HK1232767A1

HK1232767A1 - Antibodies, hybridoma producing such antibodies and uses thereof

Info

Publication number: HK1232767A1
Application number: HK17104350.7A
Authority: HK
Inventors: Cheng-Der Tony Yu; Jiann-Shiun Lai; I-Ju Chen; Cheng-Chi Wang; Yi-Chien Tsai
Original assignee: 台湾浩鼎生技股份有限公司
Priority date: 2014-04-10
Filing date: 2015-04-10
Publication date: 2018-01-19

Description

Antibodies, hybridomas producing same, and uses thereof

Cross reference to related applications

This application claims the benefit of U.S. patent application No. 61/977,824, filed on day 10, 2014, and U.S. patent application No. 62/057,381, filed on day 30, 2014, which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to antibodies to tumor-associated carbohydrate antigens, including specific portions or variants specific for at least one tumor-associated carbohydrate antigen or fragment thereof, as well as nucleic acids, complementary nucleic acids, vectors, host cells and methods of making and using the same, including therapeutic formulations and pharmaceutical compositions comprising the same. Further, methods are provided for administering to a subject an effective amount of an antibody to inhibit cancer cells.

Background

Several surface carbohydrates are expressed on malignant cells. For example, Globo H (Fuc α 1 → 2Gal β 1 → 3GalNAc β 1 → 3Gal α 1 → 4Gal β 1 → 4Glc) has been shown to over-express in a variety of epithelial cancers and is associated with tumor invasiveness and poor prognosis in breast and small cell lung cancers. Previous studies showed that Globo H and Stage-specific embryonic antigen 3(Stage-specific embryonic antigen 3, SSEA3, also known as Gb5) were observed on breast and breast cancer stem cells (WW Chang et al, "" Expression of Globo H and SSEA3 in breast cancer stem cells and involvement of fucosyltransferases 1 and 2in Globo H synthesis "" (Expression of Globo H and SSEA3 in cleavage cancer cells and the Expression of fucosyltransferases 1 and 2in Globo hsyntheses), PNAS, 105 (33): 11667 and 11672).

These findings support a rational explanation for the development of antibodies against tumor-associated carbohydrate antigens, however there is still an unmet need for effective treatment and/or prevention of cancer. The present invention provides antibodies to tumor-associated carbohydrate antigens to meet these and other needs.

Disclosure of Invention

The present invention provides antibodies, or antigen-binding portions thereof, comprising variable domains that bind to carbohydrate antigens, conjugated versions of these antibodies, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants related thereto, and methods of making and using the same, as described and enabled herein, in combination with those well known in the art to which the invention pertains. The antibody or antigen-binding portion thereof can have a dissociation constant (K) of about 10E-7M or less, about 10E-8M or less, about 10E-9M or less, about 10E-10M or less, about 10E-11M or less, or about 10E-12M or less_D). The antibody or antigen binding portion thereof can be humanized or chimeric.

In one embodiment, the invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 3.

In another embodiment, the invention provides an antibody, or antigen-binding portion thereof, comprising a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 4.

In yet another embodiment, the invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 3; and a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 4.

In a fourth embodiment, the invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region, wherein the heavy chain region comprises three Complementarity Determining Regions (CDRs), CDR1, CDR2, and CDR3 having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:5, 6, and 7, respectively. In an exemplary embodiment, the heavy chain further comprises a Framework (Framework) between the leader sequence and said CDR1 having an amino acid sequence from about 80% to about 100% homologous to SEQ ID NO: 87. In another embodiment, the heavy chain further comprises a framework between said CDR2 and CDR3 having an amino acid sequence that is about 80% to about 100% homologous to SEQ ID No. 89. In yet another exemplary embodiment, the heavy chain further comprises a framework between the CDR1 and CDR2 of a heavy chain having an amino acid sequence from about 80% to about 100% homologous to SEQ ID No. 11, wherein the framework comprises a glycine at position 9, and the antibody or antigen-binding portion thereof binds to a carbohydrate antigen, such as Globo H.

In a fifth embodiment, the invention provides an antibody, or antigen-binding portion thereof, comprising a light chain region, wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to 100% homologous to the amino acid sequences set forth in SEQ ID NOs:8, 9, and 10, respectively. In an exemplary embodiment, the light chain further comprises a framework between the leader sequence and said CDR1 having an amino acid sequence that is about 80% to about 100% homologous to SEQ ID NO: 88. In another exemplary embodiment, the light chain further comprises a framework between said CDR2 and CDR3 of the light chain having an amino acid sequence that is about 80% to about 100% homologous to SEQ ID NO: 90. In yet another exemplary embodiment, the light chain further comprises a framework between the CDRs 1 and CDR2 of the light chain having an amino acid sequence that is about 80% to about 100% homologous to SEQ ID No. 12, wherein the framework contains proline at position 12 and the antibody or antigen-binding portion thereof binds to Globo H. In yet another exemplary embodiment, the light chain further comprises a framework between the CDR1 and CDR2 of a light chain having an amino acid sequence that is about 80% to about 100% homologous to SEQ ID No. 12, wherein the framework comprises a tryptophan at position 13, and the antibody or antigen-binding portion thereof binds to a carbohydrate antigen, such as GloboH.

In a sixth embodiment, the invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region and a light chain region, wherein the heavy chain region comprises three CDRs, CDR1, CDR2, and CDR3, each having an amino acid sequence from about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:5, 6, and 7, and wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, each having an amino acid sequence from about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:8, 9, and 10.

In some embodiments, provided are antibodies, or antigen-binding portions thereof, comprising: a heavy chain region, wherein the heavy chain region comprises CDRs having an amino acid sequence from about 80% to about 100% homologous to an amino acid sequence selected from SEQ ID NOs:5, 6, or 7. In other embodiments, provided are antibodies, or antigen-binding portions thereof, comprising a light chain region, wherein the light chain region comprises CDRs having an amino acid sequence that is about 80% to about 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs 8,9, or 10.

The invention is also directed to an antibody, or antigen-binding portion thereof, comprising: a heavy chain variable domain comprising about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO. 13.

The invention also relates to an antibody, or antigen-binding portion thereof, comprising: a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 14.

The invention also relates to an antibody, or antigen-binding portion thereof, comprising: a heavy chain variable domain comprising about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO. 13; and a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 14.

An exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region, wherein the heavy chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:15, 16, and 17, respectively. Another exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a light chain region, wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:18, 19, and 20, respectively.

Another exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region and a light chain region, wherein the heavy chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:15, 16, and 17, respectively, and wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:18, 19, and 20, respectively.

In some embodiments, provided are antibodies, or antigen-binding portions thereof, comprising: a heavy chain region, wherein the heavy chain region comprises CDRs having an amino acid sequence from about 80% to about 100% homologous to an amino acid sequence selected from SEQ ID NOs:15, 16, or 17. In other embodiments, provided are antibodies, or antigen-binding portions thereof, comprising a light chain region, wherein the light chain region comprises CDRs having an amino acid sequence that is about 80% to about 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:18, 19, or 20.

One embodiment of the invention is an antibody, or antigen-binding portion thereof, comprising: a heavy chain variable domain comprising from about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 21.

Another embodiment of the invention is an antibody, or antigen-binding portion thereof, comprising: a light chain variable domain comprising about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO. 22.

In another embodiment of the invention is an antibody, or antigen-binding portion thereof, comprising: a heavy chain variable domain comprising about 80% to about 100% amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO 21; and a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 22.

An exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region, wherein the heavy chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:23, 24, and 25, respectively. Another exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a light chain region, wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:26, 27, and 28, respectively.

Another exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region and a light chain region, wherein the heavy chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:23, 24, and 25, respectively, and wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:26, 27, and 28, respectively.

In some embodiments, provided are antibodies, or antigen-binding portions thereof, comprising: a heavy chain region, wherein the heavy chain region comprises CDRs having an amino acid sequence from about 80% to about 100% homologous to an amino acid sequence selected from SEQ ID NOs:23, 24, or 25. In other embodiments, provided are antibodies, or antigen-binding portions thereof, comprising a light chain region, wherein the light chain region comprises CDRs having an amino acid sequence from about 80% to about 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:26, 27, or 28.

The invention also discloses antibodies, or antigen-binding portions thereof, comprising: a heavy chain variable domain comprising about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO. 29.

The invention also discloses antibodies, or antigen-binding portions thereof, comprising: a light chain variable domain comprising about 80% to about 100% amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO. 30.

The invention also discloses antibodies, or antigen-binding portions thereof, comprising: a heavy chain variable domain comprising about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO. 29; and a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 30.

An exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region, wherein the heavy chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:31, 32, and 33, respectively. Another exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a light chain region, wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:34, 35, and 36, respectively.

Another exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region and a light chain region, wherein the heavy chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:31, 32, and 33, respectively, and wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:34, 35, and 36, respectively.

In some embodiments, provided are antibodies, or antigen-binding portions thereof, comprising: a heavy chain region, wherein the heavy chain region comprises CDRs having an amino acid sequence from about 80% to about 100% homologous to an amino acid sequence selected from SEQ ID NOs:31, 32, or 33. In other embodiments, provided are antibodies, or antigen-binding portions thereof, comprising a light chain region, wherein the light chain region comprises CDRs having an amino acid sequence that is about 80% to about 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:34, 35, or 36.

One embodiment of the invention is an antibody, or antigen-binding portion thereof, comprising: a heavy chain variable domain comprising from about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 37.

Another embodiment of the invention provides an antibody, or antigen-binding portion thereof, comprising: a light chain variable domain comprising about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 38.

In another embodiment of the invention, there is provided an antibody, or antigen-binding portion thereof, comprising: a heavy chain variable domain comprising about 80% to about 100% amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 37; and a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 38.

An exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region, wherein the heavy chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:39, 40, and 41, respectively. Another exemplary embodiment discloses an antibody, or antigen-binding portion thereof, comprising a light chain region, wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:42, 43, and 44, respectively.

Another exemplary embodiment provides an antibody, or antigen-binding portion thereof, comprising a heavy chain region and a light chain region, wherein the heavy chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:39, 40, and 41, respectively, and wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:42, 43, and 44, respectively.

In some embodiments, provided are antibodies, or antigen-binding portions thereof, comprising: a heavy chain region, wherein the heavy chain region comprises CDRs having an amino acid sequence from about 80% to about 100% homologous to an amino acid sequence selected from SEQ ID NOs:39, 40, or 41. In other embodiments, provided are antibodies, or antigen-binding portions thereof, comprising a light chain region, wherein the light chain region comprises CDRs having an amino acid sequence that is about 80% to about 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs 42, 43, or 44.

The present invention provides pharmaceutical compositions comprising an antibody or antigen-binding portion thereof as described herein and at least one pharmaceutically acceptable carrier.

The invention also provides a method for inhibiting Globo H-expressing cancer cells, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding portion thereof as provided herein, wherein Globo H-expressing cancer cells are inhibited.

The invention also provides the compound designated as 2C2 (deposited under American Type Culture Collection (ATCC) deposit number PTA-121138); 3D7 (deposited under American type culture Collection accession number PTA-121310); 7A11 (deposited under American type culture Collection accession number PTA-121311); hybridoma clones (clones) of 2F8 (deposited under ATCC accession number PTA-121137; and 1E1 (deposited under ATCC accession number PTA-121312), and antibodies and antigen-binding portions produced therefrom.

Drawings

FIG. 1 is a line graph showing the effect of PBS, Globo H-VK9 mAbs, Globo H-2C2 mAbs and Globo H-3D7 mAb on pancreatic cancer (HPAC) volume in mice.

FIG. 2 is a line graph showing the effect of normal saline and different doses of Globo H-2C2 mAbs on breast cancer (MCF7) volume in mice.

FIGS. 3A to 3F are bar graphs showing the binding affinities of Globo H-2C2 mAb (FIG. 3A), Globo H-7A11mAb (FIG. 3B), Globo H-3D7 mAb (FIG. 3C), Globo H-2F8mAb (FIG. 3D), Globo H-1E 1mAb (FIG. 3E) and Globo H-VK9mAb (FIG. 3F) to various carbohydrate antigens.

Detailed Description

As used herein, an article "a" refers to one or more (i.e., at least one) of the grammatical object of the article. By way of example, "a component" means one component or more than one component.

As used herein, "effective amount" means a dose of the vaccine or pharmaceutical composition sufficient to reduce symptoms and signs of cancer, such as weight loss, pain, and accessible masses that are clinically accessible or radioactively detectable by various imaging devices. The terms "effective amount" and "therapeutically effective amount" are used interchangeably.

The term "subject" can refer to a vertebrate having cancer or a vertebrate believed to be in need of treatment for cancer. Subjects include all warm-blooded animals, such as mammals, such as primates, and preferably, humans. Non-human primates are also subjects. The term subject includes domesticated animals such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), as well as experimental animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.). As such, veterinary uses and pharmaceutical formulations are contemplated herein.

All numbers herein are approximate and may be modified by the term "about".

The present invention provides pharmaceutical compositions and methods for the treatment or inhibition of cancer cells. The pharmaceutical composition comprises an antibody recognizing a carbohydrate antigen, which comprises a mouse monoclonal antibody, a humanized antibody, a chimeric antibody, or any antigen binding portion thereof. These antibodies (or antigen-binding portions thereof) can neutralize carbohydrate antigens and/or inhibit cancer cells. Accordingly, the antibodies or antigen-binding portions thereof of the invention can be used for the treatment or inhibition of cancer cells.

The antibodies of the invention include any protein or peptide comprising at least one Complementarity Determining Region (CDR) of a heavy or light chain, or ligand binding portion thereof, derived from the hybridoma designated as 2C2 (deposited under american type culture collection accession number PTA-121138), as described herein; a hybridoma designated 3D7 (deposited under American type culture Collection accession number PTA-121310); the hybridoma designated 7A11 (deposited under American type culture Collection accession number PTA-121311); the hybridoma designated 2F8 (deposited under American type culture Collection accession number PTA-121137); or hybridoma designated as 1E1 (deposited under American type culture Collection accession number PTA-121312). The antibody comprises an antibody fragment, an antibody variant, a monoclonal antibody and a polyclonal antibody; and recombinant antibodies and the like. Antibodies can be produced in mice, rabbits, or humans.

Antibodies of the invention also include chimeric or humanized monoclonal antibodies generated from antibodies of the invention.

Thus, the anti-cancer antibodies of the invention include combinations of heavy or light chain variable regions, heavy or light chain constant regions, framework regions, or any portion thereof, of non-murine origin, preferably of human origin, which can incorporate the antibodies of the invention.

The antibodies of the invention are capable of modulating, reducing, antagonizing, alleviating, moderating, blocking, inhibiting, abrogating, and/or interfering with at least one Globo-H-expressed cancer cell activity in vitro, in situ, and/or in vivo.

The term "antibody" is further intended to encompass antibodies, digestion fragments, specific portions and variants thereof, including antibody mimetics or portions comprising antibodies that mimic the structure and/or function of an anti-cancer antibody or specific fragment or portion thereof, including single chain antibodies and fragments thereof, each containing at least one CDR derived from an anti-cancer antibody of the invention. Functional fragments include antigen-binding fragments that bind to Globo-H expressing cancer cells. For example, antibody fragments or portions thereof that are capable of binding to Globo-H expressing cancer cells, including, but not limited to, Fab (e.g., by papain digestion), Fab ' (e.g., by pepsin digestion and partial reduction), F (ab ')2 (e.g., by pepsin digestion), facb (e.g., by cytoplasmic digestion), pFc ' (e.g., by pepsin or cytoplasmic digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biotechnology) fragments, are encompassed by the invention (see, e.g., Colligan, Immunology, supra).

As used herein, 2C2 refers to a hybridoma clone or an antibody produced by a corresponding hybridoma clone.

The antigen-binding portion of an antibody can include a portion of an antibody that specifically binds to a carbohydrate antigen (e.g., Globo H, SSEA-3, or SSEA-4).

The humanized antibodies of the invention are antibodies from non-human species in which the amino acid sequence in the non-antigen binding region (and/or the antigen binding region) is altered so that the antibody more closely resembles a human antibody, while at the same time maintaining its original binding capacity. .

Humanized antibodies can be generated by replacing the sequences of the variable regions not directly involved in antigen binding with equivalent sequences derived from human variable regions. These methods include isolating, manipulating, and expressing nucleic acid sequences encoding all or part of the variable region from at least one of the heavy or light chains. The source of such nucleic acids is well known to those skilled in the art. Recombinant DNA encoding the humanized antibody, or fragment thereof, may then be transferred into a suitable expression vector.

The variable region of an antibody light or heavy chain comprises framework regions, called CDRs, interrupted by three hypervariable regions. In one embodiment, a humanized antibody is an antibody molecule derived from a non-human species having one, two, or all CDRs from the non-human species and one, two, or all three framework regions from a human immunoglobulin molecule.

According to one aspect of the invention, the position of the CDRs and framework residues is determined by the method disclosed in Kabat, E.A., et al, (1991) Sequences of Proteins of immunological interest (Sequences of Proteins of immunological interest), fifth edition, USA, Department of Health and public services (Department of Health and Humanservices), NIH publication No. 91-3242. According to another aspect of the invention, an antibody, or antigen-binding portion thereof, can have the following structure:

leader sequence-FW 1-CDR1-FW2-CDR2-FW3-CDR3-,

wherein, framework regions FW1, FW2, FW3 and CDRs, CDR1, CDR2 and CDR3 have the amino acid sequences disclosed in Table 1.

The humanized antibody of the present invention can be produced by a method known in the art. For example, once a non-human (e.g., murine) antibody is obtained, the variable regions can be sequenced and the positions of the CDRs and framework residues can be determined. Kabat, E.A. et al, (1991) sequence of proteins of immunological interest, fifth edition, USA, Ministry of health and public services, NIH published No. 91-3242. Chothia, C.et al, (1987), journal of molecular biology (J.mol.biol.), 196:901-917, DNA encoding the light and heavy chain variable regions can optionally be ligated to the respective constant regions and then sub-transformed into a suitable expression vector. CDR grafted antibody molecules can be produced by CDR grafting or CDR substitution. One, two or all of the CDRs of the immunoglobulin chain may be replaced. For example, all of the CDRs of a particular antibody may be from a non-human animal (e.g., a mouse such as the CDRs shown in table 1) or only some of the CDRs may be replaced. Only the CDRs needed for binding of the antibody to the predetermined carbohydrate antigen (e.g., Globo H) need be retained. Morrison, S.L., 1985, Science journal (Science), 229:1202- "1207. Oi et al, 1986, BioTechniques, 4: 214. U.S. Pat. Nos. 5,585,089 (Patent Nos.); 5,225,539; 5,693,761 and 5,693,762. EP 519596. Jones et al, 1986, Nature journal (Nature), 321: 552-525. Verhoeyan et al, 1988, journal of Science 239: 1534. Beidler et al, 1988, J.Immunol., 141: 4053-.

Also encompassed by the invention are antibodies, or antigen-binding portions thereof, comprising one or two variable regions as disclosed herein, with additional regions replaced by sequences from at least one different species, including, but not limited to, humans, rabbits, sheep, dogs, cats, cows, horses, goats, pigs, monkeys, apes, gorillas, chimpanzees, ducks, geese, chickens, amphibians, reptiles, and other animals.

Chimeric antibodies are molecules in which different portions are derived from different animal species. For example, an antibody may contain variable regions derived from a murine mAb and human immunoglobulin constant regions. Chimeric antibodies can be produced by recombinant DNA techniques. Morrison et al, Proc Natl Acad Sci 81:6851-6855 (1984). For example, the gene encoding the murine (or other species) antibody molecule is cleaved with restriction enzymes to remove the region encoding the murine Fc, and the equivalent of the gene encoding the human Fc constant region is then substituted into the recombinant DNA molecule. Chimeric antibodies can also be created by recombinant DNA technology, in which DNA encoding the murine V region can be joined to DNA encoding the human constant region. Better et al, Science, 1988, 240: 1041-. Liu et al, Proc. Natl. Acad. Sci. USA (PNAS), 1987, 84: 3439-. Liu et al, J.Immunol., 1987, 139: 3521-3526. Sun et al, Proc. Natl. Acad. Sci. USA (PNAS), 1987, 84: 214-. Nishimura et al, journal of cancer research (Canc. Res.), 1987, 47: 999-. Wood et al, Nature journal (Nature), 1985, 314: 446-449. Shaw et al, journal of the national cancer institute (J.Natl.cancer Inst.), 1988, 80: 1553-1559. International patent publication nos. WO1987002671 and WO 86/01533. European patent application No. 184,187; 171,496, respectively; 125,023; and 173,494. U.S. Pat. No. 4,816,567.

The antibody can be full length or can comprise a fragment (or a plurality of fragments) of an antibody having an antigen-binding portion, characterized by, but not limited to, Fab, F (ab ')2, Fab ', F (ab) ', Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragments (e.g., Ward et al, Nature, 341:544, 546 (1989)), isolated CDRs, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single chain antibodies produced by attaching antibody fragments using recombinant methods, or synthetic linkers, are also encompassed by the invention. Bird et al, Science journal (Science), 1988, 242: 423-. Huston et al, Proc. Natl. Acad. Sci., USA, 1988, 85: 5879-.

The antibodies of the invention or antigen-binding portions thereof can be monospecific, bispecific or multispecific. Multispecific or bispecific antibodies or fragments thereof may be specific for different epitopes of one carbohydrate of interest (e.g., Globo H), or may contain antigen binding domains specific for more than one carbohydrate of interest (e.g., antigen binding domains specific for Globo H, SSEA-3, and SSEA-4). In one embodiment, the multispecific antibody or antigen-binding portion thereof comprises at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate carbohydrate antigen or to a different epitope on the same carbohydrate antigen. Tutt et al, 1991, immunology journal (J. Immunol.), 147: 60-69. Kufer et al, 2004, Trends Biotechnol., 22:238- "244. The antibody of the invention may be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bispecific or multispecific antibody with a second binding specificity.

All antibody isotypes are encompassed by the invention, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, or IgE (all classes and subtypes are encompassed by the invention). The antibody or antigen-binding portion thereof can be a mammalian (e.g., mouse, human) antibody or antigen-binding portion thereof. The light chain of the antibody may be of the kappa or lambda type.

The variable region of an antibody or antigen-binding portion thereof of the invention can be from a non-human or human source. The framework of an antibody or antigen-binding portion thereof of the invention can be human, humanized, non-human (e.g., murine framework modified to reduce antigenicity in humans), or synthetic (e.g., consensus framework).

In one embodiment, the antibody or antigen-binding portion thereof of the invention comprises at least one heavy chain variable region and/or at least one light chain variable region.

The antibodies of the invention or antigen binding portions thereof can be less thanA dissociation constant (K) of about 10E-7M, less than about 10E-8M, less than about 10E-9M, less than about 10E-10M, less than about 10E-11M, or less than about 10E-12M_D) Specifically binds to GloboH. In one embodiment, the antibody or antigen-binding portion thereof has a dissociation constant (K) of 1 to 10x10E-9 or less_D). In another embodiment, Kd is determined by surface plasmon resonance.

The antibody has variable heavy and variable light regions that are at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the variable heavy and variable light regions of an antibody produced by clone 2C2, and can also bind to a carbohydrate antigen (e.g., Globo H). Homology can be expressed at the amino acid or nucleotide sequence level.

In some embodiments, the variable heavy chain and/or variable light chain of an antibody, for example, produced by hybridoma 2C2, hybridoma 3D7, hybridoma 7a11, hybridoma 2F8, and hybridoma 1E1, are shown in table 1.

In related embodiments, the antibody or antigen-binding portion thereof includes, for example, the CDRs of the variable heavy chain and/or the CDRs of the variable light chain of the antibody produced from hybridoma 2C2, hybridoma 3D7, hybridoma 7a11, hybridoma 2F8, and hybridoma 1E 1. The CDRs and frameworks from the variable heavy and variable light chains in these hybridoma clones are shown in table 1.

Table 1: 1-90 of SEQ ID NO

The invention also encompasses nucleic acids encoding the antibodies of the invention or antigen-binding portions thereof that specifically bind to carbohydrate antigens. In one embodiment, the carbohydrate antigen is Globo H. In another embodiment, the carbohydrate antigen is SSEA-3. In yet another embodiment, the carbohydrate antigen is SSEA-4. The nucleic acid can be expressed in a cell to produce an antibody or antigen-binding portion thereof of the invention.

In particular embodiments, an antibody or antigen-binding portion thereof of the invention includes a polypeptide comprising at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homology to the following:

a) 3 (hybridoma 2C 2);

b) 13 (hybridoma 3D 7);

c) 21 (hybridoma 7A 11);

d) 29 (hybridoma 2F 8); or

e) 37 (hybridoma 1E1) or a variable heavy chain region of the amino acid sequence of any one of SEQ ID NOs.

a) 4 (hybridoma 2C 2);

b) 14 (hybridoma 3D 7);

c) 22 (hybridoma 7A 11);

d) 30 (hybridoma 2F 8); or

e) 38 (hybridoma 1E 1).

In particular embodiments, the variable heavy chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homology to the amino acid sequence of SEQ ID No. 3, and the variable light chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, (iii), At least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the amino acid sequence of SEQ ID NO. 4.

In particular embodiments, the variable heavy chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homology to the amino acid sequence of SEQ ID No. 13, and the variable light chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, (iii), At least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the amino acid sequence of SEQ ID NO. 14.

In particular embodiments, the variable heavy chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homology to the amino acid sequence of SEQ ID No. 21, and the variable light chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, (iii), At least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the amino acid sequence of SEQ ID NO. 22.

In particular embodiments, the variable heavy chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homology to the amino acid sequence of SEQ ID No. 29, and the variable light chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, (iii), At least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the amino acid sequence of SEQ ID NO. 30.

In particular embodiments, the variable heavy chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homology to the amino acid sequence of SEQ ID No. 37, and the variable light chain region of an antibody, or antigen-binding portion thereof, comprises at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, (iii), At least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the amino acid sequence of SEQ ID NO 38.

The variable heavy chain region of an antibody, or antigen-binding portion thereof, can comprise one, two, three, or more CDRs comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the following:

a) the CDRs of the variable heavy chain region of the antibody produced by hybridoma 2C2 (SEQ ID NOs:5, 6 and 7);

b) the CDRs of the variable heavy chain region of the antibody produced by hybridoma 3D7 (SEQ ID NOs:15, 16 and 17);

c) the CDRs of the variable heavy chain region of the antibody produced by hybridoma 7A11 (SEQ ID NOs:23, 24 and 25);

d) the CDRs of the variable heavy chain region of the antibody produced by hybridoma 2F8 (SEQ ID NOs:31, 32 and 33); or

e) The amino acid sequence of any of the CDRs (SEQ ID Nos:39, 40 and 41) of the variable heavy chain region of an antibody produced by hybridoma 1E 1.

The variable light chain region of an antibody, or antigen-binding portion thereof, can comprise one, two, three, or more CDRs comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the following:

a) the CDRs of the variable light chain region of the antibody produced by hybridoma 2C2 (SEQ ID NOs:8, 9 and 10);

b) the CDRs of the variable light chain region of the antibody produced by hybridoma 3D7 (SEQ ID NOs:18, 19 and 20);

c) the CDRs of the variable light chain region of the antibody produced by hybridoma 7A11 (SEQ ID NOs:26, 27 and 28);

d) the CDRs of the variable light chain region of the antibody produced by hybridoma 2F8 (SEQ ID NOs:34, 35 and 36); or

e) The amino acid sequence of any one of the CDRs (SEQ ID NOs:42, 43 and 44) of the variable light chain region of an antibody produced by hybridoma 1E 1.

The variable heavy chain region of an antibody or antigen-binding portion thereof may comprise one, two or three or more CDRs comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homology to the CDRs (SEQ ID NOs:5, 6 and 7) of the variable heavy chain region of an antibody produced by hybridoma 2C2, or the CDRs (SEQ ID NOs:15, 16 and 17) of the variable heavy chain region of an antibody produced by hybridoma 3D7, and one of the variable heavy chain regions of an antibody or antigen-binding portion thereof, and, Two or three or more CDRs comprising an amino acid sequence of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to CDRs of the variable light chain region of an antibody produced by hybridoma 2C2 (SEQ ID NOs:8, 9, and 10) or CDRs of the variable light chain region of an antibody produced by hybridoma 3D7 (SEQ ID NOs:18, 19, and 20).

In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a framework that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to SEQ ID NO:83 (heavy chain framework 1 of the 2C2 antibody) or SED ID NO:87 (heavy chain framework 1 of the humanized 2C2 antibody, see table 1), wherein the framework is between the leader sequence and the CDR1 of the variable heavy chain region of an antibody produced by hybridoma 2C 2. In another embodiment, the antibody, or antigen-binding portion thereof, further comprises a framework that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to SEQ ID NO:84 (light chain framework 1 of 2C2 antibody) or SED ID NO:88 (light chain framework 1 of humanized 2C2 antibody, see table 1) and is between the leader sequence and the CDR1 of the variable light chain region of an antibody produced by hybridoma 2C 2.

In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a framework between the CDRs 1 and CDR2 of the variable heavy chain region of the antibody produced by hybridoma 2C2, wherein the framework can be at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID No. 11 (heavy chain framework 2in table 1). In an exemplary embodiment, the framework between CDR1 and CDR2 of the variable heavy chain region having about 80% to about 100% homology to the amino acid sequence of SEQ id No. 11 contains a glycine at position 9. The amino acid positions of SEQ ID NO. 11 are described as follows:

amino acid at position 1 of FW2(W) is a residue adjacent to CDR 1.

Amino acid at position 14 of FW2(a) is a residue adjacent to CDR 2.

In another embodiment, the antibody, or antigen-binding portion thereof, further comprises a framework between CDR1 and CDR2 of the variable light chain region of the antibody produced by hybridoma 2C2, wherein the framework is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID No. 12 (light chain framework 2in table 1). In an exemplary embodiment, the framework between the CDR1 and CDR2 of the variable light chain region of the antibody produced by hybridoma 2C2 contains proline at position 12. In another exemplary embodiment, the framework between the CDR1 and CDR2 of the variable light chain region of the antibody produced by hybridoma 2C2 contains a tryptophan at position 13. In yet another exemplary embodiment, the framework between the CDR1 and CDR2 of the variable light chain region of the antibody produced by hybridoma 2C2 contains proline at position 12 and tryptophan at position 13. The amino acid positions of SEQ ID NO 12 are described as follows:

amino acid at position 1 of FW2(W) is a residue adjacent to CDR 1.

Amino acid at position 15 of FW2(Y) is a residue adjacent to CDR 2.

In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a framework that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to SEQ ID NO:85 (heavy chain framework 3) or SED ID NO:89 (heavy chain framework 3 of a humanized antibody, see table 1), wherein the framework is between CDR2 and CDR3 of the variable heavy chain region of an antibody produced by hybridoma 2C 2. In another embodiment, the antibody, or antigen-binding portion thereof, further comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the framework of SEQ ID NO:86 (light chain framework 3) or SEQ ID NO:90 (light chain framework 3 of a humanized antibody, see table 1) and between CDR2 and CDR3 of the variable light chain region of an antibody produced by hybridoma 2C 2.

The variable heavy chain region of an antibody or antigen-binding portion thereof can comprise an amino acid sequence in which one, two, or three or more CDRs comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the CDRs (SEQ ID NOs:23, 24, and 25) of the variable heavy chain region of an antibody produced by hybridoma 7a11, and the variable light chain region of an antibody or antigen-binding portion thereof can comprise one, two, or three or more CDRs comprising at least about 70%, at least about 75%, at least about 80%, or more CDRs, At least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the amino acid sequence of the CDRs (SEQ id nos:26, 27, and 28) of the variable light chain region of an antibody produced by hybridoma 7a 11.

The variable heavy chain region of an antibody or antigen-binding portion thereof can comprise one, two, or three or more CDRs comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the CDRs (SEQ ID NOs:31, 32, and 33) of the variable heavy chain region of an antibody produced by hybridoma 2F8, or the CDRs (SEQ ID NOs:39, 40, and 41) of the variable heavy chain region of an antibody produced by hybridoma 1E1, and one of the variable heavy chain regions of an antibody or antigen-binding portion thereof, and, Two or three or more CDRs comprising an amino acid sequence of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to CDRs of a variable light chain region of an antibody produced by hybridoma 2F8 (SEQ ID NOs:34, 35, and 36) or CDRs of a variable light chain region of an antibody produced by hybridoma 1E1 (SEQ ID NOs:42, 43, and 44).

In particular embodiments, the variable regions corresponding to the variable regions in table 1 have sequence variations. For example, a heavy chain variable region in which 1, 2, 3,4, 5,6, 7, or 8 residues, or less than 40%, less than about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of the amino acid residues are substituted or deleted while retaining substantially the same immunological properties, including, but not limited to, binding to carbohydrate antigens.

In particular embodiments, the CDRs corresponding to the CDRs in table 1 have sequence variations. For example, CDRs in which 1, 2, 3,4, 5,6, 7, or 8 residues, or less than 20%, less than 30%, or less than about 40% of the total residues in the CDRs, are substituted or deleted can be present in an antibody (or antigen-binding portion thereof) that binds to a carbohydrate antigen.

The antibody or antigen-binding portion thereof can be a peptide. Such peptides may include variants, analogs, xenologs, homologs, and derivatives of the peptides that exhibit biological activity, such as binding of carbohydrate antigens. Peptides may contain analogs of one or more amino acids (which include, for example, non-naturally occurring amino acids, amino acids that occur only naturally in non-related biological systems, amino acids that are modified from mammalian systems, etc.), peptides with substituted linkages (substitated linkages), and other modifications well known in the art.

Also within the scope of the invention are antibodies, or antigen-binding portions thereof, in which specific amino acids have been substituted, deleted or added. In exemplary embodiments, these substitutions do not have a substantial effect on the biological properties of the peptide, such as binding affinity. In another exemplary embodiment, an antibody may have amino acid substitutions in the framework regions such that the binding affinity of the antibody to an antigen is enhanced. In yet another exemplary embodiment, a selected small number of framework residues of the acceptor can be replaced by the corresponding donor amino acid. The donor framework can be a mature or a monoclonal human antibody framework sequence or a corresponding sequence. Guidance regarding how to make phenotypically silent amino acid substitutions is provided in Bowie et al, Science journal (Science), 247:1306 (1990). Cunningham et al, Science, 244: 1081-. Ausubel (eds.), Current Protocols in molecular Biology (Current Protocols in molecular Biology), John Wiley and Sons, Inc. (1994). T.manitis, e.f.fritsch and j.sambrook, molecular cloning: a laboratory Manual (Molecular Cloning: laboratory Manual), Cold Spring Harbor laboratory (Cold Spring Harbor laboratory), Cold Spring Harbor Press (Cold Spring Harbor), N.Y. (1989). Pearson, journal of methods of molecular biology methods (MethodsMol. biol.), 243:307-31 (1994). Gonnet et al, Science journal (Science), 256:1443-45 (1992).

An antibody, or antigen binding portion thereof, may be derivatized or linked to another functional molecule. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, non-covalent interaction, etc.) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, a protein or peptide that can mediate a relationship to another molecule (such as a streptavidin core region or a polyhistidine tag), an amino acid linker, a signaling sequence, a carrier for immunity, or a ligand useful in protein purification, such as glutathione-S-transferase (glutaminone-S-transferase), a histidine tag, and protein a of staphylococci. One type of derivatized protein is produced by cross-linking two or more proteins (of the same type or different types). Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by a suitable spacer (e.g., M-maleimidobenzoxy-N-hydroxysuccinimide ester), or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce chemical company, Rockford, IL. Useful detectable reagents from which proteins can be derived (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting examples of fluorescent detectable agents include fluorescein, thiocyanate fluorescein, rose bengal, and phycoerythrin. Proteins or antibodies may also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase, and the like. Proteins can also be prosthetic group derivatized (e.g., streptavidin/biotin and avidin/biotin).

Nucleic acids encoding functionally active variants of the antibodies or antigen-binding portions thereof of the invention are also encompassed by the invention. These nucleic acid molecules can be hybridized to nucleic acids encoding any of the antibodies or antigen-binding portions thereof of the invention under conditions of moderate stringency, high stringency, or very high stringency. Guidance for performing shuffling reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1989. Which is incorporated herein by reference. Specific hybridization conditions referred to herein may be as follows: 1) moderately stringent hybridization conditions: 6 XSSC at about 45 ℃, followed by one or more washes with 0.2 XSSC, 0.1% SDS at 60 ℃; 2) high stringency hybridization conditions: 6 XSSC at about 45 ℃, followed by one or more washes in 0.2 XSSC, 0.1% SDS at 65 ℃; and 3) very high stringency hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65 ℃, followed by 0.2 XSSC, 1% SDS at 65 ℃ washing one or more times.

Nucleic acids encoding the antibodies or antigen-binding portions thereof of the invention can be introduced into expression vectors that can be expressed in a suitable expression system, followed by isolation or purification of the expression antibodies or antigen-binding portions thereof. Alternatively, a nucleic acid encoding an antibody or antigen-binding portion thereof of the invention can be translated into a cell-free translation system. U.S. Pat. No. 4,816,567. Queen et al, Proc Natl Acad Sci, USA, 86: 10029-.

The antibodies of the invention, or antigen-binding portions thereof, can be produced by host cells transformed with DNA encoding the light and heavy chains (or portions thereof) of the desired antibody. Antibodies can be isolated and purified from these culture supernatants and/or cells using standard techniques. For example, a host cell can be transformed with DNA encoding the light chain, heavy chain, or both of an antibody. Recombinant DNA techniques may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not required for binding, e.g., the constant region.

The nucleic acids of the invention can be expressed in a variety of suitable cells, including prokaryotic and eukaryotic cells, e.g., bacterial cells (e.g., e.coli), yeast cells, plant cells, insect cells, and mammalian cells. Many mammalian cell lines may be well known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC). Non-limiting examples of cells include all cell lines of mammalian origin or of mammalian-like character, including, but not limited to, parental cells, derivatives and/or engineered variants of monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese Hamster Ovary (CHO), NS0, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2/0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma, and lymphoma cells. Engineered variants include, for example, glycan modifications and/or site-specific integration site derivatives.

The invention also provides for a cell comprising a nucleic acid described herein. The cell may be a hybridoma or a transfectant, such as the hybridoma designated 2C 2.

Alternatively, the antibodies of the invention or antigen binding portions thereof can be synthesized by solid phase procedures well known in the art. Solid phase peptide synthesis: methods of implementation (Solid Phase Peptide Synthesis: A practical approach), E.Atherton and R.C.Shepard, IRL, Oxford university Press (1989). Methods in Molecular Biology (Methods in Molecular Biology), volume 35: peptide Synthesis procedures (Peptide Synthesis Protocols), (eds., (m.w.pennington and b.m.dunn), chapter seven). Solid phase peptide Synthesis, second edition, Pierce chemical company, Rockford, IL (1984). Barany and r.b.merrifield, peptide: analysis, Synthesis, Biology (The Peptides: Analysis, Synthesis, Biology), editors E.Gross and J.Meienhofer, volumes 1 and 2, Academic Press, New York, (1980), pages 3-254. Bodansky, principle of Peptide Synthesis (Principles of Peptide Synthesis), Springer-Verlag, Berlin (1984).

The present invention provides methods for making antibodies, or antigen-binding portions thereof, that specifically bind to carbohydrate antigens (e.g., Globo H). For example, a non-human animal is immunized with a composition comprising a carbohydrate antigen (e.g., Globo H), and then specific antibodies are isolated from the animal. The method may further comprise assessing binding of the antibody to the carbohydrate antigen.

Any of a variety of carbohydrate antigens, particularly Globo H, can be used in the present inventionIn the practice of the invention. Examples of carbohydrate antigens include, but are not limited to, Globo antigens such as Globo H, stage-specific embryonic antigen 3(SSEA-3) (also referred to as Gb5), stage-specific embryonic antigen 4(SSEA-4), Gb-4, and Gb-3, Lewis antigens such as sLe^x、Le^x、sLe^a、Le^aAnd Le^yPolysaccharide antigens such as polysialic acid (PSA), sTn (c) and Tn (c), Thomsen-Friedenreich antigens (TF (c)), gangliosides such as GD1, GD2, GD3, fucosyl GM1, GM1, GM2, GM3, GD1 α and GM2, sulpholipid antigens (sulpholipid antigen) such as 6Gal-HSO₃SiaLex and 6GluNAc-HSO₃Other carbohydrate antigens include, but are not limited to, α -galactose, α -Man-6-phosphate (α 0-Man-6-phosphate), α -L-rhamnose, α -GalNAc (Tn), α -NeuAc-OCH2C6H4-p-NHCOOCH2, Fuc α 51-2Gal α 41-4GalNAc α (H third type), NeuAc α 62-8NeuAc α, (NeuAc α 2-8)2 polysialic acid, NeuAca2-6Galb, NeuAcb2-6Gala (STn), Gala 9-3B 1-4 GalNAcb (NeuAca2-8)3, Galc α a-3 (Gal α -2) Gal β (Gal type A867-7-Gal type), Gal 867-4 GalNAc type A867 (HSO type β - β), and blood group Gal 869-3 (Gal type) Gal 7-3-4 GalNAc NAc type Gal 7-7A β (H type)₃-SiaLex、6GluNAc-HSO₃SiaLex and α 2-6 sialodiantenna-type N-glycans (α 2-6sialylated diantennary N-glycans).

In one embodiment, the anti-Globo H antibody or antigen-binding portion thereof can interact or bind to other carbohydrate antigens with high selectivity, such as those depicted in fig. 3A and 3B. Non-limiting examples of carbohydrate antigens are: SSEA-3, SSEA-4, Lewis antigen.

In one embodiment, the invention provides methods for making hybridomas that exhibit antibodies that specifically bind to carbohydrate antigens (e.g., GloboH). The method comprises the following steps: immunizing an animal with a composition comprising a carbohydrate antigen (e.g., Globo H); isolating splenocytes from the animal; producing hybridomas from splenocytes; and selecting hybridomas that produce antibodies that specifically bind to Globo H. Kohler and Milstein, Nature journal (Nature), 256:495, 1975. Harlow, e. and Lane, d., "antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press), N.Y., 1988.

In one embodiment, carbohydrate antigens are used to immunize mice subcutaneously. One or more additions may or may not be applied. The titer of antibodies in plasma can be monitored, for example, by ELISA (enzyme-linked immunosorbent assay) or flow cytometry. Mice with sufficient titers of antibodies against carbohydrate antigens were used for fusion. Mice may or may not be antigen-adjuvanted three days prior to sacrifice and removal of the spleen. Mouse splenocytes were isolated and fused with PEG to mouse myeloma cell lines. The resulting hybridomas are then screened for antigen-specific antibodies. Cells were plated and then cultured in selective media. Supernatants from individual wells were then screened by ELISA for human anti-carbohydrate antigen monoclonal antibodies. Antibody secreting hybridomas are recoated, screened again, and if still positive for anti-carbohydrate antigens, can be sub-transfected by limiting dilution.

Adjuvants may be used to increase the immunogenicity of one or more of the carbohydrate antigens. Non-limiting examples of adjuvants include aluminophosphates, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80(Tween 80), 0.5% w/v sorbitol trioleate (Span 85)), CpG-containing nucleic acids, QS21 (saponin adjuvant), alpha-galactosylceramide or synthetic analogs thereof (e.g., C34, see US 8,268,969), MPL (monophosphoryl lipid A) (MonophoroylLipid A), 3DMPL (3-O-deacylated MPL), extracts derived from Aquila (Aquila), ISCOMS (see Sjolander et al, (1998), J.Leucocyte Biol., 64:713, WO 90/03184; WO 96/11711; WO 00/48630; WO 98/36772; WO 364624/41720; WO 06/134423; and WO07/026190 mutations), LT 2, l-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukin, Freund's adjuvant (Freund's), N-acetyl-muramyl-L-threoninoyl-D-isoglutamyl-acid (thr-MDP), N-acetyl-normuramyl-L-propylaminoyl-D-isoglutamyl-acid (CGP 11637, referred to as nor-MDP), N-acetyl-muramyl-L-propylaminoyl-D-isoglutamyl-L-alanine-2- (1'-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryl) -ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria: monophosphoryl lipid A, mannose dimycolate (trehalosedocolate) and cell wall skeleton (MPL + TDM + CWS) in a 2% emulsion of squalene/Tween 80(Tween 80).

The immunized animal can be any animal capable of producing recoverable antibodies when administered an immunogen, such as, but not limited to, rabbits, mice, rats, hamsters, goats, horses, monkeys, baboons, and humans. In one aspect, the host is genetically transformed and produces human antibodies, e.g., mice expressing human immunoglobulin gene segments. U.S. patent nos. 8,236,311; 7,625,559 and 5,770,429, each of which is incorporated by reference herein in its entirety. Lonberg et al, Nature journal (Nature), 368 (6474): 856-859, 1994. Lonberg, N., (Handbook of Experimental Pharmacology), 113:49-101, 1994. Lonberg, n, and huskzar, d., international reviews of immunology (lnn. rev. immunol.), 13: 65-93, 1995. Harding, f. and Lonberg, n., new york science institute journal (ann.n.y.acad.sci.), 764: 536-.

After the host is immunized and the antibodies are generated, the antibodies are tested to determine that they are specific for the antigen of interest, and to determine whether they exhibit any interaction with other antigens. One method of performing these assays is a serum screening assay, as described in U.S. patent publication No. 2004/0126829. Anti-carbohydrate antigen antibodies can be characterized as binding to the carbohydrate by a variety of well-known techniques. For example, in an ELISA, microtiter plates are coated with toxin or toxoid antigens in PBS, and then blocked with an unrelated protein, such as Bovine Serum Albumin (BSA) diluted in PBS. Dilutions of plasma from toxin immunized mice were added to each well and cultured. The microtiter plates are washed and then incubated with a secondary antibody conjugated to an enzyme (e.g., alkaline phosphatase). After washing, the microtiter plates are developed with a matrix of enzymes (e.g., ABTS) and analyzed at a specific OD. In other embodiments, to determine whether a selected monoclonal antibody binds to a carbohydrate antigen or epitope of interest, the antibody can be biotinylated, which can then be detected with streptavidin-labeled probes. Anti-carbohydrate antigen antibodies can be tested for reactivity with carbohydrates by western blotting.

Hybridomas that produce antibodies that bind to carbohydrate antigens, preferably antibodies that bind to carbohydrate antigens with high affinity, can then be sub-transfected and further characterized. One clone, derived from each hybridoma that maintains the reactivity of the parent cell (by ELISA), can then be selected for cell banking and used for antibody purification.

The antibodies, or antigen-binding fragments, variants or derivatives thereof, of the invention may also be described or specified in terms of their binding affinity to an antigen. The affinity of an Antibody for a carbohydrate antigen can be determined experimentally using any suitable method (see, e.g., Berzofsky et al, Antibody-antigen interactions, basic Immunology, Paul, eds., ravenPress, New York, N.Y. (1984); Kuby, Janis, Immunology, W.H.Freeman and Company, New York, N.Y. (1992); and methods described herein). Measurement of affinity for a particular antibody-carbohydrate antigen interaction may vary if measured under different conditions (e.g., salt concentration, pH). Thus, affinity and other antigen binding parameters (e.g., K)_D、K_a、K_d) The measurement of (a) can preferably be performed with a standardized solution of antibody and antigen, and a standardized buffer.

The subject antibodies, or antigen-binding portions thereof, have therapeutic, prophylactic, and/or diagnostic utility in vitro and in vivo. For example, the antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, inhibit, prevent relapse, and/or diagnose cancer.

The antibody or antigen-binding portion thereof can be used on cells in culture, e.g., in vitro or ex vivo. For example, cells can be cultured in culture medium in vitro and contacted by an anti-Globo H antibody or fragment thereof. The methods can be performed on cells present in a subject as part of an in vivo procedure (e.g., therapeutic or prophylactic). For the in vivo embodiment, the contacting step is effective in a subject, and comprises administering to the subject an antitoxin antibody or portion thereof under binding conditions effective to allow the antibody or portion thereof to bind to a carbohydrate antigen (e.g., Globo H) present on one or more cancer cells, e.g., breast cancer cells, in the subject.

The antibody or antigen-binding portion thereof can be administered alone or in combination with another therapeutic agent, e.g., a second monoclonal or polyclonal antibody or antigen-binding portion thereof or a chemotherapeutic agent. The combination product may be a mixture of two compounds or it may be covalently linked. In one example, an antibody or antigen-binding portion thereof that specifically binds to Globo H can be combined with an antibody (single or multiple) or antigen-binding portion thereof that specifically binds VEGF. In another example, the second agent is a chemotherapeutic agent (e.g., cyclophosphamide, 5-fluorouracil, or actinomycin-D). The antibodies can also be administered in combination with cancer vaccines, e.g., Globo H conjugated to diphtheria toxin and saponin adjuvants.

Methods for inhibiting cancer cells

The invention also provides methods for inhibiting the growth of a cell in vitro, ex vivo, or in vivo, wherein the cell, such as a cancer cell, can be contacted with an effective amount of an antibody, or antigen-binding portion thereof, as described herein. Pathological cells or tissues such as hyperproliferative cells or tissues can be treated by contacting the cells or tissues with an effective amount of an antibody or antigen-binding portion thereof of the invention. The cells, such as cancer cells, may be primary cancer cells or may be cultured cells available from tissue banks, such as the American Type Culture Collection (ATCC). The pathological cells may be glob H-expressing cancers, gliomas, meningiomas, pituitary linear tumors, or cells that metastasize from the CNS of systemic, lung, prostate, breast, hematopoietic, or ovarian cancers. The cells may be from a vertebrate, preferably a mammal, more preferably a human. U.S. patent publication No. 2004/0087651. Balassiano et al, (2002), International journal of molecular medicine (Intern.J.mol.Med.), 10: 785-. Thorne, et al, (2004), journal of Neuroscience (Neuroscience), 127: 481-. Fernandes et al, (2005), tumor Reports (Oncology Reports), 13: 943-. Da Fonseca et al, (2008), neurosurgical journal (Surgical Neurology), 70: 259267. Da Fonseca et al, (2008), Experimental immunology and therapeutics literature (Arch. immunological. the. exp.), 56: 267-276. Hashizume et al, (2008), journal of neurooncology (Neurocollegy), 10: 112-. In one embodiment, the cancer is a Globo H-expressing cancer. In another embodiment, the cancer is a cancer that displays SSEA-3. In yet another embodiment, the cancer is a cancer that displays SSEA-4. Cancers that express Globo H, cancers that express SSEA-3, and cancers that express SSEA-4 include, but are not limited to, breast, lung, prostate, pancreatic, gastric, ovarian, and endometrial as well as colon, liver, nasopharyngeal, skin, oral, kidney, brain, cervical, and bladder cancers.

The in vitro efficacy of an antibody or antigen-binding portion thereof of the invention can be determined using methods well known in the art. For example, the cytotoxicity of an antibody, or antigen-binding portion thereof, can be studied by MTT [3- (4, 5-dimethylthiazol-2-yl) -2, 5-biphenyltetrazolium bromide ] (3- (4, 5-dimethylthiazolidin-2-yl) -2, 5-diphenyltetrazolium bromide) cytotoxicity assay. The MTT assay is based on the principle that MTT, a tetrazolium salt, is taken up by metabolically active cells, where its metabolism is a blue colored formazan product (blue colored formazan product) that can be measured spectroscopically. Journal of Immunological Methods (J.of Immunological Methods), 65: 5563, 1983. The cytotoxicity of the antibodies of the invention, or antigen-binding portions thereof, can be studied by colony formation assays. Functional detection for binding to Globo H antigen can be performed by ELISA. The cell cycle clock according to an antibody or antigen-binding portion thereof can be studied by standard Propidium Iodide (PI) staining and flow cytometry. Invasion inhibition can be studied by Boyden's Chamber (Boyden chamber). In this assay, a layer of reconstituted basement membrane, Matrigel, was coated on chemotaxis filters and served as a barrier to cell migration in the boiden chamber. Only cells with invasive capacity can cross the Matrigel barrier. Other assays include, but are not limited to, cell viability assays, apoptosis assays, and morphology assays.

The assay can also be performed in vivo using a murine model. See, e.g., b.teicher, Tumor Models for Efficacy Determination (Tumor Models for Efficacy Determination), molecular cancer therapy journal (Mol cancer), 2006; 5:2435-2443.

Pharmaceutical composition

In one embodiment, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding portion thereof described herein, and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises an isolated nucleic acid encoding an antibody or antigen-binding portion thereof of the invention, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, and the like. In one embodiment, the composition is effective to inhibit cancer cells in a subject.

Routes of administration of the pharmaceutical compositions of the present invention include, but are not limited to, intravenous, intramuscular, intranasal, subcutaneous, oral, topical, subcutaneous, intradermal, transdermal, subdermal, parenteral, rectal, spinal, or epidermal administration.

The pharmaceutical compositions of the present invention may be prepared as injectables, either as liquid solutions or suspensions, or in solid form suitable for solution or suspension in a liquid carrier prior to injection. The pharmaceutical compositions may also be prepared as the active ingredient in solid form, emulsified or encapsulated in a liposome carrier or other particulate carrier used for sustained delivery. For example, the pharmaceutical composition may be in the form of an oil emulsifier, a water-in-oil emulsion, a water-in-oil-in-water emulsionAgents, site-specific emulsions, stable emulsions, viscous emulsions, microemulsions, nanoemulsions, liposomes, microparticles, microspheres, nanospheres, nanoparticles, and a variety of natural or synthetic polymers, such as non-absorbable impermeable polymers such as ethylene vinyl acetate copolymers andcopolymers, swallowable polymers such as hydrogels, or absorbable polymers such as collagen and certain polymeric acids or polyesters, such as those used to make absorbable sutures, which allow for sustained release of the pharmaceutical composition.

The antibodies of the invention or antigen binding portions thereof are formulated into pharmaceutical compositions for delivery to a mammalian subject. The pharmaceutical composition is administered alone and/or in admixture with a pharmaceutically acceptable carrier, excipient or vehicle. Suitable carriers are such as, for example, water, saline, dextrose, glycerol, ethanol, or the like, as well as combinations thereof. In addition, the carrier may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants. A pharmaceutically acceptable carrier may contain a physiologically acceptable compound that acts such as, for example, to stabilize, or increase or decrease the rate of absorption or clearance of the pharmaceutical composition of the invention. Physiologically acceptable compounds may include, for example, sugars such as glucose, sucrose, or polyglucose; antioxidants such as ascorbic acid or glutathione; a chelating agent; low molecular weight proteins; a cleaning agent; a liposome carrier; or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives. See, for example, the twenty-first edition of the Science of rimming general pharmacology (Remington's Pharmaceutical Science), MackPublishing Company, Easton, Pa. ("Remington's"). The pharmaceutical compositions of the present invention may also include auxiliary substances, such as pharmaceutical agents, interleukins, or other biological response modifiers.

In addition, the pharmaceutical composition may be formulated as a neutral or salt form of the pharmaceutical composition. Pharmaceutically acceptable salts include acid addition salts (formed as the free amine group of the active polypeptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid; or formed with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, proparacaine, and the like.

The actual methods of preparing such dosage forms are well known or will be apparent to those skilled in the art. See, e.g., the science of rimming science of pharmacology, Mack Publishing Company, Easton, bingo, Pennsylvania, first twenty edition.

The pharmaceutical compositions can be administered in a single dose treatment or in multiple dose treatments on a schedule and for a period of time appropriate to the age, weight and condition of the subject, using the particular composition and route of administration, whether the pharmaceutical composition is used for prophylactic or therapeutic purposes, and the like. For example, in one embodiment, the pharmaceutical composition according to the invention is administered monthly, twice monthly, three times monthly, once every other week (qow), once weekly (qw), twice weekly (biw), three times weekly (tiw), four times weekly, five times weekly, six times weekly, once every other day (qod), daily (qd), twice daily (qid), or three times daily (tid).

The period of time during which the antibody according to the invention is administered, e.g., the pharmaceutical composition is administered, may vary depending on any of a variety of factors, such as the subject's response, etc. For example, the pharmaceutical composition may be administered over a period of time ranging from about one or more seconds to one or more hours, one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to 2 years, or from about 2 years to about 4 years or more.

To facilitate administration and uniformity of dosage, oral or parenteral pharmaceutical compositions in unit dosage form may be employed. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each dose contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for animal use. In one embodiment, the dose of such a compound is in a range of circulating concentrations that include ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form administered and the route of administration utilized. In another embodiment, the therapeutically effective dose can be estimated initially from cell culture assays. The dose can be formulated to achieve a circulating plasma concentration range in animal models, including IC as determined in cell culture₅₀(i.e., the concentration of test compound that achieves half-maximal inhibition of symptoms). Sonderstrup, Springer, symposium of pathology and immunology (Sem. Immunopathol.), 25:35-45, 2003. Nikula et al, inhalation toxicology (Inhal. Toxicol.)4(12) 123-53, 2000.

A therapeutically or prophylactically effective amount of an antibody, or antigen-binding portion thereof, of the invention illustratively ranges from about 0.001 to about 60mg/kg body weight, about 0.01 to about 30mg/kg body weight, about 0.01 to about 25mg/kg body weight, about 0.5 to about 25mg/kg body weight, about 0.1 to about 20mg/kg body weight, about 10 to about 20mg/kg body weight, about 0.75 to about 10mg/kg body weight, about 1 to about 10mg/kg body weight, about 2 to about 9mg/kg body weight, about 1 to about 2mg/kg body weight, about 3 to about 8mg/kg body weight, about 4 to about 7mg/kg body weight, about 5 to about 6mg/kg body weight, about 8 to about 13mg/kg body weight, about 8.3 to about 12.5mg/kg body weight, about 4 to about 6mg/kg body weight, about 4.2 to about 6.3mg/kg body weight, about 1.6 to about 2.5mg/kg body weight, From about 2 to about 3mg/kg body weight, or about 10mg/kg body weight.

The pharmaceutical composition is formulated to contain an effective amount of an antibody or antigen-binding portion thereof of the invention, wherein the amount depends on the animal to be treated and the condition being treated. In one embodiment, the antibodies of the invention, or antigen binding portions thereof, are administered in a dosage form ranging from about 0.01mg to about 10g, from about 0.1mg to about 9g, from about 1mg to about 8g, from about 2mg to about 7g, from about 3mg to about 6g, from about 10mg to about 5g, from about 20mg to about 1g, from about 50mg to about 800mg, from about 100mg to about 500mg, from about 0.01 μ g to about 10g, from about 0.05 μ g to about 1.5mg, from about 10 μ g to about 1mg of protein, from about 30 μ g to about 500 μ g, from about 40 μ g to about 300 μ g, from about 0.1 μ g to about 200 μ g, from about 0.1 μ g to about 5 μ g, from about 5 μ g to about 10 μ g, from about 10 μ g to about 25 μ g, from about 25 μ g to about 50 μ g, from about 50 μ g to about 100 μ g, from about 100 μ g to about 500 μ g, from about 500 μ g to about 1mg, from about 1mg to about 2 mg. The degree of a specific dose for any particular subject will depend upon a variety of factors including the specific peptide activity undergoing course of treatment, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination, and severity of the particular disease, and can be determined by one of skill in the art without undue experimentation.

The antibodies and antigen-binding portions thereof, pharmaceutical compositions and methods of the invention can be used in all vertebrates, e.g., mammals and non-mammals, including humans, mice, rats, guinea pigs, hamsters, dogs, cats, cows, horses, goats, sheep, pigs, monkeys, apes, gorillas, chimpanzees, rabbits, ducks, geese, chickens, amphibians, reptiles, and other animals.

The following examples of specific aspects for carrying out the invention are provided for the purpose of illustration only and are not intended to limit the scope of the invention in any way.

Examples of the invention

Example 1: hybridoma fusion and screening

A typical hybridoma fusion is performed. Mice received a primary immunization with Globo H-KLH (keyhole limpet Hemocyanin) conjugated with saponin adjuvant(immunization) and was added sequentially on days 7, 14, and 24. Bleeding tests were performed on days 10, 17, 21 and 24, and sera were tested to check the titer of anti-Globo H antibodies. Five mice were found to produce high anti-Globo H IgG and anti-Globo H IgM titers and were used for hybridoma production. Mouse hybridoma cells are used to fuse with mouse splenocytes, in accordance withAnd Milstein: (G. And Milstein C, 1975). Hybridoma supernatants were screened by affinity ELISA at 0.2. mu.g Globo H-brain amide/well. anti-Globo H Vk9mAb as a positive control group. OD of hybridoma clones with supernatant undiluted>Two background values were chosen. The first five hybridoma clones were 1E1, 2C2, 2F8, 3D7, 7a 11.

Example 2: kinetic analysis of mouse monoclonal antibodies

Kinetic binding experiments were performed at 25 ℃ with Biacore T100 (GE Healthcare) by single-cycle kinetics (SCK) and multi-cycle kinetics (MCK) methods.

Globo H was immobilized by amine coupling (immobilized) according to the manufacturer's instructions. Globo H-amine was diluted to 15mg/ml in immobilization buffer (10mM sodium acetate, pH4.5) and immobilized at 25 ℃ using a flow rate of 5. mu.l/min.

anti-Globo H antibodies (Globo H-Vk9mAb, Globo H-2C2 mAb, and Globo H-3D7 mAb) were diluted to 200nM (50nM) in running buffer. 200. mu.l of 200nM (50nM) solution can be mixed with 200. mu.l of running buffer to obtain a 100nM (25nM) solution. The dilutions were continued as the following dilution series: 200. 100, 50, 25 and 12.5nM (assay concentrations: 50nM, 25nM, 12.5nM, 6.25nM, 3.125 nM). The diluted samples were placed on shelf Positions (Rack Positions) and tested by the MCK and SCK methods. Samples for the MCK and SCK sequences were subjected to a dissociation time of 420 seconds. The surface was regenerated by injection of 10mM glycine ph2.0/1.5(v/v ═ 1) solution for 40 seconds. The SCK and MCK data were nested into the 1:1 binding model using Biacore evaluation software 2.0.

Analysis of the results dissociation constant (K)_D) A magnitude used to describe the strength of binding between an antibody and an antigen; kon (1/Ms), which is the binding rate of antibody-antigen complex formation; koff (1/s), off-rate of dissociation of the antibody-antigen complex; rmax, maximum amount of analyte reaction. Table 2 shows the hybridomas derived from: affinity and kinetic data for anti-Globo H antibodies to VK9, 2C2, and 3D 7. The 3D7 antibody and 2C2 antibody had higher binding affinity than the VK9 antibody.

TABLE 2 kinetic data for anti-Globo H antibodies

Hybridoma cell	K_D(M)	k_on(1/Ms)	k_off(1/s)	R_max	Chi²
						VK9	1.436E-7	2.994E+4	4.298E-3	68.3	8.48
3D7	7.854E-9	2.760E+5	2.168E-3	203.5	0.6514
						2C2	7.631E-9	2.810E+5	2.144E-3	187.4	0.542

Example 3: affinity assay for anti-Globo H antibodies

The following anti-Globo H antibodies can be directed against EC₅₀And percent cell binding were tested: globo H-VK9mAb, Globo H-1E 1mAb, Globo H-2C2 mAb, Globo H-2F8mAb, Globo H-3D7 mAb, and Globo H-7A11 mAb.

The method comprises the following steps:

for EC₅₀ELISA affinity of (a): wells were coated with 0.2. mu.g of Globo H-brain amide per well on ice. After blocking, between 6.2ng/ml to 51200ng/ml of test antibody was added to the wells. After incubation at room temperature for 1 hour, excess antibody was removed by washing 3 times. Goat anti-mouse IgG-HRP (1:533) was added. Color development was quantified on a disc analyzer at 490 nm. EC (EC)₅₀Determined by Prism 5.0 software.

FACS for percentage binding of cells to antibody: cancer cell lines were prepared in a total amount of 200,000 cells in 50. mu.l of FACS buffer per tube. The indicated antibodies were added to reach a final concentration of 1. mu.g/ml. After gentle shaking, the tubes were placed on ice and incubated for approximately 1 hr. After washing with FACS buffer, anti-mouse IgG-PE was added in FACS buffer to reach a final concentration of 4 μ g/ml. After gentle shaking, the tubes were placed on ice and incubated for approximately 30 min. After washing with FACS buffer, the cells tested were resuspended in 200 μ l of FACS buffer. After performing flow cytometry, the percentage of cell binding can be analyzed by WinMDI software. In the histogram, the culture of secondary antibodies was used only to define the background (M1) and binding (M2) regions. The percentage of binding region (M2) of the indicated antibody was determined based on the secondary antibody setting as background (M1) only.

Antibodies binding to Globo H in breast cancer cell line (MCF-7), lung cancer cell line (LLC1), and pancreatic cancer cell line (HPAC) can be assessed using Fluorescence Activated Cell Sorting (FACS) analysis.

As a result:table 3 summarizes EC for Globo H-2C2 mAb, Globo H-2F8mAb, Globo H-3D7 mAb, Globo H-7A11mAb, and Globo H-1E 1mAb₅₀And (4) data. The results show that Globo H-2C2 mAb, Globo H-2F8mAb, Globo H-3D7 mAb, and Globo H-7A11mAb are more effective in neutralizing Globo H antigen than Globo H-VK9 mAb. FACS analysis showed that Globo H-2C2 mAb and Globo H-3D7 mAb had higher binding affinity to Globo H antigen than Globo H VK9mAb in breast cancer cell lines. In addition, Globo H-2C2 mAb and Globo H-3D7 mAb have higher binding affinity to Globo H antigen in pancreatic cancer cell line (HPAC) than Globo H VK9 mAb. In addition, Globo H-1E 1mAb, Globo H-2C2 mAb, Globo H-3D7 mAb, and Globo H-7A11mAb have higher binding affinity to Globo H antigen than Globo H VK9mAb in lung cancer cell lines (LLC 1).

TABLE 3 EC of anti-Globo H antibodies₅₀And binding affinity

EC₅₀Values are antibody concentrations that neutralize 50% of Globo H.

Example 4: in vivo anti-tumor assessment of anti-Globo H antibodies

Nude mice weighing 33g xenografted with human pancreatic cancer (HPAC) were randomly divided into the following 6 experimental groups:

the mice were observed for tumor volume over a 29 day period, and the results were recorded and summarized in figure 1.

As a result: on day 29, tumor volume decreased in the following order: VK9 ═ 3D7<2C2 group. Tumor volume in group 2C2 was significantly reduced compared to control (P < 0.05).

Example 5: in vivo anti-tumor assessment of anti-Globo H antibodies

Nude mice with a weight of 27g xenografted with human breast cancer (MCF7) were randomly divided into the following 5 experimental groups:

the mice were observed for a period of 18 days for tumor volume, and the results were recorded and summarized in figure 2.

As a result:as shown in figure 2, after day 15, tumor volume was reduced compared to control in the following groups: 2C2(4mg/kg)>2C2(0.4 mg/kg). Tumor volume in 2C2(4mg/kg) was significantly reduced compared to the control group starting on day 8 (P)<0.05)。

Example 6: interaction of anti-Globo H antibodies

In vitro cross-over evaluations of anti-Globo H antibodies (Globo H2C 2, 7a11, 3D7, 2F8, 1E1mAb, and Globo H Vk9) were performed.

The method comprises the following steps:

GlycoDx cassette (GlycoDx cassettes) 620. mu.L of wash buffer, 100. mu.L of diluted anti-GloboH antibody, 100. mu.L of blocking buffer, 120. mu.L of conjugation buffer, and 120. mu.L of matrix buffer were added. The cassette was detected with a CCD analyzer and the data was output to an Excel spreadsheet for further analysis.

As a result: as shown in fig. 3A, Globo H2C 2 mAb binds to Globo H and shows interactive responses to other carbohydrate antigens, such as Lewis antigens (sLe)^xAnd sLe^a) And S15-S27 antigens (see table 4 for a list of carbohydrate antigens). Globo H7 a11mAb binds to Globo H and shows interactive responses to other carbohydrate antigens, such as Lewis antigens (sLe)^xAnd sLe^a) And S15-S17, S19-S22 antigens (see FIG. 3B). Globo H3D 7 mAb binds to Globo H and shows interactive responses to other carbohydrate antigens, such as Lewis antigens (sLe)^x、sLe^aAnd Le^y) And S15-S22 antigen (see FIG. 3C). Globo H2F 8mAb binds to Globo H and shows interactive responses to other carbohydrate antigens, such as Lewis antigens (sLe)^xAnd sLe^a) And S15, S17, and S21 antigens (see fig. 3D). Globo H1E 1mAb binds to Globo H and shows interactive responses to other carbohydrate antigens, such as Lewis antigens (sLe)^x) And S16, S17, and S20-S22 antigens (see FIG. 3E). In contrast, Globo H VK9mAb only bound to Globo H and showed no interaction with other carbohydrate antigens (see figure 3F).

TABLE 4 list of carbohydrate antigens in panels 3A to 3C

Example 7: binding affinity of anti-Globo H antibodies

The following in vitro evaluation of anti-Globo H humanized antibodies was performed. Table 5 lists the amino acid sequences of the heavy and light chain regions of the humanized antibody derived from hybridoma 2C 2.

TABLE 5 amino acid sequence of 2C2 humanized antibody

The results of the binding affinities determined by the ELISA method are listed in tables 6-9.

Table 6: binding affinity of antibodies with chimeric light chains and humanized heavy chains

These results show that the amino acid substitution at position 9 (from G to a) in FW2 of the heavy chain reduces the binding affinity of the antibody from 0.4 to 0.17.

Table 7: binding affinity of antibodies with humanized light and humanized heavy chains

Light chain	Heavy chain	Affinity ELISA (optical Density)
			Light chain 2	Heavy chain 2	0.42
Light chain 2-22	Heavy chain 2	0.08
			Light chain 2-23	Heavy chain 2	0.09

Table 8: binding affinity of antibodies with humanized light and humanized heavy chains

Light chain	Heavy chain	Affinity ELISA (optical Density)
			Light chain 2	Heavy chain 2-21	0.53
Light chain 2-22	Heavy chain 2-21	0.08
			Light chain 2-23	Heavy chain 2-21	0.12

Table 9: binding affinity of antibodies with humanized light and humanized heavy chains

Light chain	Heavy chain	Affinity ELISA (optical Density)
			Light chain 2	Heavy chain 2-22	0.32
Light chain 2-22	Heavy chain 2-22	0.07
			Light chain 2-23	Heavy chain 2-22	0.08

These results show that amino acid substitutions at position 12 of FW2 of the light chain (from P to L) reduced the binding affinity from 0.32-0.53 to 0.07-0.08, and amino acid substitutions at position 13 of FW2 of the light chain (from W to L) reduced the binding affinity from 0.32-0.53 to 0.08-0.12.

While particular aspects of the present invention have been illustrated and described, such aspects are to be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the appended claims. All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

List of sequence listing: SEQ ID Nos. 1 to 90

Claims

1. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 3; and/or a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 4.

2. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain region, wherein the heavy chain region comprises from about 80% to about 100% Complementarity Determining Region (CDR) amino acid sequences homologous to an amino acid sequence selected from SEQ ID NOs:5, 6, or 7.

3. The antibody, or antigen-binding portion thereof, of claim 2, further comprising a framework that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID No. 11, wherein the framework is between CDR1 and CDR2 of the heavy chain region.

4. An antibody, or antigen-binding portion thereof, comprising:

a light chain region, wherein the light chain region comprises about 80% to about 100% complementarity determining region amino acid sequences homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 9, or 10.

5. The antibody, or antigen-binding portion thereof, of claim 4, further comprising a framework that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO 12, wherein the framework is between CDR1 and CDR2 of the light chain region.

6. An antibody, or an antigen-binding portion thereof, produced by the hybridoma designated as 2C2 deposited under american type culture collection accession number PTA-121138.

7. The antibody or antigen-binding portion thereof of any one of claims 1 to 6, wherein the variable domain is capable of binding one or more carbohydrate antigens.

8. The antibody or antigen-binding portion thereof of claim 7, wherein the one or more carbohydrate antigens is Globo H, sLe^x、sTn、Tn、sLe^a、α-NeuAc-OCH₂C₆H₄-p-NHCOOCH₂、Fucα1-2Galβ1-4GalNAcβ、NeuAca2-6Galb、Gala1-3Galb1-4GlaNAcb、(NeuAca2-8)3、6Gal-HSO₃-SiaLex、6GluNAc-HSO₃-SiaLex, α 2-6 sialyldiaaeric N-glycans or polysialic acid.

9. The antibody or antigen-binding portion thereof of any one of claims 1 to 6, wherein the antibody or antigen-binding portion thereof is selected from the group consisting of: (a) a whole immunoglobulin molecule; (b) scFv; (c) a Fab fragment; (d) f (ab') 2; or (e) a disulfide bond Fv.

10. A pharmaceutical composition comprising:

the antibody or antigen binding portion thereof of any one of claims 1 to 9; and at least one pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, further comprising at least one additional therapeutic agent.

12. A method of inhibiting cancer cell proliferation, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of claims 1-9, wherein cancer cell proliferation is inhibited.

13. A hybridoma designated as 2C2 deposited under accession number PTA-121138 of american type culture collection.

14. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain variable domain comprising about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO 13; and/or a light chain variable domain comprising about 80% to about 100% amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO. 14.

15. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain region, wherein the heavy chain region comprises from about 80% to about 100% complementarity determining region amino acid sequence homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 16, or 17.

16. An antibody, or antigen-binding portion thereof, comprising:

a light chain region, wherein the light chain region comprises about 80% to about 100% complementarity determining region amino acid sequences homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:18, 19, or 20.

17. An antibody, or an antigen-binding portion thereof, produced by the hybridoma designated as 3D7 deposited under american type culture collection accession number PTA-121310.

18. The antibody or antigen-binding portion thereof of any one of claims 14 to 17, wherein the variable domain is capable of binding one or more carbohydrate antigens.

19. The antibody or antigen-binding portion thereof of claim 18, wherein the one or more carbohydrate antigens is Globo H sLe^x、sTn、Tn、sLe^a、Le^y、α-NeuAc-OCH₂C₆H₄-p-NHCOOCH₂Fuc α 1-2Gal β 1-4GalNAc β, NeuAca2-6Galb, Gala1-3Galb1-4GlaNAcb, (NeuAca2-8)3 or polysialic acid.

20. The antibody or antigen-binding portion thereof of any one of claims 14 to 17, wherein the antibody or antigen-binding portion thereof is selected from the group consisting of: (a) a whole immunoglobulin molecule; (b) scFv; (c) a Fab fragment; (d) f (ab') 2; or (e) a disulfide bond Fv.

21. A hybridoma designated as 3D7 deposited under american type culture collection accession number PTA-121310.

22. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain variable domain comprising about 80% to about 100% of the amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO 21; and/or a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 22.

23. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain region, wherein the heavy chain region comprises from about 80% to about 100% complementarity determining region amino acid sequence homologous to an amino acid sequence selected from SEQ ID NOs:23, 24, or 25.

24. An antibody, or antigen-binding portion thereof, comprising:

a light chain region, wherein the light chain region comprises about 80% to about 100% complementarity determining region amino acid sequences homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:26, 27, or 28.

25. An antibody, or an antigen-binding portion thereof, produced by the hybridoma designated as 7a11 deposited under american type culture collection accession number PTA-121311.

26. The antibody or antigen-binding portion thereof of any one of claims 22 to 25, wherein the variable domain is capable of binding to one or more carbohydrate antigens.

27. The antibody or antigen-binding portion thereof of claim 26, wherein said oneOr the saccharide antigens are Globo H, sLe^x、sTn、Tn、sLe^a、α-NeuAc-OCH₂C₆H₄-p-NHCOOCH₂Fuc α 1-2Gal β 1-4GalNAc β, NeuAca2-6Galb, Gala1-3Galb1-4GlaNAcb or (NeuAca2-8) 3.

28. The antibody or antigen binding portion thereof of any one of claims 22 to 25, wherein the antibody or antigen binding portion thereof is selected from the group consisting of (a) a whole immunoglobulin molecule; (b) scFv; (c) a Fab fragment; (d) f (ab') 2; or (e) a disulfide bond Fv.

29. A hybridoma designated as 7a11 deposited under american type culture collection accession number PTA-121311.

30. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 29; and/or a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO. 30.

31. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain region, wherein the heavy chain region comprises from about 80% to about 100% complementarity determining region amino acid sequence homologous to an amino acid sequence selected from SEQ ID NOs:31, 32, or 33.

32. An antibody, or antigen-binding portion thereof, comprising:

a light chain region, wherein the light chain region comprises about 80% to about 100% complementarity determining region amino acid sequences homologous to an amino acid sequence selected from SEQ ID NOs:34, 35, or 36.

33. An antibody, or an antigen-binding portion thereof, produced by the hybridoma designated as 2F8 deposited under american type culture collection accession number PTA-121137.

34. The antibody or antigen-binding portion thereof of any one of claims 30 to 33, wherein the variable domain is capable of binding to one or more carbohydrate antigens.

35. The antibody or antigen-binding portion thereof of claim 34, wherein the one or more carbohydrate antigens is Globo H sLe^x、Tn、sLe^aFuc α 1-2Gal β 1-4GalNAc β or Gala1-3Galb1-4 GlaNAcb.

36. The antibody or antigen binding portion thereof of any one of claims 30 to 33, wherein the antibody or antigen binding portion thereof is selected from the group consisting of (a) a whole immunoglobulin molecule; (b) scFv; (c) a Fab fragment; (d) f (ab') 2; or (e) a disulfide bond Fv.

37. A hybridoma designated as 2F8 deposited under american type culture collection accession number PTA-121137.

38. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 37; and/or a light chain variable domain comprising an amino acid sequence that is about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 38.

39. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain region, wherein the heavy chain region comprises from about 80% to about 100% complementarity determining region amino acid sequence homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:39, 40, or 41.

40. An antibody, or antigen-binding portion thereof, comprising:

a light chain region, wherein the light chain region comprises about 80% to about 100% complementarity determining region amino acid sequences homologous to an amino acid sequence selected from SEQ ID NOs:42, 43, or 44.

41. An antibody, or an antigen-binding portion thereof, produced by the hybridoma designated as 1E1 deposited under american type culture collection accession number PTA-121312.

42. The antibody or antigen-binding portion thereof of any one of claims 38 to 41, wherein the variable domain is capable of binding to one or more carbohydrate antigens.

43. The antibody or antigen-binding portion thereof of claim 42, wherein the one or more carbohydrate antigens is Globo H, sLe^x、sTn、α-NeuAc-OCH₂C₆H₄-p-NHCOOCH₂Fuc α 1-2Gal β 1-4GalNAc β, Gala1-3Galb1-4GlaNAcb, or (NeuAca2-8) 3.

44. The antibody or antigen binding portion thereof of any one of claims 38-41, wherein said antibody or antigen binding portion thereof is selected from the group consisting of (a) a whole immunoglobulin molecule; (b) scFv; (c) a Fab fragment; (d) f (ab') 2; or (e) a disulfide bond Fv.

45. A hybridoma designated as 1E1 deposited under american type culture collection accession number PTA-121312.

46. An antibody, or antigen-binding portion thereof, comprising:

a light chain region, wherein the light chain region comprises three complementarity determining regions CDR1, CDR2, and CDR3 having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:8, 9, and 10, respectively; and

a framework between CDR1 and CDR2 of a light chain having an amino acid sequence from about 80% to about 100% homologous to SEQ ID No. 12, wherein the framework comprises proline at position 12 or tryptophan at position 13 or a combination thereof, wherein the antibody or antigen-binding portion thereof binds to Globo H.

47. An antibody, or antigen-binding portion thereof, comprising:

a heavy chain region, wherein the heavy chain region comprises three complementarity determining regions CDR1, CDR2, and CDR3 having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs:5, 6, and 7, respectively; and

a framework between CDR1 and CDR2 of a heavy chain having an amino acid sequence from about 80% to about 100% homologous to SEQ ID No. 11, wherein the framework comprises a glycine at position 9, wherein the antibody or antigen-binding portion thereof binds to Globo H.

48. A humanized antibody, or an antigen-binding portion thereof, comprising:

a framework between the light chain leader sequence and CDR1 having an amino acid sequence that is about 80% to about 100% homologous to SEQ id No. 88; and

a framework between CDR2 and CDR3 of the light chain having an amino acid sequence that is about 80% to about 100% homologous to SEQ ID NO: 90.

49. A humanized antibody, or an antigen-binding portion thereof, comprising:

a heavy chain region, wherein the heavy chain region comprises three complementarity determining regions CDR1, CDR2, and CDR3 having amino acid sequences that are about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs 5,6, and 7, respectively;

a framework between the leader sequence of the heavy chain and CDR1 having an amino acid sequence from about 80% to about 100% homologous to SEQ id No. 87; and

a framework between CDR2 and CDR3 of the heavy chain having an amino acid sequence from about 80% to about 100% homologous to SEQ ID NO. 89.