesis of RNA or DNA molecules, wherein the sequence of the newly synthesized nucleic acid chain is determined by the known rules of complementary base pairing (see, e.g., Watson, 1987). Typically, vector-mediated methods include introducing nucleic acid fragments into DNA or RNA vectors, cloning amplification vectors, and recovering amplified nucleic acid fragments. Examples of such methods are provided by U.S. Patent No. 4,237,224, which is specifically incorporated herein by reference as a whole.

[0240] In another method of producing polypeptide variants, recursive sequence recombination as described in U.S. Patent No. 5,837,458 can be used. In this method, iterative cycles of recombination and screening or selection are performed to "evolve" single polynucleotide variants with, for example, increased binding affinity. Certain embodiments also provide constructs in the form of plasmids, vectors, transcripts, or expression cassettes comprising at least one polynucleotide as described herein.

[0241] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid or virus) used to transfer coding information to a host cell. The term "expression vector" refers to a vector suitable for host cell transformation and containing a nucleic acid sequence that directs and / or controls the expression of an inserted heterologous nucleic acid sequence. Expression vectors encoding antibodies or antigen-binding fragments thereof include viral vectors, such as lentiviral vectors or γ-retroviral vectors. Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex viruses type 1 and type 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (e.g., vaccinia, fowlpox and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma virus, mammalian C-type virus, B-type virus, D-type virus, HTLV-BLV group, slow virus, foamy virus (Coffin, JM, Retrovirus: Virus and its replication, "Basic Virology" (Retroviridae: The viruses and their replication, In Fundamental Virology), 3rd edition, BN Fields et al., ed., Lippincott-Raven Publishers, Philadelphia, 1996).

[0242] As used herein, "lentiviral vector" means a HIV-based lentiviral vector for gene delivery, which can be integrated or non-integrated, has a relatively large packaging capacity, and can transduce a series of different cell types. Slow viral vectors are usually produced after three (packaging, envelope and transfer) or more plasmids are transiently transfected into production cells. Like HIV, slow viral vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, viral RNA undergoes reverse transcription mediated by a viral reverse transcriptase complex. The reverse transcription product is a double-stranded linear viral DNA, which is a substrate for viral integration into the DNA of infected cells.

[0243] According to certain related embodiments, a recombinant host cell comprising one or more constructs as described herein is provided; a nucleic acid encoding an antibody or its antigen-binding fragment or its variant; and a method for producing the encoded product, the method comprising expressing the nucleic acid encoding it. Expression can be conveniently achieved by culturing a recombinant host cell containing a nucleic acid (e.g., a nucleic acid in a vector disclosed herein) under appropriate conditions. After being produced by expression, the antibody or its antigen-binding fragment can be separated and / or purified using any suitable technique and then used as desired.

[0244] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells (e.g., HEK cells, such as HEK293-c18 cells), NSO mouse melanoma cells, and many other cells. A common, preferred bacterial host is Escherichia coli.

[0245] The expression of peptides in prokaryotic cells (such as E. coli) is well established in the art. For a review, see, for example, Pluckthun, A. Bio / Technology 9:545-551 (1991). As an alternative to producing antibodies or antigen-binding fragments thereof, one skilled in the art can also culture and express in eukaryotic cells, see recent reviews, such as Ref, ME Curr. Opinion Biotech. 4:573-576; Trill JJ et al., (1995) Curr. Opinion Biotech 6:553-560.

[0246] Suitable vectors can be selected or constructed, containing suitable regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other suitable sequences. Where appropriate, the vector can be a plasmid, a virus (e.g., a phage or a phagemid). For further details, see, for example, Molecular Cloning: A Laboratory Manual: 2nd Edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulating nucleic acids (e.g., preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression and analyzing proteins) are described in detail in Current Protocols in Molecular Biology, 2nd Edition, Ausubel et al., ed., John Wiley & Sons, 1992, or subsequent updates thereof.

[0247] The term "host cell" is used to refer to a cell into which a nucleic acid sequence encoding one or more antibodies and antigen-binding fragments thereof described herein has been introduced or can be introduced, and further expresses or can express a selected target gene (such as a gene encoding any antibody or antigen-binding fragment described herein). The term includes the offspring of the parental cell, whether the offspring is identical to the original parent in morphology or genetic composition, as long as the selected gene exists. Therefore, a method is also contemplated, which includes introducing such nucleic acids into a host cell. The introduction can be carried out using any available technology. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses (such as vaccinia virus), or baculovirus for insect cells. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophages. After introduction, for example, by culturing the host cell under conditions of expressing the gene, the expression of the nucleic acid is caused or allowed. In one embodiment, the nucleic acid is integrated into the genome (such as chromosome) of the host cell. According to standard techniques, integration can be promoted by including sequences that promote recombination with the genome.

[0248] In certain embodiments, the present invention also provides a method comprising using a construct as described above in an expression system to express a specific polypeptide (such as an antibody or antigen-binding fragment thereof) as described herein. The term "transduction" is used to refer to the transfer of genes from one bacterium to another bacterium, usually by bacteriophage. "Transduction" also refers to the acquisition and transfer of eukaryotic cell sequences by retroviruses. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell is "transfected" when the exogenous DNA has been introduced into the cell membrane. Many transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory; Davis et al., 1986, BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier; and Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more foreign DNA portions into a suitable host cell.

[0249] As used herein, the term "conversion" refers to the change of the genetic characteristics of cells, and when cells are modified to contain new DNA, cells have been transformed. For example, in the case where cells are genetically modified from their native state, cells are transformed. After transfection or transduction, the transforming DNA can be physically integrated into the chromosome of the cell and recombined with the DNA of the cell, or can be maintained instantaneously as a free element without being replicated, or can be replicated independently as a plasmid. When DNA is replicated as the division of the cell, it is believed that the cell has been stably transformed. When used in combination with biomaterials (such as nucleic acid molecules, polypeptides, host cells, etc.), the term "naturally occurring" or "natural" refers to substances found in nature and not manipulated by people. Similarly, "non-naturally occurring" or "non-natural" as used herein refers to substances that are not found in nature or are structurally modified or synthesized by people.

[0250] It should be understood that, unless specifically indicated to the contrary, the implementation of the several embodiments of the present invention will employ conventional methods in virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of the art, and many of which are described below for illustrative purposes. Such techniques are fully explained in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Vols. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (N. Gait, ed., 1986). Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0251] Certain currently disclosed embodiments relate to the detection and characterization of specific immunological binding activity (e.g., antibody binding activity) to carbohydrate-defined antigenic structures. Those skilled in the art will appreciate that there are a variety of methods to produce and test carbohydrate-specific immunoreagents, including fine structural characterization of homologous carbohydrate antigens. Non-limiting examples of such techniques are described in Haji-Ghassemi et al., 2015 Glybiol. 25: 920; Dingjan et al., 2015 Mol. Immunol. 67 (2Pt A): 75-88; Soliman et al., 2017 Curr. Opin. Struct. Biol. 44: 1-8; and Hakomori, 2001 Adv. Exp. Med. Biol. 491: 369-402; and in the references cited therein. Descriptions of sources of carbohydrate-defined antigens and methods of their preparation, isolation, and structural characterization can also be found here and elsewhere. Exemplary and non-limiting applications of such methods are disclosed herein, and the disclosure is not intended to be so limited, and also contemplates the synthetic preparation of structurally defined carbohydrate antigens, including by exploiting the refined specificity of biosynthetic enzymes known in the art as glycosyltransferases. See, e.g., Wu et al., Universal phosphatase-coupled glycosyltransferase assay, Glycobiology 21(6):727-733, 2011; Becker et al., Fucose: biosynthesis and biological function in mammals, Glycobiology 13(7):41R-53R, 2003; de Vries et al., Fucosyltransferases: structure / function studies, Glycobiology 11(10):119R–128R, 2001.

[0252] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.

[0253] These and related techniques and procedures can generally be performed according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. Unless specific definitions are provided, the nomenclature used in connection with laboratory procedures and techniques of molecular biology, analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry as described herein is that which is well known and commonly used in the art. As further described herein, standard techniques can be used for recombinant techniques, molecular biology, microbiology, chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients.

[0254] Composition

[0255] In certain aspects, the disclosure provides antibodies and antigen-binding fragments thereof, as well as variants thereof and compositions comprising the same. In certain embodiments, an isolated antibody is provided, comprising an immunoglobulin heavy chain having an amino acid sequence as shown in SEQ ID NO: 10 and an immunoglobulin light chain having an amino acid sequence as shown in SEQ ID NO: 11.

[0256] In certain embodiments, an isolated antibody or antigen-binding fragment thereof is provided, comprising: an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35, and the immunoglobulin light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to:

[0257] Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen,

[0258] Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen,

[0259] Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen,

[0260] and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]

[0261] [VIII]'s Double Tentacles Y / Le B antigen,

[0262] and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A Antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV].

[0263] In other embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain variable region comprising a heavy chain complementary determining region 1 (VH CDR1), a heavy chain complementary determining region 2 (VH CDR2), and a heavy chain complementary determining region 3 (VHCDR3), wherein the heavy chain complementary determining region 1 (VH CDR1) comprises the amino acid sequence shown in SEQ ID NO:2, the heavy chain complementary determining region 2 (VH CDR2) comprises the amino acid sequence shown in SEQ ID NO:3, and the heavy chain complementary determining region 3 (VHCDR3) comprises the amino acid sequence shown in SEQ ID NO:4; and (b) an immunoglobulin light chain variable region comprising a light chain complementary determining region 1 (VL CDR1), a light chain complementary determining region 2 (VL CDR2), and a light chain complementary determining region 3 (VL CDR3), wherein the light chain complementary determining region 1 (VL CDR1) comprises the amino acid sequence shown in SEQ ID NO:6, and the light chain complementary determining region 2 (VL CDR2) comprises the amino acid sequence shown in SEQ ID NO:7. NO:7, the light chain complementary determining region 3 (VL CDR3) comprises the amino acid sequence shown in SEQ ID NO:8; wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to:

[0264] Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen,

[0265] Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen,

[0266] Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen,

[0267] and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]

[0268] [VIII]'s Double Tentacles Y / Le B antigen,

[0269] and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A Antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV].

[0270] In certain presently disclosed embodiments, the isolated antibody or antigen-binding fragment thereof is monoclonal.

[0271] In certain presently disclosed embodiments, the isolated antibodies or antigen-binding fragments thereof are humanized.

[0272] In certain embodiments, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv (ScFv) antibody, and a diabody.

[0273] In certain other embodiments, an isolated antibody or antigen-binding fragment thereof is provided, comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:5, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to:

[0274] Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen,

[0275] Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen,

[0276] Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen,

[0277] and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]

[0278] [VIII]'s Double Tentacles Y / Le B antigen,

[0279] and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A Antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV].

[0280] In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain variable region comprising a heavy chain complementary determining region 1 (VH CDR1), a heavy chain complementary determining region 2 (VH CDR2), and a heavy chain complementary determining region 3 (VHCDR3), wherein the heavy chain complementary determining region 1 comprises the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:2; the heavy chain complementary determining region 2 (VH CDR2) comprises the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:3; the heavy chain complementary determining region 3 (VH CDR3) comprises the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4 NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:4; and (b) an immunoglobulin light chain variable region comprising a light chain complementary determining region 1 (VL CDR1), a light chain complementary determining region 2 (VL CDR2) and a light chain complementary determining region 3 (VL CDR3), wherein the light chain complementary determining region 1 (VL CDR1) comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:6; the light chain complementary determining region 2 (VL CDR2) comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:7. NO:7 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical; the light chain complementary determining region 3 (VL CDR3) comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:8.

[0281] In other embodiments, the isolated antibody or antigen-binding fragment thereof comprises the following (i), (ii), (iii), (iv), (v) or (vi), or any combination thereof: (i) a VH CDR1 comprising a variant of the amino acid sequence shown in SEQ ID NO:2, wherein the variation consists of a Y→A substitution at position 33 according to Kabat numbering; (ii) a VH CDR3 comprising a variant of the amino acid sequence shown in SEQ ID NO:4, wherein the variation consists of a Y→A substitution at position 104 according to Kabat numbering; (iii) a VH CDR3 comprising a variant of the amino acid sequence shown in SEQ ID NO:4, wherein the variation consists of an H→A substitution at position 106 according to Kabat numbering; (iv) a VL CDR1 comprising a variant of the amino acid sequence shown in SEQ ID NO:6, wherein the variation consists of a Y→A substitution at position 30 according to Kabat numbering; (v) a VL CDR2 comprising a variant of the amino acid sequence shown in SEQ ID NO:7, wherein the variation consists of a G→A substitution at position 50 according to Kabat numbering; or (vi) a VH CDR3 comprising a variant of the amino acid sequence shown in SEQ ID NO:4, wherein the variation consists of an H→A substitution at position 106 according to Kabat numbering; VL CDR3 of a variant of the amino acid sequence shown in NO:8, wherein the variation consists of a T→S substitution at position 93 according to Kabat numbering.

[0282] In any of the embodiments described herein, the isolated antibody or antigen-binding fragment thereof can have reduced (e.g., reduced in a statistically significant manner) binding to the monoantennary Lewis B antigen or monoantennary Lewis Y compared to a BBC antibody comprising a VL domain having the amino acid sequence shown in SEQ ID NO:27 and a VH domain having the amino acid sequence shown in SEQ ID NO:28.

[0283] In certain other embodiments, an isolated antibody or antigen-binding fragment is provided, which comprises a CDR as described herein (i.e., a CDR having at least 90% identity to SEQ ID NOs: 2, 3, 4, 6, 7, and 8, respectively, including those CDR variants having amino acid substitutions described herein), and comprises an immunoglobulin heavy chain variable region comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 35; and an immunoglobulin light chain variable region comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 35; NO:5 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in NO:5, or consists of an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:5.

[0284] Antibodies or antigen-binding fragments thereof (such as those described herein) include but are not limited to scFv, and in certain embodiments, may be included in a fusion protein capable of specifically binding to a Lewis antigen as described herein. In some embodiments, the fusion protein can be expressed on the surface of a host cell (e.g., a T cell, a NK cell, or a NK-T cell), and comprises an extracellular component and an intracellular component, wherein the extracellular component comprises an antibody or antigen-binding fragment thereof as disclosed herein, and the intracellular component comprises an effector domain, wherein the effector domain is activated upon receiving an appropriate signal (e.g., an effector domain from the following: CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOT CH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2 or any combination thereof) can directly or indirectly promote an immune response in a cell (e.g., an immune system cell, such as a T cell), wherein the extracellular component and the intracellular component are connected by a transmembrane domain (e.g., a CD8 transmembrane domain, a CD4 transmembrane domain, a CD27 transmembrane domain or a CD28 transmembrane domain), and the intracellular component optionally includes a costimulatory domain or a portion thereof, and the costimulatory domain or a portion thereof is selected from CD27, CD28, 4-1BB (CD137), OX40 (CD134), or a combination thereof. In certain embodiments, the extracellular component of the fusion protein comprising an antibody or antigen-binding fragment of the present disclosure includes a polypeptide derived from an immunoglobulin protein; for example, IgG4 hinge-CH2-CH3.

[0285] In these and related embodiments, the antigen binding fragment may comprise an antigen binding fragment described herein (such as a scFv), and the extracellular component may further comprise a connecting region comprising a hinge; for example, in a chimeric antigen receptor molecule (CAR), which can be expressed on the cell surface of a host cell (such as a T cell, NK cell or NK-T cell) for use in cellular immunotherapy. CAR molecules and the principles involved are described in, for example: Sadelain et al., Cancer Discov., 3(4):388 (2013); Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016); Stone et al., Cancer Immunol. Immunother., 63(11):1163 (2014); Xu et al., 2018 Oncotarget 9:13991; Androulla et al., 2018 Curr. Pharm. Biotechnol. Vol. 19 (April 2018); Wu et al., 2016 Expert Opin. Biol. Ther. 16:1469; Ren et al., 2017 Protein Cell 8:634; the CAR molecules, CAR designs, and CAR design principles are incorporated herein by reference in their entirety.

[0286] Also provided herein are antibody conjugates comprising antibodies of the present disclosure or their Fabs and payload molecules connected thereto. As background, monoclonal antibodies specifically targeting antigens can be used as carrier molecules to deliver treatment or detectable payload molecules to antigen expression sites (e.g., tumor cells expressing antigens). Combination of antibody conjugates with antigens can allow, for example, targeted delivery of cytotoxic payloads or detectable moieties to diseased cells or tissues for treatment, or detection, imaging or monitoring of diseases (e.g., cancers). In certain embodiments, antibody conjugates are internalized by target cells, which express antigens after antibody conjugates are combined or once combined. Internalization to the cytosol or lysosomal compartments of target cells can allow selective release of payload molecules, such as causing cytotoxic damage to target cells.

[0287] Various techniques can be used to couple the payload molecule to the antibody or its antigen binding fragment to form the antibody conjugate of the present disclosure. In some embodiments, the antibody conjugate comprises a payload molecule covalently attached to the antibody or its antigen binding fragment by a linker. The linker in the antibody conjugate (e.g., antibody drug conjugate) for containing a cytotoxic agent or an antiproliferative agent is generally an organic compound falling into one of two groups, and the two groups are organized according to the mechanism by which the payload molecule is released from the carrier molecule. Cleavable linkers are designed to be selectively degraded or cleaved based on the intrinsic properties of the target cell: three types of cleavable linkers are protease-sensitive linkers (whereby linkers (e.g., linkers containing valine-citrulline or phenylalanine-lysine dipeptides or tetrapeptides (e.g., GFLG or ALAL)) are cleaved by proteases present in tumor cell lysosomes to release the payload molecule); pH-sensitive linkers, which contain acid-labile groups that are selectively hydrolyzed by the lower pH of endosomal and lysosomal compartments relative to cytosolic pH; and glutathione-sensitive linkers, which contain disulfide bridges that are reduced by intracellular glutathione. Non-cleavable linkers rely on nonspecific degradation of the antibody conjugate to release the payload molecule.

[0288] Disclose specific linkers, payloads, linker chemistry and related mechanisms and methods are disclosed in Nareshkumar et al., Pharm. Res. 32: 3526-3540 (2015), the compositions, methods and techniques are incorporated herein by reference in their entirety. In certain embodiments, the antibody conjugate comprises a linker selected from a cleavable linker and a non-cleavable linker. In other embodiments, the linker is a cleavable linker selected from a protease-sensitive linker, a pH-sensitive linker or a glutathione-sensitive linker. In a specific embodiment, the cleavable linker is a protease-sensitive linker comprising a valine-citrulline dipeptide.

[0289] Any suitable technique or mechanism can be used to connect or couple the linker to the antibody or its Fab. In some embodiments, the linker comprises a maleimide group (optionally PEGylated), which can react with the reducing disulfide bridges in the hinge region of the antibody or its Fab. Other sites suitable for conjugation with the linker on the carrier molecule (i.e., the antibody or its Fab) can be introduced or engineered using recombinant technology, such as introducing cysteine ​​residues or non-natural amino acids for site-specific conjugation. Methods for introducing such modifications include, for example, the methods described in Examples 6.3-7 of PCT Publication No. WO2012 / 032181.

[0290] In some embodiments, the linker further comprises a self-immolative group (also referred to as a self-immolative group or a self-immolative spacer) to facilitate the selective cleavage reaction. In certain embodiments, the self-immolative group is p-aminobenzyl alcohol (PABC).

[0291] Click chemistry for producing antibody conjugates includes those described in Meyer et al., Bioconjug. Chem. 27(12):2791-2807 (2016), and is incorporated herein by reference in its entirety.

[0292] In any one of the antibody conjugates described herein, the payload molecule may be selected from a therapeutic agent and a detectable indicator. Therapeutic agents suitable for cancer treatment include those described in Parslow et al., Biomedicines 4:14 (2016), whose payload and ADC design principles are incorporated herein by reference. In certain embodiments, the payload molecule is a therapeutic agent selected from antimitotic agents targeting tubulin, peptide-based toxins, pyrrolobenzodiazepine (PBD) dimers, antibiotics (e.g., calicheamicin), pyrimidine synthesis inhibitors (e.g., 5-fluorouracil), antimetabolites (e.g., methotrexate), DNA alkylating agents, and topoisomerase inhibitors (e.g., doxorubicin). In other embodiments, the payload molecule is selected from maytansinoid alkaloids, auristatin, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0293] In other embodiments, the payload molecule is a detectable indicator. Detectable indicators suitable for antibody conjugates, and related labeling strategies and imaging techniques (e.g., PET, MRI, NIR), including those disclosed in Friese and Wu, Mol.Immunol.67 (200): 142-152 (2015) and Moek et al., J.Nucl.Med.58: 83S-90S (2017), all of which are incorporated herein by reference. In certain embodiments, detectable indicators are selected from: radionuclides, dyes, radioactive metals, fluorescent moieties, MRI contrast agents, microbubbles, carbon nanotubes, gold particles, fluorodeoxyglucose, enzymes, chromophores, and radiopaque markers. In a specific embodiment, detectable indicators are selected from the following radionuclides: 68 Ga, 64 Cu, 86 Y. 89 Zr, 124 I. 99m Tc, 123 I. 111 In, 177 Lu,131 I. 76 Br, 78 Zr, 18 F and 124 T. In certain such embodiments, the antibody conjugate further comprises a radionuclide chelator selected from the group consisting of maleimide-labeled DOTA, N-hydroxysuccinimide-DOTA, and deferoxamine (DFO).

[0294] Also provided herein are pharmaceutical compositions comprising an antibody, antigen-binding fragment thereof, or antibody conjugate of the present disclosure; and a pharmaceutical carrier.

[0295] In another aspect, the disclosure provides isolated polynucleotides encoding antibodies or antigen-binding fragments as described herein, comprising sequence variants having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to a specific nucleotide sequence disclosed herein. In certain embodiments, polynucleotides encoding antibodies or antigen-binding fragments of the present disclosure (e.g., including scFv, Fab, F(ab'2) fragments, Fv fragments or diabodies), or polynucleotides encoding a portion of an antibody or antigen-binding fragment (e.g., heavy chain, light chain, VH, VL or CDR), have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to any one of SEQ ID NOs: 12-22 or 37. In certain embodiments, the polynucleotides are codon-optimized for expression in a host cell (e.g., a mammalian cell). In any of the above embodiments, the polynucleotides may be provided in a recombinant vector. In certain embodiments, the vector comprises an expression control sequence operably linked to a polynucleotide encoding an antibody or its antigen-binding fragment. In certain such embodiments, the vector (e.g., a recombinant vector) is an expression vector, wherein the expression control sequence comprises a promoter.

[0296] In another aspect, a host cell comprising a recombinant vector or expression vector as described herein is provided. In a related aspect, a method for producing an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to:

[0297] Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen,

[0298] Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen,

[0299] Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen,

[0300] and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]

[0301] [VIII]'s Double Tentacles Y / Le B antigen,

[0302] and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV],

[0303] The method comprises culturing the host cell of the present disclosure under conditions and for a time sufficient for the host cell to express a polynucleotide encoding the antibody or its antigen-binding fragment, thereby obtaining a culture containing the antibody or its antigen-binding fragment; and recovering the antibody or its antigen-binding fragment from the culture.

[0304] Methods and uses for detecting and treating disease

[0305] In certain embodiments, the presently disclosed antibodies, antigen-binding fragments thereof, and antibody conjugates may be used in methods of detecting or treating a disease characterized by expression (eg, overexpression) of a Lewis antigen as described herein.

[0306] As understood by those skilled in the medical field, the term "treat" or "treatment" refers to the medical management of a disease, disorder or condition of a subject (i.e., a patient, a host, which may be a human or non-human animal) (see, e.g., Stedman's Medical Dictionary). Typically, suitable dosages and treatment regimens provide one or more of an antibody, an antigen-binding fragment thereof, or an antibody conjugate in an amount sufficient to provide a therapeutic or preventive benefit. The therapeutic or preventive benefits resulting from a therapeutic treatment or prophylactic or preventive approach include, for example, improved clinical outcomes, wherein the purpose is to prevent or delay or otherwise reduce (e.g., to reduce in a statistically significant manner relative to an untreated control) an undesirable physiological change or disorder, or to prevent, delay or otherwise reduce the spread or severity of such a disease or disorder. Beneficial or desired clinical results for a subject of treatment include alleviation, reduction or alleviation of symptoms caused by or associated with the disease or condition being treated; reduction in the occurrence of symptoms; improved quality of life; longer disease-free state (i.e., reduced likelihood or propensity for a subject based on which the disease diagnosis was made will present); reduction in the extent of the disease; stabilization (i.e., not worsening) of the disease state; delay or slowing of disease progression; improvement or palliation of the disease state; and remission (whether partial or complete), whether detectable or undetectable; or overall survival.

[0307] "Treatment" can also mean to prolong survival when compared to the expected survival if the subject is not receiving treatment. Objects that need the methods and compositions described herein include those that already have a disease or condition, and objects that are prone to or are at risk of developing a disease or condition. Objects that need treatment to reduce the likelihood of the occurrence or recurrence of a disease or condition or that need preventive treatment can include objects where the severity of the disease, condition or condition is reduced or partially or completely avoided or incompletely or completely prevented (e.g., reducing the likelihood of the occurrence or recurrence of a disease or condition, which can include but does not necessarily require absolute prevention). As described in the examples, the clinical benefits provided by the compositions (and preparations comprising the compositions) and methods described herein can be evaluated by designing and conducting in vitro assays, and preclinical and clinical studies in objects to which the compositions are administered are intended to be beneficial.

[0308] For example, in certain embodiments, methods for treating or detecting cancer (i.e., cancer expressing Lewis antigens as described herein) are provided, wherein the methods include administering a pharmaceutical composition of the present disclosure to a subject in need thereof. As used herein, "cancer" refers to a condition characterized by abnormal or uncontrolled proliferation (e.g., hyperproliferation) of diseased cells, which can be characterized by malignant spread from a first tissue or site to one or more adjacent or distant tissues or sites in the body.

[0309] In a specific embodiment of the method, the subject has or is suspected of having a cancer selected from the group consisting of gastric cancer, colon cancer, breast cancer, lung cancer, lymphoma, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, uterine cancer, and squamous cell carcinoma. In other embodiments, the cancer is selected from the group consisting of gastric adenocarcinoma, mucinous gastric adenocarcinoma, undifferentiated gastric adenocarcinoma, signet ring cell gastric carcinoma, colon adenocarcinoma, invasive breast ductal carcinoma, hepatocellular carcinoma, lung adenocarcinoma, squamous cell carcinoma, metastatic lymph node adenocarcinoma, mucinous ovarian adenocarcinoma, pancreatic ductal adenocarcinoma, pancreatic papillary adenocarcinoma, prostate adenocarcinoma, and endometrioid carcinoma. Administration of the antibodies, antigen-binding fragments, or antibody conjugates described herein, in pure form or in the form of an appropriate pharmaceutical composition, can be carried out by any acceptable mode of administration for agents providing similar uses. Pharmaceutical compositions can be prepared by combining antibodies, antigen binding fragments or antibody conjugates with suitable physiologically acceptable carriers, diluents or excipients, and can be formulated into solid, semisolid, liquid or particulate (e.g., droplets) containing gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. In addition, other pharmaceutically active ingredients (including other immunosuppressants as described elsewhere herein) and / or suitable excipients (e.g., salts), buffers and stabilizers may, but are not necessarily, be present in the composition.

[0310] The exact dosage and duration of treatment can be a function of the condition or disease being treated, and can be determined empirically using known test protocols or by testing the composition in a model system known in the art and extrapolating therefrom. Controlled clinical trials can also be conducted. The dosage can also vary with the severity of the condition to be alleviated. Pharmaceutical compositions are typically formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition can be administered once, or can be divided into multiple smaller doses administered at certain time intervals. For any particular object, the specific dosage regimen can be adjusted over time according to individual needs.

[0311] The "effective amount" of the composition refers to the amount sufficient to achieve the desired clinical results or beneficial treatment as described herein in the dosage and required time period. The effective amount can be delivered in one or more administrations. If applied to a known or confirmed object with a disease or disease state, the term "therapeutic amount" can be used to relate to treatment, and "preventive effective amount" can be used to describe the effective amount of the beneficial and / or protective process used as a reduction (for example, in a statistically significant manner relative to an untreated state) of the likelihood and / or severity of the occurrence of a disease or disease state to a risk object susceptible to disease or in the development of a disease or disease state (for example, recurrence). In a statistically significant manner relative to an untreated state), the likelihood of the occurrence and / or severity of a disease or disease state.

[0312] In various embodiments, the antibody conjugates of the present disclosure include a detectable payload as described herein, and can be used for in vivo, in vitro or ex vivo detection of diseases (such as cancer). In some of these embodiments and other embodiments, the antibody (i.e., one or more antibodies) or its Fab as described herein is conjugated (e.g., covalently) to a detectable label that can be detected directly or indirectly. In the present disclosure, any of the disclosed monoclonal antibodies, Fabs thereof, and antibody conjugates can be connected to a detectable label (e.g., in addition to the detectable or therapeutic payload molecule of the antibody conjugate). In "direct detection", only one detectable antibody, i.e., a primary detectable antibody, is used. Therefore, direct detection means that the antibody itself conjugated to a detectable label can be detected without the need to add a second antibody (secondary antibody).

[0313] A "detectable label" is a molecule or material that can generate a detectable (e.g., visual, electronic or other) signal that indicates the presence and / or concentration of the label in a sample. When conjugated to a peptide, a detectable label can be used to locate and / or quantify the target to which a particular peptide is bound. Thus, the presence and / or concentration of the target in a sample can be detected by detecting the signal generated by the detectable label. Detectable labels can be detected directly or indirectly, and several different detectable labels conjugated to different specific antibodies can be used in combination to detect one or more targets.

[0314] Examples of directly detectable detectable labels include fluorescent dyes and radioactive materials and metal particles. In contrast, indirect detection requires the application of one or more additional antibodies, i.e., secondary antibodies, after the application of the primary antibody. Thus, detection is performed by detecting the binding of the secondary antibody or binding agent to the primary detectable antibody. Examples of primary detectable binding agents or antibodies that require the addition of a secondary binding agent or antibody include enzyme detectable binding agents and hapten detectable binding agents or antibodies.

[0315] In some embodiments, the detectable label is conjugated to a nucleic acid polymer comprising a first binding agent (e.g., during ISH, WISH, or FISH). In other embodiments, the detectable label is conjugated to an antibody comprising a first binding agent (e.g., during IHC).

[0316] Examples of detectable labels that can be conjugated to the antibodies, antigen-binding fragments, and antibody conjugates used in the methods of the present disclosure include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, metal particles, haptens, and dyes.

[0317] Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-formamidohexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine and dyes such as Cy2, Cy3 and Cy5, optionally substituted coumarins including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeton Red, green fluorescent protein (GFP) and its analogs, as well as conjugates of R-phycoerythrin or allophycoerythrin, inorganic fluorescent labels such as particles based on semiconductor materials, such as coated CdSe nanocrystals.

[0318] Examples of polymer particle labels include microparticles or latex particles of polystyrene, PMMA or silica that may be embedded with fluorescent dyes, or polymer micelles or capsules containing dyes, enzymes or substrates.

[0319] Examples of metal particle labels include gold particles and coated gold particles that can be transformed by silver staining. Examples of haptens include DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin. Examples of enzymatic labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosamine amidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO). Examples of commonly used substrates for horseradish peroxidase include 3,3'-diaminobenzidine (DAB), diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), Indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), α-naphtholpyronine (α-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitro blue tetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT), tetranitro blue tetrazolium (TNBT), 5-bromo-4-chloro-3-indoxyl-β-D-galactoside / ferric ferrocyanide (BCIG / FF).

[0320] Examples of commonly used substrates for alkaline phosphatase include naphthol-AS-B 1-phosphate / fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthol-AS-B1-phosphate / fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthol-AS-B1-phosphate / new fuchsin (NABP / NF), bromochloroindolyl phosphate / nitro blue tetrazolium (BCIP / NBT), and 5-bromo-4-chloro-3-indolyl-b--d-galactopyranoside (BCIG).

[0321] Examples of luminescent labels include luminol, isoluminol, acridinium esters, 1,2-dioxetanes, and pyridopyridazines. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioactive isotopes of iodine, cobalt, selenium, tritium, carbon, sulfur, and phosphorus.

[0322] The detectable label can be connected to the antibodies, antigen binding fragments and antibody conjugates described herein, or to any other molecule (e.g., antibody, nucleic acid probe or polymer) that specifically binds to the target biomarker. In addition, it will be understood by those of ordinary skill in the art that the detectable label can also be conjugated to the second, and / or third, and / or fourth, and / or fifth binding agent or antibody, etc. In addition, it will be understood by those skilled in the art that each additional binding agent or antibody used to characterize the target biomarker can be used as a signal amplification step. Biomarkers can be visually detected using, for example, an optical microscope, a fluorescent microscope, an electron microscope (wherein the detectable substance is, for example, a dye, colloidal gold particles, a luminophore reagent). Spectrophotometers can also be used to detect visually detectable substances combined with biomarkers. In the case where the detectable substance is a radioisotope, it can be detected visually by autoradiography or detected visually using a scintillation counter. See, e.g., Larsson, 1988, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.); Methods in Molecular Biology, Vol. 80, 1998, John D. Pound (ed.) (Humana Press, Totowa, NJ).

[0323] In certain embodiments, the antibodies, antigen binding fragments or antibody conjugates of the present disclosure are used to detect or diagnose a method for a disease associated with the expression of a Lewis antigen as described herein, wherein the method comprises contacting the antibody, antigen binding fragment or antibody conjugate with a sample from an object suspected of having the disease or at risk of having the disease, and detecting the formation of an antibody: antigen complex and / or detecting specific binding of the antibody, antigen binding fragment or antibody conjugate in the sample. In certain embodiments, the sample comprises blood (e.g., peripheral blood), tissue, tumor, or any combination thereof. In certain embodiments, the diagnosis or detection method is performed ex vivo or in vitro.

[0324] Methods for in vivo detection of antibody conjugates with a detectable payload or detectable label include those described / disclosed in Friese and Wu, Mol. Immunol. 67(200):142-152(2015) and Moek et al., J. Nucl. Med. 58:83S-90S(2017), all incorporated herein by reference. In certain embodiments, detection of the antibody, antigen binding fragment, or antibody conjugate comprises performing positron emission tomography (PET), magnetic resonance imaging (MRI), near infrared imaging (NRI), x-ray computed tomography (CT), single photon emission computed tomography (SPECT), optical imaging, ultrasonography, or any combination thereof.

[0325] The preferred mode of administration depends on the nature of the condition to be treated, which in certain embodiments will refer to a harmful or clinically undesirable condition, the extent, severity, likelihood of occurrence and / or duration of which can be reduced (e.g., reduced in a statistically significant manner relative to an appropriate control situation (e.g., an untreated control)) according to certain methods provided herein. After administration, the following amounts are considered effective: detectably reduce, inhibit, at least partially prevent, reduce the severity or likelihood of such conditions occurring, or delay the severity or likelihood of such conditions occurring, such as a partial or complete reduction in tumor burden or a partial or complete reduction in metastatic spread. Those skilled in the relevant art will be familiar with the many diagnostic, surgical and / or other clinical criteria that may indicate the clinical appropriateness of administering the compositions described herein and / or may adapt them. See, e.g., Hanahan and Weinberg, 2011 Cell 144:646; Hanahan and Weinberg 2000 Cell 100:57; Cavallo et al., 2011 Canc. Immunol. Immunother. 60:319; Kyrigideis et al., 2010 J. Carcinog. 9:3; Park et al., 2009 Molec. Therap. 17:219; Cheever et al., 2009 Clin Cancer Res 15(17):5323-5337; Lu et al., 2013 Curr. Pharm. Biotechnol. 14:714-22; Layke et al., 2004 Am. Fam. Physician 69:1133049; Bunn, 2012 Arch. Pathol. Lab. Med. 136:1478-81; Manne et al., 2005 Drug Discov. Today 10:965; Schmoll et al. (eds.), 2009 ESMO Handbook of Cancer Diagnosis and Treatment Evaluation, CRC Press, Boca Raton, FL; Faix, 2013 Crit. Rev. Clin. Lab. Sci. 50(1):23-36 (“Biomarkers of Sepsis”); Wiersinga et al., 2014 Virulence 5(1):36-44 (“Host innate immune responses to sepsis”). sepsis)"); Hotchkiss et al., 2013 Nat. Rev. Immunol. 13:862; Aziz et al., 2013 J.Leukoc.Biol.93(3):329; Beyrau et al., 2012 Open Biol.2:120134; Fry, 2012 Amer.Surg.78:1; Kellum et al., 2007 Arch.Intern.Med.167(15):1655; Remick, 2007 Am.J.Pathol.170(5):1435; Hotchkiss et al., 2003 New Engl.J.Med.348:138-150; Humar et al., Atlas of Organ Transplantation, 2006, Springer; Kuo et al., Comprehensive Atlas of Transplantation, 2004 Lippincott, Williams & Wilkins; Gruessner et al., Living Donor Organ Transplantation, 2007 McGraw-Hill Professional; Antin et al., Manual of Stem Cell and Bone Marrow Transplantation, 2009 Cambridge University Press; Wingard et al. (eds.), Hematopoietic Stem Cell Transplantation: A Handbook for Clinicians, 2009 American Association of Blood Banks; and references cited therein. .

[0326] Therefore, the typical routes of administering these and related pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhaled, parenteral, sublingual, oral, rectal, vaginal and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. The pharmaceutical composition according to certain embodiments of the present invention is formulated to allow the active ingredient contained therein to be bioavailable when the composition is administered to the patient. The composition to be administered to the object or patient can take the form of one or more dosage units, wherein, for example, a tablet can be a single dosage unit, and the container of the antibody, its antigen-binding fragment or antibody conjugate in the form of an aerosol as described herein can accommodate multiple dosage units. The actual method of preparing such dosage forms is known to those skilled in the art or will be obvious; for example, see Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000). In any case, the composition to be administered will contain a therapeutically effective amount of the antibody, its antigen-binding fragment or antibody conjugate of the present disclosure for treating the disease or condition of interest according to the teachings herein. In certain embodiments, administering comprises administering by a route selected from the group consisting of intravenous, parenteral, intragastric, intrapleural, intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, oral, topical, transdermal, intracranial, intrathecal, intranasal, and intramuscular.

[0327] The pharmaceutical composition can be in solid or liquid form. In one embodiment, the carrier is particulate, so that the composition is, for example, in tablet or powder form. The carrier can be a liquid, and the composition can be, for example, an oral oil, an injectable liquid or an aerosol, which can be used, for example, for inhalation administration. When used for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are included in the forms considered as solid or liquid herein.

[0328] As a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gum, flakes, etc. Such solid compositions usually contain one or more inert diluents or edible carriers. In addition, one or more of the following substances may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch, lactose or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as mint, methyl salicylate or orange flavoring; and coloring agents. When the pharmaceutical composition is in the form of a capsule (e.g., a gelatin capsule), it may contain a liquid carrier (e.g., polyethylene glycol or oil) in addition to the above-mentioned types of substances.

[0329] The pharmaceutical composition can be in liquid form, such as elixir, syrup, solution, emulsion or suspension. As two examples, the liquid can be used for oral administration or delivered by injection. When intended for oral administration, the preferred composition contains one or more sweeteners, preservatives, dyes / colorants and flavor enhancers in addition to the compounds of this invention. In the composition intended to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents can be included.

[0330] Liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils (such as synthetic monoglycerides or diglycerides, which can be used as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral preparations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0331] Liquid pharmaceutical compositions for parenteral or oral administration should contain a certain amount of antibodies, Fabs thereof or antibody conjugates disclosed herein, so as to obtain a suitable dosage. Typically, the amount is at least 0.01% of the antibody or Fab in the composition. When intended for oral administration, the amount can be varied from 0.1% to about 70% of the weight of the composition. Some oral pharmaceutical compositions contain about 4% to about 75% of antibodies, Fabs thereof or antibody conjugates. In certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that the parenteral dosage unit contains 0.01-10% by weight of antibodies, Fabs thereof or antibody conjugates before dilution.

[0332] The pharmaceutical composition can be intended for topical administration, in which case the carrier can suitably include a solution, an emulsion, an ointment or a gel matrix. For example, the matrix can include one or more of: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, a diluent (such as water and alcohol) and an emulsifier and a stabilizer. A thickener can be present in a topical pharmaceutical composition. If intended for transdermal administration, the composition can include a transdermal patch or an iontophoresis device. The pharmaceutical composition can be intended for rectal administration, such as in the form of a suppository, which will melt and release the drug in the rectum. Compositions for rectal administration can contain an oleaginous matrix as a suitable non-irritating excipient. This matrix includes but is not limited to lanolin, cocoa butter and polyethylene glycol.

[0333] Pharmaceutical compositions can include various materials that change the physical form of solid or liquid dosage units. For example, the composition can include materials that form a coating shell around the active ingredient. The material forming the coating shell is usually inert and can be selected from, for example, sugar, shellac and other enteric coating agents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule. Pharmaceutical compositions in solid or liquid form can include reagents that are combined with antibodies, antigen-binding fragments thereof or antibody conjugates of the present invention to facilitate the delivery of the compound. Suitable reagents that can work in this capacity include monoclonal antibodies or polyclonal antibodies, one or more proteins or liposomes. The pharmaceutical composition can be essentially composed of dosage units that can be administered as an aerosol. The term aerosol is used to represent a variety of systems from those of colloidal nature to systems consisting of pressurized packaging. Delivery can be carried out by liquefied or compressed gas or by a suitable pump system that distributes the active ingredient. Aerosols can be delivered in a single-phase, two-phase or three-phase system to deliver the active ingredient. The delivery of an aerosol includes necessary containers, activators, valves, sub-containers, etc., which together can form a test kit. Preferred aerosols can be determined by one of ordinary skill in the art without undue experimentation.

[0334] Pharmaceutical compositions can be prepared by methods well known in the pharmaceutical field. For example, pharmaceutical compositions intended to be administered by injection can be prepared by combining a composition comprising one or more of an antibody, an Fab or an antibody conjugate as described herein and an optional salt, a buffer and / or a stabilizer with sterile distilled water to form a solution. Surfactants can be added to promote the formation of a uniform solution or suspension. Surfactants are compounds that non-covalently interact with the peptide composition to promote the dissolution or uniform suspension of antibodies, Fab or an antibody conjugate in an aqueous delivery system.

[0335] The composition is administered in a therapeutically effective amount, which will vary according to a variety of factors (including the activity of the specific compound used; the metabolic stability and duration of action of the compound; the age, weight, general health, sex and diet of the patient; the mode and time of administration; the rate of excretion; the combination of drugs; the severity of a particular disorder or condition; and the subject being treated). Typically, the therapeutically effective daily dose (for a 70 kg mammal) is about 0.001 mg / kg (i.e. 0.07 mg) to about 100 mg / kg (i.e. 7.0 g); preferably, the therapeutically effective dose (for a 70 kg mammal) is about 0.01 mg / kg (i.e. 0.7 mg) to about 50 mg / kg (i.e. 3.5 g); more preferably, the therapeutically effective dose (for a 70 kg mammal) is about 1 mg / kg (i.e. 70 mg) to about 25 mg / kg (i.e. 1.75 g).

[0336] Compositions comprising antibodies, antigen-binding fragments thereof or antibody conjugates of the present disclosure may also be administered simultaneously with one or more other therapeutic agents, before one or more other therapeutic agents are administered, or after one or more other therapeutic agents are administered. Such combination therapies may include administering a single pharmaceutical dosage formulation containing a compound of the present invention and one or more additional active agents, and administering a composition comprising an antibody, antigen-binding fragment thereof or antibody conjugate of the present invention and each active agent in its own separate pharmaceutical dosage formulation. For example, antibodies, antigen-binding fragments thereof or antibody conjugates and other active agents as described herein may be administered to a patient together in a single oral dosage composition (such as in a tablet or capsule), or each agent may be administered in a separate oral dosage formulation. Similarly, antibodies, antigen-binding fragments thereof or antibody conjugates and other active agents as described herein may be administered to a patient together in a single parenteral dosage composition (such as in a saline solution or other physiologically acceptable solution), or each agent may be administered in a separate parenteral dosage formulation. Where separate dosage formulations are used, the composition comprising the antibody and one or more additional active agents may be administered at essentially the same time (i.e., simultaneously), or at separately staggered times (i.e., sequentially and in any order); combination therapy is understood to include all of these regimens.

[0337] Therefore, in certain embodiments, it is also contemplated that the antibodies, Fabs thereof or antibody conjugates disclosed herein are administered in combination with one or more other therapeutic agents. Such therapeutic agents may be accepted in the art as standard treatments for specific disease states (such as cancer) as described herein. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic agents, immune checkpoint inhibitors, interfering RNA, stimulatory immune checkpoint molecule agonists, another antibody, Fabs or antibody conjugates or other activities and adjuvants targeting cancer.

[0338] As used herein, the term "immune suppression agent" refers to one or more cells, proteins, molecules, compounds or complexes that provide an inhibitory signal to help control or suppress an immune response. For example, immunosuppressants include those molecules that: partially or completely block immune stimulation; reduce, prevent or delay immune activation; or increase, activate or upregulate immune suppression. Exemplary immunosuppressive agents that are targeted (e.g., with immune checkpoint inhibitors) include PD-1, PD-L1, PD-L2, LAG3, CTLA4, B7-H3, B7-H4, CD244 / 2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.

[0339] Immunosuppressants Inhibitors (also referred to as immune checkpoint inhibitors) can be compounds, antibodies, antibody fragments or fusion polypeptides (e.g., Fc fusions, such as CTLA4-Fc or LAG3-Fc), antisense molecules, ribozymes or RNAi molecules or low molecular weight organic molecules. In any embodiment disclosed herein, the method may be intended to include administering an antibody, antigen binding fragment or antibody conjugate of the present disclosure alone or in any combination with an inhibitor of any of the following immunosuppressive components.

[0340] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with a PD-1 inhibitor, for example, a PD-1-specific antibody such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP-224, BMS-936558 or an antigen-binding fragment thereof, or any combination thereof. In other embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with a PD-L1-specific antibody such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A or an antigen-binding fragment thereof, or any combination thereof.

[0341] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with a LAG3 inhibitor (such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof).

[0342] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with inhibitors of CTLA4. In specific embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with CTLA4-specific antibodies or their binding fragments, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept) or any combination thereof.

[0343] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with a B7-H3 specific antibody or antigen-binding fragment thereof, such as embulizumab (MGA271), 376.96 or both. The B7-H4 antibody binding fragment can be a scFv or a fusion protein thereof, such as those described, for example, in Dangaj et al., Cancer Res. 73:4820, 2013, and those described in U.S. Pat. No. 9,574,000 and PCT Patent Publication Nos. WO 2016 / 40724A1 and WO 2013 / 025779A1.

[0344] In certain embodiments, the antibodies, antigen binding fragments or antibody conjugates of the present disclosure are used in combination with inhibitors of CD244. In certain embodiments, the antibodies, antigen binding fragments or antibody conjugates of the present disclosure are used in combination with inhibitors of BLTA, HVEM, CD160 or any combination thereof. Anti-CD160 antibodies are described in, for example, PCT Publication No. WO2010 / 084158.

[0345] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with inhibitors of TIM3. In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with inhibitors of Gal9.

[0346] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with inhibitors of adenosine signaling (e.g., decoy adenosine receptors). In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with inhibitors of A2aR.

[0347] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with inhibitors of KIR, such as riluumab (BMS-986015). In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with inhibitory cytokines (typically cytokines other than TGFβ) or inhibitors of Treg development or activity.

[0348] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with an IDO inhibitor, such as: L-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indomod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; April 6-10, 2013), 1-methyl-tryptophan (1-MT)-tirapazamine, or any combination thereof.

[0349] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with an arginase inhibitor, such as: N(ω)-nitro-L-arginine methyl ester (L-NAME), N-ω-hydroxy-nor-1-arginine (deNOHA), L-NOHA, 2(S)-amino-6-borohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0350] In certain embodiments, an antibody, antigen-binding fragment, or antibody conjugate of the present disclosure is conjugated to an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0351] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with an inhibitor of TIGIT such as, for example, COM902 (Compugen, Toronto, Ontario, Canada), an inhibitor of CD155 such as, for example, COM701 (Compugen), or both.

[0352] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication No. WO 2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication No. WO 2017 / 021526.

[0353] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with a LAIR1 inhibitor.

[0354] In certain embodiments, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure are used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0355] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an agent that increases the activity of a stimulatory immune checkpoint molecule (ie, is an agonist). For example, the antibodies, antigen-binding fragments or antibody conjugates of the present disclosure can be used in combination with a CD137 (4-1BB) agonist (such as, for example, uretulumab), a CD134 (OX-40) agonist (such as, for example, MEDI6469, MEDI6383 or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as, for example, CDX-1127), a CD28 agonist (such as, for example, TGN1412, CD80 or CD86), a CD40 agonist (such as, for example, CP-870, 893, rhuCD40L or SGN-40), a CD122 agonist (such as, for example, IL-2), an agonist of GITR (such as, for example, a humanized monoclonal antibody described in PCT Patent Publication No. WO 2016 / 054638), an agonist of ICOS (CD278) (such as, for example, GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos314-8, or any combination thereof). In any of the embodiments disclosed herein, the method may include administering an antibody, antigen-binding fragment, or antibody conjugate of the present disclosure alone or in any combination with one or more agonists of a stimulatory immune checkpoint molecule (including any of the foregoing).

[0356] In certain embodiments, the combination therapy comprises an antibody, antigen-binding fragment, or antibody conjugate of the present disclosure and a second therapy comprising one or more of the following: another antibody, antigen-binding fragment, or antibody conjugate thereof that is specific for a cancer antigen (i.e., the same or a different antigen) expressed by the cancer, radiation therapy, surgery, a chemotherapeutic agent, cytokine, RNAi, or any combination thereof.

[0357] In certain embodiments, the combined treatment method comprises administering an antibody, antigen binding fragment or antibody conjugate of the present disclosure, and further administering radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy (such as γ-radiation) and radiopharmaceutical therapy. Surgery and surgical techniques suitable for treating a given cancer in a subject are well known to those of ordinary skill in the art. Proton therapy is reviewed in Thariat et al., Bull. Cancer pii: S0007-4551 (1) 300001-8 (2018).

[0358] In certain embodiments, the combined treatment method comprises administering an antibody, antigen binding fragment or antibody conjugate of the present disclosure and further administering a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitory drugs, DNA damaging agents, antimetabolites (such as folic acid antagonists, pyrimidine analogs, purine analogs and sugar-modified analogs), DNA synthesis inhibitors, DNA interactors (such as intercalators) and DNA repair inhibitors. Exemplary chemotherapeutic agents include, but are not limited to, the following groups: antimetabolites / anticancer agents such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine); antiproliferative / antimitotic agents, which include natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and vinorelbine, epipodophyllotoxins (etoposide, teniposide, glycosides), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, carbodine, dactinomycin, daunomycin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, paclitaxel, taxotere, temozolomide, teniposide, triethylenethiophosphoramide, and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunomycin, doxorubicin, The drugs include the following: adriamycin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (which systemically metabolize L-asparagine and deprive cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (dichloromethyl diethylamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamines (altretinoin and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozotocin), triazene-dacarbazine (trazenes-dac arbazinine, DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytics (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents;Antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (retinoic acid); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin) ), amsacrine, camptothecin, daunomycin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers; toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin or diphtheria toxin, and caspase activators; and chromatin disruptors. ;

[0359] Cytokines can be used to manipulate host immune responses to achieve anticancer activity. See, e.g., Floros & Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines used to promote immune anticancer or antitumor responses include, e.g., IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, alone or in combination with an antibody, antigen-binding fragment, or antibody conjugate of the present disclosure.

[0360] Cellular immunotherapy (including T cells, NK cells or NK-T cells involving expression of natural or recombinant TCRs and CARs specific for cancer antigens, and including adoptive transfer of such cells to recipients) is an emerging treatment for cancer (see, e.g., Bonini and Mondino, Eur. J. Immunol. 45(9): 2457-69 (2015) and Metha and Rezvani, Front. Immunol. 9: 283 (2018)). In certain embodiments, the subject receiving the antibody, antigen-binding fragment or antibody conjugate (or pharmaceutical composition) of the present disclosure has or is receiving (i.e., concurrently, simultaneously or sequentially) cellular immunotherapy targeting cancer.

[0361] Also provided herein are any of the currently disclosed antibodies, antigen binding fragments, antibody conjugates, polynucleotides, vectors, host cells, and compositions for treating, detecting, or diagnosing a disease characterized by expression (e.g., overexpression) of a Lewis antigen as described herein. In certain embodiments, the disease is cancer, such as any cancer disclosed herein. In certain embodiments, the antibody, antigen binding fragment, antibody conjugate, or composition is used in any combination therapy described herein.

[0362] Also provided herein are any of the currently disclosed antibodies, antigen binding fragments, antibody conjugates, polynucleotides, vectors, host cells, and compositions for the preparation of a drug for treating a disease characterized by expression (e.g., overexpression) of a Lewis antigen as described herein. In certain embodiments, the disease is cancer, such as any cancer disclosed herein. In certain embodiments, the drug comprises any combination therapy described herein or is used in any combination therapy described herein.

[0363] Unless the content clearly indicates otherwise, the singular forms "a / an", and "the" used in this specification and the appended claims include plural references. In addition, the use of alternatives (such as "or") should be understood to mean one, two, or any combination of the alternatives.

[0364] In this specification, unless the context requires otherwise, the words "comprise", "have" or variations such as "has / having", "comprises / comprising" will be understood to imply the inclusion of the stated elements or integers, or groups of elements or integers, but not the exclusion of any other elements or integers, or groups of elements or integers. In this specification, unless otherwise stated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the range, as well as its fractions (such as one tenth and one hundredth of an integer) where appropriate. In addition, unless otherwise stated, any numerical range related to any physical feature described herein (such as polymer subunits, size or thickness) should be understood to include any integer within the range.

[0365] As used herein, unless otherwise indicated, the term "about" means no more than ±20% of the indicated range, value, or structure, or in certain embodiments no more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6% or 5% of the indicated range, value, or structure.

[0366] In addition, it should be understood that each compound or group of compounds derived from the various combinations of structures and substituents described herein is disclosed by this application to the same extent as if each compound or group of compounds were set forth individually. Therefore, the selection of a specific structure or a specific substituent is within the scope of this disclosure.

[0367] The term "consisting essentially of" is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or those materials or steps that do not materially affect the basic characteristics of the claimed subject matter.

[0368] Unless explicitly stated otherwise, every embodiment in this specification shall necessarily apply to every other embodiment. Example

[0369] Example 1

[0370] Generation of chimeric BBC antibodies

[0371] Isolation of antibody variable region cDNA

[0372] Hybridoma cells expressing the murine IMH2 / BBC antibody comprising a VL domain having the amino acid sequence shown in SEQ ID NO: 27 and a VH domain having the amino acid sequence shown in SEQ ID NO: 28 were obtained from Dr. S. Hakomori (Cancer Research 52: 3739-3745 (1992)). To prepare RNA for cDNA synthesis, 9×10 6 hybridoma cells and then purified using the "Total RNA Miniprep Purification Kit" according to the manufacturer's protocol. TM (GeneMark TM , GMbiolab Co. Ltd., Taichung, Taiwan, China) for RNA isolation. Then, the SMART RACE cDNA amplification kit was used TM The antibody gene encoding IMH2 was cloned from purified RNA (Takara / BD Biosciences-Clontech, Palo Alto, CA) with minor modifications compared to the protocol recommended by the supplier.

[0373] In brief, after first-strand cDNA synthesis and dC tailing, the cDNA of the light chain variable region of IMH2 specific coding was isolated by two rounds of PCR using primers provided by the kit and specific primers designed in the constant region based on known mouse kappa chain sequences. The first PCR was performed as follows: 5 cycles of 30 seconds at 94°C and 1 minute at 72°C; followed by 5 cycles of 30 seconds at 94°C, 30 seconds at 67°C, and 1 minute at 72°C. Twenty-seven (27) additional PCR reaction cycles (including 30 seconds at 94°C, 30 seconds at 62°C, and 1 minute at 72°C) were added to ensure successful amplification. A second set of PCR was then performed to further improve fidelity, and the second nested PCR included the following steps: a step of preheating at 94°C for 5 minutes, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 54°C, and 1 minute at 72°C, and a final extension step of 3 minutes at 72°C.

[0374] The cloning strategy for the heavy chain gene was slightly different. First, a single-stranded cDNA template for PCR reaction was prepared from RNA to constant domain 1 (CH1) using mouse IgG3-specific primers. Gene isolation was then performed by a single round of PCR as follows: preheating at 94°C for 5 minutes, including 35 PCR reaction cycles at 94°C for 30 seconds, at 62°C for 30 seconds, and at 72°C for 1 minute, followed by NUP primers (SMART TM A final extension step of 5 min at 72°C was performed in the presence of a nested primer for the mouse CH1 domain using a RACE amplification kit (GeneMark TM cDNA encoding the variable regions of the light and heavy chain fragments was purified by GMbiolab Co. Ltd. and introduced into the yT&A cloning vector (Yeastern Biotech TM , Taipei, Taiwan, China) for positive clone identification and sequence determination.

[0375] Construction of antibody expression plasmid

[0376] In order to construct an expression plasmid for producing an antibody referred to herein as "BBC" antibody, only cDNA encoding mature (no leader peptide) heavy chain variable region and light chain variable region was prepared using PCR primers and antibody genes cloned in the above-mentioned yT&A vector. PCR was performed as follows: preheated at 94°C for 5 minutes, including 35 PCR reaction cycles of 30 seconds at 94°C, 30 seconds at 65°C, and 60 seconds at 72°C, and a final extension step of 3 minutes at 72°C. During the PCR reaction, restriction endonuclease recognition sequences were incorporated into the 5' (NheI) and 3' (ApaI) ends of VH cDNA and the 5' (NheI) and 3' (BsiWI) ends of VL cDNA to facilitate subsequent expression plasmid engineering. The amplified cDNA fragments were then digested with restriction enzymes ApaI / NheI for heavy chain and NheI / BsiWI for light chain genes. After gel purification, the recovered VH and VL cDNAs were ligated to pGNX-RhcG1 ​​(VH) or pGNX-Rhck vector (VL) at the same restriction enzyme cloning site to obtain expression vectors pGNX-RhcG1-BBC and pGNX-Rhck-BBC, respectively. The inserted cDNA sequence was confirmed using primers upstream of the multiple cloning site.

[0377] Transient production of antibodies

[0378] To transiently produce chimeric BBC antibodies, HEK293-c18 cells were co-transfected with pGNX-RhcG1-BBC and pGNX-Rhck-BBC expression plasmids encoding the heavy and light chains in the presence of polyethyleneimine. Culture supernatants were collected at the end of day 7 post-transfection for analysis.

[0379] Example 2

[0380] Generation of humanized BBC antibodies

[0381] To generate a humanized version of the BBC antibody (as described in Example 1 above), homologous human antibody sequences (human acceptors) were selected for CDR transplantation. Briefly, potential human acceptor sequences were identified by searching the NCBI protein database to locate sequences that exhibited the highest homology with the heavy chain (SEQ ID NO: 28) variable region and light chain (SEQ ID NO: 27) variable region of the BBC antibody. Human acceptor frameworks CAD89404.1 (Vh) and AAS01771.1 (VI) ( Figure 1 ). However, inserting non-human CDR sequences directly into the human acceptor framework may result in loss of binding affinity. Binding affinity can be restored after transferring the framework residues from the human acceptor back to the non-human donor sequence. Preferred reverse mutations restore binding affinity by maintaining the original CDR conformation.

[0382] To restore the binding affinity after CDR grafting, we first used Accelrys Discovery Studio based on the BBC crystal structure data. TM The 3-D model of the antibody was established using the software of BIOVIA (San Diego, CA). The reverse mutation of key amino acids was then predicted by examining the structure as follows:

[0383] 1. Calculate the mutation energy (stability) of the residues changed on the humanized framework. Positive values ​​of mutation energy correspond to a destabilizing effect of the mutation, and vice versa.

[0384] 2. Check the spatial distance between framework residues and CDR regions. Consider the residues closest to the CDR ( Inside).

[0385] 3. Check the residues located at the interface of the heavy chain variable region and the light chain variable region. These residues contribute to the assembly of the heavy and light chains and therefore may have a significant impact on the antibody structure.

[0386] Ten (10) influential positions (3 in the light chain and 7 in the heavy chain) were initially selected for back mutation based on predicted criteria. In addition, the methionine (M) residue at position 70 (according to Kabat numbering) of the selected human heavy chain template appeared to be an uncommon motif and was therefore replaced by a highly conserved isoleucine (I) at this site ( Figure 1 ; Residues that reverse mutated (human receptor → hBBC.8) are underlined in Table 3 below; Met → Ile residues are underlined in bold italics in Table 3, Figure 1 These amino acid conversions generate the "hBBC.8" humanized antibody.

[0387] Additional changes were introduced to further improve the antibodies. First, two positions (R66 and F71) in the hBBC.8 light chain that varied between the mouse and human sequences were mutated to generate "hBBC.9" (containing the F71Y mutation) and "hBBC.10" (containing the F71Y and R66G mutations) ( Figure 2A ; in Table 3, residues are bold and italicized, not underlined). Figure 2B Binding of BBC and generated humanized variants hBBC.9 and hBBC.10 to AGS cells is shown. Briefly, the AGS human gastric cancer cell line (ATCCCRL-1739; ATCC, Manassas, VA) was routinely maintained in F12 medium supplemented with 10% dialyzed fetal bovine serum. For cell binding studies, approximately 3×10 5Each cell was mixed with an equal volume of diluted antibody. After incubation at room temperature for 1 hour, 2 ml of PBS was added to each sample to rinse away unbound antibody. After centrifugation, the recovered cell pellet was directly resuspended in 200 μl of fluorescein (FITC)-AffiniPure TM Goat anti-human IgG (specific for Fcγ fragment diluted 1:200 in PBS) (Jackson ImmunoResearch, West Grove, PA, Cat. 109-095-098). After incubation at room temperature for 30 minutes, PBS washes were repeated to eliminate unbound secondary antibody. The collected cells were resuspended in 200 μl PBS and analyzed on a BD FACSCanto TM The proteins were analyzed on a flow cytometer system (BD Biosciences, San Jose, CA). Further analysis was performed to identify potential immunogenic sequences.

[0388] Other mutations in the heavy or light chain produce other variants "hBBC.9.1" and "hBBC.10.1" (in Table 3 below, residues are bold and underlined, without italics). Specifically, the crystal structures of the BBC antibody and the hBBC.8 antibody and the simulated antigen / antibody complex were analyzed. Several residues in the light chain CDR region and the heavy chain CDR region (positions specified according to Kabat numbering) were identified for single-site amino acid replacement. Amino acid conversion at the specified position of the antibody light or heavy chain gene was performed by two rounds of PCR reactions with specifically designed primers. In order to facilitate the insertion of the mutated antibody cDNA fragment into the expression vector, restriction sites were incorporated at each end during the PCR reaction (5'NheI and 3'ApaI for the cDNA chain and 5'NheI and 3'BsiWI for the light chain cDNA). DNA fragments were generated after the second PCR reaction, cut with NheI / ApaI or NheI / BsiWI, and ligated into the same sites of pGNX-RhcG1 ​​and pGNX-Rhck vectors for heavy and light chain gene construction, respectively. Table 1 shows the mutation sites and changes.

[0389] Table 1. Single-site mutations in hBBC.8

[0390]

[0391] In the AGS cell binding assay, many mutants exhibited in vitro binding activities equal to or greater than 1 / 2 of the activity of the chimeric antibody (BBC) ( Figure 2C, see also Table 2). In addition, as shown in Table 2, some mutants also exhibited improved specificity in the ELISA assay by reduced cross-reactivity with monovalent Lewis B structures (equal to or less than 1 / 8 intensity compared to BBC). Monovalent Lewis B is a blood group antigen expressed in normal human tissues.

[0392] Table 2. Binding activity of BBC CDR mutants

[0393]

[0394] (=) No change in binding activity relative to BBC

[0395] (-) Compared with BBC, the binding activity is reduced

[0396] (+) Increased binding activity relative to BBC

[0397] Since hBBC.8 showed approximately 1 / 3 of the AGS cell binding activity compared to BBC ( Figure 2B ), and showed very good tumor inhibition in a xenograft mouse model (see Example 6), these single amino acid substituted antibody mutants have the potential to exhibit anti-tumor activity. The positions and amino acid substitutions of the clones analyzed are summarized in Table 1. In order to further confirm their ability to improve affinity and / or specificity, a total of seven (7) single mutation sites (Table 2) were selected for evaluation in humanized antibody templates. These included 5 single residue substitutions (three in the light chain and two in the heavy chain) that were tested for their effect on antibody binding to AGS cells ( Figure 2C ), tested for antibody binding specificity (with purified Le B Compared to the two heavy chain single residue substitutions that affected AGS cells), and two heavy chain double residue substitutions analyzed similarly ( Figure 2D ).

[0398] exist Figure 2E A further comparison of the various humanized BBC formats constructed to the original chimeric format in terms of affinity (AGS cell binding assay) and specificity (Lewis b ELISA assay) is shown in FIG.

[0399] Epibase analysis of hBBC.9 (Applied Protein Services, Lonza Biologics, Cambridge, UK) predicted regions in the heavy chain region to have a strong risk of immunogenicity. To minimize potential immunogenicity issues, six additional amino acid changes were added to the heavy chain framework 3 region (positions 78, 80, 82–84, and 86) to match the reference humanized antibody at these positions ( Figure 3). These conversions to "hBBC.10.1FQ" provided the heavy chain framework. Table 3 summarizes the amino acid sequences of the various heavy and light chain variable regions described in this example.

[0400] Table 3. Amino acid sequences of antibody VH and VL regions

[0401]

[0402]

[0403] Example 3

[0404] Characterization of identified HBBC epitopes

[0405] BBC and the variants described herein were designed as glycan-binding monoclonal antibodies. Based on the published specificity data of the parent IMH2 (BBC) antibody (Ito et al., Cancer Res. 52:3739, 1992), it was assumed that the target epitope of the generated BBC antibody was identical to that of Le B Antigen and Le Y Antigenic Correlation The following epitope characterization studies were performed using hBBC.10.1, also referred to as "hBBC" in the following examples and figures.

[0406] Immunopurification

[0407] GSL-derived glycans from the colorectal cancer cell line Colo-205 were isolated and immunopurified using BBC. Figure 4A and Figure 4B As shown in , glycans in the unbound fraction and the eluted fraction were permethylated and characterized by MALDI-MS analysis. The glycan profile of the unbound fraction was similar to the input glycan profile, indicating that most GSL-derived glycans from COLO 205 were not bound to the BBC. However, in the eluted (BBC-bound) fraction, Fuc4(LacNAc)3Lac glycans were purified exclusively by the BBC. Specifically, it was surprisingly found that based on the Le 4-terminal chromatin observed on the I-branch antigen in the MS / MS experiment, the glycan profile of the unbound fraction was similar to the input glycan profile, indicating that most GSL-derived glycans from COLO 205 were not bound to the BBC. B Fingerprint fragments and Le Y Fingerprint fragment, hBBC purified glycans carry biantennary Le B / Y (i.e., Le B / Le B ,Le B / Le Y or Le Y / Le Y ), while the glycans in the unbound fraction are linear structures (probably Le B -Le A -Le A-Lac). This result shows that the biantennary Le on the I antigen B / Y It is the table position of BBC.

[0408] This result was also supported by immunopurification of BBC-bound glycans from GSL-derived glycans from the cell lines NCI-N87 and SW1116, which were selected for biantennary Le on I antigen. B / Y As expected, BBC bound to the tetrafucosylated GSL-derived glycan Fuc4(LacNAc)3Lac, but not to the monofucosylated, difucosylated, and trifucosylated glycans ( Figure 5A-Figure 5B ). By MSMS sequencing, it was confirmed that the BBC-binding glycans of NCI-N87 and SW1116 GSLs were biantennary Le carrying I antigen. B / Y ( Fig. 6A and Figure 6B ).

[0409] In addition to Fuc4(LacNAc)3Lac, a panel of GSL-derived glycans from NCI-N87 and SW1116 were purified that were characterized by multiple fucosylation (four fucose residues are the minimum requirement for BBC binding). The two dominant BBC-binding glycans of NCI-N87 GSL, Fuc4(LacNAc)4Lac and Fuc6(LacNAc) 4→5 Lac was sequenced and determined to have biantennary or triantennary Le Y The glycan carrying I antigen ( Fig. 7A and Figure 7B ). In addition, when the released N-glycans from the AGS cell line were immunopurified with BBC, the enriched N-glycans were found to be those with bi- or tri-antennary Le Y The structure of Fig. 8A and Figure 8B ). This result is consistent with the enrichment of I antigen.

[0410] These data show that Le B / Y It is a combined unit of the BBC. However, the single-antenna Lewis B / Y Not sufficient to provide strong binding to BBC. Unique I antigen and N-glycans with complete terminal fucosylation provide multivalent Le B / Y , which was indicated as a strong binding epitope for BBC. Comparable immunopurification studies were also performed on hBBC and showed similar glycan enrichment specificity as BBC (data not shown), suggesting that hBBC and BBC share similar epitopes.

[0411] Isothermal titration calorimetry

[0412] Isothermal titration calorimetry (ITC) was performed to analyze the hBBC and Le Y -Gal, Le B -Gal, Le A -Gal and Le X -Gal linear glycans, and Le Y / Le Y -ASGP, Le X / Le X -ASGP, H-ASGP, Le Y / Le Y -I antigen and Le Y / Le B -I antigen. The BR96 antibody (described in U.S. Pat. No. 5,491,088A; variants described in U.S. Pat. No. 5,792,456A) was included as a control. Because the glycans characterized by the hBBC immunopurification experiments were present in limited amounts, the various Le B and Le Y The relevant glycans were obtained from commercial sources (Elycityl SA, Crolles, France) or enzymatically synthesized in-house according to methods established in the art (e.g., Wu et al., 2011 Glycobiology 21(6):727-733; Becker et al., 2003 Glycobiology 13(7):41R-53R; de Vries et al., 2001 Glycobiology 11(10):119R–128R).

[0413] In short, for isothermal titration calorimetry (ITC), filtered PBS pH7.2 was prepared internally, and the same batch of buffer was used within one ITC injection cycle. mAb was pre-exchanged in filtered PBS pH7.2 buffer at a protein concentration of 50 μM. The amount of glycan antigen was quantified by high-performance anion exchange pulsed amperometric detection (HPAEC-PAD, such as Rothenhofer et al., 2015 J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 988: 106), with galactose as the monosaccharide analysis of the calculation standard. The quantitative glycan antigen was dissolved in filtered PBS pH7.2 (the same batch as the mAb solution preparation), and 60 μL solution was used for each injection.

[0414] ITC experiments were performed on a MicroCal iTC200 system (Malvern Instruments Ltd, Malvern, UK). After the mAb solution (50 μM) was loaded into the sample cell of the iTC200, the system temperature was set to 25 °C. After the system temperature reached 25 °C, the glycan antigen solution was loaded into the syringe and slowly lowered into the mAb-filled sample cell. The experimental parameters were as follows: injection #: 20; cell temperature: 25 °C; reference power: 6 μcal / s; initial delay: 60 s; sample cell concentration: 50 μM; stirring speed: 750 rpm, syringe concentration input, and glycan antigen concentration were measured. The injection parameters were as follows: injection volume: 2 μL; duration: 4 s; interval: 150 s; fill time: 5 s; and the first injection volume was adjusted to 1 μL. When all settings were confirmed, the experiment was started and the resulting data from the iTC200 were processed by Origin. The titration curve was fitted using the One Set of Sites fitting model and repeated 100 times to obtain the best fitting result. D .

[0415] Fig.9A , Fig. 9B , Figure 10A-10C and Figures 11A-11F ITC titration plots of various glycan antigens with hBBC antibodies are shown. Figure 12A-12C Figure 2 shows the ITC titration plots of various glycan antigens with the BR96 antibody. In brief, ITC analysis showed that hBBC B -Gal (KD = 26.2 μM) than Le Y -Gal (KD = 80.6 μM) has a higher specific binding affinity. In addition, hBBC has a higher specific binding affinity for the biantennary structure (Le Y / Le Y -ASGP, Le Y / Le Y -I antigen and Le Y / Le B -I antigen) shows the alignment of single-chain Le Y -Gal antigen has a stronger binding affinity. hBBC has a stronger binding affinity to Le Y / Le Y -ASGP, Le Y / Le Y -I antigen and Le B / Le Y -I antigens appear to have similar binding affinities, suggesting that for biantennary glycan epitopes, Le Y and Le B The side chains contribute considerably to the specific binding. Fig.11A-F, glycan antigen lacking a tetrafucosylated LacNAc moiety, either in single-chain or biantennary form (Le X -Gal, Le A -Gal, type I H antigen, type II H antigen, H-ASGP and Le X / Le X -ASGP), none of which showed specific binding to hBBC. These results suggest that tetrafucosylated LacNAc with type I or type II linkage is essential for specific binding of hBBC. In summary, consistent with the immunopurification data, ITC results show that hBBC binds to proteins including those containing Le Y and / or Le B The epitope of the structure of the biantennary Le Y / Le Y -I antigen and biantennary Le Y / Le Y -ASGP( Figure 12A-12C ) compared to the BR96 control, which showed similar results to the single-chain Le Y -Gal binding affinity is slightly higher, indicating that BR96 has a high affinity for single-chain and biantennary Le Y There is no specific selectivity between glycans. Therefore, hBBC has a unique epitope specificity compared to BR96.

[0416] Table 4. Isothermal titration calorimetry results

[0417]

[0418]

[0419] These results partially characterize the hBBC epitope. However, the interaction between free-flowing hBBC and immobilized glycans provides additional information about the epitope; therefore, hBBC was tested with a range of immobilized glycans in surface plasmon resonance (SPR) and ELISA experiments. As shown in Table 5 (same glycan description as in Table 4), the hBBC was expressed using a streptavidin-coated analytical surface and biotin-conjugated glycans (Le Y -Gal-biotin, Le B -Gal-biotin, Le Y / Le Y -ASGA-Biotin and Le B / Le B -ASGA-biotin for SPR; Le Y -Gal-biotin, Le B -Gal-biotin, LeY / Le Y -ASGA-Biotin, Le B / Le B -ASGA-Biotin, 3-Le Y / 6-LeB-ASGA-biotin and 3-Le B / 6-Le Y -ASGA-biotin for ELISA).

[0420] Table 5. Structure and MW of glycan antigens used in SPR and ELISA

[0421]

[0422]

[0423] Surface plasmon resonance

[0424] hBBC vs Le Y -Gal-biotin, Le B -Gal-biotin and Le Y / Le Y -ASGA-biotin binding affinity is analyzed by surface plasmon resonance (SPR) on a streptavidin-coated chip. In short, Biacore T100 is used as a running buffer together with HBS-EP+ buffer (GE Healthcare). Biotinylated polysaccharides are diluted to 10pM and fixed on a sensor chip SA (GE Healthcare) according to standard procedures. Polysaccharides are fixed for 60s at a flow rate of 10μL / min. HBBC or BR96 buffer is exchanged to HBS-EP+ buffer with Zeba desalting spin column (7K MWCO, 0.5mL, ThermoFisher) and continuously diluted to 480, 240, 120, 60 and 30nM with HBS-EP+ buffer. Single cycle kinetic analysis is performed. Antibodies are associated with polysaccharides for 150s at a flow rate of 30μL / min and dissociated for 300s. The chip is regenerated for 120s at a flow rate of 50μL / min with 2M MgCl2. Data were evaluated using Biacore T200 evaluation software (GE Healthcare). The two-state response was used for curve fitting and the best fit values ​​for ka, kd and KD were obtained (Table 6). Fig.13A and Fig. 13B SPR sensorgrams showing hBBC and BR96 binding to various glycan antigens are provided.

[0425] Table 6. Summary of SPR results

[0426] Antigen name hBBC BR96 <![CDATA[Le Y -Gal-Biotin]]> 40~59μM 270nM <![CDATA[Le B -Gal-Biotin]]> 2.3~3.1μM n / a <![CDATA[Le Y / Le Y -ASGA-Biotin]]> 2~3μM 1μM <![CDATA[Le B / Le B -ASGA-Biotin]]> 1μM n / a

[0427] SPR showed that the single-chain Le Y -Gal-biotin compared to biantennary Le Y / Le Y -ASGA-biotin showed a higher affinity for hBBC, which is consistent with the ITC results. B -Gal-biotin compared to biantennary Le B / Le B -ASGA-biotin showed a higher affinity for hBBC, which is consistent with the Y The observed trend of hBBC for Le B -Gal-biotin showed a higher Y -Gal-biotin affinity, which is comparable to the ITC results. SPR also showed that hBBC B / Le B -ASGA-biotin has a higher binding affinity than Le Y / Le Y -ASGA-biotin, which binds to hBBC based on Le B Antigen alignment based on Le Y This is consistent with the observation that the antigen has a higher affinity. B / Le B -ASGA and Le Y / Le Y The comparable KD values ​​obtained for hBBC with -ASGA further suggest that hBBC specifically associates with polyfucosylated (i.e., bivalent Lewis B structures or Lewis Y structures). B / Le B -ASGA response (sensogram) compared to Le B -Gal's response is stronger. B / Le B -ASGA appears to be the structure with the highest affinity to hBBC.

[0428] By comparison, BR96 is better than Le Y -Gal-biotin and Le Y / Le Y -ASGA-biotin has a slightly higher affinity than the structurally similar antigen Le Y / Le Y In conclusion, the SPR data are highly consistent with the ITC experimental data.

[0429] Indirect ELISA

[0430] To evaluate antibody binding affinity by ELISA, biotinylated glycan antigens were diluted to an appropriate concentration in PBS buffer. To evaluate antibody binding affinity by ELISA, biotinylated glycan antigens were diluted to a sufficient concentration in PBS buffer. 100 μL of diluted antigen solution was applied to a 96-well assay plate coated with streptavidin and incubated in a 37°C shaker for 3.5 hours. The plate was washed with PBST (0.05% Tween-20 in PBS buffer) to remove excess glycan antigen. Primary antibodies titrated continuously in diluent (0.1% BSA in PBS buffer) were applied to the assay plate and incubated in a 37°C shaker for 1 hour. Subsequently, PBST washing was performed, and 100 μL of HRP-conjugated anti-human IgG antibody solution (SouthernBiotech, diluted 1:15,000 in diluent) was incubated in an assay plate in a shaker at 37°C for 1 hour. After washing away excess secondary antibody, 100 μL of TMB reagent was applied and incubated at 37°C for 15 min, followed by quenching with 50 μL of 0.5N HCl. OD values ​​were detected at 450 nm in a VERSA max microplate reader (Molecular Devices), and OD values ​​at 650 nm were subtracted. Data were processed in Softmax Pro (Molecular Devices).

[0431] Le Y / Le Y -ASGA relative to Le Y -Gal

[0432] In Le Y -Gal-biotin-coated and Le Y / Le Y The binding activity of hBBC and BR96 was tested on ELISA plates coated with streptavidin-ASGA-biotin. The amount of coated antigen was Fig.14A and Fig. 14B As shown in the BBC's Y / Le Y -ASGA's combination comparison with Le Y -Gal binds much more strongly. BR96 shows the opposite pattern to hBBC, as it binds Le Y -Gal's ability is better than Le Y / Le Y -ASGA was much better. In conclusion, the indirect ELISA provided similar binding affinity results as the ITC and SPR experiments.

[0433] Le B / Le B -ASGA relative to Le Y / Le Y -ASGA and Le B -Gal

[0434] Next, we tested hBBC on Le B / Le B-ASGA-Biotin, Le Y / Le Y -ASGA-Biotin and Le B -Gal-biotin-coated streptavidin-coated ELISA plates. The amount of coated antigen is Fig.15 As shown in the results of indirect ELISA, hBBC showed B / Le B -ASGA binding is significantly stronger than Le Y / Le Y -Gal and LeB-Gal combination.

[0435] Antigen binding ELISA

[0436] Developed antigen binding ELISA to assess the relative binding affinity of hBBC to different glycan antigens. In short, hBBC was titrated continuously and applied to a 96-well assay plate functionalized with streptavidin coated with different glycan antigens. The binding of hBBC to glycan antigens was detected with a human IgG-specific antibody conjugated with horseradish peroxidase (HRP), and then developed in TMB reagent. The absorbance at 450nm using a microplate reader is directly proportional to the amount of hBBC bound to its antigen. Relative affinity is determined by plotting OD as a function of hBBC concentration.

[0437] Antigen preparation

[0438] Biotinylated Lewis Y pentose, Le Y -Gal-sp3-biotin was purchased from Elicityl (Crolls, France). Lewis B pentose was purchased from Elicityl and further biotinylated in-house (Le B -Gal-LC-biotin). The other four biotinylated glycans: Le Y / Le Y -ASGA-Biotin, Le B / Le B -ASGA-Biotin, 3-Le Y / 6-Le B -ASGA-Biotin(Le Y / Le B -ASGA-biotin) and 3-Le B / 6-Le Y -ASGA-Biotin(Le B / Le Y-ASGA-biotin) was synthesized by a series of enzymatic glycosylation reactions followed by chemical biotinylation reactions (Table 5). Biotinylated glycans were analyzed by thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI-MS) to ensure that the purity was at least 95% and that there was no unconjugated biotin. HPAEC-PAD monosaccharide analysis was used for the quantification of biotinylated glycans.

[0439] Antibody

[0440] hBBC was used in antigen binding ELISA.

[0441] ELISA

[0442] Biotinylated glycan antigens were diluted to 18.7 nM in PBS buffer. 100 μL of the diluted antigen solution was applied to a 96-well assay plate coated with streptavidin and incubated in a shaker at 37°C for 3.5 hours. The plate was washed with PBST (0.05% Tween-20 in PBS buffer) to remove excess glycan antigen. Primary antibodies titrated continuously in diluent (0.1% BSA in PBS buffer) were added to the assay plate and incubated in a shaker at 37°C for 1 hour. After PBST washing, 100 μL of HRP-conjugated anti-human IgG antibody solution (SouthernBiotech, diluted 1:10,000 in diluent) was incubated in the assay plate on a shaker at 37°C for 1 hour. After washing away excess secondary antibodies, 100 μL of TMB reagent was applied and incubated at 37°C for 15 min, followed by quenching with 50 μL of 0.5N HCl. OD values ​​were measured at 450 nm in a VERSA max microplate reader (Molecular Devices), and the OD value at 650 nm was subtracted. Data were processed in Softmax Pro (Molecular Devices).

[0443] result

[0444] Data such as Fig.16A In the antigen binding ELISA, when hBBC is combined with Le B / Le B -ASGA, Le Y / Le Y -ASGA, 3-Le Y / 6-Le B -ASGA-Biotin and 3-Le B / 6-Le Y -ASGA-biotin binding, OD values ​​increased within a narrow range of low antibody concentrations (0.4-4 μg / mL), while hBBC and LeY -Gal and Le B -Gal binding was significantly weaker, indicating the presence of a divalent Le in one glycan Y ,Le B ,Le B / Le Y or Le Y / Le B , providing a single Lewis Y / B Part of the higher binding affinity to hBBC.

[0445] Direct comparison of antigen binding affinity: hBBC.10.1 versus BBC

[0446] The purpose of the experiment was to compare the antigen affinity of hBBC.10.1 and ancestral BBC antibodies by antigen binding ELISA.

[0447] Antigen preparation

[0448] Biotinylated Lewis Y pentose, Le Y -Gal-sp3-biotin was purchased from Elicityl. Lewis B pentose was purchased from Elicityl and further biotinylated in-house (Le B -Gal-LC-biotin). The other four biotinylated glycans: Le Y / Le Y -ASGA-Biotin, Le B / Le B -ASGA-Biotin, 3-Le Y / 6-Le B -ASGA-Biotin(Le Y / Le B -ASGA-biotin) and 3-Le B / 6-Le Y -ASGA-Biotin(Le B / Le Y -ASGA-biotin) was synthesized by a series of enzymatic glycosylation reactions followed by chemical biotinylation reactions (Table 5). Biotinylated glycans were analyzed by TLC and ESI-MS to ensure that the purity was at least 95% and that there was no unconjugated biotin. HPAEC-PAD monosaccharide analysis was used for the quantification of biotinylated glycans.

[0449] Antibody

[0450] BBC and hBBC.10.1 were used in antigen binding ELISA.

[0451] Antigen binding ELISA

[0452] 96-well EvenCoat TM Streptavidin microtiter plates (R&D Biosystems, MN, catalog number: CP004) were incubated with various biotinylated polysaccharides in PBS at 3.73 pmol / well overnight at 4°C. After washing three times with PBS / 0.05% Tween 20 (Sigma, catalog number: P1379-500mL), 100 μL of diluted BBC or hBBC.10.1 antibodies were added to the antigen-coated wells and incubated at 37°C for 1 hr. The wells were washed three times with PBS / 0.05% Tween 20 and then incubated with 100 μL of 10000× diluted mouse anti-human IgG (Fc)-HRP (Southern Biotech, catalog number: 9040-05) at 37°C for 1 hr. After washing, 100 μl SureBlue TM Reverse TMB (KPL, catalog number: 53-00-03) was developed at 37°C for 15 minutes. 0.5N HCl was added to terminate the reaction. TM The absorbance was read at a wavelength of 450 nm with a reference wavelength of 650 nm on a microplate reader (Molecular Devices, San Jose, CA) and the color was analyzed by SoftmaxPro TM Data were processed using the ELISA software (Molecular Devices).

[0453] result:

[0454] BBC( Fig. 16B ) and hBBC.10.1( Fig. 16C ) antigen binding ELISA showed that hBBC.10.1 had a stronger affinity for single-chain glycan antigen (Le B -Gal and Le Y -Gal) and both antibodies have a low affinity for the biantennary glycan antigen (Le B / Le B -ASGA, Le Y / Le Y -ASGA, 3-Le Y / 6-Le B -ASGA and 3-Le B / 6-Le Y These data suggest that hBBC.10.1 has better binding selectivity between single-chain and biantennary antigens than BBC.

[0455] Summarize

[0456] Epitope characterization experiments described in this example demonstrate that hBBC binds to bi- and tri-antennary Le Y / B I antigen-specific binding, and biantennary and triantennary Le Y N-glycan specific binding. In addition, the epitope recognized by hBBC contains a bifucosylated LacNAc backbone. B / Le B -ASGA is the antigen with the strongest binding affinity to hBBC. B -Gal and Le Y -Gal antigen compared to biantennary Le Y / Le Y -ASGA, Le Y / Le B -ASGA and Le B / Le Y -ASGA also showed high affinity for hBBC. Similarly, regardless of whether the antigen was a single chain or a biantennary structure, the binding affinity of Le B The affinity of antigens for hBBC was higher than that based on Le Y Finally, the binding behavior (kinetics) and epitope of hBBC are different from those of BR96.

[0457] Example 4

[0458] Internalization of HBBC in target cells

[0459] Antibodies can be used as carriers to deliver functional payloads to desired sites. For example, some cancer therapies use antigen-specific antibodies to deliver cytotoxic drugs (i.e., antibody-drug conjugates or ADCs) to tumor cells via endocytosis, also known as internalization. Some current ADCs include cleavable linkers that are cleaved in the lysosomal compartment, thereby selectively releasing the drug after internalization, with the added benefit of increasing the stability of the drug in serum.

[0460] To test whether hBBC is efficiently internalized into cancer cells, the following experiment was performed. First, in a conventional internalization assay, hBBC antibodies were added to cultured AGS gastric cancer cells and allowed to bind for 1 hr at 4°C to allow specific antibody / receptor interactions but prevent endocytosis. Then, nonspecifically bound antibodies were washed away and the cells were moved to 37°C to allow normal endocytosis. At 0min and 30min, the cells were fixed and hBBC was detected using Alexa488-conjugated anti-human IgG antibodies ( Fig.17 , left). F-actin was labeled with phalloidin rhodamine (right; co-distributed staining is shown as yellow fluorescent signal). Fig.17As shown in, hBBC stains the cell membrane at 0 min, and then internalizes and locates in the cytosolic vesicles around the nucleus after incubation at 37 ° C (30 min, permeabilized cells). The cytosolic signal was further confirmed by the non-permeabilized control, in which only a weak membrane signal can be detected (30 min, non-permeabilized cells). These data show that hBBC is effectively internalized into AGS cells within 30 minutes.

[0461] In the second experiment, the real-time intracellular localization of hBBC was examined. Briefly, hBBC antibodies were added to AGS cells and incubated at 37°C for 4 or 8 hours to promote internalization. The cells were then fixed and the real-time localization of hBBC was examined using Alexa488-conjugated anti-human IgG antibodies ( Fig.18 , left panel shows anti-human IgG). Lysosomes were labeled with anti-Lamp-1 antibody and then with labeled anti-rabbit IgG antibody (shown in the middle panel). Fig.18 As shown in, after 4 or 8 hours of incubation, hBBC colocalizes with Lamp-1 (merge; right figure), indicating that internalized hBBC is localized in the lysosomal compartment. In addition, hBBC can be stabilized in the lysosomal compartment without obvious degradation for at least 8 hours. These results show that hBBC has the utility of being used as a conjugate (such as ADC) for targeting antigens.

[0462] Example 5

[0463] HBBC immunostaining in human tissue samples

[0464] Healthy and cancerous tissue samples were obtained and immunostaining for hBBC 10.1 was performed on formalin-fixed paraffin-embedded tissue sections according to standard protocols. Tables 7 and 8 summarize the staining results for healthy and cancerous tissues, respectively.

[0465] Table 7. hBBC.10.1 immunostaining of various normal human tissues.

[0466]

[0467]

[0468] Table 8. hBBC immunostaining in human cancer tissues.

[0469]

[0470]

[0471] In addition, various human cancer cell lines were stained with hBBC.10.1 to determine the epitope expression pattern. The results are summarized in Table 9.

[0472] Table 9. hBBC epitope expression on human cancer cell lines.

[0473]

[0474] Example 6

[0475] Antitumor activity of HBBC in a colon cancer xenograft model

[0476] A series of in vivo animal studies were conducted to evaluate the tumor inhibitory effects of hBBC in xenograft SCID models, including the introduction of human cancer cells from gastric cancer cell lines AGS, TSGH9201 and colon cancer cell lines COLO 201, COLO 205, DLD-1. hBBC.10.1 has shown strong to moderate binding levels to all cell lines except COLO 205 (Table 9 of Example 5). hBBC.10.1 was used in all xenograft studies and BR96 was used as a control.

[0477] In vivo antitumor activity of hBBC.10.1 in DLD-1 and COLO 205 xenograft models

[0478] The purpose of this study is to examine the tumor inhibitory effect of high doses of hBBC.10.1 in DLD-1 and COLO 205 xenograft models. In brief, human colon cancer cell lines DLD-1 and COLO 205 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in RPMI 1640 culture medium supplemented with 10% fetal bovine serum (FBS), and cell cultures were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere. Cells at passages 5-8 were used for tumor inoculation.

[0479] Specific pathogen-free (SPF) female CB17 severe combined immunodeficient (SCID) mice were purchased from BioLASCO (Taiwan, China) and acclimatized for at least one week before any experimental manipulation. The mice were placed in a temperature-controlled environment (22 ± 2 ° C) with a humidity of 50 ± 10% in an individually ventilated cage (IVC) in a 12:12 h light-dark cycle. All experiments were conducted in compliance with relevant regulations and animal protection laws.

[0480] Cancer cells were collected at a rate of 5×10 6The cells were resuspended in ice-cold serum-free medium containing 50% BD Matrigel (Catalog No.: 354248) at a density of 100 μL / 200 μL and injected subcutaneously into the flank region of SCID mice aged 6-8 weeks. Tumor size was measured weekly with a vernier caliper (Laser Tools and Technics (LTT), Hsinchu City, Taiwan Province, China, 150×0.05 mm), and tumor weight was estimated as “weight (mg) = (width 2 × length) mm 3 / 2" (Ito et al., (1992) Cancer Res. 52: 3739). When the tumor weight reached 150-200 mg, the mice were randomly divided into 9 groups (n=6 in each group), each group having comparable tumor size, and antibody treatment was started. Tumor-bearing SCID mice were injected intraperitoneally with hBBC.10.1 (batch: 17001) at 50 mg / kg once a week for 6 weeks. Tumor-bearing SCID mice injected intraperitoneally with saline were used as negative controls.

[0481] The results of DLD-1 and COLO 205 xenograft experiments are shown in Fig.19A and Fig.19B hBBC.10.1 was able to effectively inhibit DLD-1 tumor growth relative to controls, but not COLO 205 tumor growth. These data are consistent with the observed ability of hBBC.10.1 to bind to DLD-1 cells but not COLO 205.

[0482] Antitumor activity of hBBC in a gastric adenocarcinoma xenograft model

[0483] The purpose of this study was to compare the anti-tumor efficacy of hBBC.10.1 and BR96 at a low dose range of 0.008 to 1 mg / kg in the AGS xenograft model. In brief, the human gastric adenocarcinoma cell line AGS (CRL-1739) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). AGS cells were grown in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS), and the cell culture was maintained in a humidified incubator with a 5% CO2 atmosphere at 37°C. AGS cells at passages 5-8 were used for tumor inoculation.

[0484] Specific pathogen-free (SPF) female CB17 severe combined immunodeficient (SCID) mice were purchased from BioLASCO (Taiwan, China) and acclimatized for at least one week before any experimental manipulation. The mice were placed in a temperature-controlled environment (22 ± 2 ° C) with a humidity of 50 ± 10% in an individually ventilated cage (IVC) in a 12:12 h light-dark cycle. All experiments were conducted in compliance with relevant regulations and animal protection laws.

[0485] AGS cells were cultured at 5 × 10 6 The cells were resuspended at a density of 100 μL / 200 μL in ice-cold serum-free medium containing 50% BD matrix (Catalog No. 354248) and injected subcutaneously into the flank region of SCID mice aged 6-8 weeks. Tumor size was measured weekly with a vernier caliper (Laser Tools and Technics (LTT), Hsinchu City, Taiwan Province, China, 150×0.05 mm), and tumor weight was estimated as “weight (mg) = (width 2 × length) mm 3 / 2" (Ito et al., (1992) Cancer Res. 52: 3739). When the tumor weight reached 150-200 mg, the mice were randomly divided into 9 groups (n=6 in each group), each group having comparable tumor size, and antibody treatment was started. Tumor-bearing SCID mice were injected intraperitoneally with hBBC.10.1 (batch: 17001) or BR96 (batch: 17001) at a dose of 1, 0.2, 0.04 or 0.008 mg / kg once a week for six weeks, and the first dose was given at 1.5 times the predetermined dose. Tumor-bearing SCID mice injected intraperitoneally with saline were used as negative controls.

[0486] The results of hBBC.10.1-treated and BR96-treated mice are shown in Fig. 20A and Fig. 20B When administered at doses of 1 and 0.2 mg / kg per week, both hBBC.10.1 and BR96 significantly inhibited tumor growth compared to controls. However, neither antibody showed an inhibitory effect at lower doses (0.04 and 0.008 mg / kg). For ethical considerations, mice were sacrificed when the tumor burden was greater than 10% of body weight. Some mice receiving saline or lower doses of antibodies reached this endpoint 60 days after inoculation; therefore, only data generated until day 60 were statistically analyzed. In contrast, in AGS tumor-bearing mice administered high doses (1 and 0.2 mg / kg) of hBBC or BR96, tumor growth was effectively delayed to 10% of body weight. 10.1. This dose-response study shows that hBBC.10.1 has comparable antitumor efficacy to BR96 in AGS tumor-bearing mice.

[0487] The anti-tumor efficacy of hBBC.10.1 was compared with that of other generated hBBC antibodies disclosed herein. In one experiment, harvested AGS cells were washed twice with PBS and cultured at 5×10 6 Resuspend the cells at a density of 100 cells / 200 μL in 25% BD Matrigel. TM(BD Biosciences, catalog number: 354248) in PBS. Then, 200 μL of AGS cell suspension was subcutaneously injected (5×10 6 The tumor size was measured weekly with a vernier caliper (Laser 150×0.05 mm), and the tumor weight was estimated as “weight (mg) = (width 2 × length) mm 3 / 2" [(Hisashi Ito et al., (1992)]. When the tumor weight reached 150-200 mg, the mice were randomly divided into 6 groups (n=6 in each group) with comparable tumor size and antibody treatment was started. Tumor-bearing SCID mice were injected intraperitoneally with hBBC.8 or mutants (hBBC.9, hBBC.9.1, hBBC.10.1) at a dose of 0.25 mg / kg twice a week for 6 weeks. In addition, hBBC.8 was tested at a higher dose (2 mg / kg). Tumor-bearing SCID mice injected intraperitoneally with saline were used as negative controls. Data are shown in Fig. 20C Although the antitumor effects of hBBC.8, hBBC.9, and hBBC.9.1 were weaker than those of hBBC.10.1, antitumor effects were still observed. At a higher dose (2 mg / kg), hBBC.8 was observed to have an antitumor activity comparable to that of hBBC.10.1 (0.25 mg / kg).

[0488] The antitumor activity of hBBC.10.1 was also compared with that of hBBC.10.1FQ. Briefly, harvested AGS cells were washed twice with PBS and cultured at 5 × 10 6 Resuspend the cells at a density of 100 cells / 200 μL in 25% BD Matrigel. TM (Catalog No.: 354248) in PBS. Then, 200 μL of AGS cell suspension was subcutaneously injected (5×10 6 The tumor size was measured weekly with a vernier caliper (Laser 150×0.05 mm), and the tumor weight was estimated as “weight (mg) = (width 2 × length) mm 3 / 2" [(Hisashi Ito et al., (1992)]. When the tumor weight reached 150-200 mg, the mice were randomly divided into 3 groups (n=8 in each group), each with comparable tumor size, and antibody treatment was started. Tumor-bearing SCID mice were injected intraperitoneally with hBBC.10.1 or hBBC.10.1FQ at a dose of 0.25 mg / kg once a week for six weeks, and the first dose was given at 1.5 times the predetermined dose. Tumor-bearing SCID mice injected intraperitoneally with saline were used as negative controls. Data ( Fig.20D ) showed that hBBC.10.1FQ had slightly stronger anti-tumor activity.

[0489] In subsequent experiments, xenograft AGS (xAGS) cells isolated from AGS xenograft tumors were shown to express at least 2×hBBC epitopes compared to AGS cell line cells (data not shown). xAGS cells also elicited more potent ADCC and CDC activity via hBBC compared to the parental cell line (data not shown). Fig.21 As shown in , the direct killing effect of hBBC on target xAGS cells was weaker than that of BR96 (PI staining).

[0490] Antitumor activity of hBBC in a gastric cancer xenograft model

[0491] The purpose of this study was to compare the antitumor efficacy of hBBC.10.1 and BR96 at a low dose range of 0.04 to 10 mg / kg in the TSGH 9201 xenograft model. Briefly, the human gastric cancer cell line TSGH 9201 (Catalog No.: 60146) was obtained from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute, FIRDI, Hsinchu City, Taiwan Province, China. The cells were stained with anti-hBBC (Batch: B24) followed by 200-fold dilution of fluorescein (FITC)-AffiniPure TM The cells were stained with goat anti-human IgG (Fcγ fragment specific) (Jackson ImmunoResearch Inc., West Grove, PA, catalog number: 109-095-098) and the cells were separated by two rounds of fluorescence activated cell sorting (FACS) (BD FACSJAZZ TMCell sorter, BD Biosciences, Singapore, catalog number: 655486) was used to enrich TSGH 9201 cells that highly expressed the hBBC epitope. Compared with the parental TSGH 9201 cells, the expression level of the hBBC epitope was increased by nearly 4 times in the enriched cells, named TSGH 9201 (2s) (not shown). Both the parental and enriched cells were grown in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1mM sodium pyruvate. The cell culture was maintained at 37°C in a humidified incubator under a 5% CO2 atmosphere. TSGH 9201 (2s) cells of passages 5-8 were used for tumor inoculation.

[0492] Specific pathogen-free (SPF) female CB17 severe combined immunodeficient (SCID) mice were purchased from BioLASCO (Taiwan, China) and acclimatized for at least one week before any experimental manipulation. The mice were placed in a temperature-controlled environment (22 ± 2 ° C) with a humidity of 50 ± 10% in an individually ventilated cage (IVC) in a 12:12 h light-dark cycle. All experiments were conducted in compliance with relevant regulations and animal protection laws.

[0493] TSGH 9201 (2s) cells were cultured at 5×10 6 The cells were resuspended at a density of 100 μL / 200 μL in ice-cold serum-free medium containing 25% BD Matrigel (Catalog No. 354248), and 200 μL of the cell suspension was injected subcutaneously into the flank region of SCID mice aged 6-8 weeks. Tumor size was measured weekly with a vernier caliper (Laser Tools and Technics (LTT), Hsinchu City, Taiwan Province, China, 150×0.05 mm), and tumor weight was estimated as “weight (mg) = (width 2 × length) mm 3 / 2”(Ito et al., (1992) Cancer Res.52:3739). When the tumor weight reached 150-200 mg, the mice were randomly divided into 3 groups (n=5 per group), each with comparable tumor size, and antibody treatment was started. To evaluate the in vivo efficacy of hBBC.10.1 and BR96, tumor-bearing SCID mice were injected intraperitoneally with 10 mg / kg once a week for six weeks, with the first dose given at 1.5 times the designated dose. In a second study comparing the anti-tumor activity of low-dose hBBC.10.1 and BR96, tumor-bearing SCID mice were injected intraperitoneally with 10, 1, 0.2 or 0.04 mg / kg once a week for six weeks, with the first dose given at 1.5 times the designated dose. Tumor-bearing SCID mice injected intraperitoneally with saline were used as negative controls.

[0494] The results are shown in Fig. 22 Compared with the saline group, hBBC.10.1 and BR96 both significantly inhibited tumor growth at a dose of 10 mg / kg per week.

[0495] Antitumor activity of hBBC.10.1 in a colorectal adenocarcinoma xenograft model

[0496] The objectives of this study were: 1) to examine the ability of hBBC.10.1 to inhibit the growth of COLO 201 xenograft tumors; and 2) to compare the antitumor activity of hBBC and BR96 in the COLO 201 xenograft model over a dose range of 0.008 to 1 mg / kg.

[0497] Briefly, the human colorectal adenocarcinoma cell line COLO 201 (CCL-224) derived from ascites was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). COLO 201 cells were grown in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% sodium pyruvate. Cell cultures were maintained at 37° C. in a humidified incubator under a 5% CO2 atmosphere. COLO 201 cells at passages 5-8 were used for tumor inoculation.

[0498] Specific pathogen-free (SPF) female CB17 severe combined immunodeficient (SCID) mice were purchased from BioLASCO (Taiwan, China) and allowed to acclimate for at least one week before any experimental manipulation. The mice were placed in a temperature-controlled environment (22 ± 2 ° C) with a humidity of 50 ± 10% in an individually ventilated cage (IVC) in a 12:12 h light-dark cycle. All experiments were conducted in compliance with relevant regulations and animal protection laws.

[0499] COLO 201 cells were cultured at 2×10 6 The cells were resuspended in ice-cold serum-free medium at a density of 100 μL / 200 μL, and 200 μL of the cell suspension was subcutaneously injected into the flank region of 6-8 week-old SCID mice. Tumor size was measured weekly with a vernier caliper (Laser 150×0.05 mm), and tumor weight was estimated as “weight (mg) = (width 2 × length) mm 3 / 2" (Ito et al., (1992) Cancer Res. 52:3739). When the tumor weight reached 150-200 mg, the mice were randomly divided into control and treatment groups (n=6 in each group), each with comparable tumor size, and antibody treatment was started. Two studies were conducted to evaluate the in vivo efficacy of hBBC in inhibiting COLO 201 tumor growth.

[0500] In the first study, hBBC.10.1 (Batch: B26) was injected intraperitoneally into tumor-bearing SCID mice at a dose range of 2 to 50 mg / kg twice a week for six consecutive weeks, with the first dose given at 1.5 times the designated dose. The results of the first study were shown in Fig.23 Compared with the saline group, treatment with 2-50 mg / kg of hBBC.10.1 significantly inhibited tumor growth. Only one week after hBBC administration (i.e., two weeks after tumor inoculation), the tumors shrank rapidly and eventually disappeared.

[0501] In a second study, the antitumor activity of low doses of hBBC.10.1 and BR96 was further compared. Tumor-bearing SCID mice were injected intraperitoneally with hBBC.10.1 (Batch: 17001) or BR96 (Batch: 17001) at 1, 0.2, 0.04, and 0.008 mg / kg once a week (starting on day 7) for 6 weeks, with the first dose given at 1.5 times the designated dose. Tumor-bearing SCID mice injected intraperitoneally with saline were used as negative controls. Purified antibody hTKH2.2 (Batch: 1020429) was included as a negative control antibody, which is a humanized anti-STn antibody produced in-house by transient expression in HEK293 cells. The results of the second study are shown in Fig.24A and Fig. 24BWeekly doses of 1 or 0.2 mg / kg of hBBC.10.1 and BR96 both significantly inhibited tumor growth compared to saline and hTKH2.2 control groups, while lower doses (0.04 and 0.008 mg / kg of hBBC.10.1; 0.04 mg / kg of BR96) showed a delayed tumor growth effect. For ethical considerations, mice were killed when the tumor burden was greater than 10% of body weight. Some mice receiving saline or lower doses of hBBC.10.1 or BR96 reached this endpoint 49 days after inoculation. Therefore, only data obtained until 49 days were statistically analyzed.

[0502] This dose-response study demonstrated that hBBC.10.1 had comparable antitumor efficacy to BR96 in COLO 201 tumor-bearing mice.

[0503] Example 7

[0504] Immunostaining of HBBC in primate and human tissues

[0505] hBBC.10.1 immunostaining was performed on corresponding healthy tissues from humans and cynomolgus monkeys (Macaca fascicularis). Briefly, formalin-fixed paraffin-embedded tissue sections were stained with 2 μg / ml of hBBC.10.1 according to standard protocols. The results are summarized in Table 10 and show similar staining patterns between human and cynomolgus monkey tissues.

[0506] Table 10. hBBC.10.1 immunostaining of human and cynomolgus monkey tissues

[0507]

[0508]

[0509] Example 8

[0510] Safety and tolerability study of HBBC.10.1 in primate models

[0511] In order to evaluate the tolerance and acceptable dose range of HBBC.10.1 in cynomolgus monkeys after a single intravenous (IV) bolus injection, a single dose tolerance and dose range determination study (non-GLP) was conducted. In short, male and female cynomolgus monkeys (Macaca fascicularis) were divided into four groups, one male and one female in each group. The HBBC.10.1 dose levels received by animals in groups 1 to 4 were 0, 50, 200 and 300 mg / kg, respectively. Animals in groups 2 and 3 were administered once via slow intravenous injection. The bolus injection lasted at least 5 minutes. Animals in groups 1 and 4 were administered once via intravenous infusion using a pump and perfusion infusion line every 20 minutes (± 1 minute). Approximately 24 hours after administration, autopsy was performed. During the autopsy, gross observations and organ weights were recorded, and tissues were collected for histopathological examination. Some of the collected tissues were processed into slides and examined under a microscope.

[0512] The results show that hBBC.10.1 is well tolerated in the 50-300 mg / kg dose range in cynomolgus monkeys. Minimal bleeding was observed in the cecum and / or colon of animals receiving 200 and 300 mg / kg, which may be related to the test article. Other possible changes related to the test article are chronic and acute inflammation of the stomach in one animal receiving 200 mg / kg, neutrophil infiltration of the duodenum crypts in one animal receiving 200 mg / kg, and villous atrophy in the ileum of one animal receiving 300 mg / kg. No abnormal findings were observed in animals receiving 50 mg / kg (Group 2).

[0513] The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet are incorporated herein by reference in their entirety. If it is necessary to adopt the concepts of various patents, applications, and publications to provide other embodiments, then various aspects of the embodiments can be modified.

[0514] These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be interpreted to limit the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full scope of equivalents required by such claims. Therefore, the claims are not limited by the disclosure. Sequence Listing <110> GlycoNex, Inc. Chang, Tong-Hsuan Yang Mei-Chun Liu, Liahng-Yirn Jerry Ting Chang, Shu-Yen Chen, Yen-Ying Lin, Yu-Yu Tang Shu-Lun <120> Therapeutic antibodies <130> 400100.401WO <140> PCT <141> 2019-05-30 <150> US 62 / 678,890 <151> 2018-05-31 <160> 51 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - heavy chain variable (VH) domain of antibody hBBC.10.1FQ <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Thr Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Leu Gly Tyr Ile His Tyr Thr Gly Asn Thr Lys Tyr Ser Pro Ser Leu 50 55 60 Lys Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Glu Ala Leu Arg Gly Tyr Asp Ala Gly Phe Trp Phe Thr Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequences - Heavy chain complementarity determining region 1 (VHCDR1) of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, <400> 2 Ser Gly Tyr Thr Trp His 1 5 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequences - VH CDR2 of antibody hBBC.9.1, antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 3 Tyr Ile His Tyr Thr Gly Asn Thr Lys Tyr Ser Pro Ser Leu Lys Ser 1 5 10 15 <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequences - VH CDR3 of antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 4 Glu Ala Leu Arg Gly Tyr Asp Ala Gly Phe Trp Phe Thr Tyr 1 5 10 <210> 5 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequences - light chain variable (VL) domains of antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - light chain complementary determining region 1 (VLCDR1) of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, <400> 6 Thr Ala Ser Glu Asp Ile Tyr Asn Arg Leu Thr 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequences - VL CDR2 of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 7 Gly Ala Thr Ser Leu Asp Thr 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequences - VL CDR3 of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 8 Gln Gln Tyr Trp Thr Thr Pro Trp Thr 1 5 <210> 9 <211> 278 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - Single chain variable region fragment (scFv) derived from antibody hBBC.10.1 in the [VL-VH] direction <220> <221> VARIANT <222> (113)...(157) <223> Any or all of amino acids 113-157 may be present or absent <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln 145 150 155 160 Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser 165 170 175 Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly Tyr Thr Trp 180 185 190 His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Leu Gly Tyr 195 200 205 Ile His Tyr Thr Gly Asn Thr Lys Tyr Ser Pro Ser Leu Lys Ser Arg 210 215 220 Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys Leu 225 230 235 240 Ser Ser Val Thr Thr Glu Asp Thr Ala Val Tyr Tyr Cys Gly Arg Glu 245 250 255 Ala Leu Arg Gly Tyr Asp Ala Gly Phe Trp Phe Thr Tyr Trp Gly Gln 260 265 270 Gly Thr Leu Val Thr Val 275 <210> 10 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - Full length heavy chain (HC) of antibody hBBC.10.1 <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Thr Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Leu Gly Tyr Ile His Tyr Thr Gly Asn Thr Lys Tyr Ser Pro Ser Leu 50 55 60 Lys Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Glu Ala Leu Arg Gly Tyr Asp Ala Gly Phe Trp Phe Thr Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 11 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequences - Full length light chain (LC) of antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 12 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence - Heavy chain variable (VH) domain of antibody hBBC.10.1 <400> 12 caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagccagac cctgagcctg 60 acctgcaccg tgagcggcta tagcattacc agcggctata cctggcattg gattcgccag 120 catccgggca aaggcctgga atggctgggc tatattcatt ataccggcaa caccaaatat 180 agcccgagcc tgaaaagccg cctgagcatt agccgcgata ccagcaaaaa ccagttcttc 240 ctgaaactga gcagcgtgac caccgaagat accgcggtgt attattgcgg ccgcgaagcg 300 ctgcgcggct atgatgctgg cttctggttt acctattggg gccaaggcac cctggtgacc 360 gtg 363 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence - Heavy chain complementarity determining region 1 (VHCDR1) of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9 <400> 13 agcggctata cctggcat 18 <210> 14 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequences - VH CDR2 of antibody hBBC.9.1, antibody hBBC.10 and antibody hBBC.10.1FQ <400> 14 tatattcatt ataccggcaa caccaaatat agcccgagcc tgaaaagc 48 <210> 15 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequences - VH CDR3 of antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 15 gaagcgctgc gcggctatga tgctggcttc tggtttacct at 42 <210> 16 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequences - variable light chain (VL) domains of antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 16 gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60 attacctgca ccgcgagcga agatattttat aaccgcctga cctggtatca gcagaaaccg 120 ggcaaagtgc cgcgtctgct gatttctggc gcgaccagcc tggataccgg cgtgccgagc 180 cgctttagcg gcagcggcag cggcaccgat tacaccctga ccattagcag cctgcagccg 240 gaagatgtgg cgacctatta ttgccagcag tattggacca ccccgtggac ctttggccag 300 ggcaccaaac tggaaattaa a 321 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequences - Light chain complementarity determining region 1 (VLCDR1) of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9 <400> 17 accgcgagcg aagatattta taaccgcctg acc 33 <210> 18 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequences - VL CDR2 of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 18 ggcgcgacca gcctggatac c 21 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequences - VL CDR3 of antibody IMH2 / BBC, antibody hBBC.8, antibody hBBC.9, antibody hBBC.9.1, antibody hBBC.10, antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 19 cagcagtatt ggaccacccc gtggacc 27 <210> 20 <211> 834 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence - Single chain variable region fragment (scFv) derived from antibody hBBC.10.1 in the [VL-(L)-VH] orientation <220> <221> misc_feature <222> (337)...(471) <223> Any or all of nucleotides 337-471 may be present or absent. <400> 20 gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60 attacctgca ccgcgagcga agatattttat aaccgcctga cctggtatca gcagaaaccg 120 ggcaaagtgc cgcgtctgct gatttctggc gcgaccagcc tggataccgg cgtgccgagc 180 cgctttagcg gcagcggcag cggcaccgat tacaccctga ccattagcag cctgcagccg 240 gaagatgtgg cgacctatta ttgccagcag tattggacca ccccgtggac ctttggccag 300 ggcaccaaac tggaaattaa aggtggaggc ggttctggtg gaggcggttc tggtggaggc 360 ggttctggtg gaggcggttc tggtggaggc ggttctggtg gaggcggttc tggtggaggc 420 ggttctggtg gaggcggttc tggtggaggc ggttctggtg gaggcggttc tcaggtgcag 480 ctgcaggaaa gcggcccggg cctggtgaaa ccgagccaga ccctgagcct gacctgcacc 540 gtgagcggct atagcattac cagcggctat acctggcatt ggattcgcca gcatccgggc 600 aaaggcctgg aatggctggg ctatattcat tataccggca acaccaaata tagcccgagc 660 ctgaaaagcc gcctgagcat tagccgcgat accagcaaaa accagttctt cctgaaactg 720 agcagcgtga ccaccgaaga taccgcggtg tattattgcg gccgcgaagc gctgcgcggc 780 tatgatgctg gcttctggtt tacctattgg ggccaaggca ccctggtgac cgtg 834 <210> 21 <211> 1362 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence - full-length heavy chain (HC) of antibody hBBC.10.1 <400> 21 caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagccagac cctgagcctg 60 acctgcaccg tgagcggcta tagcattacc agcggctata cctggcattg gattcgccag 120 catccgggca aaggcctgga atggctgggc tatattcatt ataccggcaa caccaaatat 180 agcccgagcc tgaaaagccg cctgagcatt agccgcgata ccagcaaaaa ccagttcttc 240 ctgaaactga gcagcgtgac caccgaagat accgcggtgt attattgcgg ccgcgaagcg 300 ctgcgcggct atgatgctgg cttctggttt acctattggg gccaaggcac cctggtgacc 360 gtgtcgagcg cttccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420 acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 480 acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 540 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600 acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362 <210> 22 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence - full-length light chain (LC) of antibody hBBC.10.1 and antibody hBBC.10.1FQ <400> 22 gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60 attacctgca ccgcgagcga agatatttat aaccgcctga cctggtatca gcagaaaccg 120 ggcaaagtgc cgcgtctgct gatttctggc gcgaccagcc tggataccgg cgtgccgagc 180 cgctttagcg gcagcggcag cggcaccgat tacaccctga ccattagcag cctgcagccg 240 gaagatgtgg cgacctatta ttgccagcag tattggacca ccccgtggac ctttggccag 300 ggcaccaaac tggaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttaa 645 <210> 23 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence - Illustrative Spacer Amino Acid Sequence <400> 23 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp 1 5 10 <210> twenty four <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequences - Illustrative Spacer Amino Acid Sequences <400> twenty four Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - Flexible multi-linker amino acid sequence <400> 25 Gly Gly Gly Gly Ser 1 5 <210> 26 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - Flexible multi-linker amino acid sequence <400> 26 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - VL domain of antibody IMH2 / BBC <400> 27 Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Asn Val Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Lys Asp Tyr Ala Leu Ser Ile Thr Ser Leu Gln Thr 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 28 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - VH domain of antibody IMH2 / BBC <400> 28 Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Thr Trp His Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Leu Gly Tyr Ile His Tyr Ser Gly Asn Thr Lys Tyr Ser Pro Ser Leu 50 55 60 Lys Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Gly Arg Glu Ala Leu Arg Gly Tyr Asp His Gly Phe Trp Phe Thr Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 29 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - VL domain of human acceptor framework AAS01771.1 <400> 29 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 30 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - VH domain of human receptor framework CAD89404.1 <400> 30 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Ser Met Ser Arg Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Pro Tyr Tyr Asp Ser Pro Arg Pro Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 31 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - VL domain of antibody hBBC.8 <400> 31 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 32 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequences - VH domains of antibodies hBBC.8, hBBC.9 and hBBC.10 <400> 32 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Thr Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Leu Gly Tyr Ile His Tyr Ser Gly Asn Thr Lys Tyr Ser Pro Ser Leu 50 55 60 Lys Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Glu Ala Leu Arg Gly Tyr Asp His Gly Phe Trp Phe Thr Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 33 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequences - VL domains of antibodies hBBC.9 and hBBC.9.1 <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 34 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - VH domain of antibody hBBC.9.1 <400> 34 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Thr Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Leu Gly Tyr Ile His Tyr Thr Gly Asn Thr Lys Tyr Ser Pro Ser Leu 50 55 60 Lys Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Glu Ala Leu Arg Gly Ala Asp His Gly Phe Trp Phe Thr Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 35 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence - VH Domain of Antibody hBBC.10.1 <400> 35 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Thr Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Leu Gly Tyr Ile His Tyr Thr Gly Asn Thr Lys Tyr Ser Pro Ser Leu 50 55 60 Lys Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Glu Ala Leu Arg Gly Tyr Asp Ala Gly Phe Trp Phe Thr Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 36 <211> 278 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - Single chain variable region fragment (scFv) derived from antibody hBBC.10.1 in the [VH-VL] direction <220> <221> VARIANT <222> (127)...(171) <223> Any or all of amino acids 127-171 may be present or absent. <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Thr Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Leu Gly Tyr Ile His Tyr Thr Gly Asn Thr Lys Tyr Ser Pro Ser Leu 50 55 60 Lys Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Glu Ala Leu Arg Gly Tyr Asp Ala Gly Phe Trp Phe Thr Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr Cys Thr Ala Ser Glu Asp Ile Tyr Asn Arg Leu Thr Trp Tyr Gln 195 200 205 Gln Lys Pro Gly Lys Val Pro Arg Leu Leu Ile Ser Gly Ala Thr Ser 210 215 220 Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 225 230 235 240 Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr 245 250 255 Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp Thr Phe Gly Gln Gly 260 265 270 Thr Lys Leu Glu Ile Lys 275 <210> 37 <211> 834 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence - single chain variable region fragment (scFv) derived from antibody hBBC.10.1 in the [VH-(L)-VL] orientation <220> <221> misc_feature <222> (393)...(513) <223> Any or all of nucleotides 393-513 may be present or absent. <400> 37 caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagccagac cctgagcctg 60 acctgcaccg tgagcggcta tagcattacc agcggctata cctggcattg gattcgccag 120 catccgggca aaggcctgga atggctgggc tatattcatt ataccggcaa caccaaatat 180 agcccgagcc tgaaaagccg cctgagcatt agccgcgata ccagcaaaaa ccagttctttc 240 ctgaaactga gcagcgtgac caccgaagat accgcggtgt attattgcgg ccgcgaagcg 300 ctgcgcggct atgatgctgg cttctggttt acctattggg gccaaggcac cctggtgacc 360 gtgggtggag gcggttctgg tggaggcggt tctggtggag gcggttctgg tggaggcggt 420 tctggtggag gcggttctgg tggaggcggt tctggtggag gcggttctgg tggaggcggt 480 tctggtggag gcggttctgg tggaggcggt tctgatattc agatgaccca gagcccgagc 540 agcctgagcg cgagcgtggg cgatcgcgtg accattacct gcaccgcgag cgaagatatt 600 tataaccgcc tgacctggta tcagcagaaa ccgggcaaag tgccgcgtct gctgatttct 660 ggcgcgacca gcctggatac cggcgtgccg agccgcttta gcggcagcgg cagcggcacc 720 gattacaccc tgaccattag cagcctgcag ccggaagatg tggcgaccta ttattgccag 780 cagtattgga ccaccccgtg gacctttggc cagggcacca aactggaaat taaa 834 <210> 38 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthesized sequence - hBBC.10.1 <400> 38 Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Val Tyr Tyr Cys Gly Arg 20 25 30 <210> 39 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - Heavy chain framework region 3 hBBC.10.1FQ <400> 39 Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Thr Phe Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Thr Thr Glu Asp Thr Ala Val Tyr Tyr Cys Gly Arg 20 25 30 <210> 40 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - heavy chain framework region 3 reference 1 <400> 40 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 41 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - heavy chain framework region 3 reference 2 <400> 41 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg 20 25 30 <210> 42 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - IMH2 / hBBC CDRH2 <400> 42 Tyr Ile His Tyr Ser Gly Asn Thr Lys Tyr Ser Pro Ser Leu Lys Ser 1 5 10 15 <210> 43 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - IMH2 / hBBC CDRH2 <400> 43 Glu Ala Leu Arg Gly Tyr Asp His Gly Phe Trp Phe Thr 1 5 10 <210> 44 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - human receptor VH 1 <400> 44 Val Gln Leu Gln Glu Trp Gly Pro Gly Leu Val Lys Pro Ser Gln Thr 1 5 10 15 Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Tyr Asn Phe Gly 20 25 30 His Tyr Trp Ser Trp Ile Arg His Tyr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Leu Thr Ile Ser Ala Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Glu Leu Asn Ser Met Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Gly Gly Ser Ala Ala Gly Thr His Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val 115 120 <210> 45 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - human receptor VH 2 <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Asn Ser Ser 20 25 30 Gly Phe Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Met Phe Tyr Gly Gly Ser Pro Tyr Asn Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Tyr Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Ala Phe Asp Tyr Ser Ala Ser Gly Ser Phe Tyr Phe Gly 100 105 110 Ser Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 46 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence - Human Receptor VH 3 <400> 46 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Ser Met Ser Arg Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Pro Tyr Tyr Asp Ser Pro Arg Pro Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 47 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - human receptor VH 4 <400> 47 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Ser Gly 20 25 30 Gly Thr His Trp Ser Trp Ile Arg Gln Leu Pro Gly Gln Gly Leu Glu 35 40 45 Trp Leu Gly Tyr Leu Tyr Asn Ser Arg Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Glu Ser Arg Leu Thr Ile Ser Ala Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Asn Leu Ser Thr Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Lys Ser Gly Phe Arg Glu Phe Asp Leu Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val 115 <210> 48 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - human receptor VH 5 <400> 48 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Thr Ser Ile Ser Thr Gly 20 25 30 Gly Tyr His Trp Thr Trp Ile Arg Gln Gln Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Gly Tyr Ile Tyr His Ser Gly Ser Ser Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Asn Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asn Ser Gly Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val 115 <210> 49 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence - human receptor VH 6 <400> 49 Gln Leu Gln Leu Gln Glu Ser Gly Pro Arg Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Ile Ser Gly Ala 20 25 30 Tyr His Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Gly Tyr Ile Tyr Tyr Thr Gly Asn Thr Tyr Phe Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Met Asn Ser Val Thr Val Ala Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Ile Ala Leu Pro Gly Arg Gly Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 50 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence - Human Receptor VH 7 <400> 50 Leu Glu Gln Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser 1 5 10 15 Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly Tyr Trp Asn 20 25 30 Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met Gly Tyr Ile 35 40 45 Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Leu Ser Leu Arg Ser Arg Ile 50 55 60 Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu Gln Leu Asn 65 70 75 80 Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Leu Ile Thr 85 90 95 Thr Thr Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val <210> 51 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence - Human Receptor VH 8 <400> 51 Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Asn Gly 20 25 30 Phe Trp Ile Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Gln Asn Gln Phe Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Gly Thr Tyr Tyr Cys Ala 85 90 95 Cys Arg Ser Tyr Gly Arg Thr Pro Tyr Tyr Phe Asp Phe Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val 115

Claims

1. An isolated antibody or antigen-binding fragment thereof comprising: (a) an immunoglobulin heavy chain variable region, which comprises a heavy chain complementary determining region 1 (VH CDR1), a heavy chain complementary determining region 2 (VHCDR2), and a heavy chain complementary determining region 3 (VH CDR3), wherein the heavy chain complementary determining region 1 (VH CDR1) consists of the amino acid sequence shown in SEQ ID NO: 2, the heavy chain complementary determining region 2 (VH CDR2) consists of the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain complementary determining region 3 (VH CDR3) consists of the amino acid sequence shown in SEQ ID NO: 4; and (b) an immunoglobulin light chain variable region, comprising a light chain complementary determining region 1 (VL CDR1), a light chain complementary determining region 2 (VLCDR2) and a light chain complementary determining region 3 (VL CDR3), wherein the light chain complementary determining region 1 (VL CDR1) consists of the amino acid sequence shown in SEQ ID NO: 6, the light chain complementary determining region 2 (VL CDR2) consists of the amino acid sequence shown in SEQ ID NO: 7, and the light chain complementary determining region 3 (VL CDR3) consists of the amino acid sequence shown in SEQ ID NO: 8; Wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to: Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen, Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen, Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen, and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc] [VIII]'s Double Tentacles Y / Le B antigen, and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A Antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV].

2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

5.

3. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 92% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 92% identical to the amino acid sequence shown in SEQ ID NO:

5.

4. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

5.

5. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO:

5.

6. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:

5.

7. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody is monoclonal.

8. The isolated antibody or antigen-binding fragment thereof of claim 7, wherein the antibody is a humanized antibody.

9. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv (ScFv) antibody and a diabody.

10. The isolated antibody or antigen-binding fragment thereof according to claim 7, which is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv (ScFv) antibody and a diabody.

11. The isolated antibody or antigen-binding fragment thereof according to claim 8, which is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv (ScFv) antibody and a diabody.

12. An isolated antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein: (i) the VH comprises: a VH complementary determining region (CDR) 1 consisting of a variant of the amino acid sequence set forth in SEQ ID NO: 2, wherein the variation consists of a Y→A substitution at position 33 of SEQ ID NO: 35; a heavy chain complementary determining region 2 (VH CDR2) consisting of the amino acid sequence set forth in SEQ ID NO: 3; and a heavy chain complementary determining region 3 (VH CDR3) consisting of the amino acid sequence set forth in SEQ ID NO: 4; and the VL comprises: a light chain complementary determining region 1 (VL CDR1) consisting of the amino acid sequence set forth in SEQ ID NO: 6; a light chain complementary determining region 2 (VL CDR2) consisting of the amino acid sequence set forth in SEQ ID NO: 7; and a light chain complementary determining region 3 (VL CDR3) consisting of the amino acid sequence set forth in SEQ ID NO: 8 (ii) the VH comprises: a VH CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:2; a VH CDR2 consisting of a variant of the amino acid sequence set forth in SEQ ID NO:3, wherein the variant consists of a T→S substitution at position 55 of SEQ ID NO:35; and a VH CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:4; and the VL comprises: a VL CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:6; a VL CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:7; and a VL CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:8; (iii) the VH comprises: a VH CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 2; a VH CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a VH CDR3 consisting of a variant of the amino acid sequence shown in SEQ ID NO: 4, wherein the variant consists of a Y→A substitution at position 104 of SEQ ID NO: 35, or an A→H substitution at position 106 of SEQ ID NO: 35; and the VL comprises: a VL CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 6; a VL CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 7; and a VLCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 8; (iv) the VH comprises: a VH CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:2; a VH CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:3; and a VH CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:4; and the VL comprises: a VL CDR1 consisting of a variant of the amino acid sequence set forth in SEQ ID NO:6, wherein the variation consists of a Y→A substitution at position 30 of SEQ ID NO:5; a VL CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:7; and a VL CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:8; (v) the VH comprises: a VH CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:2; a VH CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:3; and a VH CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:4; and the VL comprises: a VL CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:6; a VL CDR2 consisting of a variant of the amino acid sequence set forth in SEQ ID NO:7, wherein the variant consists of a G→A substitution at position 50 of SEQ ID NO:5; and a VL CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:8; (vi) the VH comprises: a VH CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:2; a VH CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:3; and a VH CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:4; and the VL comprises: a VL CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:6; a VL CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:7; and a VL CDR3 consisting of a variant of the amino acid sequence set forth in SEQ ID NO:8, wherein the variation consists of a T→S substitution at position 93 of SEQ ID NO:5; (vii) the VH comprises: a VH CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:2; a VH CDR2 consisting of a variant of the amino acid sequence set forth in SEQ ID NO:3, wherein the variant consists of a T→S substitution at position 55 of SEQ ID NO:35; and a VH CDR3 consisting of a variant of the amino acid sequence set forth in SEQ ID NO:4, wherein the variant consists of a Y→A substitution at position 104 of SEQ ID NO:35, or consists of an A→H substitution at position 106 of SEQ ID NO:35; and the VL comprises: a VL CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:6; a VL CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:7; and a VL CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:8; Wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to: Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen, Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen, Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen, and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc] [VIII]'s Double Tentacles Y / Le B antigen, and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A Antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV].

13. The isolated antibody or antigen-binding fragment thereof of claim 12, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

5.

14. The isolated antibody or antigen-binding fragment thereof of claim 12, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 92% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 92% identical to the amino acid sequence shown in SEQ ID NO:

5.

15. The isolated antibody or antigen-binding fragment thereof of claim 12, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

5.

16. The isolated antibody or antigen-binding fragment thereof of claim 12, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO:

5.

17. An isolated antibody or antigen-binding fragment thereof as described in claim 12, wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:35, and the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:

5.

18. The isolated antibody or antigen-binding fragment thereof of any one of claims 12-17, wherein the antibody is monoclonal.

19. The isolated antibody or antigen-binding fragment thereof of claim 18, wherein the antibody is a humanized antibody.

20. The isolated antibody or antigen-binding fragment thereof according to any one of claims 12 to 17, which is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv (ScFv) antibody and a diabody.

21. The isolated antibody or antigen-binding fragment thereof of claim 18, which is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv (ScFv) antibody, and a diabody.

22. The isolated antibody or antigen-binding fragment thereof of claim 19, which is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv (ScFv) antibody, and a diabody.

23. An isolated antibody or antigen-binding fragment thereof, comprising: an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 35, and the immunoglobulin light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 5, wherein the antibody or antigen-binding fragment thereof can specifically bind to: Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen, Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen, Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen, and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc] [VIII]'s Double Tentacles Y / Le B antigen, and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A Antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV].

24. The isolated antibody or antigen-binding fragment thereof of claim 23, wherein the antibody is monoclonal.

25. The isolated antibody or antigen-binding fragment thereof of claim 24, wherein the antibody is a humanized antibody.

26. The isolated antibody or antigen-binding fragment thereof of claim 24, which is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single-chain Fv (ScFv) antibody, and a diabody.

27. An isolated antibody comprising an immunoglobulin heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 10 and an immunoglobulin light chain consisting of the amino acid sequence shown in SEQ ID NO: 11, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to: Biantennary Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen, Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen, Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen, and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc] [VIII]'s Double Tentacles Y / Le B antigen, and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A Antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV].

28. The isolated antibody of claim 27 which is monoclonal.

29. The isolated antibody of claim 28, wherein the antibody is a humanized antibody.

30. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-26, or the antibody of any one of claims 27-29.

31. The isolated polynucleotide of claim 30, wherein the polynucleotide is codon optimized for expression in a host cell.

32. A recombinant vector comprising the polynucleotide of claim 30 or claim 31.

33. The recombinant vector of claim 32, comprising an expression control sequence operably linked to a polynucleotide encoding an antibody or antigen-binding fragment thereof.

34. The recombinant vector of claim 33, which is an expression vector, wherein the expression control sequence comprises a promoter.

35. A host cell comprising the recombinant vector of claim 32.

36. A host cell comprising the recombinant vector of claim 34.

37. A method for producing an antibody or antigen-binding fragment thereof that is capable of specifically binding to: Biantenna Le comprising structure [I] or structure [II] B / Le B antigen, Biantennary Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen, Biantennary Le containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] B / Le Y antigen, and containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc] [VIII]'s Double Tentacles Y / Le B antigen, and wherein the antibody or antigen-binding fragment thereof does not specifically bind to: a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX] X antigen, or a biantennary Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] X antigen, or a monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A antigen, or a monoantennary type 2 H antigen comprising Fucα1-2Galβ1-4GlcNAc[XII], or a biantennary type 2 H antigen comprising (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type 1 H antigen comprising Fucα1-2Galβ1-3GlcNAc[XIV], The method comprises: Cultivating the host cell of claim 35 under conditions and for a time sufficient for the host cell to express a polynucleotide encoding the antibody or antigen-binding fragment thereof, thereby obtaining a culture containing the antibody or antigen-binding fragment thereof; and The antibody or antigen-binding fragment thereof is recovered from the culture.

38. An antibody conjugate comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1-26 or the antibody of any one of claims 27-29, and a payload molecule linked thereto.

39. The antibody conjugate of claim 38, wherein the payload molecule is covalently linked to the antibody or antigen-binding fragment thereof via a linker.

40. The antibody conjugate of claim 39, wherein the linker is a cleavable linker. The antibody conjugate of claim 39 , wherein the linker is a non-cleavable linker.

42. The antibody conjugate of claim 40, wherein the cleavable linker is a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker.

43. The antibody conjugate of claim 42, wherein the cleavable linker is a protease-sensitive linker comprising a valine-citrulline dipeptide.

44. The antibody conjugate of any one of claims 40-43, wherein the linker comprises a maleimide group.

45. The antibody conjugate of claim 44, wherein the antibody or antigen-binding fragment thereof comprises a reduced disulfide bridge in the hinge region, and the reduced disulfide bridge is coupled to a maleimide group.

46. ​​The antibody conjugate of any one of claims 40-43, wherein the linker further comprises a self-immolative group. The antibody conjugate of claim 46 , wherein the self-immolative group is p-aminobenzyl alcohol (PABC).

48. The antibody conjugate of any one of claims 38-43, wherein the payload molecule is selected from a therapeutic agent and a detectable indicator.

49. The antibody conjugate of claim 48, wherein the payload molecule is a detectable indicator.

50. The antibody conjugate of claim 49, wherein the detectable indicator is selected from the group consisting of a radionuclide, a dye, a radiometal, a fluorescent moiety, an MRI contrast agent, a microbubble, a carbon nanotube, a gold particle, fluorodeoxyglucose, an enzyme, a chromophore, and a radiopaque marker.

51. The antibody conjugate of claim 50, wherein the detectable indicator is a radionuclide selected from: 68 Ga, 64 Cu, 86 Y. 89 Zr, 124 I. 99m Tc, 123 I. 111 In, 177 Lu, 131 I. 76 Br, 78 Zr, 18 F and 124 T.

52. The antibody conjugate of claim 50 or claim 51, further comprising a radionuclide chelator selected from the group consisting of maleimide-labeled DOTA, N-hydroxysuccinimide-DOTA, and deferoxamine (DFO).

53. A pharmaceutical composition comprising: the isolated antibody or antigen-binding fragment thereof of any one of claims 1-26, the isolated antibody of any one of claims 27-29, or the antibody conjugate of any one of claims 38-52, and a pharmaceutical carrier.

54. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-26, the antibody of any one of claims 27-29, the polynucleotide of claim 30 or 31, the recombinant vector of any one of claims 32-34, the host cell of claim 35 or 36, the antibody conjugate of any one of claims 38-52, or the pharmaceutical composition of claim 53 in the preparation of a medicament for treating, diagnosing or detecting gastric cancer or colon cancer in a subject, wherein the gastric cancer or colon cancer expresses a gene selected from the group consisting of biantennary Le B / Le B , Double Tentacles Y / Le Y , Double Tentacles B / Le Y and Double-Tentacle Le Y / Le B One or more biantennary Lewis antigens.

55. The use according to claim 54, wherein the gastric cancer or colon cancer is selected from gastric adenocarcinoma and colon adenocarcinoma.

56. The method of claim 54 or 55, wherein the drug is administered by a route selected from the group consisting of intravenous, parenteral, intragastric, intrapleural, intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, oral, topical, transdermal, intracisternal, intrathecal, intranasal, and intramuscular.

57. The use of claim 54 or 55, wherein the subject is receiving or has previously received: (a) Immunosuppressive therapy; (b) stimulatory immune checkpoint molecules; (c) radiotherapy; (d) chemotherapy; (e) cellular immunotherapy; or (f) Any combination of (a)-(e).

58. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-26, the antibody of any one of claims 27-29, the polynucleotide of claim 30 or 31, the recombinant vector of any one of claims 32-34, the host cell of claim 35 or 36, the antibody conjugate of any one of claims 38-52, or the pharmaceutical composition of claim 53 in the preparation of a medicament for treating gastric cancer or colon cancer in a subject, wherein the gastric cancer or colon cancer expresses a gene selected from the group consisting of biantennary Le B / Le B , Double Tentacles Y / Le Y , Double Tentacles B / Le Y and Double-Tentacle Le Y / Le B One or more biantennary Lewis antigens.

59. The use according to claim 58, wherein the gastric cancer or colon cancer is selected from gastric adenocarcinoma and colon adenocarcinoma.

60. The use of claim 58 or 59, wherein the medicament is administered by a route selected from the group consisting of intravenous, parenteral, intragastric, intrapleural, intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, oral, topical, transdermal, intracisternal, intrathecal, intranasal, and intramuscular.

61. The use of claim 58 or 59, wherein the subject is receiving or has previously received: (a) Immunosuppressive therapy; (b) stimulatory immune checkpoint molecules; (c) radiotherapy; (d) chemotherapy; (e) cellular immunotherapy; or (f) Any combination of (a)-(e).

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