Therapeutic antibodies that bind to the bivalent antenna-type Lewis B and Lewis Y antigens

Abstract

To provide therapeutic antibodies binding to biantennary Lewis B and Lewis Y antigens.SOLUTION: Disclosed herein are humanized antibodies, antigen-binding fragments thereof, and antibody conjugates, that are capable of specifically binding to certain biantennary Lewis antigens, which antigens are expressed in a variety of cancers. The presently disclosed antibodies are useful to target antigen-expressing cells for treatment or detection of disease, including various cancers. Also provided are polynucleotides, vectors, and host cells for producing the disclosed antibodies and antigen-binding fragments thereof. Pharmaceutical compositions, methods of treatment and detection, and uses of the antibodies, antigen-binding fragments, antibody conjugates, and compositions are also provided.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 678,890, filed on May 31, 2018, under 35 U.S.C. § 119(e), which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding the Sequence Listing The sequence listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference. The name of the text file containing the sequence listing is 400100_401WO_SEQUENCE_LISTING.txt. The text file is 43.3 KB, was created on May 30, 2019, and was electronically submitted via EFS - Web.

[0003] Embodiments of the present disclosure generally relate to antibodies and antigen - binding fragments thereof that are capable of specifically binding to certain Lewis antigens, as well as methods for their production and use. 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 of the bivalent Le B / Le B 、Le Y / Le Y 、Le B / Le Y 、and Le Y / Le B antigens and are useful in treating or detecting diseases characterized by the expression of such antigens, such as cancer.

Background Art

[0004] Immunotherapy is an emerging modality for treating a variety of diseases, including various cancers. For example, the clinical efficacy of monoclonal antibodies (mAbs) with antitumor activity has been demonstrated since the late 1990s (see, e.g., Topalian et al., J. Clin. Oncol. 29(36):4828-4836 (2011)), and several blockbuster cancer drugs are mAbs (e.g., rituximab, trastuzumab, and bevacizumab).

[0005] Antibody-based therapies can specifically target cells expressing an antigen, such as tumor cells, and can result in cytotoxic activity through several means, such as by inducing antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and T cell-mediated cytotoxicity. Other approaches include using an antibody as a carrier vehicle to selectively deliver a cytotoxic or anti-proliferative agent to kill target cells and / or inhibit the growth and metastatic spread of such cells.

[0006] Suitable criteria for monoclonal antibody therapy include, for example, the ability of the antibody to specifically recognize and bind to the desired antigen, rather than binding indiscriminately to one or more other possible epitopes, such as proteins or glycans, expressed on healthy cells. The antibody should also not be immunogenic to the patient. In this regard, antibodies specific for human disease antigens are often generated in non-human hosts, such as mice and rabbits, and may have amino acid sequences and / or carbohydrate motifs of the host species that can be immunogenic in human patients. Thus, antibodies preferred for use in therapy lack immunogenic properties or, if present, are minimal. The characteristics of the target antigen must also be considered. The antigen should preferably be selectively expressed or highly overexpressed in a particular disease state (e.g., cancer) so that the antibody-mediated therapy does not have unwanted and harmful "on-target, off-tumor" effects on healthy tissue.

[0007] Aberrant glycosylation (e.g., overexpression or misexpression of glycoproteins and glycolipids) is a feature common to many cancers (see, e.g., Blanas et al., Front. Oncol. 8:39 (2018). Without wishing to be bound by theory, aberrant glycosylation can affect several cellular processes that cause cancer progression and metastasis (e.g., cell proliferation and metabolism, angiogenesis, cell-matrix interactions, and cell-cell adhesion). Exemplary carbohydrate antigens aberrantly expressed in cancer include type I and type II Lewis antigens, which are fucosylated carbohydrate epitopes at the terminus (non-reducing end) of the Lewis antigen system. Type I and type II Lewis antigens include the structurally related H1, H2, and Lewis A, B, X, and Y antigens, all of which share three monosaccharide units: (reducing) terminal N-acetylglucosamine (GlcNac); galactose (Gal); and fucose (Fuc). Type I Lewis antigens differ from type II in the type of glycosidic bond (Galβ1→3GlcNac: type I; Galβ1→4GlcNac: type II) in their lactosamine core chain and exist in diverse 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., gastrointestinal and genital epithelia), but are overexpressed on the cell surface of several solid cancers, including 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 lymphoma).

[0008] Some therapies targeting Lewis antigens for treating cancer are known, such as the discontinued Seattle Genetics / Bristol Meyers Squibb antibody-drug conjugate "cBR96-Dox," which used the BR96 monoclonal antibody as a carrier molecule to Le YDoxorubicin is delivered to tumor cells expressing an antigen (Hellstrom et al., Cancer Res. 50(7):2183 - 2190 (1990)). Clearly, there is still a need for additional therapies in the art to treat cancers such as cancers expressing Lewis antigen as a tumor - associated antigen or a tumor - specific antigen. The embodiments disclosed herein address this need and provide other related advantages.

Prior Art Documents

Non - Patent Documents

[0009]

Non - Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0010] According to certain embodiments disclosed herein, an isolated antibody is provided that comprises an immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 10 and an immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 11.

[0011] In certain embodiments, an isolated antibody or an antigen - binding fragment thereof that comprises an immunoglobulin heavy - chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 35; and an immunoglobulin light - chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5, wherein the antibody or the antigen - binding fragment thereof binds to a biantennary - type Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1 - 2Galβ1 - 3(Fucα1 - 4)GlcNAc]2[II] B / Le B antigen, a biantennary - type Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1 - 2Galβ1 - 4(Fucα1 - 3)GlcNAc]2[IV] Y / Le Y antigen, A bivalent Le antigen 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 an antibody or antigen-binding fragment thereof that can specifically bind to a bivalent Le antigen containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B antigen, and does not specifically bind to a monovalent Le antigen containing Galβ1→4(Fucα1→3)GlcNAc[IX], a bivalent Le antigen containing [Galβ1→4(Fucα1→3)GlcNAc]2[X], a monovalent Le antigen containing Galβ1-3(Fucα1-4)GlcNAc[XI], a monovalent H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], a bivalent H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monovalent H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV]. An isolated antibody or antigen-binding fragment thereof is provided. A monovalent Le antigen containing Galβ1→4(Fucα1→3)GlcNAc[IX], X a bivalent Le antigen containing [Galβ1→4(Fucα1→3)GlcNAc]2[X], X a monovalent Le antigen containing Galβ1-3(Fucα1-4)GlcNAc[XI], A a monovalent H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], a bivalent H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monovalent H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV].

[0012] In certain embodiments, an isolated antibody or antigen-binding fragment thereof comprising: (a) an immunoglobulin heavy chain variable region comprising a heavy chain complementarity determining region 1 (VH CDR1) having the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain complementarity determining region 2 (VH CDR2) having the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain complementarity determining region 3 (VH CDR3) having the amino acid sequence set forth in SEQ ID NO: 4; and (b) an immunoglobulin light chain variable region comprising a light chain complementarity determining region 1 (VL CDR1) having the amino acid sequence set forth in SEQ ID NO: 6, a light chain complementarity determining region 2 (VL CDR2) having the amino acid sequence set forth in SEQ ID NO: 7, and a light chain complementarity determining region 3 (VL CDR3) having the amino acid sequence set forth in SEQ ID NO: 8, wherein the antibody or antigen-binding fragment thereof bivalent antennal type Le containing Fuc4(Galβ1→3GlcNAc)2 [I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2 [II] B / Le B to an antigen bivalent antennal type Le containing Fuc4(Galβ1→4GlcNAc)2 [III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2 [IV] Y / Le Y to an antigen bivalent antennal type 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 to an antigen and bivalent antennal type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)] [VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc] [VIII] Y / Le B to an antigen is capable of specifically binding, An antibody or an antigen-binding fragment thereof specifically binds to a monoantennary type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX], x but does not specifically bind to a biantennary type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X], x a monoantennary type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI], A a monoantennary type H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], a biantennary type H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantennary type H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV]. There is provided an isolated antibody or an antigen-binding fragment thereof.

[0013] In certain embodiments described herein, the isolated antibody can be a monoclonal antibody. In certain embodiments, the isolated antibody or an antigen-binding fragment thereof is a humanized antibody. In certain embodiments, the isolated antibody or an antigen-binding fragment thereof is selected from Fab fragments, F(ab’)2 fragments, Fv fragments, single-chain Fv (scFv) antibodies, and diabodies.

[0014] The isolated antibody or an antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region having an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 35; and an immunoglobulin light chain variable region having an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 5, and the antibody or an antigen-binding fragment thereof does not specifically bind to a biantennary type Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II], B / Le B antigen, A biantenna-type Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen, A biantenna-type 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 a biantenna-type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B antigen is capable of specifically binding, An antibody or an antigen-binding fragment thereof also specifically binds to a monoantenna-type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX] x antigen, and a biantenna-type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] x antigen, and a monoantenna-type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A Embodiments are also provided herein that do not specifically bind to a monoantenna-type H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], a biantenna-type H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monoantenna-type H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV].

[0015] In certain embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising: (a) a variable heavy chain region of an immunoglobulin comprising a heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 2; a heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 4; and (b) a variable light chain region of an immunoglobulin comprising a light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 6; a light chain complementarity determining region 2 (VL CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 7; and a light chain complementarity determining region 3 (VL CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0016] In certain further embodiments, the following (i), (ii), (iii), (iv), (v), or (vi): (i) a VH CDR1 comprising a variant of the amino acid sequence set forth in SEQ ID NO: 2, wherein the mutation 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 set forth in SEQ ID NO: 4, wherein the mutation 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 set forth in SEQ ID NO: 4, wherein the mutation 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 set forth in SEQ ID NO: 6, wherein the mutation 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 set forth in SEQ ID NO: 7, 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 set forth in SEQ ID NO: 8, wherein the mutation consists of a T→S substitution at position 93 according to Kabat numbering, or any combination thereof, an isolated antibody or antigen-binding fragment thereof according to the present disclosure is provided.

[0017] In certain further embodiments, an immunoglobulin heavy chain variable region comprising a complementarity determining region (CDR) described herein (i.e., a CDR having at least 90% identity to each of SEQ ID NOs: 2, 3, 4, 6, 7, and 8, including CDR variants having the amino acid substitutions described herein), and comprising or consisting of an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth 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 percent identity to the amino acid sequence set forth in SEQ ID NO: 5 are provided, an isolated antibody or antigen-binding fragment thereof.

[0018] 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 disclosure, as disclosed herein, when compared to a "BBC" antibody comprising a VL domain having the amino acid sequence set forth in SEQ ID NO: 27 and a VH domain having the amino acid sequence set forth in SEQ ID NO: 28, may exhibit reduced (e.g., statistically significantly reduced as determined using accepted methods in the art) binding to monoantennary Lewis B or monoantennary Lewis Y antigen (including, in certain embodiments, no binding). Monovalent Lewis B is a blood group antigen expressed in normal human tissues, and thus the antibodies, antigen-binding fragments, and antibody-drug conjugates disclosed herein have improved specificity for cancer antigens and reduced binding to monoantennary Lewis B antigen expressed in normal human tissues compared to the BBC antibody. In certain embodiments, the antibodies, antigen-binding fragments, or antibody-drug conjugates disclosed herein have an increase in binding to an antigen expressed on the cell surface by any one or more of Formulas [I]-[VIII] herein that is 1-fold, 2-fold, 3-fold, 5-fold, 6-fold, 7-fold, 8-fold, or greater compared to binding to the monoantennary Lewis B antigen.

[0019] In certain embodiments, an antibody, antigen-binding fragment, or antibody-drug conjugate binds to the monoantennary Lewis B antigen with an affinity that is 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, 1 / 3, or 1 / 2 of the affinity of BR96, as measured, for example, in an ELISA binding assay.

[0020] 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, binds to a monoantennary Le recognized by the antibody BR96 Y binds to an antigen different from the antigen, and, without being bound by theory, may enable safer and more specific targeting of cancer cells in therapeutic applications as compared to BR96. 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, binds to cancer cells expressing the antigen, efficiently and stably internalizes into the lysosomes of such cells, and is safely tolerated at therapeutic doses in non-human primate models.

[0021] In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the polynucleotide is codon-optimized for expression in a host cell. 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 linked to the polynucleotide encoding the antibody or antigen-binding fragment. In specific embodiments, the recombinant vector is an expression vector in which the expression control sequence comprises a promoter.

[0022] In another embodiment, a host cell comprising the recombinant and / or expression vector of the present disclosure is provided. In certain other embodiments, related methods of producing an antibody or an antigen-binding fragment thereof disclosed herein, wherein the antibody or antigen-binding fragment thereof is a biantenna-type Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen, a biantenna-type Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen, a biantenna-type 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 a biantenna-type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B antigen is capable of specifically binding to, the antibody or antigen-binding fragment thereof is also a monoantenna-type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX] x antigen, and also a biantenna-type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] x antigen, and also a monoantenna-type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] AIt does not specifically bind to an antigen, nor to a monoantennary type 2 H antigen containing Fucα1-2Galβ1-4GlcNAc[XII], nor to a diantennary type 2 H antigen containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], nor to a monoantennary type 1 H antigen containing Fucα1-2Galβ1-3GlcNAc[XIV]. A method is provided that comprises culturing a host cell described herein under conditions and for a time sufficient for expression by the host cell of a polynucleotide encoding an antibody or an antigen-binding fragment thereof, thereby obtaining a culture containing the antibody or the antigen-binding fragment thereof; and recovering the antibody or the antigen-binding fragment thereof from the culture.

[0023] In another embodiment, an antibody conjugate is provided that comprises an isolated antibody of the present disclosure or an antigen-binding fragment thereof; and a payload molecule conjugated thereto. In certain embodiments, the payload molecule is covalently linked to the antibody or an antigen-binding fragment thereof by 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 that includes a valine-citrulline peptide. In some embodiments, the linker includes a maleimide group. In certain embodiments, the antibody or an antigen-binding fragment thereof disclosed herein includes a reduced disulfide bridge in the hinge region, and the reduced disulfide bridge is coupled to the maleimide group. Embodiments are also provided herein where the linker further includes a self-immolative group, such as para-aminobenzyl alcohol (PABC), etc. In certain embodiments, the antibody conjugate includes an antibody or antigen-binding fragment disclosed herein, and a payload molecule selected from a therapeutic agent and a detectable indicator. In certain embodiments, the payload molecule is a tubulin-targeting anti-mitotic agent, a peptide-based toxin, a pyrrolobenzodiazepine (PBD) dimer, an antibiotic, a pyrimidine synthesis inhibitor, an antimetabolite, a DNA alkylating agent, and a topoisomerase inhibitor. In certain embodiments, the payload molecule is selected from mayntansinoid, auristatin, doxorubicin, calicheamicin, PBD dimer, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF). In certain other embodiments, the payload molecule is a detectable indicator. In certain further embodiments, the detectable indicator is selected from a radionuclide, a dye, a radioactive metal, a fluorescent moiety, an MRI contrast agent, a microbubble, a carbon nanotube, a gold particle, fluorodeoxyglucose, an enzyme, a chromophore, and a radiopaque marker.In certain embodiments, the detectable indicator is... 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 a radionuclide selected from T. In certain embodiments, the antibody conjugate comprises a chelator of a radionuclide selected from DOTA labeled with maleimide, N-hydroxysuccinimide-DOTA, and desferrioxamine (DFO).

[0024] Pharmaceutical compositions are also provided herein, and in some embodiments, comprise an isolated antibody, antigen-binding fragment, or antibody conjugate disclosed herein; and a pharmaceutical carrier.

[0025] In certain embodiments, the present disclosure also provides methods of treatment or detection that include the use of the antibodies, antigen-binding fragments, and / or antibody conjugates disclosed herein. In some embodiments, methods of treating or detecting cancer are provided that include administering a pharmaceutical composition disclosed herein to a subject in need thereof. In certain embodiments, the subject has or is suspected of having cancer selected from gastric cancer, colorectal cancer, breast cancer, lung cancer, lymphatic cancer, 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 carcinoma, colorectal adenocarcinoma, invasive ductal carcinoma of the breast, hepatocellular carcinoma, lung adenocarcinoma, squamous cell carcinoma, metastatic lymph node adenocarcinoma, mucinous ovarian adenocarcinoma, pancreatic ductal adenocarcinoma, papillary adenocarcinoma of the pancreas, prostatic adenocarcinoma, and endometrial carcinoma. In certain embodiments, the composition disclosed herein (e.g., comprising an antibody or antigen-binding fragment thereof disclosed herein) is administered to the subject by a route selected from intravenous, parenteral, intragastric, intrathoracic, intralung, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, oral, topical, transdermal, intracapsular, intrathecal, intranasal, and intramuscular. In certain further embodiments of the methods disclosed herein for detecting or treating cancer, the subject has received 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).

[0026] These and other aspects and embodiments of the present disclosure will become apparent by reference to the following detailed description and the accompanying drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was individually incorporated. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0051] Brief Description of the Sequences SEQ ID NO: 1 is the amino acid sequence of the heavy chain variable (VH) domain of the antibody hBBC.10.1FQ:

Chemical Formula

[0052] SEQ ID NO: 2 is the amino acid sequence of the heavy chain complementarity determining region 1 (VH CDR1) of the antibodies IMH2 / BBC, hBBC.8, hBBC.9, hBBC.9.1, hBBC.10, hBBC.10.1, and hBBC.10.1FQ:

Chemical Formula

[0053] SEQ ID NO: 3 is the amino acid sequence of the VH CDR2 of the antibodies hBBC.9.1, hBBC.10.1, and hBBC.10.1FQ:

Chemical Formula

[0054] SEQ ID NO: 4 is the amino acid sequence of the VH CDR3 of the antibodies hBBC.10.1 and hBBC.10.1FQ:

Chemical Formula

[0055] SEQ ID NO: 5 is the amino acid sequence of the light chain variable (VL) domain of antibodies hBBC.10, hBBC.10.1, and hBBC.10.1FQ:

Chemical formula

[0056] SEQ ID NO: 6 is the amino acid sequence of the light chain complementarity determining region 1 (VL CDR1) of antibodies IMH2 / BBC, hBBC.8, hBBC.9, hBBC.9.1, hBBC.10, hBBC.10.1, and hBBC.10.1FQ:

Chemical formula

[0057] SEQ ID NO: 7 is the amino acid sequence of the VL CDR2 of antibodies IMH2 / BBC, hBBC.8, hBBC.9, hBBC.9.1, hBBC.10, hBBC.10.1, and hBBC.10.1FQ:

Chemical formula

[0058] SEQ ID NO: 8 is the amino acid sequence of the VL CDR3 of antibodies IMH2 / BBC, hBBC.8, hBBC.9, hBBC.9.1, hBBC.10, hBBC.10.1, and hBBC.10.1FQ:

Chemical formula

[0059] SEQ ID NO: 9 is the amino acid sequence of the single chain variable fragment (scFv) derived from antibody hBBC.10.1 in the [VL-VH] direction:

Chemical formula

Chem.

[0060] SEQ ID NO: 10 is the amino acid sequence of the full-length heavy chain (HC) of antibody hBBC.10.1:

Chem.

[0061] SEQ ID NO: 11 is the amino acid sequence of the full-length light chain (LC) of antibodies hBBC.10, hBBC.10.1, and hBBC.10.1FQ:

Chem.

[0062] SEQ ID NO: 12 is the nucleotide sequence encoding the heavy chain variable (VH) domain of antibody hBBC.10.1:

Chem.

[0063] SEQ ID NO: 13 is the nucleotide sequence encoding the heavy chain complementarity determining region 1 (VH CDR1) of antibodies IMH2 / BBC, hBBC.8, hBBC.9, hBBC.9.1, hBBC.10, hBBC.10.1, and hBBC.10.1FQ:

Chem.

[0064] SEQ ID NO: 14 is the nucleotide sequence encoding the VH CDR2 of antibodies hBBC.9.1, hBBC.10, and hBBC.10.1FQ:

Chem.

[0065] SEQ ID NO: 15 is the nucleotide sequence encoding the VH CDR3 of antibody hBBC.10.1 and antibody hBBC.10.1FQ:

Chem.

[0066] 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:

Chem.

[0067] SEQ ID NO: 17 is the nucleotide sequence encoding the light chain complementarity 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:

Chem.

[0068] 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:

Chem.

[0069] 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:

Chem.

[0070] Sequence number 20 is a nucleotide sequence encoding a single-chain variable fragment (scFv) derived from antibody hBBC.10.1 in the [VL-(L)-VH] direction:

Chemical formula

Chemical formula

[0071] Sequence number 21 is a nucleotide sequence encoding the full-length heavy chain (HC) of antibody hBBC.10.1:

Chemical formula

[0072] Sequence number 22 is a nucleotide sequence encoding the full-length light chain (LC) of antibody hBBC.10.1 and antibody hBBC.10.1FQ:

Chemical formula

Chemical formula

[0073] Sequence number 23 is an exemplary spacer amino acid sequence

Chemical formula

[0074] Sequence number 24 is an exemplary spacer amino acid sequence

Chemical formula

Chemical formula

[0075] Sequence number 26 is a flexible polylinker amino acid sequence

Chemical formula

[0076] Sequence number 27 is the amino acid sequence of the VL domain of antibody IMH2 / BBC:

Chemical formula

[0077] Sequence number 28 is the amino acid sequence of the VH domain of antibody IMH2 / BBC:

Chemical formula

[0078] Sequence number 29 is the amino acid sequence of the VL domain of human acceptor framework AAS01771.1:

Chemical formula

[0079] Sequence number 30 is the amino acid sequence of the VH domain of human acceptor framework CAD89404.1:

Chemical formula

[0080] Sequence number 31 is the amino acid sequence of the VL domain of antibody hBBC.8:

Chemical formula

[0081] Sequence number 32 is the amino acid sequence of the VH domain of antibody hBBC.8, antibody hBBC.9, and antibody hBBC.10:

Chem.

[0082] Sequence number 33 is the amino acid sequence of the VL domain of antibody hBBC.9 and antibody hBBC.9.1:

Chem.

[0083] Sequence number 34 is the amino acid sequence of the VH domain of antibody hBBC.9.1:

Chem.

[0084] Sequence number 35 is the amino acid sequence of the VH domain of antibody hBBC.10.1:

Chem.

[0085] Sequence number 36 is the amino acid sequence of the single-chain variable fragment (scFv) derived from antibody hBBC.10.1 in the [VH-VL] direction:

Chem.

Chem.

[0086] Sequence number 37 is the nucleotide sequence encoding the single-chain variable fragment (scFv) derived from antibody hBBC.10.1 in the [VH-(L)-VL] direction: [Chemistry] In the formula, "x" is the array shown within the brackets [Chemistry] may be one, or 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repetitions thereof.

[0087] These and other arrays are provided in the accompanying sequence listing.

[0088] Detailed Description The present disclosure relates to humanized antibodies and antigen-binding fragments thereof that are capable of specifically binding to certain Lewis antigens expressed in various cancers. More specifically, as first described herein and presented in more detail below, the chimeric, humanized antibodies, unexpectedly, bind with excellent specificity to certain bivalent-type Lewis B / Y antigens expressed on cancer cells and have robust antitumor activity in vivo. In addition, the antibodies disclosed herein, advantageously, have surprisingly reduced (e.g., decreased in a statistically significant manner) binding to the monovalent-type Lewis B antigen (which is expressed in healthy tissues) when compared to the BBC antibody having a VL domain with the amino acid sequence set forth in SEQ ID NO: 27 and a VH domain with the amino acid sequence set forth in SEQ ID NO: 28. The antibodies disclosed herein bind to an antigen different from the monovalent-type Le Y antigen recognized by the antibody BR96, and without wishing to be limited by theory, the present antibodies may enable safer and more specific targeting of cancer cells in therapeutic applications. Furthermore, the antibodies and antigen-binding fragments of the present disclosure also bind to cancer cells expressing the antigen, efficiently and stably internalize into the lysosomes of such cells, and are safely tolerated at therapeutic doses in non-human primate models.

[0089] According to certain preferred embodiments, and without being limited to theory, the beneficial uses of the antibodies and antigen-binding fragments thereof disclosed herein relate to methods of diagnosing and / or treating cancer, such as various gastric cancers, ovarian cancers, lung cancers, prostate cancers, pancreatic cancers, and other cancers. These and related embodiments are disclosed in more detail herein.

[0090] Polypeptides and Proteins 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 terms do not exclude modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The term "polypeptide" or "protein" may mean one or more chains of amino acids, wherein each chain is covalently linked by peptide bonds, and the polypeptide or protein may be a native protein, i.e., one that occurs naturally and in particular has the sequence of a protein produced by a non-recombinant cell, or a genetically engineered or recombinant cell, and may include multiple chains non-covalently and / or covalently linked to each other by peptide bonds, a molecule having the amino acid sequence of a native protein, or a molecule having one or more amino acid deletions, additions, and / or substitutions from the native sequence. Thus, a "polypeptide" or "protein" may include one (referred to as a "monomer") or more (referred to as a "multimer") amino acid chains. The terms "peptide," "polypeptide," and "protein" specifically encompass the antibodies and antigen-binding fragments of the present disclosure, or sequences having one or more amino acid deletions, additions, and / or substitutions from the antibody or its antigen-binding fragment.

[0091] "Amino acid", as used herein, refers to naturally occurring 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 include those encoded by the genetic code and those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., a compound having an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium). Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds having a structure different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0092] "Mutation", as used herein, refers to a change in the sequence of a nucleic acid molecule or a polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations can result in several different types of changes in the sequence, including substitutions, insertions, or deletions of nucleotides or amino acids.

[0093] 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. "Contiguous amino acids," as used herein, refers to covalently linked amino acids corresponding to an uninterrupted linear portion of the disclosed amino acid sequence. In certain embodiments, the polypeptide fragment may comprise an amino acid chain that is at least 5 to about 500 amino acids in length. In certain embodiments, the fragment is understood to be 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.

[0094] As used herein, the terms "isolated protein" and "isolated polypeptide" mean that the protein or polypeptide of interest: (1) does not contain at least some of the other proteins or polypeptides that are typically found together in nature; (2) does not substantially contain other proteins or polypeptides from the same source, e.g., from the same species; (3) is expressed by cells from different species; (4) is separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials that associate in nature; (5) is not associated (by covalent or non-covalent interactions) with portions of proteins or polypeptides that can associate with the "isolated protein" or "isolated polypeptide" in nature; (6) is operably associated (by covalent or non-covalent interactions) with a polypeptide that does not associate 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, or may be of synthetic origin according to any of several well-known chemical properties related to peptide and protein synthesis, or any combination thereof. In certain embodiments, the isolated protein or polypeptide is substantially free of proteins or polypeptides or other contaminants found in its natural environment that may interfere with its use (therapeutic, diagnostic, prophylactic, investigational, or otherwise).

[0095] The polypeptide may include a signal (or leader) sequence at the N-terminus of the protein that directs the movement of the protein co-translationally or post-translationally. The polypeptide may also be fused in-frame or conjugated to 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, a "fusion protein" or "fusion polypeptide" refers to a protein having at least two distinct domains in a single chain, where the domains are not naturally found together in a protein. The polynucleotide encoding the fusion protein can be constructed using PCR, recombinantly produced, etc., or such fusion proteins can be synthesized. The fusion protein may further contain other components, such as tags, linkers, or transduction markers. The fusion domain polypeptide may be joined to the polypeptide at the N-terminus and / or C-terminus, and non-limiting examples include sequences derived from immunoglobulins, such as Ig constant region sequences or portions thereof, affinity tags, such as His tags (e.g., hexahistidine or other polyhistidine), FLAG™ or myc or other peptide affinity tags, detectable polypeptide moieties, such as green fluorescent protein (GFP) or variants thereof (e.g., yellow fluorescent protein (YFP), blue fluorescent protein (BFP), other aequorins or derivatives thereof, etc.), or other detectable polypeptide fusion domains, enzymes or portions thereof, such as glutathione-S-transferase (GST) or other known enzymes for detection and / or reporter fusion domains. Additional detectable moieties are discussed herein.

[0096] Cysteine-containing peptides can be used as fusion peptides that can be conjugated to the N and / or C termini of a polypeptide, such as an antibody or an antigen-binding fragment thereof of the present disclosure, so as to allow for the immediate assembly of such polypeptides into disulfide-bridged dimers, trimers, tetramers or higher-order multimers according to established methods. For example, fusion polypeptides containing sequences derived from members of the immunoglobulin gene superfamily that contain cysteine residues capable of forming inter-chain disulfide bridges are also well-known for other strategies for engineering S-S 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). Also contemplated is an alternative approach for grafting peptide sequences that promote the assembly of multimers as fusion domains onto desired polypeptides such as the antibodies and antigen-binding fragments described herein (e.g., Fan et al., 2008 FASEB J. 22:3795).

[0097] Polypeptide modifications may be carried out biosynthetically and / or chemically according to a variety of well-known methods, and may also include conjugation to carrier proteins (e.g., keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA) or other molecules), and covalent or non-covalent immobilization to solid supports. Chemical or biosynthetic conjugation to a carrier is contemplated, according to certain embodiments, for the generation of multivalent conjugates against the antibodies or antigen-binding fragments thereof described herein.

[0098] Detectable indicator moieties (sometimes referred to as reporter moieties), such as detectable labeling using fluorophores (e.g., FITC, TRITC, Texas Red, etc.) are also contemplated. Examples of a wide range of detectable indicators (including colorimetric indicators) that can be selected for specific purposes are described in Haugland, 2005 The Handbook: A Guide to Fluorescent Probes and Labeling Technologies - Tenth Ed., Invitrogen Corp. / Molecular Probes (trademark), Eugene, OR; Mohr, 1999 J. Mater. Chem., 9: 2259 - 2264; Suslick et al., 2004 Tetrahedron 60:11133 - 11138; and U.S. Patent 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.) Detectable indicators can be fluorescent indicators, luminescent indicators, phosphorescent indicators, radiometric indicators, dyes, enzymes, substrates of enzymes, energy transfer molecules, or affinity labels.

[0099] 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. For such affinity approaches, reagents for immunometric assays, such as suitably labeled antibodies or lectins, may be prepared, e.g., with radionuclides (e.g., 76 Br, 78 Zr, 18In (Fl), those prepared as fluorophores, with affinity tags, labeled with biotin or a biotin mimetic sequence, or as antibody-enzyme conjugates (e.g., Weir, D.M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston; Scouten, W.H., 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-Tenth Ed., 2005 Invitrogen Corp. / Molecular Probes™, Eugene, OR; Scopes, R.K., Protein Purification: Principles and Practice, 1987, Springer-Verlag, NY; Hermanson, G.T. et al., Immobilized Affinity Ligand Techniques, 1992, Academic Press, Inc., NY; Luo et al., 1998 J. Biotechnol. 65:225 and references cited therein).

[0100] Peptide linker / spacer sequences can also be used, if desired, to separate the multiple polypeptide components at a distance sufficient for each polypeptide to fold reliably into its secondary and / or tertiary structure. Such peptide linker sequences can be incorporated into the fusion polypeptide using standard techniques well known in the art.

[0101] Certain peptide spacer sequences can be selected, for example, based on (1) the ability to assume a flexible, extended conformation; (2) the inability to assume a secondary structure capable of interacting with functional epitopes on the first and second polypeptides; and / or (3) the lack of residues having hydrophobicity or charge capable of reacting with functional epitopes of the polypeptide. In certain embodiments, the peptide spacer sequence contains, for example, Gly, Asn, and Ser residues. Other near-neutral amino acids, such as Thr and Ala, may also be included in the spacer sequence. Other amino acid sequences that can be usefully employed as spacers include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Patent No. 4,935,233, and U.S. Patent No. 4,751,180. Other exemplary and non-limiting examples of spacers 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. USA 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)).

[0102] In some embodiments, if the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric interference, a spacer sequence is not necessary. The two coding sequences may be fused directly without any spacer, or for example, by using a flexible polylinker composed of the pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 25) if present in a single repeat, or repeated 1 to 5 times or more, or more; see, for example, SEQ ID NO: 26. In certain exemplary and non-limiting embodiments, the peptide spacer can be between 1 and 5 amino acids, between 5 and 10 amino acids, between 5 and 25 amino acids, between 5 and 50 amino acids, between 10 and 25 amino acids, between 10 and 50 amino acids, between 10 and 100 amino acids, or any range of amino acids therebetween. In other exemplary embodiments, the peptide spacer comprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids in length.

[0103] 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. A "conservative substitution" refers to an amino acid substitution that does not significantly affect or alter a particular characteristic (e.g., binding activity such as specific binding activity) of a particular protein. Generally, a conservative substitution is a substitution in which the substituted amino acid residue is replaced with an amino acid residue having 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 classified into groups of conservative substitutions by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, the aliphatic classification can include Gly, Ala, Val, Leu, and Ile for substitution purposes. Other groups of conservative substitutions include sulfur-containing: Met and cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, non-polar 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 non-polar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0104] For example, it may be desirable to improve the binding affinity and / or other biological properties of an antibody or an antigen-binding fragment thereof. Amino acid sequence variants can be prepared, for example, by introducing appropriate nucleotide changes into the encoding polynucleotide or by peptide synthesis. Such modifications include, for example, deletions of residues within the amino acid sequence of the antibody or an antigen-binding fragment thereof, and / or insertions into it, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions can be made to reach the final antibody or antigen-binding fragment variant provided that the final construct has the desired characteristics (e.g., while retaining specific binding to the bivalent Lewis antigens described herein, does not detectably bind to the monovalent Le X or Le A or H antigen or shows a statistically significant decrease in binding thereto). Amino acid changes can also alter post-translational processes of the antibody or an antigen-binding fragment thereof, for example, by changing the number or location of glycosylation sites.

[0105] Determination of the three-dimensional structure of a representative antibody or an antigen-binding fragment thereof can be carried out via conventional techniques such that one or more amino acid substitutions, additions, deletions, or insertions with selected natural or unnatural amino acids can be substantially modeled for the purpose of determining whether the resulting structural variant retains the space-filling properties of the species disclosed herein. For example, 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. See also the references cited therein. For example, as additional non-limiting examples of computer algorithms that can be used in these and related embodiments for the rational design of the antibodies and antigen-binding fragments provided herein, VMD can be mentioned, which is a molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3D graphics and built-in scripting (see the website regarding the Theoretical and Computational Biophysics Group, University of Illinois at Urbana-Champagne at ks.uiuc.edu / Research / vmd / ).

[0106] Many other computer programs are known in the art and available to those skilled in the art, and these enable the determination of atomic dimensions (van der Waals radii) from space-filling models of energy-minimized conformations; searching for regions of high affinity for different chemical groups and thereby strengthening bonds, such as GRID, Monte Carlo searches for calculating 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) for evaluating force field calculations and analyses (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). Computer programs using various suitable computers are also commercially available, for example, from Schroedinger (Munich, Germany).

[0107] antibody Certain preferred embodiments of the present invention are bivalent antenna-type Le containing Fuc4(Galβ1→3GlcNAc)2 [I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2 [II] b / Le b antigen, bivalent antenna-type Le containing Fuc4(Galβ1→4GlcNAc)2 [III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2 [IV] Y / Le Y antigen, A bivalent antenna-type 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 a bivalent antenna-type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B antigen An antibody or an antigen-binding fragment thereof that specifically binds to, wherein the antibody or the antigen-binding fragment thereof does not contain a monovalent antenna-type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX], x nor to an antigen containing [Galβ1→4(Fucα1→3)GlcNAc]2[X], a bivalent antenna-type Le, x nor to an antigen containing Galβ1-3(Fucα1-4)GlcNAc[XI], a monovalent antenna-type Le, A nor to an antigen containing Fucα1-2Galβ1-4GlcNAc[XII], a monovalent antenna-type H antigen type 2, nor to an antigen containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], a bivalent antenna-type H antigen type 2, nor to an antigen containing Fucα1-2Galβ1-3GlcNAc[XIV], a monovalent antenna-type H antigen type 1, and in a certain further particularly preferred embodiment, does not specifically bind to certain other bivalent antenna-type or monovalent antenna-type Lewis antigens described herein, relating to an antibody or an antigen-binding fragment thereof.

[0108] 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, so long as they exhibit the desired biological activity, e.g., retain the ability to specifically bind to the bivalent Lewis antigens disclosed herein. The term "immunoglobulin" (Ig) is used herein synonymously with "antibody".

[0109] The basic antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by at least one (and typically one) covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (V H ) at the N-terminus, followed by three constant domains (C H ) for each of the α and γ chains and four C H domains for the μ and ε isotypes. Each L chain has a variable domain (V L ) at the N-terminus and a constant domain (C L ) at the other terminus. V L is aligned with V H , and C L is aligned with the first constant domain (C H 1) of the heavy chain. Certain amino acid residues are thought to form the interface between the light and heavy chain variable domains. The pairing of V H and V L together forms a single antigen-binding site.

[0110] L chains from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant domains (C L ). The constant domains of their heavy chains (CH ) According to the amino acid sequence, 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 named alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in sequence and function. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. It is understood that mammals encoding multiple Ig isotypes can undergo isotype class switching. H Based on relatively minor differences in sequence and function, they are divided into subclasses. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. It is understood that mammals encoding multiple Ig isotypes can undergo isotype class switching.

[0111] IgM antibodies consist of five basic heterotetrameric units together with an additional polypeptide called the J chain, and therefore contain 10 antigen-binding sites. However, secreted IgA antibodies can polymerize to form multivalent aggregates containing 2 to 5 of the basic four-chain units together with the J chain. In the case of IgG, the four-chain unit generally has a molecular weight of about 150,000 daltons. For the structure and properties of different classes of antibodies, 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, page 71, and Chapter 6.

[0112] The variable (V) domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. H The gene sequence encoding the V domain has multiple copies of variable (V), diversity (D), and joining (J) segments. L The gene sequence encoding the V domain contains multiple copies of V and J segments. H and V LThe region undergoes gene rearrangement (i.e., somatic recombination) to develop diverse antigen specificities in antibodies. The term "variable" refers to the fact that certain segments of the V domain vary widely between antibodies in sequence.

[0113] However, variability is not uniformly distributed over the 110 - amino - acid span of the variable domain. Instead, the V region consists of relatively invariant stretches called framework regions (FRs) of 15 - 30 amino acids separated by short regions of extreme variability called "hypervariable regions". These hypervariable regions are the result of somatic hypermutation during the affinity - maturation process and are typically 9 - 18 amino acids long each. However, they have been found to range in length from 4 - 28 amino acids depending on the particular epitope. For example, CDR3 regions up to at least 22 or 23 amino acids in length have been described. See, for example, Morea V, et al., J Mol Biol. 275(2):269 - 94 (1998) and Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91 - 3242 (1991). Antibody amino - acid positions (e.g., CDR sequences) can be determined according to known numbering schemes such as Kabat, Chothia, IMGT, and / or EU numbering schemes.

[0114] The variable domains of the native heavy and light chains each contain four framework regions (FRs), most of which adopt a β-sheet conformation and are connected by three highly variable regions (also known as complementarity-determining regions (CDRs) and further defined below) that form loops that connect, and in some cases form part of, the β-sheet structure. The highly variable regions in each chain are held together in proximity, sometimes by FRs, with highly variable regions from other chains and contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to antigen but exhibit the participation of the antibody in various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), or other mechanisms that may involve interaction of the constant region domain with cell surface Fc receptors (FcRs).

[0115] The term "highly variable region", as used herein, refers to the amino acid residues of the antibody that are involved in antigen binding. The highly variable regions generally include the amino acid residues from "complementary determining regions" or "CDRs" (e.g., as determined according to Kabat numbering, approximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in V L and approximately residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in V Happroximately 28 - 36 (H1), 50 - 65 (H2), and 95 - 102 (H3) in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); see also, and / or by methods known in the art for identifying CDRs as defined by Kabat, e.g., as 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, pages 422 - 438), and / or residues from "hypervariable loops" (e.g., V L having residues 26 - 32 (L1), 50 - 52 (L2), and 91 - 96 (L3), and V H having 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3); Chothia and Lesk, J. Mol. Biol. 196:901 - 917 (1987)).

[0116] "Isolated antibody" refers to an antibody that has been separated and / or recovered from the components of its natural environment. The contaminating components of its natural environment are materials that may interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is (1) greater than 95% by weight of the antibody as determined by the Bradford method, most preferably greater than 99% by weight; (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie Blue or silver staining. An isolated antibody includes an antibody in situ within a recombinant cell because at least one component of the antibody's natural environment is absent. However, usually, an isolated antibody is prepared by at least one purification step.

[0117] An "intact" antibody is an antibody that includes an antigen-binding site, as well as C L and at least the heavy chain constant domains, C H 1, C H 2 and C H 3. The constant domains can be the constant domains of the native sequence (e.g., the constant domains of the human native sequence) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

[0118] An "antibody fragment" is a polypeptide that comprises 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. Patent 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.

[0119] Papain digestion of an antibody yields two identical antigen-binding fragments called “Fab” fragments and the remaining “Fc” fragment, so named because of its ability to readily crystallize. The Fab fragment consists of the entire L chain, along with the variable domain of the H chain (V H ), and the first constant domain (C H 1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab’)2 fragment that roughly corresponds to two disulfide-linked Fab fragments with divalent antigen-binding activity and is still capable of cross-linking antigens. Both Fab and F(ab’)2 are examples of “antigen-binding fragments”. The Fab’ fragment differs from the Fab fragment by having a few additional residues at the carboxy terminus of the C H 1 domain that includes one or more cysteines from the hinge region of the antibody. Fab’-SH is the name of the Fab’ herein where the cysteine residue of the constant domain has 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.

[0120] The “Fc” fragment includes the carboxy-terminal portions of both H chains (i.e., the CH2 and CH3 domains of IgG) held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc region. The Fc domain is the part of the antibody that is recognized by cell receptors, such as FcR, and to which C1q, a complement-activating protein, binds. As discussed herein, modifications (e.g., amino acid substitutions) can be made to the Fc domain to modify (e.g., improve, reduce, or ablate) one or more functionalities of an Fc-containing polypeptide (e.g., an antibody of the disclosure).

[0121] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable domain in a strong non-covalent association. From the folding of these two domains, six highly variable loops (three loops each from the H and L chains) are generated that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three CDRs specific for the antigen) has the ability to recognize and bind the antigen, but typically with a lower affinity than the entire binding site.

[0122] "Single-chain Fv" is also abbreviated as "sFv" or "scFv" and is an antibody fragment that contains the V H and V L antibody domains connected in a single polypeptide chain. Preferably, the sFv polypeptide further contains a polypeptide linker between the V H and V L domains that enables the sFv to form the desired structure for antigen binding. For a general review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, see below.

[0123] The term "diabody" refers to a small antibody fragment prepared by constructing an sFv fragment (see the above paragraph) with a short linker (about 5-10 residues) between the V H and V L domains such that inter-chain, but not intra-chain, pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are composed of the V H and V LIt is a heterodimer of two "crossed" sFv fragments present on polypeptide chains with different domains. Diabodies are described in detail, for example, in EP404,097; WO93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Other antibody fragments and molecules containing them include, for example, linear antibodies, tandem scFv, scFv-Fc, tandem scFv-Fc, scFv dimers, scFv-zippers, diabody-Fc, diabody-CH3, sc diabodies, sc diabody-Fc, sc diabody-CH3, nanobodies, TandAb, minibodies, miniantibodies, triabodies, tetra-bodies, scFab, Fab-scFv, Fab-scFv-Fc, scFv-CH-CL-scFv, and F(ab’)2-scFv2, all of which are also contemplated herein.

[0124] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are multispecific antibodies, such as bispecific or trispecific antibodies. Forms of bispecific antibodies are disclosed, for example, in Spiess et al., Mol. Immunol. 67(2):95 (2015), and Brinkmann and Kontermann, mAbs 9(2):182-212 (2017), the bispecific forms and methods of making them are incorporated herein by reference, for example, bispecific T cell engagers (BiTE), DART, Knobs-Into-Holes (KIH) assembly, scFv-CH3-KIH assembly, KIH common light chain antibodies, TandAb, Triple Body, TriBi minibody, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2-scFv2, tetravalent HCab, Intrabody, CrossMab, dual action Fab (DAF) (two-in-one or four-in-one), DutaMab, DT-IgG, Charge Pair, Fab-arm exchange, SEED body, Triomab, LUZ-Y assembly, Fcab, κλ-body, 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).

[0125] As used herein, the term "polyclonal antibody" refers to an antibody obtained from a population of antigen-specific antibodies that recognize more epitopes than one specific antigen. "Antigen" or "immunogen" refers to a peptide, lipid, polysaccharide, or polynucleotide that is recognized by the adaptive immune system. The antigen may be a self or non-self molecule. Examples of antigens include, but are not limited to, bacterial cell wall components, pollen, and the rh factor. The region of an antigen that is specifically recognized by a specific antibody is an "epitope" or "antigenic determinant". A single antigen may have multiple epitopes.

[0126] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for potential naturally occurring variations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that contain different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope of the antigen. Monoclonal antibodies are advantageous in that, in addition to their specificity, they can be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacteria, eukaryotic animals, or plant cells (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibody" may 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).

[0127] As used herein, a monoclonal antibody includes a "chimeric antibody" in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody from a different species or belonging to a different antibody class or subclass, and also includes fragments of such antibodies so long as they exhibit the desired biological activity (see U.S. Patent 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. Further, a chimeric antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further refine the performance of the antibody. 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 include primatized and humanized antibodies.

[0128] A "humanized antibody" is generally considered to be a human antibody having one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are typically obtained from the variable domains. Humanization has conventionally been performed by replacing the corresponding sequences of a human antibody with non-human variable sequences according to the methods 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)). Thus, such "humanized" antibodies are chimeric antibodies in which substantially fewer than intact human variable domains are replaced by the corresponding sequences from non-human species (U.S. Patent Nos. 4,816,567; 5,530,101 and 7,498,415). In some cases, "humanized" antibodies are produced by non-human cells or animals and contain human sequences, e.g., the H C domain.

[0129] A "human antibody" is an antibody that contains only sequences that are present in antibodies naturally produced by humans. However, a human antibody, as used herein, may contain residues or modifications not found in naturally occurring human antibodies, including the modified and variant sequences described herein. These are typically made to further refine or enhance the performance of the antibody. In some cases, human antibodies are produced by transgenic animals. See, e.g., U.S. Patent Nos. 5,770,429; 6,596,541 and 7,049,426.

[0130] The "effector function" of an antibody refers to the biological activities that can be attributed to the Fc region of the antibody (the Fc region of the native sequence or the Fc region of an amino acid sequence variant), and varies depending on 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 receptor); and B cell activation. Amino acid modifications (e.g., substitutions) for modifying (e.g., improving, reducing, or eliminating) Fc functionality include, for example, T250Q / M428L, M252Y / S254T / T256E, H433K / N434F, M428L / N434S, E233P / L234V / L235A / G236+A327G / A330S / P331S, E333A, S239D / A330L / I332E, P257I / 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 are 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 and are incorporated herein by reference.

[0131] The phrase "functional fragment or analog" of an antibody or antigen-binding fragment thereof refers to a compound having qualitative biological activities common to a full-length antibody or antigen-binding fragment thereof.

[0132] An antibody or antigen-binding fragment thereof having the "biological characteristics" of a specified antibody or antigen-binding fragment thereof is one having one or more of the biological characteristics of that antibody or antigen-binding fragment thereof that distinguish it from other antibodies or binding fragments derived from antibodies. For example, in certain embodiments, an antibody or antigen-binding fragment thereof having the biological characteristics of a specified antibody binds to the same epitope to which the specified antibody binds and / or has effector functions common to the specified antibody.

[0133] An antibody, as used herein, at a detectable level, preferably, greater than or equal to about 10 4 M -1 or equal thereto, or greater than or equal to about 10 5 M -1 or equal thereto, greater than or equal to about 10 6 M -1 or equal thereto, greater than or equal to about 10 7 M -1 or equal thereto, or 10 8 M -1 or equal thereto, affinity constant K a when reacting with an antigen, is said to be "immunologically specific", "specific" or "specifically binds" to the antigen. The affinity of an antibody for its cognate antigen is also usually expressed as the dissociation constant K D and, in certain embodiments, an antibody or antigen-binding fragment thereof has a K -4 less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to about 10 -7 M, less than or equal to 10 -8 M, or less than or equal to 10 DWhen binding with [the relevant entity], it specifically binds to the Lewis antigen of the present disclosure. The affinity of the antibody and antigen-binding fragment can be easily determined using conventional techniques, such as those described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)), or by surface plasmon resonance (SPR) (e.g., Hearty et al., 2012 Meths. Mol. Biol. 907:411), isothermal titration calorimetry (ITC) (e.g., Dam et al., 2008 J. Biol. Chem. 283: 31366), enzyme-linked immunosorbent assay (ELISA) (e.g., Bobrovnik, 2003 J. Biochem. Biophys. Meths. 75(3): 213), or other methods well known to those skilled in the art.

[0134] The binding characteristics of the antibody to its antigen, cell, or tissue can generally be determined and evaluated using immunodetection methods, such as, for example, immunofluorescence-based assays, such as immunohistochemistry (IHC) and / or fluorescence-activated cell sorting (FACS). Other methods for determining the binding of the antibody to the antigen include, for example, enzyme-linked immunosorbent assay (ELISA), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) techniques.

[0135] "Carrier", as used herein, includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the dosages and concentrations used. Physiologically acceptable carriers are often pH buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphoric acid, citric acid, 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 dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions that form salts such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN®), polyethylene glycol (PEG), and poloxamer (PLURONICS®).

[0136] Polynucleotides, Vectors, and Host Cells In a further aspect, the disclosure provides, in certain embodiments, an isolated polynucleotide encoding the antibodies and antigen-binding fragments thereof described herein, and also provides a vector containing the same. The nucleic acid containing the polynucleotide may comprise DNA or RNA and may be wholly or partially synthetic.

[0137] As used herein, the term "polynucleotide" means a single-stranded or double-stranded nucleic acid polymer, specifically including single-stranded and double-stranded forms of DNA. Polynucleotides can be generated, for example, by polymerase chain reaction (PCR) or by in vitro translation, and fragments can be generated by any of ligation, excision, endonuclease action, or exonuclease action. In certain embodiments, the polynucleotides of the present disclosure are produced by PCR. Polynucleotides may be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (such as in the form of α-enantiomers of naturally occurring nucleotides), or a combination of both. In further embodiments, the nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide.

[0138] The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides having modified or substituted sugar groups (such as modified with bromouridine, arabinoside, or 2'3'-dideoxyribose), etc. The term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphorothioanilato, phosphororaniladate, phosphoramidate, etc. For example, their disclosures are incorporated herein by reference for all purposes, 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 See 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. Patent No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543. An oligonucleotide may contain a detectable label in order to enable detection of the oligonucleotide or its hybridization.

[0139] As used herein, the term "isolated polynucleotide" means a polynucleotide of genomic, cDNA, or synthetic origin, or a combination of parts thereof, and depending on its origin, the isolated polynucleotide is (1) not associated with all or part of the polynucleotide in which it is found in nature, (2) ligated to a polynucleotide to which it is not ligated in nature, or (3) not present in nature as part of a larger sequence.

[0140] References to nucleotide sequences herein include, unless the context requires otherwise, DNA molecules having the specified sequences and RNA molecules having the specified sequences with T replaced by U.

[0141] As used herein, the term "operably linked" means that the components to which the term applies are in a relationship that enables them to perform their inherent functions under suitable conditions. For example, a transcriptional control sequence is "operably linked" to a protein coding sequence if it is ligated thereto such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

[0142] As used herein, the term "control sequence" refers to a polynucleotide sequence capable of affecting the expression, processing, or intracellular localization of a coding sequence to which it is ligated or operably linked. The nature of such control sequences may depend on the host organism. In certain embodiments, transcriptional control sequences for prokaryotes may include a promoter, ribosome binding site, and transcription termination sequence. In other specific embodiments, transcriptional control sequences for eukaryotes may include a promoter that includes one or more recognition sites for transcription factors, transcriptional enhancer sequences, transcription termination sequences, and polyadenylation sequences. In certain embodiments, a "control sequence" may include a leader sequence and / or a fusion partner sequence. Expression control sequences include appropriate transcription initiation, termination promoters, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize mRNA in the cytoplasm; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and optionally sequences that enhance protein secretion. Expression control sequences may be operably linked if they are contiguous with the gene of interest and, in trans, or act distally to control the expression of the gene of interest.

[0143] Expression includes, but is not limited to, processes such as transcription, translation, and, where introns are present, RNA splicing.

[0144] As will be understood by those skilled in the art, polynucleotides include genomic sequences, extra-genomic, and plasmid-encoded sequences that can express or be adapted to express proteins, polypeptides, peptides, etc., as well as smaller engineered gene segments. Such segments may be isolated naturally or synthetically modified by those skilled in the art.

[0145] As will be recognized by those skilled in the art, the polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be a DNA (genomic, cDNA or synthetic) or RNA molecule. Examples of RNA molecules 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 may or may not be present, but may be present within the polynucleotides according to the present disclosure, and the polynucleotides may or may not be linked to other molecules and / or support materials. The polynucleotide may contain native sequences or may contain sequences encoding variants or derivatives of such sequences.

[0146] Accordingly, according to these and related embodiments, the present disclosure also provides polynucleotides encoding the antibodies or antigen-binding fragments thereof described herein.

[0147] In other related embodiments, the polynucleotide variant may have substantial identity to the polynucleotide sequence encoding the antibody or antigen-binding fragment thereof described herein. For example, the polynucleotide may be at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity when compared to a reference polynucleotide sequence such as the sequence encoding the antibody or antigen-binding fragment thereof described herein using the methods described herein (e.g., BLAST analysis using standard parameters as described below). Those skilled in the art recognize that these values can be appropriately adjusted to determine the corresponding identity of the proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc.

[0148] Typically, the polynucleotide variant preferably contains one or more substitutions, additions, deletions, and / or insertions such that the binding affinity of the antibody or antigen-binding fragment thereof encoded by the variant polynucleotide is not substantially reduced compared to the antibody or antigen-binding fragment thereof encoded by the polynucleotide sequences specifically described herein (e.g., compared to the antibody referred to herein as hBBC.10.1, which includes an immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 10 and an immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 11).

[0149] In certain other related embodiments, the polynucleotide fragment may comprise or consist essentially of successive stretches of various lengths of a sequence that is identical or complementary to the sequence encoding the antibodies or antigen-binding fragments thereof described herein. For example, at least about 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, 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 consecutive nucleotides of the sequence encoding the antibodies or antigen-binding fragments thereof or variants thereof disclosed herein, as well as all intermediate lengths therebetween are provided. "Intermediate length" in this context means any length between the recited values, e.g., 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers ranging from 200 to 500; 500 to 1,000, etc. The polynucleotide sequences described herein may be extended at one or both ends by additional nucleotides not found in the native sequence. This 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 or both ends of the polynucleotide encoding the antibodies or antigen-binding fragments thereof described herein.

[0150] In another embodiment, provided are polynucleotide sequences that can hybridize to the antibodies or antigen-binding fragments thereof, or variants thereof, provided herein, or fragments thereof, or complementary sequences thereof, under medium to high stringency conditions. Hybridization techniques are well known in the field of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide provided herein to other polynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing overnight at 50°C to 60°C in 5×SSC; and subsequently washing twice at 65°C for 20 minutes each with 2×, 0.5×, and 0.2×SSC containing 0.1% SDS. One of ordinary skill in the art will understand that the stringency of hybridization can be readily manipulated, for example, by changing the salt content of the hybridization solution and / or the temperature at which the hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, except that the temperature of hybridization is raised to, for example, 60°C to 65°C or 65°C to 70°C.

[0151] In certain embodiments, the polynucleotides described above, such as polynucleotide variants, fragments, and hybridizing sequences, encode an antibody or an antigen-binding fragment thereof that binds to the bivalent Lewis antigens described herein. In other embodiments, such polynucleotides encode an antibody or an antigen-binding fragment thereof that binds to the Lewis antigens disclosed herein, or a variant thereof, and an antibody or an antigen-binding fragment thereof specifically described herein (e.g., antibody hBBC.10.1), with at least about 50%, at least about 70%, and in certain embodiments, at least about 90% identity. In further embodiments, such polynucleotides encode an antibody or an antigen-binding fragment thereof, or a variant thereof, that binds to the Lewis antigens disclosed herein with a greater affinity than the antibodies or antigen-binding fragments thereof specifically described herein, e.g., an antibody or an antigen-binding fragment thereof that binds quantitatively with at least about 105%, 106%, 107%, 108%, 109%, or 110% identity, and an antibody or an antigen-binding fragment thereof specifically described herein.

[0152] As described elsewhere in this specification, determination of the three-dimensional structure of an antibody or antigen-binding fragment thereof disclosed herein can be performed via conventional techniques such that one or more amino acid substitutions, additions, deletions or insertions with selected natural or non-natural amino acids can be substantially modeled for the purpose of determining whether the resulting structural variant retains the space-filling properties of the species disclosed herein. For example, a variety of computer programs are known to those of skill in the art for determining appropriate amino acid substitutions (or appropriate polynucleotides encoding the amino acid sequences) within an antibody or antigen-binding fragment thereof such that affinity is maintained or improved affinity is achieved. See, for example, 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 See Science 355:201, and the references cited therein.

[0153] The polynucleotides or fragments thereof described herein may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., regardless of the length of the coding sequence itself, and as a result, their overall lengths may vary greatly. Accordingly, it is contemplated that nucleic acid fragments of almost all lengths can be used, and the overall length is preferably limited by ease of preparation and use in the intended recombinant DNA protocol. For example, exemplary polynucleotide segments having an overall length of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length (including all intermediate lengths) are contemplated to be useful.

[0154] When comparing polynucleotide sequences, two sequences are said to be "identical" if they are the same when aligned so that the nucleotide sequences in the two sequences are maximally matched, as described below. The comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein refers to a segment of at least about 20, usually 30 to about 75, 40 to about 50 consecutive positions, and after optimally aligning the two sequences, the sequences can be compared to a reference sequence of the same number of consecutive positions.

[0155] Optimal alignment of sequences for comparison can be performed using the Megalign program in the DNASTAR® Lasergene package of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships。Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 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, D.G. and Sharp, P.M., CABIOS 5:151-153 (1989);Myers, E.W. and Muller W., CABIOS 4:11-17 (1988);Robinson, E.D., Comb. Theor 11:105 (1971);Santou, N. Nes, M., Mol. Biol. Evol. 4:406-425 (1987);Sneath, P.H.A. and Sokal, R.R., Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA (1973);Wilbur, W.J. and Lipman, D.J., Proc. Natl. Acad., Sci. USA 80:726-730 (1983)。

[0156] Alternatively, the optimal alignment of arrays for comparison can be performed by the local identity algorithm of Smith and Waterman, Add. APL. Math 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 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.

[0157] One preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity 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 percent sequence identity between two or more polynucleotides. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, the cumulative score can be calculated for nucleotide sequences using parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of word hits in each direction is halted when the cumulative alignment score drops by amount X from its maximum achieved value; when the cumulative score goes to zero or below due to the cumulative alignment of one or more residues with negative scores; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignment, (B) of 50, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands.

[0158] In certain embodiments, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, and for optimal alignment of the two sequences, a portion of the polynucleotide sequence in the comparison window may include 20 percent or less, usually 5 - 15 percent, or 10 - 12 percent additions or deletions (i.e., gaps) when compared to the reference sequence (without addition or deletion). The percentage is calculated by determining the number of positions in both sequences where identical nucleobases are present to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0159] As a result of the degeneracy of the genetic code, it will be appreciated by those skilled in the art that there are many nucleotide sequences encoding the antibodies or antigen-binding fragments thereof described herein. Some of these 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 frequency are expressly contemplated by the present disclosure. In certain embodiments, sequences that are codon-optimized for mammalian expression are particularly contemplated. Codon optimization can be performed using known techniques and tools, for example, using the GenScript® OptimiumGene™ tool. Codon-optimized sequences include partially codon-optimized sequences (i.e., at least one codon is optimized for expression in the host cell) and fully codon-optimized sequences.

[0160] Therefore, in another embodiment, a mutagenesis approach, such as site-directed mutagenesis, can be used to prepare variants and / or derivatives of the antibodies or antigen-binding fragments thereof described herein. This approach allows specific modifications in the polypeptide sequence to be made via mutagenesis of the underlying polynucleotide encoding them. These techniques provide a straightforward approach for preparing and testing sequence variants incorporating one or more of the foregoing considerations, for example, by introducing one or more nucleotide sequence changes into the polynucleotide.

[0161] Site-directed mutagenesis enables the production of mutants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation and a sufficient number of adjacent nucleotides to provide primer sequences of sufficient size and sequence complexity to form stable double-strands flanking the deletion junctions to be traversed. Mutations can be used in selected polynucleotide sequences to improve, alter, reduce, modify, or otherwise change the properties of the polynucleotide itself and / or the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0162] In certain embodiments, the inventors contemplate mutagenesis of the polynucleotide sequences encoding the antibodies or antigen-binding fragments thereof, or variants thereof, disclosed herein to alter one or more properties of the encoded polypeptide, such as binding affinity to a Lewis antigen, in accordance with the present disclosure. Techniques for site-directed mutagenesis are well known in the art and are widely used to create variants of both polypeptides and polynucleotides. For example, site-directed mutagenesis is often used to alter specific portions of DNA molecules. In such embodiments, primers typically containing about 14 to about 25 nucleotides or thereabouts in length are used with about 5 to about 10 residues on either side of the junction of the sequence to be altered.

[0163] As will be appreciated by those skilled in the art, site-directed mutagenesis techniques have often utilized phage vectors that exist in both single-stranded and double-stranded forms. Typical vectors useful in site-specific mutagenesis include vectors such as the M13 phage. These phages are readily commercially available. Double-stranded plasmids are also customarily used in site-directed mutagenesis that eliminates the step of transferring the gene of interest from the plasmid to the phage.

[0164] Generally, site-directed mutagenesis according to this specification is performed by first obtaining a single-stranded vector or melting the two strands of a double-stranded vector containing a DNA sequence encoding the desired peptide within the sequence. Oligonucleotide primers having the desired mutated sequence are generally prepared by synthesis. This primer is then annealed to the single-stranded vector and exposed to a DNA polymerase, such as the E. coli polymerase I Klenow fragment, to complete the synthesis of the mutated strand. In this way, a heteroduplex is formed in which one strand encodes the original unmutated sequence and the second strand has the desired mutation. This heteroduplex vector is then used to transform a suitable cell, such as an E. coli cell, and clones containing recombinant vectors with the mutated sequence compilation are selected.

[0165] The preparation of sequence variants of a DNA segment encoding a selected peptide using site-directed mutagenesis provides a means of producing useful potential species, but this does not mean limitation as there are other methods of obtaining sequence variants of peptides and the DNA sequences encoding them. For example, a recombinant vector encoding a desired peptide sequence may be treated with a mutagen, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, which are incorporated herein by reference for that purpose.

[0166] The term "oligonucleotide-directed mutagenesis procedure" as used herein refers to a template-dependent process and vector-mediated propagation that results in an increase in the concentration of a specific nucleic acid molecule or an increase in the concentration of a detectable signal, such as amplification, compared to its initial concentration. The term "oligonucleotide-directed mutagenesis procedure" as used herein is intended to refer to a process that includes the template-dependent extension of a primer molecule. The term template-dependent process refers to nucleic acid synthesis of an RNA or DNA molecule, in which case the sequence of the newly synthesized strand of nucleic acid is defined by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector-mediated techniques include the introduction of a nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. An example of such a technique is provided by U.S. Patent No. 4,237,224, which is specifically incorporated herein by reference in its entirety.

[0167] In another approach for the production of polypeptide variants, iterative sequence recombination can be used as described in U.S. Patent No. 5,837,458. In this approach, iterative cycles of recombination and screening or selection are performed to "evolve" individual polynucleotide variants having, for example, increased binding affinity. Certain embodiments also provide constructs in the form of plasmids, vectors, transcription or expression cassettes that include at least one polynucleotide described herein.

[0168] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information into a host cell. The term "expression vector" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of an inserted heterologous nucleic acid sequence. Examples of expression vectors encoding an antibody or an antigen-binding fragment thereof include viral vectors, such as lentiviral vectors or γ-retroviral vectors. Examples of 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), positive-strand RNA viruses, such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 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, mammalian type C, type B viruses, type D viruses, HTLV-BLV group, lentiviruses, spumaviruses (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0169] As used herein, "lentiviral vector" means an HIV-based lentiviral vector for gene delivery, which may be either integrative or non-integrative, has a relatively large packaging capacity, and can transduce a variety of different cell types. Lentiviral vectors are typically generated after transient transfection of producer cells with three (packaging, envelope, and transfer) or more plasmids. Like HIV, lentiviral vectors enter target cells via the interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which serves as a substrate for viral integration into the DNA of the infected cell.

[0170] According to certain related embodiments, provided are recombinant host cells comprising one or more constructs described herein; nucleic acids encoding antibodies or antigen-binding fragments thereof or variants thereof; and methods of producing the encoded products, which methods include expressing the nucleic acids encoding therefor. Expression can conveniently be achieved by culturing recombinant host cells containing the nucleic acids (e.g., in the vectors of the present disclosure) under appropriate conditions. After production by expression, the antibody or antigen-binding fragment thereof can be isolated and / or purified using any suitable technique and then used as required.

[0171] Systems for the cloning and expression of 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 the expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells (e.g., HEK cells, e.g., HEK293-c18 cells), NSO mouse melanoma cells, and the like. A common preferred bacterial host is E. coli.

[0172] The expression of peptides in prokaryotic cells such as E. coli is well established in the art. For a review, see, e.g., Pluckthun, A. Bio / Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for the production of antibodies or antigen-binding fragments thereof. For recent reviews, see, e.g., Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

[0173] Suitable vectors containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences, can be selected or constructed as required. The vector can be, if desired, a plasmid, a virus such as a phage, or a phagemid. For further details, see, e.g., Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for the manipulation of nucleic acids, mutagenesis, sequencing, introduction of DNA into cells and gene expression, as well as the analysis of proteins, for example in the preparation of nucleic acid constructs, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, or subsequent updates thereof.

[0174] The term "host cell" is used to refer to a cell into which, or into which it is possible to introduce, a nucleic acid sequence encoding one or more of the antibodies and antigen-binding fragments thereof described herein, and which further expresses, or is capable of expressing, a selected gene of interest, for example a gene encoding any of the antibodies or antigen-binding fragments described herein. The term includes the progeny of a parent cell and, so long as the selected gene is present, whether or not the progeny are identical to the original parent in morphology or genetic constitution. Thus, methods including the introduction of such nucleic acids into host cells are also contemplated. The introduction can be effected using any available technique. In the case of eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, retroviruses or other viruses such as vaccinia, or, in the case of insect cells, baculovirus, liposome-mediated transfection and transduction. In the case of bacterial cells, suitable techniques include calcium chloride transformation, electroporation and transfection using bacteriophage. Following introduction, expression from the nucleic acid may or may not be effected, for example by culturing the host cell under conditions for gene expression. In one embodiment, the nucleic acid is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by inclusion of sequences that promote recombination with the genome according to standard techniques.

[0175] In certain embodiments, the present invention also provides a method that includes using a construct as described above in an expression system to express a particular polypeptide, such as an antibody or an antigen-binding fragment thereof described herein. The term "transduction" is commonly used to refer to the transfer of genes from one bacterium to another by a phage. "Transduction" also refers to the acquisition and transfer of eukaryotic cell sequences by a retrovirus. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell is "transfected" when exogenous DNA has been introduced into the interior of the cell membrane. Several transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories; 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 exogenous DNA moieties into a suitable host cell.

[0176] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell, and a cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed when it is genetically modified from its native state. After transfection or transduction, the transforming DNA may be recombined with the cell's DNA by physical integration into the cell's chromosome, or it may be transiently maintained as an episomal element without replication, or it may replicate independently as a plasmid. A cell is considered to be stably transformed when the DNA is replicated with cell division. The terms "naturally occurring" or "native" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, etc., refer to materials found in nature and not manipulated by humans. Similarly, "non-naturally occurring" or "non-native" as used herein refers to materials not found in nature or that have been structurally modified or synthesized by humans.

[0177] The practice of some embodiments of the invention employs, unless specifically indicated to the contrary, conventional methods in virology, immunology, microbiology, molecular biology, and recombinant DNA techniques within the skill of the art, many of which are exemplified below for purposes of illustration. Such techniques are explained in detail in the literature. For example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3 rdSee, e.g., Ausubel et al., Current Protocols in Molecular Biology (2nd ed., Wiley & Sons, 1995); Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid 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.

[0178] Certain embodiments disclosed herein relate to the detection and characterization of specific immunological binding activities (e.g., antibody binding activity) directed to antigenic structures defined by carbohydrates. Those skilled in the art will understand that there are various methods for generating and testing carbohydrate-specific immunological reagents, including the precise structural characterization of cognate carbohydrate antigens. Non-limiting examples of such techniques are Haji-Ghassemi et al., 2015 Glybiol. 25:920; Dingjan et al., 2015 Mol. Immunol. 67(2 Pt 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 are described in the references cited therein. Therein, and elsewhere, can also be found a description of sources of antigens defined by carbohydrates, and methods for their preparation, isolation, and structural characterization. Exemplary and non-limiting applications of such approaches are disclosed herein, and the present disclosure is not intended to be so limited, and also contemplates the preparation by synthesis of structurally defined carbohydrate antigens, including by utilization of the excellent specificity of biosynthetic enzymes known as glycosyltransferases in the art. For example, Wu et al., Universal phosphatase-coupled glycosyltransferase assay, see 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.

[0179] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques can be performed according to the specifications of the manufacturer, or as generally accomplished in the art, or as described herein.

[0180] These and related techniques and procedures can generally be carried out according to methods well known in the conventional art and as described in various general and more specific references cited and discussed throughout this specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly employed in the art. Standard techniques can be used for recombinant techniques, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, and delivery, as well as for the treatment of patients, and are further described herein.

[0181] Composition In certain embodiments, the present 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 that comprises an immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 10 and an immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 11.

[0182] In certain embodiments, an isolated antibody or antigen-binding fragment thereof that comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 35; and an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5, wherein the antibody or antigen-binding fragment thereof contains a biantennary type Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B to an antigen contains a biantennary type Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y to an antigen A biantenna-type 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 a biantenna-type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B antigen is capable of specifically binding, and an antibody or an antigen-binding fragment thereof does not specifically bind to a monoantenna-type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX], x nor to a biantenna-type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X], x nor to a monoantenna-type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI], A nor to a monoantenna-type H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], nor to a biantenna-type H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], nor to a monoantenna-type H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV]. There is provided an isolated antibody or an antigen-binding fragment thereof.

[0183] In other embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (VH CDR1) having the amino acid sequence set forth in SEQ ID NO: 2; a heavy chain complementarity determining region 2 (VH CDR2) having the amino acid sequence set forth in SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (VH CDR3) having the amino acid sequence set forth in SEQ ID NO: 4; and (b) a light chain variable region comprising a light chain complementarity determining region 1 (VL CDR1) having the amino acid sequence set forth in SEQ ID NO: 6; a light chain complementarity determining region 2 (VL CDR2) having the amino acid sequence set forth in SEQ ID NO: 7; and a light chain complementarity determining region 3 (VL CDR3) having the amino acid sequence set forth in SEQ ID NO: 8, and the antibody or antigen-binding fragment thereof specifically binds to a biantennary-type Le containing Fuc4(Galβ1→3GlcNAc)2 [I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2 [II] B / Le B antigen, specifically binds to a biantennary-type Le containing Fuc4(Galβ1→4GlcNAc)2 [III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2 [IV] Y / Le Y antigen, specifically binds to a biantennary-type 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 specifically binds to a biantennary-type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)] [VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc] [VIII] Y / Le B antigen and is capable of specifically binding thereto, The antibody or its antigen-binding fragment specifically binds to a monoantennary type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX], x also to a biantennary type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X], x also to a monoantennary type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI], A and does not specifically bind to a monoantennary type H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], nor to a biantennary type H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], nor to a monoantennary type H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV].

[0184] In certain embodiments disclosed herein, the isolated antibody or its antigen-binding fragment is monoclonal.

[0185] In certain embodiments disclosed herein, the isolated antibody or its antigen-binding fragment is humanized.

[0186] In certain embodiments, the isolated antibody or its antigen-binding fragment is selected from a Fab fragment, an F(ab’)2 fragment, an Fv fragment, a single-chain Fv (ScFv) antibody, and a diabody.

[0187] In certain further embodiments, an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 35; and an immunoglobulin light chain variable region comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 5, wherein the antibody or its antigen-binding fragment, A bivalent type Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen, A bivalent type Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen, A bivalent type 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 a bivalent type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B antigen is capable of specifically binding, An antibody or an antigen-binding fragment thereof contains a monovalent type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX] x antigen, and also a bivalent type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] x antigen, and also a monovalent type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A There is provided an isolated antibody or an antigen-binding fragment thereof that does not specifically bind to a monovalent type H antigen 2 type containing Fucα1-2Galβ1-4GlcNAc[XII], nor to a bivalent type H antigen 2 type containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], nor to a monovalent type H antigen 1 type containing Fucα1-2Galβ1-3GlcNAc[XIV].

[0188] In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises (a) a heavy-chain complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 2; a heavy-chain complementarity-determining region 2 (VH CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 3; a heavy-chain complementarity-determining region 3 (VH CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 4, in an immunoglobulin heavy-chain variable region; and (b) a light-chain complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 6; a light-chain complementarity-determining region 2 (VL CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 7; and a light-chain complementarity-determining region 3 (VL CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 8, in an immunoglobulin light-chain variable region.

[0189] In other embodiments, the isolated antibody or antigen-binding fragment thereof is any of the following (i), (ii), (iii), (iv), (v), or (vi): (i) a VH CDR1 comprising a variant of the amino acid sequence set forth in SEQ ID NO: 2, wherein the mutation 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 set forth in SEQ ID NO: 4, wherein the mutation 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 set forth in SEQ ID NO: 4, wherein the mutation 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 set forth in SEQ ID NO: 6, wherein the mutation 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 set forth in SEQ ID NO: 7, wherein the variant consists of a G→A substitution at position 50 according to Kabat numbering; CDR2; or (vi) a VL CDR3 comprising a variant of the amino acid sequence set forth in SEQ ID NO: 8, wherein the mutation consists of a T→S substitution at position 93 according to Kabat numbering, or any combination thereof. In any of the embodiments described herein, the isolated antibody or antigen-binding fragment thereof may have reduced (e.g., statistically significantly decreased) binding to the monoantennary form of the Lewis B antigen or the monoantennary form of the Lewis Y when compared to a BBC antibody comprising a VL domain having the amino acid sequence set forth in SEQ ID NO: 27 and a VH domain having the amino acid sequence set forth in SEQ ID NO: 28.

[0190]

[0191] ​In certain further embodiments, an immunoglobulin heavy chain variable region is provided that comprises a CDR as described herein (i.e., a CDR having at least 90% identity to SEQ ID NO: 2, 3, 4, 6, 7, and 8, including CDR variants having the amino acid substitutions described herein) and comprises or consists of an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 35; and an immunoglobulin light chain variable region that comprises or consists of an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 5, wherein an isolated antibody or antigen-binding fragment thereof is provided.

[0192] Antibodies or antigen-binding fragments thereof, including but not limited to scFv, such as those described herein, may in certain embodiments be included in a fusion protein capable of specifically binding to a Lewis antigen described herein. In some embodiments, the fusion protein is capable of being expressed on the surface of a host cell, such as a T cell, NK cell, or NK-T cell, and includes an extracellular component comprising an antibody or antigen-binding fragment thereof disclosed herein, and an intracellular component that, upon receiving an appropriate signal, is capable of directly or indirectly promoting an immunological response in a cell (e.g., an immune system cell, such as a T cell), the effector domain (e.g., 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, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or an effector domain derived from any combination thereof), the extracellular and intracellular components 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 co-stimulatory domain or a portion thereof 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 is a polypeptide derived from an immunoglobulin protein; e.g., comprising IgG4 hinge-CH2-CH3.

[0193] In these and related embodiments, the antigen-binding fragment may include an antigen-binding fragment described herein, such as an scFv, and the extracellular component may further include a linker region including a hinge, for example, in a chimeric antigen receptor molecule (CAR), and such molecules can be expressed on the cell surface of host cells such as T cells, NK cells, or NK-T cells for use in cellular immunotherapy. The CAR molecules and principles of design are described, for example, in 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. Volume 19 (April 2018); Wu et al., 2016 Expert Opin. Biol. Ther. 16:1469; Ren et al., 2017 Protein Cell 8:634, and those CAR molecules, CAR designs, and principles of CAR design are hereby incorporated by reference in their entirety.

[0194] Also provided herein are antibody conjugates comprising an antibody or antigen-binding fragment thereof of the present disclosure; and a payload molecule linked thereto. As background, monoclonal antibodies that specifically target an antigen can be used as carrier molecules for delivering a therapeutic or detectable payload molecule to the site of antigen expression, such as a tumor cell expressing the antigen. Binding of the antibody conjugate to the antigen can enable, for example, targeted delivery of a cytotoxic payload or detectable moiety to diseased cells or tissues for the treatment, detection, imaging, or monitoring of a disease, such as cancer. In certain embodiments, the antibody conjugate is internalized by the target cell expressing the antigen after or upon binding by the antibody conjugate. Internalization into the cytosolic or lysosomal compartments of the target cell can allow, for example, selective release of the payload molecule to the target cell to cause cytotoxic damage to the target cell.

[0195] A variety of techniques can be used to couple a payload molecule to an antibody or an antigen-binding fragment thereof to form the antibody conjugates of the present disclosure. In some embodiments, the antibody conjugate comprises a payload molecule covalently linked to an antibody or an antigen-binding fragment thereof by a linker. Linkers used in antibody conjugates (e.g., antibody-drug conjugates) containing a cytotoxic or anti-proliferative agent are typically organic compounds that fall into one of two groups 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 according to the unique properties of the target cell: The three types of cleavable linkers are protease-sensitive linkers (by which cleavage of the linker, e.g., a linker containing a valine-citrulline or phenylalanine-lysine dipeptide or tetrapeptide (e.g., GFLG or ALAL), by proteases present in tumor cell lysosomes releases the payload molecule); pH-sensitive linkers containing acid-labile groups that are selectively hydrolyzed by endosomal and lysosomal compartments at a lower pH compared to the pH of the cytosol; and glutathione-sensitive linkers containing disulfide bridges that are reduced by intracellular glutathione. Non-cleavable linkers rely on non-specific degradation of the antibody conjugate to release the payload molecule.

[0196] Specific linkers, payloads, chemical properties of the linkers, and related mechanisms and methods are disclosed in Nareshkumar et al., Pharm. Res. 32:3526-3540 (2015), and their compositions, methods, and techniques are hereby incorporated by reference in their entirety. In certain embodiments, the antibody conjugate comprises a linker selected from cleavable linkers and non-cleavable linkers. In further embodiments, the linker is a cleavable linker selected from protease-sensitive linkers, pH-sensitive linkers, or glutathione-sensitive linkers. In a specific embodiment, the cleavable linker is a protease-sensitive linker comprising a valine-citrulline dipeptide.

[0197] The linker can be connected or coupled to the antibody or an antigen-binding fragment thereof using any suitable technique or mechanism. In some embodiments, the linker comprises a maleimide group (optionally pegylated) capable of reacting with a reduced disulfide bridge in the hinge region of the antibody or an antigen-binding fragment thereof. Other sites on the carrier molecule (i.e., the antibody or an antigen-binding fragment thereof) suitable for conjugation to the linker can be introduced or engineered using recombinant techniques, such as introducing cysteine residues or unnatural 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 WO2012 / 032181.

[0198] In some embodiments, the linker further comprises a self-destructive group, also referred to as a self-immolative group or self-immolative spacer, to assist in a selective cleavage reaction. In certain embodiments, the self-destructive group is para-aminobenzyl alcohol (PABC).

[0199] Click chemistry useful for generating antibody conjugates includes Meyer Examples include those described in et al., Bioconjug. Chem. 27(12):2791-2807 (2016), which are hereby incorporated by reference in their entirety.

[0200] In any of the antibody conjugates described herein, the payload molecule can be selected from a therapeutic agent and a detectable indicator. Suitable therapeutic agents for cancer therapy include those disclosed in Parslow et al., Biomedicines 4 :14 (2016), the principles of whose payload and ADC design are hereby incorporated by reference. In certain embodiments, the payload molecule is a tubulin-targeting anti-mitotic agent, a peptide-based toxin, a pyrrolobenzodiazepine (PBD) dimer, an antibiotic (e.g., calicheamicin), a pyrimidine synthesis inhibitor (e.g., 5-fluorouracil), an antimetabolite (e.g., methotrexate), a DNA alkylating agent, and a topoisomerase inhibitor (e.g., doxorubicin). In further embodiments, the payload molecule is selected from maytansinoids, auristatins, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF).

[0201] In other embodiments, the payload molecule is a detectable indicator. Suitable detectable indicators for use in antibody conjugates, as well as related labeling strategies and imaging techniques (e.g., PET, MRI, NIR), include those of 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, the detectable indicator is selected from 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 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 is a radionuclide selected from. In certain such embodiments, the antibody conjugate further comprises a chelator of a radionuclide selected from DOTA labeled with maleimide, N-hydroxysuccinimide-DOTA, and desferrioxamine (DFO).

[0202] Also provided herein are pharmaceutical compositions comprising an antibody, an antigen-binding fragment thereof, or an antibody conjugate of the present disclosure; and a pharmaceutical carrier.

[0203] In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment described herein, which, when compared to the specific nucleotide sequences disclosed herein, has 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 and includes sequence variants. In certain embodiments, the polynucleotide encoding an antibody or antigen-binding fragment of the present disclosure (e.g., including scFv, Fab, F(ab’2) fragment, Fv fragment, or diabody) or a part of an antibody or antigen-binding fragment (e.g., heavy chain, light chain, VH, VL, or CDR) has 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 when compared to any one of SEQ ID NOs: 12-22 or 37. In certain embodiments, the polynucleotide is codon-optimized for expression in a host cell, such as a mammalian cell. In any of the above embodiments, the polynucleotide can be provided in a recombinant vector. In certain embodiments, the vector includes 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 in which the expression control sequence includes a promoter.

[0204] In another aspect, a host cell containing a recombinant vector or expression vector described herein is provided. In a related aspect, a method for producing an antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is biantennary type Le containing Fuc4(Galβ1→3GlcNAc)2 [I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2 [II]B / Le B to the antigen a biantennary type Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y to the antigen a biantennary type 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 to the antigen and a biantennary type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B to the antigen is capable of specifically binding an antibody or an antigen-binding fragment thereof to a monoantennary type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX] x also to a biantennary type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] as an antigen x also to a monoantennary type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] as an antigen A does not specifically bind to a monoantennary type H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], nor to a biantennary type H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], nor to a monoantennary type H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV] as an antigen A method is provided that comprises culturing a host cell of the present disclosure under conditions and for a time sufficient for expression by the host cell of a polynucleotide encoding an antibody or an antigen-binding fragment thereof, thereby obtaining a culture comprising the antibody or the antigen-binding fragment thereof; and recovering the antibody or the antigen-binding fragment thereof from the culture.

[0205] Methods and uses for detecting and treating diseases In certain embodiments, the antibodies, antigen-binding fragments thereof, and antibody conjugates disclosed herein are useful in methods for detecting or treating diseases characterized by the expression (e.g., overexpression) of the Lewis antigens described herein.

[0206] As will be understood by those of ordinary skill in the medical arts, the terms “treating” and “treatment” refer to the medical management of a disease, disorder, or condition in a subject (i.e., a patient, host which can be a human or non-human animal) (see, e.g., Stedman's Medical Dictionary). Generally, appropriate 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 prophylactic benefit. Therapeutic benefits resulting from a therapeutic treatment or prophylactic or preventative method include, for example, improved clinical outcomes, the goal being to prevent, retard, or otherwise reduce an undesirable physiological change or disorder (e.g., to reduce it in a statistically significant manner compared to an untreated control), or to prevent, retard, or otherwise reduce the spread or severity of such a disease or disorder. Beneficial or desired clinical results from treating a subject include elimination, diminishment, or alleviation of symptoms caused by or associated with the disease or disorder to be treated; a decrease in the appearance of symptoms; an improvement in quality of life; a longer disease-free state (i.e., reducing the likelihood or tendency for the subject to exhibit symptoms that would form the basis for a diagnosis of the disease); a reduction in the extent of the disease; a stabilized (i.e., non-worsening) disease state; a delay or slowing of disease progression; an amelioration or remission of the disease state; and remission (whether partial or complete) whether detectable or undetectable; or alternatively overall survival.

[0207] "Treatment" also may mean prolonging survival as compared to survival expected if the subject had not received treatment. Subjects in need of the methods and compositions described herein include subjects already having a disease or disorder, as well as subjects having a tendency to have or being at risk of developing a disease or disorder. Subjects in need of treatment to reduce the likelihood of the appearance or recurrence of a disease or disorder, or in need of prophylactic treatment, include subjects that reduce the severity of, or partially or completely avoid, or inadequately or completely prevent a disease, condition, or disorder (e.g., reducing the likelihood of the appearance or recurrence of a disease or disorder, which may include, but is not necessarily required, complete prevention). The clinical benefits provided by the compositions (and preparations containing the compositions) and methods described herein can be evaluated by in vitro assays, preclinical studies, and the design and conduct of clinical studies in subjects in whom administration of the composition is intended to provide a benefit, as described in the examples.

[0208] For example, in certain embodiments, a method for treating or detecting cancer (i.e., cancer expressing a Lewis antigen described herein) is provided, the method comprising administering a pharmaceutical composition of the disclosure to a subject in need thereof. "Cancer" as used herein refers to a condition characterized by abnormal or unregulated growth (e.g., hyperplasia) of affected cells, which may be characterized by malignant spread from a first tissue or site to adjacent or distant tissue(s) or site(s) in the body.

[0209] In certain embodiments of the method, the subject has or is suspected of having cancer selected from gastric cancer, colon cancer, breast cancer, lung cancer, lymphoma, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, uterine cancer, and squamous cell carcinoma. In further embodiments, the cancer is selected from gastric adenocarcinoma, mucinous gastric adenocarcinoma, undifferentiated gastric adenocarcinoma, signet ring cell gastric carcinoma, colon adenocarcinoma, invasive ductal carcinoma of the breast, hepatocellular carcinoma, lung adenocarcinoma, squamous cell carcinoma, metastatic lymph node adenocarcinoma, mucinous ovarian adenocarcinoma, pancreatic ductal adenocarcinoma, papillary adenocarcinoma of the pancreas, prostate adenocarcinoma, and endometrial carcinoma. Administration of the antibodies, antigen-binding fragments, or antibody conjugates described herein, in pure form or in a suitable pharmaceutical composition, can be effected via any of the recognized modes of administration of agents for providing similar utility. The pharmaceutical compositions can be prepared by combining the antibody, antigen-binding fragment, or antibody conjugate with a suitable physiologically acceptable carrier, diluent or excipient, and formulated into preparations in the form of solid, semi-solid, liquid or gaseous microparticles (e.g., microdroplets), e.g., tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmaceutically active ingredients (including other immunosuppressive agents as described elsewhere herein) and / or suitable excipients, such as salts, buffers and stabilizers, may or may not be present, but may be present within the composition.

[0210] 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 known model systems in the art and extrapolating therefrom. Controlled clinical trials may be performed. The dosage can also be varied depending on the severity of the condition to be alleviated. The pharmaceutical compositions are generally formulated and administered such that a therapeutically useful effect is achieved while minimizing undesirable side effects. The composition may be administered in a single dose or may be divided into several smaller doses administered at intervals. For any particular subject, the specific dosage regimen can be adjusted over time according to the individual need.

[0211] An "effective amount" of a composition refers to an amount sufficient, within the dosage and for the period required, to achieve the desired clinical result or beneficial treatment described herein. The effective amount can be delivered in one or multiple administrations. When the administration is to a subject already known or confirmed to have a disease or disease state, the term "therapeutic amount" can be used in reference to the treatment, whereas a "prophylactically effective amount" is a beneficial and / or protective process that reduces the likelihood and / or severity of the occurrence of a disease or disease state (e.g., to be statistically significant compared to the untreated state), and / or can be used to describe administering an effective amount to a subject at risk of developing or having a disease or disease state (e.g., recurrence).

[0212] In various embodiments, the antibody conjugates of the present disclosure include a detectable payload described herein and can be used to detect diseases such as cancer, either in vivo, in vitro, or ex vivo. In certain of these and other embodiments, the antibodies (i.e., one or more antibodies) or antigen-binding fragments thereof described herein are 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, their antigen-binding fragments, and antibody conjugates may be linked 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 detectable primary antibody, is used. Thus, direct detection means that the antibody conjugated to the detectable label can detect itself without the need for the addition of a second antibody (secondary antibody).

[0213] A "detectable label" is a molecule or material that can produce a detectable signal (e.g., visually, electronically, or otherwise) indicating the presence and / or concentration of a label in a sample. A detectable label, when conjugated to a peptide, can be used to identify and / or quantify the location of a target to which the specific peptide binds. Thereby, the presence and / or concentration of a target in a sample can be detected by detecting the signal produced 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.

[0214] Examples of detectable labels that can be detected directly include fluorescent dyes, radioactive substances, 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 binder to the detectable primary antibody. Examples of detectable primary binders or antibodies that require the addition of a secondary binder or antibody include enzyme-detectable binders and hapten-detectable binders or antibodies.

[0215] In some embodiments, the detectable label is conjugated to a nucleic acid polymer that includes a first binder (e.g., in an ISH, WISH, or FISH process). In other embodiments, the detectable label is conjugated to an antibody that includes a first binder (e.g., in an IHC process).

[0216] 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.

[0217] Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamidocaproic 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 allophycocyanin (APC), Texas Red, Princeton Red, green fluorescent protein (GFP) and its analogs, and conjugates of R-phycoerythrin or allophycocyanin, inorganic fluorescent labels, for example particles based on semiconductor materials such as coated CdSe nanocrystals.

[0218] Examples of polymer particle labels include microparticles or latex particles of polystyrene, PMMA or silica, or polymer micelles or capsules containing dyes, enzymes or substrates, into which fluorescent dyes can be incorporated.

[0219] Examples of metal particle labels include gold particles and coated gold particles, which can be converted by silver staining. Examples of haptens include DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin. Examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosamimidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO). Examples of substrates commonly used for horseradish peroxidase include 3,3'-diaminobenzidine (DAB), nickel-enhanced diaminobenzidine, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), indophenol blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), alpha-naphthol pyronin (alpha-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT), tetranitroblue tetrazolium (TNBT), 5-bromo-4-chloro-3-indoxyl-beta-D-galactoside / ferro-ferricyanide (BCIG / FF).

[0220] Examples of substrates commonly used for alkaline phosphatase include naphthol-AS-B1-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 fuchsine (NABP / NF), bromochloroindolyl phosphate / nitroblue tetrazolium (BCIP / NBT), 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (BCIG).

[0221] Examples of luminescent labels include luminol, isoluminol, acridinium ester, 1,2-dioxetane, and pyridopyridazine. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioisotopes of iodide, cobalt, selenium, tritium, carbon, sulfur, and phosphorus.

[0222] Detectable labels may be linked to the antibodies, antigen-binding fragments, and antibody conjugates described herein, or to any other molecule that specifically binds to a biological marker of interest, such as an antibody, nucleic acid probe, or polymer. Furthermore, one of ordinary skill in the art may recognize that detectable labels may also be conjugated to a second and / or third and / or fourth and / or fifth binding agent or antibody, etc. Furthermore, one of ordinary skill in the art may recognize that each additional binding agent or antibody used to characterize a biological marker of interest may serve as a signal amplification step. Biological markers may be detected visually, for example, using light microscopy, fluorescence microscopy, electron microscopy, and detectable substances include, for example, dyes, colloidal gold particles, luminescent reagents. Visually detectable substances bound to biological markers can also be detected using a spectrophotometer. If the detectable substance is a radioisotope, detection may be visual by autoradiography or non-visual using a scintillation counter. See, for example, Larsson, 1988, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.); Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) See (Humana Press, Totowa, N.J.).

[0223] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in methods for detecting or diagnosing a disease associated with the expression of a Lewis antigen described herein, the method comprising contacting the antibody, antigen-binding fragment, or antibody conjugate with a sample from a subject suspected of having or at risk of having the disease, and detecting the formation of an antibody:antigen complex in the sample, 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 diagnostic or detection method is performed ex vivo or in vitro.

[0224] Methods for in vivo detection of antibody conjugates with a detectable payload or detectable label include those described 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, detecting 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, ultrasound, or any combination thereof.

[0225] The preferred mode of administration is determined by the nature of the condition to be treated, which in certain embodiments refers to a detrimental or clinically undesirable condition whose degree, severity, likelihood of occurrence and / or duration can be reduced (e.g., reduced to be statistically significant compared to the situation of an appropriate control such as an untreated control) according to certain methods provided herein. After administration, an amount that detectably reduces, inhibits, at least partially prevents, reduces the severity or likelihood of occurrence of, or delays such a condition, e.g., a partial or complete reduction of tumor burden, or a partial or complete reduction of metastatic spread, is considered effective. Those of ordinary skill in the relevant art are familiar with numerous diagnostic, surgical and / or other clinical criteria that can demonstrate and / or adapt to the clinical validity of the administration of the compositions described herein. For example, 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”); 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. (Ed.), Hematopoietic Stem Cell Transplantation: A Handbook for Clinicians, 2009 American Association of Blood Banks; and references cited therein.

[0226] Thus, typical routes of administration of these and related pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. Pharmaceutical compositions, according to certain embodiments of the invention, are formulated such that the active ingredient contained therein is bioavailable upon administration of the composition to a patient. Compositions administered to a subject or patient may be in the form of one or more dosage units; for example, a tablet may be a single dosage unit, or a container of an antibody, antigen-binding fragment thereof, or antibody conjugate described herein in aerosol form may hold multiple dosage units. The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition administered, in each case, contains a therapeutically effective amount of an antibody, antigen-binding fragment thereof, or antibody conjugate of the present disclosure for the treatment of the disease or condition of interest in accordance with the teachings herein. In certain embodiments, administering comprises administering by a route selected from intravenous, parenteral, intragastric, intrathoracic, intralung, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, oral, topical, transdermal, intracapsular, intrathecal, intranasal, and intramuscular.

[0227] The pharmaceutical composition may be in solid or liquid form. In one embodiment, the carrier is a microparticle such that the composition is in the form of, for example, a tablet or powder. The carrier may be a liquid and the composition may be, for example, an oral oil, an injectable liquid, or an aerosol useful, for example, in inhalation administration. When oral administration is intended, the pharmaceutical composition is preferably in either solid or liquid form, and semi-solid, semi-liquid, suspension, and gel forms are included herein as forms considered to be either solid or liquid.

[0228] As a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gums, cachets, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, 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 and odor-masking agents such as peppermint, methyl salicylate or orange flavor; and coloring agents. When in the form of capsules, such as gelatin capsules, the pharmaceutical composition may contain, in addition to the materials of the above types, a liquid carrier such as polyethylene glycol or an oil.

[0229] The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid can be, as two examples, for delivery by oral administration or injection. When oral administration is intended, the preferred composition contains, in addition to the present compound, one or more of a sweetening agent, a preservative, a coloring agent / colorant and a flavor enhancer. In the composition intended to be administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer and an isotonic agent may be included.

[0230] Liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may contain one or more of the following adjuvants: a sterile diluent, such as water for injection, a saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, an inert oil, such as synthetic mono- or diglycerides that can serve as a solvent or suspending medium, polyethylene glycol, glycerin, propylene glycol or other solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetic acid, citric acid or phosphoric acid, and an agent for adjusting tonicity, such as sodium chloride or dextrose. The parenteral preparation may be enclosed in an ampoule, a disposable syringe, or a multiple-dose vial made of glass or plastic. Physiological saline is the preferred adjuvant. The pharmaceutical composition for injection is preferably sterilized.

[0231] A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of an antibody, an antigen-binding fragment thereof, or an antibody conjugate disclosed herein such that a suitable dosage is obtained. Typically, this amount is at least 0.01% antibody or antigen-binding fragment in the composition. When oral administration is intended, this amount can be varied to be between 0.1% and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% antibody, an antigen-binding fragment thereof, or an antibody conjugate. In certain embodiments, the pharmaceutical compositions and preparations according to the invention are prepared such that, prior to dilution, a parenteral dosage unit contains between 0.01% and 10% by weight of an antibody, an antigen-binding fragment thereof, or an antibody conjugate.

[0232] The pharmaceutical composition may be intended for topical administration, in which case the carrier may preferably include a base of a liquid, an emulsion, an ointment or a gel. The base may include, for example, one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. A thickening agent may be present in the pharmaceutical composition for topical administration. When transdermal administration is intended, the composition may include a transdermal patch or an iontophoresis device. The pharmaceutical composition may be intended for rectal administration, for example, in the form of a suppository, which melts in the rectum and releases the drug. The composition for rectal administration may contain an oily base as a suitable excipient with little irritation. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

[0233] The pharmaceutical composition may contain various materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may contain a material that forms a coating shell around the active ingredient. The material forming the coating shell is typically inert and can be selected, for example, from sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. The pharmaceutical composition in solid or liquid form may contain an agent that binds to the antibody, antigen-binding fragment, or antibody conjugate of the present invention, thereby assisting in the delivery of the compound. Suitable agents that can act in this capacity include monoclonal or polyclonal antibodies, one or more proteins, or liposomes. The pharmaceutical composition may consist essentially of a dosage unit that can be administered as an aerosol. The term aerosol is used to represent a wide variety of systems ranging from colloidal nature to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas or by a suitable pump system for dispensing the active ingredient. The aerosol may be delivered in a single-phase, two-phase, or three-phase system for delivering the active ingredient. Aerosol delivery includes the necessary container, activator, valve, sub-container, etc., which can be combined to form a kit. One skilled in the art can determine the preferred aerosol without undue experimentation.

[0234] The pharmaceutical composition can be prepared by methods well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining the antibody, antigen-binding fragment, or antibody conjugate described herein, and one or more salts, buffers, and / or stabilizers as required, with sterile distilled water such that a solution is formed. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. A surfactant is a compound that interacts non-covalently with the peptide composition to facilitate the dissolution or homogeneous suspension of the antibody, antigen-binding fragment, or antibody conjugate in an aqueous delivery system.

[0235] The composition is administered in a therapeutically effective amount, and the therapeutically effective amount varies depending on 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; drug combinations; the severity of the particular disorder or condition; and the therapy the subject is receiving. Generally, a therapeutically effective daily dose is (for a 70 kg mammal) from 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 is (for a 70 kg mammal) from 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 is (for a 70 kg mammal) from about 1 mg / kg (i.e., 70 mg) to about 25 mg / kg (i.e., 1.75 g).

[0236] Compositions comprising an antibody, antigen-binding fragment, or antibody conjugate of the present disclosure can be administered simultaneously with, prior to, or subsequent to the administration of one or more other therapeutic agents. Such combination therapies include the administration of a single pharmaceutical dosage formulation containing a compound of the invention and one or more additional active agents, as well as the administration of a composition comprising an antibody, antigen-binding fragment, or antibody conjugate of the invention and each active agent in separate pharmaceutical dosage formulations. For example, the antibodies, antigen-binding fragments, or antibody conjugates described herein and other active agents may be administered to a patient together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate oral dosage formulations. Similarly, the antibodies, antigen-binding fragments, or antibody conjugates described herein and other active agents 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 separate parenteral dosage formulations. When separate dosage formulations are used, the compositions comprising the antibody and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at staggered times, i.e., sequentially, in any order; combination therapies are understood to include all of these regimens.

[0237] Accordingly, in certain embodiments, administration of an antibody, antigen-binding fragment, or antibody conjugate of this disclosure in combination with one or more other therapeutic agents is also contemplated. Such therapeutic agents may be acceptable in the art as standard treatments for the particular disease states described herein, such as cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, immune checkpoint inhibitors, interfering RNAs, agonists of stimulatory immune checkpoint molecules, another antibody, antigen-binding fragment, or antibody conjugate that targets cancer, or other active and adjuvant agents.

[0238] As used herein, the term "immunosuppressive agent" or "immunosuppressant" refers to one or more cells, proteins, molecules, compounds, or complexes that provide inhibitory signals to assist in the control or suppression of the immune response. For example, immunosuppressive agents include molecules that partially or completely block immune stimulation; reduce, prevent, or delay immune activation; or increase, activate, or upregulate immunosuppression. Exemplary immunosuppressive agents for targeting (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.

[0239] Inhibitors of immunosuppressive agents (also referred to as immune checkpoint inhibitors) can be compounds, antibodies, antibody fragments, or fusion polypeptides (e.g., Fc fusions, e.g., CTLA4-Fc or LAG3-Fc), antisense molecules, ribozymes, or RNAi molecules, or low molecular weight organic molecules. 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 inhibitors of any one of the following immunosuppressive components.

[0240] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with a PD-1 inhibitor, such as 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 further embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an antibody specific for PD-L1, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or an antigen-binding fragment thereof, or any combination thereof.

[0241] 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.

[0242] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of CTLA4. In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an antibody specific for CTLA4 or a binding fragment thereof, such as ipilimumab, tremelimumab, a CTLA4-Ig fusion protein (e.g., abatacept, belatacept), or any combination thereof.

[0243] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an antibody specific for B7-H3 or an antigen-binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. The B7-H4 antibody binding fragment is described, for example, in Dangaj et al., As described in Cancer Res. 73:4820, 2013, it may be the scFv or fusion protein thereof, and may also be those described in U.S. Patent No. 9,574,000 and PCT Patent Publications WO2016 / 40724 A1 and WO2013 / 025779 A1.

[0244] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of CD244. In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT Publication WO2010 / 084158.

[0245] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of TIM3. In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of Gal9.

[0246] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor. In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of A2aR.

[0247] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015). In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitory cytokine (typically a cytokine other than TGFβ) or an inhibitor of the generation or activity of Tregs.

[0248] 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 levo-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (1-MT)-tilapazamine, or any combination thereof.

[0249] 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(omega)-nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoylethyl)-L-cysteine (BEC), or any combination thereof.

[0250] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0251] 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 COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as COM701 (Compugen), or both.

[0252] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication WO2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication WO2017 / 021526.

[0253] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the present disclosure are used in combination with an inhibitor of LAIR1.

[0254] 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.

[0255] In certain embodiments, the antibodies, antigen-binding fragments, or antibody conjugates of the disclosure are used in combination with an agent that increases the activity of a stimulatory immune checkpoint molecule (i.e., is an agonist). For example, the antibodies, antigen-binding fragments, or antibody conjugates of the disclosure can be combined with a CD137 (4-1BB) agonist (such as urelumab), a CD134 (OX-40) agonist (such as MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as CDX-1127), a CD28 agonist (such as TGN1412, CD80, or CD86), a CD40 agonist (such as CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (such as IL-2), an agonist of GITR (such as a humanized monoclonal antibody described in PCT Patent Publication WO2016 / 054638), an agonist of ICOS (CD278) (such as GSK3359609, mAb88.2, JTX-2011, Icos 145-1, Icos 314-8, or any combination thereof). In any of the embodiments disclosed herein, the method can include administering the antibodies, antigen-binding fragments, or antibody conjugates of the disclosure with one or more agonists of stimulatory immune checkpoint molecules, including any of the foregoing, alone or in any combination.

[0256] In certain embodiments, the combination therapy includes a secondary therapy that includes one or more of the antibodies, antigen-binding fragments, or antibody conjugates of the disclosure, and another antibody, antigen-binding fragment, or antibody conjugate specific for a cancer antigen (i.e., the same or a different antigen) expressed by the cancer, radiation treatment, surgery, chemotherapeutic agents, cytokines, RNAi, or any combination thereof.

[0257] In certain embodiments, the methods of combination therapy include administering an antibody, antigen-binding fragment, or antibody conjugate of the present disclosure, and further administering radiation treatment or surgery. Radiation therapy is well-known in the art and includes X-ray therapy, such as gamma radiation, and radiopharmaceutical therapy. Suitable surgeries and surgical techniques 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).

[0258] In certain embodiments, the method of combination therapy 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, inhibitors of chromatin function, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interacting agents (e.g., intercalating agents), and DNA repair inhibitors. Exemplary chemotherapeutic agents include, without limitation, the following groups: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folic acid antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, including natural products, such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilone, and navelbine, epipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, melphalan, mechlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide, and etoposide (VP16)); antibiotics, such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin;Enzymes (L-asparaginase, which metabolizes L-asparagine throughout the body and deprives cells lacking the ability to synthesize asparagine on their own); antiplatelet agents; anti-proliferative / anti-mitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates - busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes - dacarbazinine (DTIC); anti-proliferative / anti-mitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (e.g., tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti-migratory agents; anti-secretory agents (breveldin); immunosuppressive drugs (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic 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, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signal transduction 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.

[0259] Cytokines can be used to manipulate the host immune response against anti-cancer activity. See, for example, Floros & Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for promoting the anti-cancer or anti-tumor response of the immune system include, for example, 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 any combination with the antibodies, antigen-binding fragments, or antibody conjugates of this disclosure.

[0260] Cell immunotherapy, including those containing T cells, NK cells, or NK-T cells expressing natural or recombinant TCRs and CARs specific for cancer antigens, and including adoptive transfer of such cells into recipients, is an emerging therapy for cancer (e.g., Bonini (see also Mondino, Eur. J. Immunol. 45(9):2457-69 (2015) and Metha and Rezvani, Front. Immunol. 9:283 (2018)). In certain embodiments, a subject receiving an antibody, antigen-binding fragment, or antibody conjugate (or pharmaceutical composition) of the present disclosure has received or is receiving (i.e., concurrently, simultaneously, or sequentially) a cellular immunotherapy targeting cancer.

[0261] Provided herein is any of the antibodies, antigen-binding fragments, antibody conjugates, polynucleotides, vectors, host cells, and compositions disclosed herein for use in treating, detecting, or diagnosing a disease characterized by the expression (e.g., overexpression) of a Lewis antigen 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 of the combination therapies described herein.

[0262] Provided herein is any of the antibodies, antigen-binding fragments, antibody conjugates, polynucleotides, vectors, host cells, and compositions disclosed herein for use in the preparation of a medicament for treating a disease characterized by the expression (e.g., overexpression) of a Lewis antigen described herein. In certain embodiments, the disease is cancer, such as any cancer disclosed herein. In certain embodiments, the medicament comprises or is used with any of the combination therapies described herein.

[0263] As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural referents unless the context clearly dictates otherwise. Further, the use of the alternative conjunction (e.g., "or") should be understood to mean either one of the alternatives, both of them, or any combination thereof.

[0264] Throughout this specification, unless the context requires otherwise, the words "comprise", "have", "has", "having", "comprises" or "comprising" and variations thereof are to be understood to imply the inclusion of stated elements or integers or groups of elements or integers but not the exclusion of any other element or integer or group of elements or integers. In this description, any range of concentrations, percentage ranges, ratio ranges, or integer ranges are to be understood to include any integer value within the recited range and, where appropriate, fractions thereof (for example, one tenth and one hundredth of an integer), unless otherwise specified. Also, any numerical range recited herein with respect to any physical characteristic, such as the subunits of a polymer, size or thickness, is to be understood to include any integer within the recited range, unless otherwise specified.

[0265] The term "about", as used herein, unless otherwise specified, means within ±20% of the indicated range, value, or structure, or in certain embodiments, within ±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, unless otherwise specified.

[0266] In addition, it should be understood that individual compounds or groups of compounds derived from various combinations of the structures and substituents described herein are disclosed by this application to the same extent as if each compound or group of compounds were individually recited. Thus, the selection of a particular structure or particular substituent is within the scope of this disclosure.

[0267] The term "consisting essentially of" is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or materials or steps that do not materially affect the basic characteristics of the claimed subject matter.

[0268] Each embodiment in this specification shall be applied by making necessary modifications to any other embodiment, unless explicitly stated otherwise.

Example

[0269] (Example 1) Generation of chimeric BBC antibody Isolation of cDNA of antibody variable region Hybridoma cells expressing a mouse IMH2 / BBC antibody containing a VL domain having the amino acid sequence set forth in SEQ ID NO: 27 and a VH domain having the amino acid sequence set forth 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 individual hybridoma cells were first collected by low-speed centrifugation (300 g for 5 minutes), and then RNA was isolated using the "total RNA miniprep purification kit" (trademark) (GeneMark (trademark), GMbiolab Co., Ltd., Taichung City, Taiwan, ROC) according to the manufacturer's protocol. Subsequently, the antibody gene encoding IMH2 was cloned from the purified RNA using the SMART RACE cDNA Amplification Kit (trademark) (Takara / BD Biosciences-Clontech, Palo Alto, CA) with minor modifications compared to the supplier's recommended protocol.

[0270] Briefly, after cDNA synthesis and dC tail addition of the first strand, the cDNA specifically encoding the light chain variable region of IMH2 was isolated by two rounds of PCR using primers supplied by the kit and specific primers designed based on the known mouse kappa chain sequence in the constant region. The first PCR was performed for 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. An additional 27 cycles of PCR reaction containing 30 seconds at 94°C, 30 seconds at 62°C and 1 minute at 72°C were added to confirm the success of amplification. Then, a second nested PCR was performed including a preheating step 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 at 72°C for 3 minutes to further improve the fidelity.

[0271] The cloning strategy for the heavy chain gene was slightly different. First, the single-stranded cDNA template used for the PCR reaction was prepared from RNA using mouse IgG3-specific primers for the constant domain 1 (CH1). Gene isolation was then performed by a single round of PCR as follows: a preheating at 94°C for 5 minutes, a PCR reaction of 35 cycles including 30 seconds at 94°C, 30 seconds at 62°C, 1 minute at 72°C, followed by a final extension step at 72°C for 5 minutes in the presence of NUP primers (SMART (trademark) RACE amplification kit, Clontech, Palo Alto, CA) and nested primers for the mouse CH1 domain. Then, the cDNA encoding the variable regions of both the light and heavy chain fragments was purified using a PCR purification kit (GeneMark (trademark) GMbiolab Co., Ltd., ROC) and introduced into the yT&A cloning vector (Yeastern Biotech (trademark), Taipei, Taiwan, ROC) for identification and sequencing of positive clones.

[0272] Construction of antibody expression plasmid To construct an expression plasmid for producing the antibodies referred to in this specification as "BBC" antibodies, only the cDNAs encoding the mature (leader peptide-free) heavy and light chain variable regions were prepared using PCR primers and the antibody genes cloned into the yT&A vector described above. PCR was performed as follows: a 5-minute preheating at 94 °C, 35 cycles of PCR reaction including 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 enzyme recognition sequences were incorporated at the 5' (NheI) and 3' (ApaI) ends of the VH cDNA and at the 5' (NheI) and 3' (BsiWI) ends of the VL cDNA to facilitate subsequent manipulation of the expression plasmid. The amplified cDNA fragments were then sequentially digested with the restriction enzymes ApaI / NheI for the heavy chain and NheI / BsiWI for the light chain gene. After gel purification, the recovered VH and VL cDNAs were ligated into the pGNX-RhcG1(VH) or pGNX-Rhck vector (VL) at the same restriction enzyme cloning sites to obtain the expression vectors pGNX-RhcG1-BBC and pGNX-Rhck-BBC, respectively. The inserted cDNA sequences were confirmed using primers upstream of the multiple cloning site.

[0273] Transient production of antibodies For transient production of chimeric BBC antibodies, HEK293-c18 cells were co-transfected with the pGNX-RhcG1-BBC and pGNX-Rhck-BBC expression plasmids encoding the heavy and light chains in the presence of polyethyleneimine. For analysis, the culture supernatant was collected at the end of day 7 after transfection.

[0274] (Example 2) Generation of humanized BBC antibodies To produce a humanized form of the BBC antibody (described above in Example 1), homologous human antibody sequences (human acceptors) were selected and CDR grafting was performed. Briefly, potential human acceptor sequences were identified by searching the NCBI protein database to identify the positions of sequences that showed the greatest homology to the variable regions of the heavy chain (SEQ ID NO: 28) and light chain (SEQ ID NO: 27) of the BBC antibody. The human acceptor frameworks CAD89404.1 (Vh) and AAS01771.1 (Vl) were selected (Figure 1). However, directly inserting non-human CDR sequences into the human acceptor framework can result in loss of binding affinity. The binding affinity can be restored after moving framework residues from the human acceptor back to the non-human donor sequence. Preferred revertant mutations restore binding affinity by maintaining the original CDR conformation.

[0275] To restore binding affinity after CDR grafting, based on the BBC crystal structure data, a 3D model of the antibody was first constructed using Accelrys Discovery Studio (TM) (BIOVIA, San Diego, CA) software. Then, amino acids important for revertant mutations were predicted by examining the structure as follows:

[0276] 1. The mutational energy (for stability) of the changed residues in the humanized framework was calculated. A positive value of the mutational energy corresponds to the destabilizing effect of the mutation, and vice versa.

[0277] 2. The spatial distance between framework residues and the CDR regions was examined. Residues closest to the CDR were considered (within 4 Å).

[0278] 3. Residues located at the interface between the heavy chain variable region and the light chain variable region were examined. These residues contribute to the assembly of the heavy and light chains and could therefore have a significant impact on the antibody structure.

[0279] First, 10 influential positions (3 in the light chain and 7 in the heavy chain) were selected for back mutations based on prediction criteria. In addition, the methionine (M) residue at position 70 (according to Kabat numbering) of the selected human heavy chain template seemed to be a low-frequency motif, and thus that site was replaced with highly conserved isoleucine (I) (Figure 1; the back-mutated residues (human acceptor→hBBC.8) are underlined in Table 3 below; the Met→Ile residues are shown in bold italic and underlined in Table 3 and are circled in Figure 1). These amino acid conversions produced the "hBBC.8" humanized antibody.

[0280] Additional changes were introduced to further improve the antibody. First, two positions (R66 and F71) in the hBBC.8 light chain that vary between the mouse and human sequences were mutated to produce "hBBC.9" (containing the F71Y mutation) and "hBBC.10" (containing the F71Y and R66G mutations) (Figure 2A; residues shown in bold and italic and without underline in Table 3). Figure 2B shows the binding of BBC as well as the generated humanized variants hBBC.9 and hBBC.10 to AGS cells. Briefly, the AGS human gastric cancer cell line (ATCC CRL-1739; ATCC, Manassas, VA) was regularly maintained in F12 medium supplemented with 10% dialyzed fetal bovine serum. To perform cell binding studies, approximately 3×10 in 100 μl of PBS 5Individual cells were mixed with an equal volume of diluted antibody. After incubation for 1 hour at room temperature, 2 ml of PBS was added to each sample to wash away unbound antibody. Following centrifugation, the recovered cell pellet was directly resuspended in 200 μl of fluorescein (FITC)-AffiniPure™ goat anti-human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, West Grove, PA, catalog number 109-095-098) diluted 1:200 in PBS. After incubation for 30 minutes at room temperature, PBS washes were repeated to remove unbound secondary antibody. The collected cells were resuspended in 200 μl of PBS and analyzed on a BD FACSCanto™ flow cytometer system (BD Biosciences, San Jose, CA). Further analysis was performed to identify sequences that might be immunogenic.

[0281] Additional mutations in the heavy or light chains produced additional variants “hBBC.9.1” and “hBBC.10.1” (residues underlined in bold and not italicized in Table 3 below). Specifically, the crystal structures of the BBC and hBBC.8 antibodies, as well as the simulated antigen / antibody complexes, were analyzed. Several residues in the light and heavy chain CDR regions (positions identified according to Kabat numbering) were identified for single-site amino acid substitutions. Amino acid switches at the designated positions in the antibody light or heavy chain genes were performed by two PCR reactions using specifically designed primers. To facilitate insertion of the mutated antibody cDNA fragments into the expression vectors, restriction sites (5’ NheI and 3’ ApaI for the cDNA strand, 5’ NheI and 3’ BsiWI for the light chain cDNA) were incorporated at each end during the PCR reactions. After the second PCR reaction, DNA fragments were produced, cut with NheI / ApaI or NheI / BsiWI, and ligated into the same sites of the pGNX-RhcG1 and pGNX-Rhck vectors for heavy and light chain gene construction, respectively. Table 1 shows the mutated sites and changes. Table 1. Single-site mutations of hBBC.8

Table 1

[0282] Several variants showed in vitro binding activity equal to or greater than 1 / 2 of the activity of the chimeric antibody (BBC) in the AGS cell binding assay (see also Figure 2C, Table 2). In addition, as shown in Table 2, some variants also showed improved specificity in the ELISA assay by reduction of cross-reactivity with the monovalent Lewis B structure (equal to or less than 1 / 8 the intensity compared to BBC). Monovalent Lewis B is a blood group antigen expressed in normal human tissues. Table 2. Binding activity of BBC CDR variants

Table 2

[0283] hBBC.8 showed approximately 1 / 3 of the AGS cell binding activity compared to BBC (Figure 2B) and showed very good tumor inhibition in the xenograft mouse model (see Example 6), so these antibody variants replaced with these single amino acids have the potential to exhibit anti-tumor activity. Table 1 summarizes the positions and amino acid replacements of these analyzed clones. To further confirm their ability to improve affinity and / or specificity, a total of seven single mutation sites (Table 2) were selected and evaluated in the humanized antibody template. These were five single residue substitutions tested for their effect on antibody binding to AGS cells (3 in the light chain and 2 in the heavy chain) (Figure 2C), purified Le B They included two single residue substitutions in the heavy chain tested for their effect on the specificity of antibody binding to AGS cells compared to and two double residue replacements in the heavy chain analyzed similarly (Figure 2D).

[0284] Figure 2E shows further comparison of the various humanized BBC versions constructed with respect to affinity (AGS cell binding assay) and specificity (Lewis b ELISA assay) with the original chimeric form.

[0285] Epibase analysis of hBBC.9 (Applied Protein Services, Lonza Biologics, Cambridge, UK) predicted regions with strong immunogenic risk in the heavy chain region. To minimize potential immunogenicity issues, these positions were matched with the reference humanized antibody, including six additional amino acid changes (at positions 78, 80, 82 - 84, and 86) in the framework 3 region of the heavy chain (Figure 3). These conversions yielded the heavy chain framework for "hBBC.10.1FQ". Table 3 summarizes the amino acid sequences of the various heavy and light chain variable regions described in this example. Table 3. Amino acid sequences of antibody VH and VL regions

Table 3-1

Table 3-2

[0286] (Example 3) Characterization of HBBC epitopes BBC and the variants described herein were designed as glycan-binding monoclonal antibodies. Based on the published specificity data of the parental IMH2 (BBC) antibody (Ito et al., Cancer Res. 52:3739, 1992), it was hypothesized that the target epitope of the generated BBC antibody is related to the Le B and Le Y antigens. The following epitope characterization studies were performed using hBBC.10.1, which is also referred to as "hBBC" in the following examples and referenced figures.

[0287] Immunopurification GSL-derived glycans were isolated from the colorectal cancer cell line Colo-205 and immunopurified using BBC. The glycans in the unbound and eluted fractions were permethylated and profiled using MALDI-MS analysis as shown in Figures 4A and 4B. The glycan profile of the unbound fraction was similar to the input glycan profile, indicating that most GSL-derived glycans from COLO205 did not bind to BBC. However, in the eluted (BBC-bound) fraction, the Fuc4(LacNAc)3Lac glycan was purified exclusively by BBC. Specifically, surprisingly, the hBBC-purified glycan was Le B and Le Y Based on the observation of both fingerprint fragments, the biantenna-type Le B / Y (i.e., Le B / Le B , Le B / Le Y , or Le Y / Le Y ) was present, whereas the glycans in the unbound fraction had a linear structure, potentially Le B -Le A -Le A -Lac. These results indicated that the biantenna-type Le B / Y in the I antigen is an epitope of BBC.

[0288] This result was further supported by the immunopurification of BBC-binding glycans from GSL-derived glycans of the cell lines NCI-N87 and SW1116, which were selected for the glycolipid expression of the biantenna-type Le B / Y in the I antigen. As expected, BBC bound to the tetrafucosylated GSL-derived glycan Fuc4(LacNAc)3Lac rather than mono-, bi-, and trifucosylated glycans (Figures 5A - 5B). Through MSMS sequencing, the BBC-binding glycans of NCI-N87 and SW1116 GSLs were the I antigen-bearing biantenna-type Le B / Ywas confirmed (Figs. 6A and 6B).

[0289] In addition to Fuc4(LacNAc)3Lac, a group of GSL-derived glycans from NCI-N87 and SW1116 featuring multiple fucosylation (four fucose residues are the minimum requirement for binding by BBC) were purified. Two dominant BBC-binding glycans of NCI-N87 GSL, Fuc4(LacNAc)4Lac and Fuc6(LacNAc) 4→5 Lac were sequenced using MSMS and determined to be I antigen-bearing glycans with bi- or triantennary Le Y (Figs. 7A and 7B). Furthermore, when the released N-glycans from the AGS cell line were immunopurified with BBC, the enriched N-glycans were found to have a structure with bi- or triantennary Le Y (Figs. 8A and 8B). This result is consistent with the enriched I antigen.

[0290] These data indicate that Le B / Y is the binding unit of BBC. However, monoantennary Lewis B / Y is not sufficient to obtain strong binding to BBC. The unique I antigen and fully terminal fucosylated N-glycans provided multivalent Le B / Y which indicates that this is a strong binding epitope of BBC. Comparative immunopurification studies were also performed with hBBC and similar glycan enrichment specificities were shown (data not shown), which indicates that hBBC and BBC share similar epitopes.

[0291] Isothermal titration calorimetry Isothermal titration calorimetry (ITC) was performed to determine the binding affinities between hBBC and a series of linear glycans of Le Y -Gal, Le B -Gal, Le A -Gal, and Le X -Gal, as well as Le Y / Le Y -ASGP, Le X / Le X -ASGP, H-ASGP, Le Y / Le Y -I antigen, and Le Y / Le B -The binding affinity between the branched glycan of I antigen was analyzed. The BR96 antibody (described in US Patent No. 5,491,088 A; variant described in US Patent No. 5,792,456 A) was included as a control. Since the characterized glycans from the hBBC immunopurification experiments were present in limited amounts, various Le B and Le Y related glycans were obtained from a commercial source (Elycityl SA, Crolles, France) or enzymatically synthesized in-house according to established methods 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).

[0292] Briefly, for isothermal titration calorimetry (ITC), filtered PBS at pH 7.2 was prepared in-house and the same batch of buffer was used within one ITC injection. The mAb was pre-exchanged with filtered PBS buffer at pH 7.2 containing a protein concentration of 50 μM. Galactose was used as a standard for calculation for pulsed amperometric detection (HPAEC-PAD, e.g., Rothenhofer et al., 2015 J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 988:106) The amount of glycan antigen was quantified by high performance anion exchange in monosaccharide analysis. The quantified glycan antigen was dissolved in filtered PBS at pH 7.2 (the same batch as for mAb solution preparation) and 60 μL of the solution was used for each injection.

[0293] The ITC experiment was performed on a MicroCal iTC200 system (Malvern Instruments Ltd, Malvern, UK). After loading the mAb solution (50 μM) 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 sample cell filled with mAb. The experimental parameters were as follows: number of injections: 20; cell temperature: 25 °C; reference power: 6 μcal / s; initial delay: 60 s; sample cell concentration: 50 μM; stirring speed: 750 rpm, and the syringe concentration input with the measured glycan antigen concentration was used. The injection parameters were as follows: injection volume: 2 μL; duration: 4 s; interval: 150 s; filling period: 5 s, and the first injection volume was adjusted to 1 μL. After confirming all the settings, the experiment was started and the obtained data was processed by Origin for the iTC200. The titration curve was fitted using the One Set of Sites fitting model with 100 repetitions to obtain the best fitting result. As a result, K and K D were calculated.

[0294] Figures 9A, 9B, 10A - 10C, and 11A - 11F show the ITC titration graphs of various glycan antigens using the hBBC antibody. Figures 12A - 12C show the ITC titration graphs of various glycan antigens using the BR96 antibody. Briefly, the ITC analysis showed that hBBC had a higher specific binding affinity for Le B -Gal (KD = 26.2 μM) than for Le Y -Gal (KD = 80.6 μM). Furthermore, hBBC showed a stronger binding affinity for biantenna-type structures (Le Y / Le Y -ASGP, Le Y / Le Y -I antigen, and Le Y / Le B -I antigen) than for the single-chain Le Y -Gal antigen. Le Y / Le Y -ASGP, Le Y / LeY -I antigen and Le B / Le Y -Since the binding affinities of hBBC for the I antigen appeared to be similar, for the biantenna-type glycan epitope, Le Y and Le B side chains are suggested to have an equivalent contribution to specific binding. Furthermore, as shown in FIGS. 11A - F, glycan antigens lacking the tetrafucosylated LacNAc moiety did not show specific binding to hBBC, whether in the single-chain or biantenna-type form (Le X -Gal, Le A -Gal, H-antigen type I, H antigen type II, H-ASGP, and Le X / Le X -ASGP). These results indicate that the tetrafucosylated LacNAc having either type I or type II linkage is required for specific binding by hBBC. In summary, consistent with the immunopurification data, the ITC results showed that hBBC binds to epitopes containing structures with Le Y and / or Le B (Table 4; glycans are depicted as follows: white circle = Gal; filled circle = Man; filled square = GlcNac; closed triangle = Fuc). The BR96 control showed a similar but slightly higher binding affinity for single-chain Le Y / Le Y -I antigen and biantenna-type Le Y / Le Y -ASGP compared to biantenna-type Le Y -Gal (FIGS. 12A - 12C), suggesting that BR96 does not have specific selectivity between single-chain and biantenna-type Le Y glycans. Thus, hBBC has unique epitope specificity compared to BR96. Table 4. Results of isothermal titration calorimetry

Table 4-1

Table 4-2

[0295] These results partially characterize the hBBC epitope. However, the interaction between freely flowing hBBC and immobilized glycans provides additional information about the epitope; therefore, hBBC was tested with a series of immobilized glycans in surface plasmon resonance (SPR) and ELISA experiments. As shown in Table 5, the analysis surface coated with streptavidin and biotin-conjugated glycans (for SPR, Le Y -Gal-biotin, Le B -Gal-biotin, Le Y / Le Y -ASGA-biotin, and Le B / Le B -ASGA-biotin; for ELISA, Le Y -Gal-biotin, Le B -Gal-biotin, Le Y / Le Y -ASGA-biotin, Le B / Le B -ASGA-biotin, 3-Le Y / 6-Le B -ASGA-biotin, and 3-Le B / 6-Le Y -ASGA-biotin) were utilized (the same glycan depictions as in Table 4). Table 5. Structures and MW of glycan antigens used in SPR and ELISA

Table 5-1

Table 5-2

[0296] Surface plasmon resonance Le Y -Gal-biotin, Le B -Gal-biotin and Le Y / Le YThe binding affinity of hBBC for -ASGA-biotin was analyzed by surface plasmon resonance (SPR) on a chip coated with streptavidin. Briefly, Biacore T100 was used with HBS-EP+ buffer (GE Healthcare) as the running buffer. Biotinylated glycan was diluted to 10 pM and immobilized on a sensor chip SA (GE Healthcare) according to standard procedures. The glycan was immobilized for 60 s at a flow rate of 10 μL / min. The buffer of hBBC or BR96 was exchanged to HBS-EP+ buffer using a Zeba desalting spin column (7K MWCO, 0.5 mL, ThermoFisher) and serially diluted to 480, 240, 120, 60, and 30 nM with HBS-EP+ buffer. A single-cycle kinetic analysis was performed. The antibody was associated with the glycan for 150 s at a flow rate of 30 μL / min and dissociated for 300 s. The chip was regenerated with 2 M MgCl2 for 120 s at a flow rate of 50 μL / min. Data were evaluated using Biacore T200 evaluation software (GE Healthcare). A two-state reaction was used for curve fitting to obtain the best-fit values of ka, kd, and KD (Table 6). Figures 13A and 13B provide SPR sensorgrams showing the binding of hBBC and BR96 to various glycan antigens. Table 6. Summary of SPR results [Table 6]

[0297] SPR is the Biantenna-type Le Y / Le Y -ASGA-biotin showed a higher affinity for hBBC compared to single-chain Le Y -Gal-biotin, which was consistent with the results of ITC. In addition, the Biantenna-type Le B / Le B -ASGA-biotin showed a higher affinity for hBBC compared to single-chain Le B -Gal-biotin. This was also consistent with the trend observed for the Le Y antigen. hBBC is LeY -Gal-biotin compared to Le B -Gal-biotin showed a higher affinity, which was comparable to the results of ITC. SPR showed that hBBC was Le B / Le B -ASGA-biotin for Le Y / Le Y -ASGA-biotin also showed a higher binding affinity, which was consistent with the observation that hBBC had a higher affinity for the Le B base antigen than the Le Y base antigen. Le B / Le B -ASGA and Le Y / Le Y -ASGA. The comparable KD values of hBBC obtained from -ASGA further indicated that hBBC specifically associated with multiply fucosylated, i.e., divalent Lewis B or Lewis Y structures. Notably, similar KD values were obtained from the two antigens, but Le B / Le B -ASGA for hBBC response was stronger than the Le B -Gal response (sensorgram). Le B / Le B -ASGA is considered to be the structure with the highest affinity for hBBC.

[0298] By comparison, BR96 showed a slightly higher affinity for Le Y -Gal-biotin and Le Y / Le Y -ASGA-biotin, but when it became the structurally similar antigen Le Y / Le Y -ASGP, it was comparable to the results from ITC. Overall, the SPR data was in good agreement with the data from the ITC experiments.

[0299] Indirect ELISA To evaluate antibody binding affinity by ELISA, biotinylated glycan antigen was diluted to a sufficient concentration with PBS buffer. 100 μL of the diluted antigen solution was applied to a 96-well assay plate coated with streptavidin and incubated at 37 °C for 3.5 hours in a shaker. The plate was washed with PBST (0.05% Tween®-20 in PBS buffer) to remove excess glycan antigen. Primary antibody serially titrated with diluent (0.1% BSA in PBS buffer) was applied to the assay plate and incubated at 37 °C for 1 hour in a shaker. Following PBST washing, 100 μL of HRP-conjugated anti-human IgG antibody solution (SouthernBiotech, 1:15,000 dilution with diluent) was incubated at 37 °C for 1 hour in the assay plate in a shaker. After washing off the excess secondary antibody, 100 μL of TMB reagent was applied, incubated at 37 °C for 15 minutes, and subsequently quenched with 50 μL of 0.5 N HCl. VERSA The OD value was detected at 450 nm and the value at 650 nm was subtracted using a VERSAmax microplate reader (Molecular Devices). The data was processed with Softmax Pro (Molecular Devices).

[0300] Le Y / Le Y -ASGA vs Le Y -Gal The binding activities of hBBC and BR96 were tested on ELISA plates coated with Le Y -Gal-biotin and Le Y / Le Y -ASGA-biotin, and coated with streptavidin. The amount of coated antigen was as shown in FIGS. 14A and 14B. hBBC bound much more strongly to Le Y -Gal than to Le Y / Le Y -ASGA. BR96 bound much better to Le Y / Le Y -ASGA than to Le YAt the point of binding to α-Gal, it showed a pattern opposite to that of hBBC. In short, indirect ELISA provided binding affinity results similar to those of ITC and SPR experiments.

[0301] Le B / Le B -ASGA vs Le Y / Le Y -ASGA and Le B -Gal Next, hBBC was tested for binding to streptavidin-coated ELISA plates coated with Le B / Le B -ASGA-biotin, Le Y / Le Y -ASGA-biotin, and Le B -Gal-biotin. The amount of coating antigen was as shown in Figure 15. The results of indirect ELISA showed that hBBC had a much stronger binding to Le B / Le B -ASGA than to Le Y / Le Y -Gal and Le B -Gal.

[0302] Antigen-binding ELISA To evaluate the relative binding affinity of hBBC to different glycan antigens, an antigen-binding ELISA was developed. Briefly, hBBC was titrated serially and applied to streptavidin-functionalized 96-well assay plates coated with different glycan antigens. The binding of hBBC to the glycan antigens was detected using a human IgG-specific antibody conjugated to horseradish peroxidase (HRP), followed by color development in TMB reagent. The absorbance at 450 nm using a microplate reader was proportional to the amount of hBBC bound to the antigen. The relative affinity was determined by plotting the OD as a function of the hBBC concentration.

[0303] Antigen preparation Biotinylated Lewis Y pentasaccharide, Le Y-Gal-sp3-biotin was purchased from Elicityl (Crolles, 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) were synthesized via a series of enzymatic glycosylations followed by chemical biotinylation (Table 5). The biotinylated glycans were analyzed by thin-layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI-MS) to confirm that the purity reached at least 95% and that they contained no unconjugated biotin. HPAEC-PAD monosaccharide analysis was applied for the quantification of the biotinylated glycans.

[0304] antibody hBBC was used in the antigen-binding ELISA.

[0305] ELISA The biotinylated glycan antigen was diluted to 18.7 nM with 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 h. The plate was washed with PBST (0.05% Tween®-20 in PBS buffer) to remove the excess glycan antigen. Primary antibodies serially titrated with diluent (0.1% BSA in PBS buffer) were applied to the assay plate and incubated in a shaker at 37 °C for 1 h. Following PBST washes, 100 μL of HRP-conjugated anti-human IgG antibody solution (SouthernBiotech, 1:10,000 dilution with diluent) was incubated in the assay plate in a shaker at 37 °C for 1 h. After washing away the 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 0.5 N HCl. OD values were detected at 450 nm and the values at 650 nm were subtracted using a VERSA max microplate reader (Molecular Devices). The data were processed with Softmax Pro (Molecular Devices).

[0306] Results Data are shown in Fig. 16A. In the antigen-binding ELISA, the OD values increased within a narrow range of low antibody concentrations (0.4 - 4 μg / mL) when hBBC bound to 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. In contrast, the binding of hBBC to Le Y -Gal and Le B -Gal appeared to be weaker, which may be due to the divalent Le Y , Le B , Le B / Le Y , or Le Y / LeB resulted in a higher binding affinity to hBBC than that brought about by a single Lewis Y / B moiety, indicating that.

[0307] Direct comparison of antigen binding affinities: hBBC.10.1 vs BBC The aim of the experiment was to compare the antigen affinity of hBBC.10.1 with that of the parental BBC antibody by antigen-binding ELISA.

[0308] Antigen preparation Biotinylated Lewis Y pentasaccharide, Le Y -Gal-sp3-biotin was purchased from Elicityl. Lewis B pentasaccharide 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) were synthesized via a series of enzymatic glycosylations followed by chemical biotinylation (Table 5). The biotinylated glycans were analyzed by TLC and ESI-MS to confirm that the purity reached at least 95% and that they contained no unconjugated biotin. HPAEC-PAD monosaccharide analysis was applied for quantification of the biotinylated glycans.

[0309] Antibodies BBC and hBBC.10.1 were used in the antigen-binding ELISA.

[0310] Antigen-binding ELISA A 96-well EvenCoat™ streptavidin microplate (R&D Biosystems, MN, catalog number CP004) was incubated overnight at 4°C with various biotinylated glycans in PBS at a quantity of 3.73 pmol / well. After washing three times with PBS / 0.05% Tween® 20 (Sigma, catalog number P1379-500mL), 100 μL of diluted BBC or hBBC.10.1 antibody was added to the wells coated with antigen and then incubated at 37°C for 1 hour. The wells were washed three times with PBS / 0.05% Tween® 20 and subsequently incubated at 37°C for 1 hour with 100 μL of 10,000-fold diluted mouse anti-human IgG (Fc)-HRP (Southern Biotech, catalog number 9040-05). After washing, 100 μl of SureBlue™ Reverse TMB (KPL, catalog number 53-00-03) was added at 37°C for 15 minutes for color development. The reaction was stopped by adding 0.5N HCl. Absorbance was read at a wavelength of 450 nm using a reference at 650 nm with a VERSAMAX™ microplate reader (Molecular Devices, San Jose, CA), and the data was processed with SoftmaxPro™ software (Molecular Devices).

[0311] Results: The antigen-binding ELISAs of BBC (Figure 16B) and hBBC.10.1 (Figure 16C) showed that hBBC.10.1 had a lower affinity for the single-chain glycan antigens (Le B -Gal and Le Y -Gal) compared to BBC, and that both antibodies had a higher affinity for the biantennary-type glycan antigens (Le B / Le B -ASGA, Le Y / Le Y -ASGA, 3-Le Y / 6-Le B -ASGA, and 3-Le B / 6-Le Y-ASGA), indicating high affinity. From these data, it was suggested that hBBC.10.1 had better binding selectivity between single-chain antigens and biantenna antigens than BBC.

[0312] Summary From the epitope characterization experiments described in this example, hBBC specifically bound to biantenna and triantenna Le Y / B I antigens from cancer cell lines, as well as to biantenna and triantenna Le Y N-glycans. Furthermore, the epitope recognized by hBBC contained a difucosylated LacNAc backbone. Biantenna Le B / Le B -ASGA was the antigen with the strongest binding affinity to hBBC. Biantenna Le Y / Le Y -ASGA, Le Y / Le B -ASGA, and Le B / Le Y -ASGA showed high affinity for hBBC compared to single-chain Le B -Gal and Le Y -Gal antigens. Also, antigens based on Le B alone showed higher affinity for hBBC than antigens based on Le Y alone, regardless of whether the antigen had a single-chain structure or a biantenna structure. Finally, the binding behavior (dynamics) and epitope of hBBC were different from those of BR96.

[0313] (Example 4) Internalization of hBBC in target cells 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 contain cleavable linkers that are cleaved in the lysosomal compartment after internalization, thereby selectively releasing the drug and having the additional benefit of increasing the stability of the drug in serum.

[0314] To test whether hBBC was effectively internalized into cancer cells, the following experiment was performed. First, in a conventional internalization assay, hBBC antibody was added to AGS gastric cancer cells in culture, and binding was carried out at 4 °C for 1 hour to allow specific antibody / receptor interactions to occur while stopping endocytosis. Next, non-specifically bound antibody was washed away, and the cells were transferred to 37 °C to allow normal endocytosis to occur. At 0 and 30 minutes, the cells were fixed and hBBC was detected using an Alexa488-conjugated anti-human IgG antibody (Figure 17, left panel). F-actin was labeled with phalloidin rhodamine (right panel; co-distributed staining appeared as a yellow fluorescence signal). As shown in Figure 17, hBBC stained the cell membrane at 0 minutes and then underwent internalization and, after incubation at 37 °C (30 minutes, permeabilized cells), localized to vesicles around the nucleus in the cytosol. The cytosolic signal was further demonstrated by non-permeabilized controls that could detect very little membrane signal (30 minutes, non-permeabilized cells). These data indicate that hBBC is effectively internalized into AGS cells within 30 minutes.

[0315] In the second experiment, the real-time intracellular localization of hBBC was examined. Briefly, hBBC antibody was 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 an Alexa488-conjugated anti-human IgG antibody (anti-human IgG shown in the left panel of Figure 18). Lysosomes were labeled with an anti-Lamp-1 antibody and subsequently with an anti-rabbit IgG antibody (shown in the central panel). As shown in Figure 18, after 4 or 8 hours of incubation, hBBC co-localized with Lamp-1 (merge; right panel), indicating that internalized hBBC is localized in the lysosomal compartment. Furthermore, hBBC can be stabilized in the lysosomal compartment without apparent degradation for at least 8 hours. These results indicate that hBBC has utility for use in antigen-targeted conjugates, such as ADCs and the like.

[0316] (Example 5) Immunostaining of hBBC in human tissue samples Samples of healthy and cancerous tissues were obtained and immunostaining with hBBC10.1 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. Table 7. hBBC.10.1 immunostaining of various human normal tissues [Table 7-1] [Table 7-2]

[0317] Table 8. hBBC immunostaining in human cancerous tissues [Table 8]

[0318] In addition, various human cancer cell lines were stained with hBBC.10.1 to determine the epitope expression pattern. Table 9 summarizes the results. Table 9. hBBC Epitope Expression in Human Cancer Cell Lines

Table 9

[0319] (Example 6) Antitumor Activity of hBBC in a Xenograft Model of Colorectal Cancer A series of in vivo animal studies were conducted to evaluate the tumor inhibitory effect of hBBC in a xenograft SCID model. The studies included introducing human cancer cells derived from the gastric cancer cell line AGS, TSGH9201, and the colorectal cancer cell lines COLO201, COLO205, DLD-1. hBBC.10.1 showed strong to moderate binding levels to all cell lines except COLO205 (Table 9 in Example 5). hBBC.10.1 was used in all xenograft studies, and BR96 was used as a control.

[0320] In vivo Antitumor Activity of hBBC.10.1 in the DLD-1 and COLO205 Xenograft Models vivo The purpose of this study was to examine the tumor inhibitory effect of high-dose hBBC.10.1 in the DLD-1 and COLO205 xenograft models. Briefly, the human colorectal cancer cell lines DLD-1 and COLO205 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), and the cell cultures were maintained at 37°C in a humidified incubator under a 5% CO2 atmosphere. Cells at passages 5 - 8 were used for tumor inoculation.

[0321] Specific pathogen-free (SPF) female CB17 severe combined immunodeficiency (SCID) mice were purchased from BioLASCO (Taiwan) and allowed to acclimate for at least 1 week before any experimental procedures. The mice were housed in individually ventilated cages (IVCs) in a temperature-controlled environment (22 ± 2 °C) with 50 ± 10% humidity under a 12:12 hour light-dark cycle. All experiments were performed in accordance with the regulations and animal protection laws defined by the Taiwan Agricultural Council.

[0322] Cancer cells were resuspended in ice-cold serum-free medium containing 50% BD Matrigel (catalog number 354248) at a cell density of 5 × 10 6 / 200 μL and subcutaneously injected into the flank region of 6 - 8-week-old SCID mice. Tumor size was measured weekly with calipers (Laser Tools and Technics (LTT), Hsinchu City, Taiwan, 150 × 0.05 mm), and tumor weight was estimated as "weight in 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 nine groups (n = 6 per group) with equivalent tumor sizes in each group, and antibody treatment was initiated. SCID mice with tumors were intraperitoneally injected with hBBC.10.1 (lot: 17001) at 50 mg / kg once a week for 6 weeks. SCID mice with tumors injected intraperitoneally with saline were used as negative controls.

[0323] The results from the DLD-1 and COLO205 xenograft experiments are shown in Figures 19A and 19B, respectively. hBBC.10.1 was able to effectively inhibit DLD-1 tumor growth but not COLO205 tumor growth compared to the control. These data are consistent with the observed ability of hBBC.10.1 to bind to DLD-1 rather than COLO205 cells.

[0324] Anti-tumor activity of hBBC in a xenograft model of gastric adenocarcinoma The purpose of this study was to compare the antitumor efficacy of hBBC.10.1 and BR96 at low doses in the range of 0.008 - 1 mg / kg in an AGS xenograft model. Briefly, 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 cultures were maintained at 37°C in a humidified incubator under a 5% CO2 atmosphere. AGS cells at passages 5 - 8 were used for tumor inoculation.

[0325] Specific pathogen-free (SPF) female CB17 severe combined immunodeficient (SCID) mice were purchased from BioLASCO (Taiwan) and allowed to acclimatize for at least 1 week prior to any experimental procedures. The mice were housed in individually ventilated cages (IVCs) in a temperature-controlled environment (22 ± 2°C) with 50 ± 10% humidity under a 12:12 hour light-dark cycle. All experiments were carried out in accordance with the regulations and animal protection laws set by the Taiwan Agricultural Council.

[0326] AGS cells were resuspended in ice-cold serum-free medium containing 50% BD Matrigel (catalog number 354248) at a cell density of 5×10 6 / 200 μL and subcutaneously injected into the flank region of 6 - 8-week-old SCID mice. Tumor size was measured weekly with calipers (Laser Tools and Technics (LTT), Hsinchu City, Taiwan, 150×0.05 mm), and tumor weight was calculated as "weight in mg = (width 2 × length) mm 3Estimated as " / 2" [Ito et al. (1992) Cancer Res. 52:3739]. When the tumor weight reached 150 - 200 mg, the mice were randomly divided into nine groups (n = 6 per group) with each group having an equivalent tumor size, and antibody treatment was initiated. SCID mice bearing tumors were intraperitoneally injected once a week for 6 weeks with either hBBC.10.1 (lot: 17001) or BR96 (lot: 17001) at 1, 0.2, 0.04, or 0.008 mg / kg. The first dose was given at 1.5 times the predetermined dose. SCID mice bearing tumors injected with saline were used as negative controls.

[0327] The results for mice treated with hBBC.10.1 and BR96 are shown in Figures 20A and 20B, respectively. When the doses of 1 and 0.2 mg / kg were administered weekly, both hBBC.10.1 and BR96 significantly inhibited tumor growth compared to the control. However, neither antibody was able to show an inhibitory effect at the lower doses (0.04 and 0.008 mg / kg). Due to ethical considerations, mice were sacrificed when the tumor burden exceeded 10% of the body weight. Statistical analysis was performed only on the data generated up to day 60 since some mice that received saline or the lower doses of antibody reached this endpoint by 60 days post - inoculation. In contrast, in mice bearing AGS tumors administered the higher doses (1 and 0.2 mg / kg) of hBBC.10.1 or BR96, tumor growth to 10% of the body weight was effectively delayed. This dose - response study indicates that hBBC.10.1 has anti - tumor efficacy equivalent to BR96 in mice bearing AGS tumors.

[0328] The anti - tumor efficacy of hBBC.10.1 was compared with that of other generated hBBC antibodies of the present disclosure. In one experiment, the collected AGS cells were washed twice with PBS and resuspended in PBS containing 25% BD Matrigel™ (BD Biosciences, catalog number 354248) at 5×10 6It was resuspended at a cell density of / 200 μL. Subsequently, 200 μL of the AGS cell suspension was subcutaneously injected into the flank region of female SCID mice (6 - 8 weeks old) (5×10 6 cells / mouse). Tumor size was measured weekly with calipers (Laser, 150×0.05 mm), and tumor weight was estimated as "weight in 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 six groups (n = 6 per group) with each group having an equivalent tumor size, and antibody treatment was initiated. SCID mice bearing tumors were intraperitoneally injected 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 of 2 mg / kg. SCID mice bearing tumors injected intraperitoneally with saline were used as negative controls. The data are shown in Figure 20C. Antitumor effects were observed with hBBC.8, hBBC.9, and hBBC.9.1, but the effects were slightly weaker than those of hBBC.10.1. Antitumor activity equivalent to that of hBBC.10.1 (0.25 mg / kg) was observed with hBBC.8 at a higher dose (2 mg / kg).

[0329] hBBC.10.1 was also compared with hBBC.10.1FQ regarding antitumor activity. Briefly, the collected AGS cells were washed twice with PBS and resuspended at a cell density of 5×10 6 / 200 μL in PBS containing 25% BD Matrigel (trademark) (catalog number 354248). Subsequently, 200 μL of the AGS cell suspension was subcutaneously injected into the flank region of female SCID mice (6 - 8 weeks old) (5×10 6 cells / mouse). Tumor size was measured weekly with calipers (Laser, 150×0.05 mm), and tumor weight was estimated as "weight in mg = (width 2 × length) mm 3Estimated as "[Hisashi Ito et al. (1992)]. When the tumor weight reached 150 - 200 mg, the mice were randomly divided into three groups (n = 8 per group) with each group having an equivalent tumor size, and antibody treatment was initiated. SCID mice bearing tumors were intraperitoneally injected once a week for 6 weeks with either hBBC.10.1 or hBBC.10.1FQ at a dose of 0.25 mg / kg. The first dose was given at 1.5 times the predetermined dose. SCID mice bearing tumors injected with saline were used as negative controls. The data (Figure 20D) indicate that hBBC.10.1FQ had slightly stronger antitumor activity.

[0330] In subsequent experiments, xenograft AGS (xAGS) cells isolated from AGS xenograft tumors were shown to express at least twice as many hBBC epitopes as AGS cell line cells when compared (data not shown). xAGS cells also induced more potent ADCC and CDC activities by hBBC when compared to the parental cell line (data not shown). hBBC had a somewhat weaker direct killing effect (PI staining) against target xAGS cells compared to BR96, as shown in Figure 21.

[0331] Antitumor activity of hBBC in a xenograft model of gastric cancer The aim of this study was to compare the antitumor efficacy of hBBC.10.1 and BR96 at low doses in the range of 0.04 - 10 mg / kg in the TSGH9201 xenograft model. Briefly, the human gastric cancer cell line TSGH9201 (catalog number 60146) was obtained from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan). Twice fluorescence-activated cell sorting (FACS) (BD FACSJAZZ (trademark) cell sorter, BD Biosciences, Singapore, catalog number 655486), followed by the use of anti-hBBC (lot: B24), and then staining with 200-fold diluted fluorescein (FITC)-AffiniPure (trademark) goat anti-human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch Inc., West Grove, PA, catalog number 109-095-098) were used to enrich TSGH9201 cells with a higher level of hBBC epitope expression. Compared with the parental TSGH9201 cells, the expression level of the hBBC epitope was enriched approximately 4-fold in the enriched cells named TSGH9201(2s) (not shown). Both the parental and enriched cells were grown in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate. The cell cultures were maintained at 37 °C in a humidified incubator under a 5% CO2 atmosphere. TSGH9201(2s) cells at passages 5 - 8 were used for tumor inoculation.

[0332] Specific pathogen-free (SPF) female CB17 severe combined immunodeficiency (SCID) mice were purchased from BioLASCO (Taiwan) and allowed to acclimate for at least 1 week before any experimental procedures. The mice were housed in individually ventilated cages (IVCs) in a temperature-controlled environment (22 ± 2°C) with 50 ± 10% humidity under a 12:12-hour light-dark cycle. All experiments were performed in accordance with the regulations and animal protection laws set by the Taiwan Agricultural Council.

[0333] TSGH9201(2s) cells were resuspended in ice-cold serum-free medium containing 25% BD Matrigel (catalog number 354248) at a cell density of 5×10 6 / 200 μL, and 200 μL of the cell suspension was subcutaneously injected into the flank region of 6- to 8-week-old SCID mice. Tumor size was measured weekly with calipers (Laser Tools and Technics (LTT), Hsinchu City, Taiwan, 150×0.05 mm), and tumor weight was estimated as "weight in 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 three groups (n = 5 per group) with equal tumor sizes, and antibody treatment was initiated. To evaluate the in vivo efficacy of hBBC.10.1 and BR96, SCID mice bearing tumors were intraperitoneally injected with either hBBC.10.1 (lot: B24) or BR96 (lot: T05) at 10 mg / kg once a week for 6 weeks. The first dose was given at 1.5 times the indicated dose. In a second study to compare the antitumor activities of hBBC.10.1 and BR96 at lower doses, SCID mice bearing tumors were intraperitoneally injected with either hBBC.10.1 (lot: 17001) or BR96 (lot: 17001) at 10, 1, 0.2, or 0.04 mg / kg once a week for 6 weeks. The first dose was given at 1.5 times the indicated dose. SCID mice bearing tumors injected intraperitoneally with saline were used as negative controls.

[0334] Figure 22 shows the results. Both hBBC.10.1 and BR96 significantly inhibited tumor growth at a weekly dose of 10 mg / kg compared to the saline group.

[0335] Antitumor activity of hBBC.10.1 in a xenograft model of colorectal adenocarcinoma The objectives of this study were: 1) to examine the ability of hBBC.10.1 to inhibit the tumor growth of COLO201 xenografts; and 2) to compare the antitumor activities of hBBC and BR96 at doses ranging from 0.008 to 1 mg / kg in the COLO201 xenograft model.

[0336] Briefly, the human colorectal adenocarcinoma cell line COLO201 (CCL-224) derived from ascites was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). COLO201 cells were grown in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate. The cell cultures were maintained in a humidified incubator at 37 °C under a 5% CO2 atmosphere. COLO201 cells at passages 5 - 8 were used for tumor inoculation.

[0337] Specific pathogen-free (SPF) female CB17 severe combined immunodeficient (SCID) mice were purchased from BioLASCO (Taiwan) and acclimated for at least 1 week before any experimental procedures. The mice were housed in individually ventilated cages (IVCs) in a temperature-controlled environment (22 ± 2 °C) with 50 ± 10% humidity under a 12:12-hour light-dark cycle. All experiments were performed in accordance with the regulations and animal protection laws defined by the Taiwanese Council of Agriculture.

[0338] COLO201 cells were resuspended in ice-cold serum-free medium at a cell density of 2×10 6 / 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 calipers (Laser, 150 × 0.05 mm), and tumor weight was calculated as "weight in mg = (width2 × length) mm 3 Estimated as “ / 2” [Ito et al. (1992) Cancer Res. 52:3739]. When the tumor weight reached 150 - 200 mg, the mice were randomly divided into a control group and a treatment group (n = 6 per group) with each group having an equivalent tumor size, and antibody treatment was initiated. Two studies were performed to evaluate the in vivo efficacy of hBBC against COLO201 tumor growth.

[0339] In the first study, SCID mice bearing tumors were intraperitoneally injected with hBBC.10.1 (lot: B26) at doses ranging from 2 - 50 mg / kg twice a week for 6 weeks. The first dose was given at 1.5 times the indicated dose. Figure 23 shows the results from the first study. Treatment with hBBC.10.1 at 2 - 50 mg / kg significantly inhibited tumor growth compared to the saline group. The tumors rapidly shrank and ultimately disappeared within just 1 week after hBBC administration, i.e., 2 weeks after tumor inoculation.

[0340] In a second study, a further comparison of the anti-tumor activities of hBBC.10.1 and BR96 at lower doses was performed. SCID mice bearing tumors were intraperitoneally injected once a week for 6 weeks (starting on day 7) with either hBBC.10.1 (lot: 17001) or BR96 (lot: 17001) at 1, 0.2, 0.04, and 0.008 mg / kg. The first dose was given at 1.5 times the indicated dose. SCID mice bearing tumors injected with saline were used as a negative control. The purified antibody hTKH2.2 (lot: 1020429) was included as a negative control antibody, which is a humanized anti-STn antibody produced in-house by transient expression in HEK293 cells. Figures 24A and 24B show the results from the second study. Both hBBC.10.1 and BR96 significantly inhibited tumor growth at weekly doses of 1 or 0.2 mg / kg compared to the saline and hTKH2.2 control groups, but lower doses (0.04 and 0.008 mg / kg of hBBC.10.1; 0.04 mg / kg of BR96) showed an effect on delaying tumor growth. Due to ethical considerations, mice were sacrificed when the tumor burden became greater than 10% of body weight. Some mice receiving saline or lower doses of hBBC.10.1 or BR96 reached this endpoint at 49 days post-inoculation, so statistical analysis was performed only on the data obtained up to day 49.

[0341] This dose-response study shows that hBBC.10.1 has anti-tumor efficacy equivalent to BR96 in mice bearing COLO201 tumors.

[0342] (Example 7) Immunostaining of hBBC in Primate and Human Tissues hBBC.10.1 immunostaining was performed on corresponding healthy tissues from human and cynomolgus monkey (Macaca fascicularis). Briefly, using formalin-fixed paraffin-embedded tissue sections, staining was performed with 2 μg / ml of hBBC.10.1 according to a standard protocol. The results are summarized in Table 10 and showed similar staining patterns between human and cynomolgus monkey tissues. Table 10. hBBC.10.1 Immunostaining of Human and Cynomolgus Monkey Tissues [Table 10]

[0343] (Example 8) Safety and Tolerability Study of hBBC.10.1 in Primate Models To evaluate the tolerability and acceptable dose range of hbbc.10.1 after a single intravenous (iv) bolus injection in cynomolgus monkeys, a single-dose tolerability and dose range finding study (non-GLP) was performed. Briefly, male and female cynomolgus monkeys (Macaca fascicularis) were assigned to four groups with one male and one female in each group. Animals in groups 1 - 4 received dose levels of hbbc.10.1 of 0, 50, 200, and 300 mg / kg, respectively. Animals in groups 2 and 3 were dosed once via a slow i.v. bolus injection over a duration of at least 5 minutes. Animals in groups 1 and 4 were dosed once by a 20-minute (±1 minute) i.v. infusion using a pump and primed infusion line. Approximately 24 hours after dosing, necropsy was performed. During necropsy, overall observations and organ weights were recorded and tissues were collected for histopathology. A portion of the collected tissues was processed into slides and examined microscopically.

[0344] From the results, it was shown that hBBC.10.1 was well tolerated in cynomolgus monkeys in the dose range of 50 - 300 mg / kg. 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. Changes related to other possible test articles were chronic and acute inflammation in the stomach of one animal receiving 200 mg / kg, neutrophil infiltration in the crypts of the duodenum 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).

[0345] Additional embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the application data sheet are hereby incorporated by reference in their entirety. Aspects of the embodiments can be modified as necessary so that the concepts of the various patents, applications, and publications are used to provide further embodiments.

[0346] These and other variations can be made into embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in this specification and in the claims, but rather the claims are to be construed to include all possible embodiments together with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure. The present invention provides, for example, the following items. (Item 1) An immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 35; and an immunoglobulin light chain variable region comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 5, wherein the antibody or antigen-binding fragment thereof bivalent antenna-type Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B to the antigen bivalent antenna-type Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y to the antigen bivalent antenna-type 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 to the antigen and bivalent antenna-type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B to the antigen is capable of specifically binding, the antibody or antigen-binding fragment thereof also binds specifically to a monoantennary-type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX] x and to a bivalent antenna-type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] xThe antigen also includes a monoantennary type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A An isolated antibody or antigen-binding fragment thereof that does not specifically bind to the antigen, nor to the monoantennary type H antigen 2 containing Fucα1-2Galβ1-4GlcNAc[XII], nor to the biantennary type H antigen 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], nor to the monoantennary type H antigen 1 containing Fucα1-2Galβ1-3GlcNAc[XIV]. (Item 2) (a) A heavy chain complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 2; a heavy chain complementarity-determining region 2 (VH CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 3; a heavy chain complementarity-determining region 3 (VH CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 4, and (b) The light chain complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 6; the light chain complementarity-determining region 2 (VL CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 7; and the light chain complementarity-determining region 3 (VL CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 8, of an immunoglobulin light chain variable region The isolated antibody or antigen-binding fragment thereof according to item 1, comprising (Item 3) One of the following (i), (ii), (iii), (iv), (v), or (vi): (i) VH CDR1 comprising a variant of the amino acid sequence set forth in SEQ ID NO: 2, wherein the mutation consists of a Y→A substitution at position 33 according to Kabat numbering, VH CDR1; and / or (ii) VH CDR3 comprising a variant of the amino acid sequence set forth in SEQ ID NO: 4, wherein the mutation consists of a Y→A substitution at position 104 according to Kabat numbering, VH CDR3; (iii) VH CDR3 comprising a variant of the amino acid sequence set forth in SEQ ID NO: 4, wherein the mutation consists of an H→A substitution at position 106 according to Kabat numbering, VH CDR3; (iv) VL CDR1 comprising a variant of the amino acid sequence set forth in SEQ ID NO: 6, wherein the mutation consists of a Y→A substitution at position 30 according to Kabat numbering, VL CDR1; (v) VL CDR2 comprising 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 according to Kabat numbering, VL CDR2; or (vi) A VL CDR3 comprising a variant of the amino acid sequence set forth in SEQ ID NO: 8, wherein the mutation consists of a T→S substitution at position 93 according to Kabat numbering, or an isolated antibody or antigen-binding fragment thereof according to item 1 or 2, comprising any combination thereof. (Item 4) (a) An immunoglobulin heavy chain variable region comprising a heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2; a heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 4; and (b) a light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 6; a light chain complementarity determining region 2 (VL CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 7; and a light chain complementarity determining region 3 (VL CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 8, an isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof bivalent antenna-type Le containing Fuc4(Galβ1→3GlcNAc)2 [I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2 [II] B / Le B to the antigen, bivalent antenna-type Le containing Fuc4(Galβ1→4GlcNAc)2 [III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2 [IV] Y / Le Y to the antigen, bivalent antenna-type 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 to the antigen, and a biantenna-type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B antigen, is capable of specifically binding to, the antibody or an antigen-binding fragment thereof, a monoantenna-type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX] x antigen, and a biantenna-type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] x antigen, a monoantenna-type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A antigen, a monoantenna-type H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], a biantenna-type H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], and a monoantenna-type H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV], and is an isolated antibody or an antigen-binding fragment thereof that does not specifically bind to them. (Item 5) an isolated antibody or an antigen-binding fragment thereof containing an immunoglobulin heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 35; and an immunoglobulin light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 5, wherein the antibody or an antigen-binding fragment thereof a biantenna-type Le containing Fuc4(Galβ1→3GlcNAc)2[I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2[II] B / Le B antigen, a biantenna-type Le containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV] Y / Le Y antigen, A bivalent Le antigen 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 an antibody or antigen-binding fragment thereof that can specifically bind to a bivalent Le antigen containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B antigen, and does not specifically bind to a monovalent Le antigen containing Galβ1→4(Fucα1→3)GlcNAc[IX], a bivalent Le antigen containing [Galβ1→4(Fucα1→3)GlcNAc]2[X], a monovalent Le antigen containing Galβ1-3(Fucα1-4)GlcNAc[XI], a monovalent H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], a bivalent H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monovalent H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV]. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to a bivalent Le antigen containing Fuc2(Galβ1→3GlcNAc)[Fuc2(Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI] x antigen, and also binds specifically to a monovalent Le antigen containing Galβ1→4(Fucα1→3)GlcNAc[IX], a bivalent Le antigen containing [Galβ1→4(Fucα1→3)GlcNAc]2[X], a monovalent Le antigen containing Galβ1-3(Fucα1-4)GlcNAc[XI], a monovalent H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], a bivalent H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monovalent H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV]. x An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to a bivalent Le antigen containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] A antigen, and also binds specifically to a monovalent Le antigen containing Galβ1→4(Fucα1→3)GlcNAc[IX], a bivalent Le antigen containing [Galβ1→4(Fucα1→3)GlcNAc]2[X], a monovalent Le antigen containing Galβ1-3(Fucα1-4)GlcNAc[XI], a monovalent H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], a bivalent H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], or a monovalent H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV]. (Item 6) An isolated antibody comprising an immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 10 and an immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 11. (Item 7) The isolated antibody according to Item 6, which is monoclonal. (Item 8) The isolated antibody according to any one of Items 1 to 5, or an antigen-binding fragment thereof, which is monoclonal. (Item 9) The isolated antibody according to item 6 or 7, which is a humanized antibody, or the isolated antibody according to any one of items 1 to 5 or 8, or an antigen-binding fragment thereof. (Item 10) An isolated antibody according to any one of items 1 to 5, 8, or 9, or an antigen-binding fragment thereof, selected from a Fab fragment, an F(ab’)2 fragment, an Fv fragment, a single-chain Fv (ScFv) antibody, and a diabody. (Item 11) An isolated polynucleotide encoding the antibody according to any one of items 1 to 10, or an antigen-binding fragment thereof. (Item 12) The isolated polynucleotide according to item 11, which is codon-optimized for expression in a host cell. (Item 13) A recombinant vector comprising the polynucleotide according to item 11 or item 12. (Item 14) The recombinant vector according to item 13, comprising an expression control sequence operably linked to the polynucleotide encoding the antibody or an antigen-binding fragment thereof. (Item 15) The recombinant vector according to item 14, wherein the expression control sequence is an expression vector comprising a promoter. (Item 16) A host cell comprising the recombinant vector according to item 13. (Item 17) A host cell comprising the expression vector according to item 15. (Item 18) A method for producing an antibody or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof is against a biantenna-type Le B / Le B antigen, is against a biantenna-type Le Y / Le Y antigen containing Fuc4(Galβ1→4GlcNAc)2[III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2[IV], A biantenna type 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 a biantenna type Le containing Fuc2(Galβ1-4GlcNAc)[Fuc2(Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] Y / Le B antigen is capable of specifically binding to, wherein the antibody or an antigen-binding fragment thereof binds specifically to a monoantenna type Le containing Galβ1→4(Fucα1→3)GlcNAc[IX] x antigen, also to a biantenna type Le containing [Galβ1→4(Fucα1→3)GlcNAc]2[X] x antigen, also to a monoantenna type Le containing Galβ1-3(Fucα1-4)GlcNAc[XI] A antigen, also does not specifically bind to a monoantenna type H antigen type 2 containing Fucα1-2Galβ1-4GlcNAc[XII], nor to a biantenna type H antigen type 2 containing (Fucα1-2Galβ1-4GlcNAc)2[XIII], nor to a monoantenna type H antigen type 1 containing Fucα1-2Galβ1-3GlcNAc[XIV], the method is culturing the host cell according to item 16 under conditions and for a time sufficient for expression of the polynucleotide encoding the antibody or an antigen-binding fragment thereof by the host cell, thereby obtaining a culture containing the antibody or an antigen-binding fragment thereof; and recovering the antibody or an antigen-binding fragment thereof from the culture A method comprising (Item 19) An isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 10; and an antibody conjugate comprising a payload molecule linked thereto. (Item 20) An isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 10, or an antibody conjugate according to item 19 or items 21 to 35; and a pharmaceutical composition comprising a pharmaceutical carrier. (Item 21) The antibody conjugate according to item 19, wherein the payload molecule is covalently linked to the antibody or antigen-binding fragment thereof by a linker. (Item 22) The antibody conjugate according to item 21, wherein the linker is selected from a cleavable linker and a non-cleavable linker. (Item 23) The antibody conjugate according to item 22, wherein the cleavable linker is a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. (Item 24) The antibody conjugate according to item 23, wherein the cleavable linker is a protease-sensitive linker comprising a valine-citrulline peptide. (Item 25) The antibody conjugate according to any one of items 22 to 24, wherein the linker contains a maleimide group. (Item 26) The antibody conjugate according to any one of items 22 to 25, wherein the antibody or antigen-binding fragment thereof contains a reduced disulfide bridge in the hinge region, and the reduced disulfide bridge is coupled to the maleimide group. (Item 27) The antibody conjugate according to any one of items 22 to 26, wherein the linker further contains a self-destructive group. (Item 28) The antibody conjugate according to item 27, wherein the self-destructive group is para-aminobenzyl alcohol (PABC). (Item 29) The antibody conjugate according to any one of items 19 or 21 to 28, wherein the payload molecule is selected from a therapeutic agent and a detectable indicator. (Item 30) The antibody conjugate according to item 29, wherein the payload molecule is a therapeutic agent selected from a tubulin-targeting anti-mitotic agent, a peptide-based toxin, a pyrrolobenzodiazepine (PBD) dimer, an antibiotic, a pyrimidine synthesis inhibitor, an antimetabolite, a DNA alkylating agent, and a topoisomerase inhibitor. (Item 31) The antibody conjugate according to item 30, wherein the payload molecule is selected from maytansinoid, auristatin, doxorubicin, calicheamicin, PBD dimer, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF). (Item 32) The antibody conjugate according to item 29, wherein the payload molecule is a detectable indicator. (Item 33) The antibody conjugate according to item 32, wherein the detectable indicator is selected from a radionuclide, a dye, a radioactive metal, a fluorescent moiety, an MRI contrast agent, a microbubble, a carbon nanotube, a gold particle, fluorodeoxyglucose, an enzyme, a chromophore, and a radiopaque marker. (Item 34) The detectable indicator is 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 The antibody conjugate according to item 33, which is a radionuclide selected from T. (Item 35) The antibody conjugate according to any one of item 33 or item 34, further comprising a chelating agent for a radionuclide selected from DOTA labeled with maleimide, N-hydroxysuccinimide-DOTA, and desferrioxamine (DFO). (Item 36) A method for treating or detecting cancer, comprising administering the pharmaceutical composition according to item 20 to a subject in need thereof. (Item 37) The method according to item 36, wherein the subject has or is suspected of having cancer selected from gastric cancer, colon cancer, breast cancer, lung cancer, lymphoma, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, uterine cancer, and squamous cell carcinoma. (Item 38) The method according to item 37, wherein the cancer is selected from gastric adenocarcinoma, mucinous gastric adenocarcinoma, undifferentiated gastric adenocarcinoma, signet ring cell gastric carcinoma, colon adenocarcinoma, invasive ductal carcinoma of the breast, hepatocellular carcinoma, lung adenocarcinoma, squamous cell carcinoma, metastatic lymph node adenocarcinoma, mucinous ovarian adenocarcinoma, pancreatic ductal adenocarcinoma, papillary adenocarcinoma of the pancreas, prostate adenocarcinoma, and endometrial carcinoma. (Item 39) The method according to any one of items 36 to 38, wherein the administration comprises administration by a route selected from intravenous, parenteral, intragastric, intrathoracic, intratracheal, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, oral, topical, transdermal, intracapsular, intrathecal, intranasal, and intramuscular. (Item 40) The subject is (a) immunosuppressive therapy; (b) stimulatory immune checkpoint molecules; (c) radiotherapy; (d) chemotherapy (e) cellular immunotherapy; or (f) any combination of (a) to (e) The method according to any one of items 36 to 39, wherein the subject is receiving or has previously received (Item 41) The antibody according to any one of items 1 to 10, the host cell according to item 16 or 17, the antibody conjugate according to any one of items 19 or 21 to 35, or the composition according to item 20 for use in the treatment, diagnosis, or detection of cancer. (Item 42) The antibody according to any one of items 1 to 10, the polynucleotide according to item 11 or 12, the recombinant vector according to any one of items 13 to 15, the host cell according to item 16 or 17, the antibody conjugate according to any one of items 19 or 21 to 35, or the composition according to item 20 for use in the preparation of a medicament for the treatment, diagnosis, or detection of cancer.

Claims

1. An antibody conjugate comprising an isolated antibody or an antigen-binding fragment thereof, and a payload molecule linked to the isolated antibody or an antigen-binding fragment thereof, wherein the isolated antibody or an antigen-binding fragment thereof is (a) an immunoglobulin heavy chain variable region (VH) comprising a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, wherein the variant consists of a Y→A substitution at position 33 of SEQ ID NO: 35, complementarity-determining region 1 (VH CDR1); a complementarity-determining region 2 (VH CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, wherein the variant consists of a T→S substitution at position 55 of SEQ ID NO: 35, complementarity-determining region 2 (VH CDR2); a complementarity-determining region 3 (VH CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof, wherein the variant consists of a Y→A substitution at position 104 of SEQ ID NO: 35, an A→H substitution at position 106 of SEQ ID NO: 35, or both, complementarity-determining region 3 (VH CDR3); an immunoglobulin heavy chain variable region (VH), and (b) an immunoglobulin light chain variable region (VL) comprising a complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, wherein the variant consists of a Y→A substitution at position 30 of SEQ ID NO: 5, complementarity-determining region 1 (VL CDR1); a complementarity-determining region 2 (VL CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof, wherein the variant consists of a G→A substitution at position 50 of SEQ ID NO: 5, and complementarity-determining region 2 (VL CDR2); and a complementarity-determining region 3 (VL CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof, wherein the variant consists of a T→S substitution at position 93 of SEQ ID NO: 5, complementarity-determining region 3 (VL CDR3) an immunoglobulin light chain variable region (VL) comprising, wherein the antibody or antigen-binding fragment thereof is (i) to (iv) (i) a biantenna-type LeB / LeB antigen comprising Fuc 4 (Galβ1→3GlcNAc)2 [I] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]2 [II], (ii) a biantenna-type LeY / LeY antigen comprising Fuc 4 (Galβ1→4GlcNAc)2 [III] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc]2 [IV], (iii) a biantenna-type LeB / LeY antigen comprising Fuc 2 (Galβ1→3GlcNAc)[Fuc 2 (Galβ1→4GlcNAc)][V] or [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][VI], and (iv) a biantenna-type LeY / LeB antigen comprising Fuc 2 (Galβ1-4GlcNAc)[Fuc 2 (Galβ1-3GlcNAc)][VII] or [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc][Fucα1-2Galβ1-3(Fucα1-4)GlcNAc][VIII] An antibody conjugate capable of specifically binding to at least one of.

2. The isolated antibody or antigen-binding fragment thereof comprises the VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, the VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, the VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4, the VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, the VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and the VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

8. The antibody conjugate according to claim 1.

3. The antibody or antigen-binding fragment thereof is the biantenna-type Le Ｂ / Le ＢAntigen [I] or [II], the biantennary Le Ｙ / Le Ｙ Antigen [III] or [IV], the biantennary Le Ｂ / Le Ｙ Antigen [V] or [VI] and biantennary Le Ｂ / Le Ｙ 3. An antibody conjugate as described in claim 1 or claim 2, capable of specifically binding to antigen [VII] or [VIII].

4. The antibody or antigen-binding fragment thereof is a monoantennary Le comprising Galβ1→4(Fucα1→3)GlcNAc[IX]. ｘ The antigen also contains [Galβ1→4(Fucα1→3)GlcNAc]. ２ Bi-antenna type Le containing [X] ｘ The antigen also contains monoantennary Le containing Galβ1-3(Fucα1-4)GlcNAc[XI]. Ａ The monoantennary H antigen type 2, which contains Fucα1-2Galβ1-4GlcNAc[XII], also contains (Fucα1-2Galβ1-4GlcNAc) ２ The antibody conjugate according to any one of claims 1 to 3, which does not specifically bind to either biantennary H antigen type 2 comprising [XIII] or monoantennary H antigen type 1 comprising Fucα1-2Galβ1-3GlcNAc[XIV].

5. the VH comprises an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:35; The VL comprises an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:5; The antibody conjugate according to any one of claims 1 to 4.

6. The VH contains an amino acid sequence having at least 97% identity to the amino acid sequence set forth in SEQ ID NO: 35, and the VL contains an amino acid sequence having at least 97% identity to the amino acid sequence set forth in SEQ ID NO: 5, the antibody conjugate according to claim 5.

7. The VH contains the amino acid sequence set forth in SEQ ID NO: 35, and the VL contains the amino acid sequence set forth in SEQ ID NO: 5, the antibody conjugate according to claim 5.

8. The antibody or an antigen-binding fragment thereof further contains an Fc fragment, the antibody conjugate according to any one of claims 1 to 7.

9. The Fc fragment contains a human IgG1 subclass, the antibody conjugate according to claim 8.

10. The antibody contains an immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 10 and an immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 11, the antibody conjugate according to any one of claims 1 to 9.

11. The antibody contains an immunoglobulin heavy chain having an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 10, and / or the antibody contains an immunoglobulin light chain having an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 11, the antibody conjugate according to any one of claims 1 to 9.

12. The antibody conjugate according to any one of claims 1 to 9, wherein the antibody comprises an immunoglobulin heavy chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 10, and wherein the antibody comprises an immunoglobulin light chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:

11.

13. The antibody conjugate according to claim 11 or 12, wherein the immunoglobulin heavy chain and / or the immunoglobulin light chain comprises a natural amino acid replaced with a non-native cysteine amino acid or a non-native unnatural amino acid configured for site-specific conjugation.

14. The antibody conjugate according to any one of claims 1 to 9, wherein the antibody or an antigen-binding fragment thereof comprises a non-native cysteine or a non-native unnatural amino acid configured for site-specific conjugation.

15. The antibody conjugate according to any one of claims 1 to 7, wherein the antigen-binding fragment is selected from a Fab fragment, an F(ab')2 fragment, an Fv fragment, a single-chain Fv (scFv) antibody, and a diabody.

16. The antibody conjugate according to any one of claims 1 to 15, wherein the payload molecule is covalently linked to the antibody or an antigen-binding fragment thereof by a linker.

17. The antibody conjugate according to claim 16, wherein the linker is a cleavable linker.

18. The antibody conjugate according to claim 17, wherein the cleavable linker is a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker.

19. The antibody conjugate according to claim 16, wherein the linker is a non-cleavable linker. **Claim 20** The antibody conjugate according to any one of claims 1 to 19, wherein the payload molecule is a therapeutic agent, a detectable indicator, or a combination thereof. **Claim 21** The antibody conjugate according to claim 20, wherein the therapeutic agent is selected from tubulin-targeting antimicrotubule agents, peptide-based toxins, pyrrolobenzodiazepine (PBD) dimers, antibiotics, pyrimidine synthesis inhibitors, antimetabolites, DNA alkylating agents, and topoisomerase inhibitors. **Claim 22** The antibody conjugate according to claim 21, wherein the therapeutic agent is selected from maytansinoids, auristatins, doxorubicin, calicheamicin, PBD dimers, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF). **Claim 23** The antibody conjugate according to claim 22, wherein the therapeutic agent is monomethyl auristatin E (MMAE), and the payload molecule is covalently linked to the antibody or an antigen-binding fragment thereof by a protease-sensitive linker. **Claim 24** The antibody conjugate according to claim 23, wherein the protease-sensitive linker comprises a valine-citrulline dipeptide. **Claim 25** The antibody conjugate according to claim 20, wherein the detectable indicator is selected from radionuclides, dyes, radioactive metals, fluorescent moieties, MRI contrast agents, microbubbles, carbon nanotubes, gold particles, fluorodeoxyglucose, enzymes, chromophores, and radiopaque markers. **Claim 26** The detectable indicator is ６８ Ga, ６４ Cu, ８６ Y, ８９ Zr, １２４ I, ９９ｍTc, １２３ I, １１１ In, １７７ Lu, １３１ I, ７６ Br, ７８ Zr, １８ F, and １２４ The antibody conjugate according to claim 25, which is a radionuclide selected from T.

27. The antibody conjugate according to claim 25, wherein the antibody conjugate comprises a chelator of a radionuclide selected from DOTA labeled with maleimide, N-hydroxysuccinimide-DOTA, and desferrioxamine (DFO).

28. A pharmaceutical composition comprising the antibody conjugate according to any one of claims 1 to 27 and a pharmaceutical carrier.

29. In a subject in need of treatment for cancer, a biantenna-type Le Ｂ / Le Ｂ biantenna-type Le Ｙ / Le Ｙ biantenna-type Le Ｂ / Le Ｙ and the biantenna-type Le Ｙ / Le Ｂ The pharmaceutical composition according to claim 28, for treating cancer that expresses one or more biantenna-type Lewis antigens selected from the group consisting of.

30. The pharmaceutical composition according to claim 29, wherein the cancer is colorectal adenocarcinoma, gastric adenocarcinoma or lung cancer.

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