HK1232767B - Antibodies, hybridoma producing such antibodies and uses thereof - Google Patents

Description

Antibodies, hybridomas producing the same, and uses thereof

Cross-reference to related applications

This application claims the benefit of U.S. Patent Application No. 61/977,824, filed April 10, 2014, and U.S. Patent Application No. 62/057,381, filed September 30, 2014, which are hereby incorporated by reference in their entireties.

Technical Field

The present invention relates to antibodies against tumor-associated carbohydrate antigens, comprising antibodies specifically targeting at least one tumor-associated carbohydrate antigen or a specific portion or variant thereof, as well as nucleic acids encoding these antibodies, complementary nucleic acids, vectors, host cells, and methods for producing and using the same, including therapeutic formulations and pharmaceutical compositions containing the antibodies. Furthermore, methods are provided for administering an effective amount of the antibodies to a subject to inhibit cancer cells.

Background Art

Several surface carbohydrates are expressed on malignant tumor cells. For example, Globo H (Fuc α1→2Galβ1→3GalNAcβ1→3Gal α1→4Galβ1→4Glc) has been shown to be overexpressed in a variety of epithelial cancers and is associated with tumor aggressiveness and poor prognosis in breast cancer and small cell lung cancer. Previous studies have shown that Globo H and stage-specific embryonic antigen 3 (SSEA3, also known as Gb5) are observed in breast cancer and breast cancer stem cells (WW Chang et al., "Expression of Globo H and SSEA3 in breast cancer stem cells and the involvement of fucosyl transferases 1 and 2 in Globo H synthesis", PNAS, 105(33): 11667-11672).

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

Summary of the 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 thereof, and methods of making and using the same, as described and enabled herein, and in combination with known methods in the art. The antibodies, or antigen-binding portions thereof, may have a dissociation constant (KD) of about 10E-7 M or less, about 10E-8 M or less, about 10E-9 M or less, about 10E-10 M or less, about 10E-11 M or less, or about 10E-12 _M or less. The antibodies, or antigen-binding portions thereof, may 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 present invention provides an antibody, or an antigen-binding portion thereof, comprising a heavy chain variable domain comprising an amino acid sequence 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 about 80% to about 100% homologous to the amino acid sequence set forth in SEQ ID NO:4.

In a fourth embodiment, the present 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, each having an amino acid sequence about 80% to about 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 5, 6, and 7. In an exemplary embodiment, the heavy chain further comprises a framework between the leader sequence and the CDR1 having an amino acid sequence about 80% to about 100% homologous to SEQ ID NO: 87. In another embodiment, the heavy chain further comprises a framework between the CDR2 and CDR3 having an amino acid sequence 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 the heavy chain having an amino acid sequence about 80% to about 100% homologous to SEQ ID NO: 11, wherein the framework contains 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 present invention provides an antibody, or an antigen-binding portion thereof, comprising a light chain region, wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, each having an amino acid sequence about 80% to 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10. In an exemplary embodiment, the light chain further comprises a framework between the leader sequence and the CDR1 having an amino acid sequence about 80% to about 100% homologous to SEQ ID NO: 88. In another exemplary embodiment, the light chain further comprises a framework between the CDR2 and CDR3 of the light chain having an amino acid sequence 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 CDR1 and CDR2 of the light chain having an amino acid sequence about 80% to about 100% homologous to SEQ ID NO: 12, wherein the framework contains a 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 the light chain having an amino acid sequence about 80% to about 100% homologous to SEQ ID NO: 12, wherein the framework contains a tryptophan at position 13, and the antibody, or antigen-binding portion thereof, binds to a carbohydrate antigen, such as Globo H.

In a sixth embodiment, the present invention provides an antibody, or an 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 approximately 80% to approximately 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively, and wherein the light chain region comprises three CDRs, CDR1, CDR2, and CDR3, having amino acid sequences approximately 80% to approximately 100% homologous to the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, 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 that is 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 SEQ ID NOs: 8, 9, or 10.

The present invention is also directed to an antibody, or an 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:13.

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

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

Another exemplary embodiment provides an antibody, or an 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 approximately 80% to approximately 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 approximately 80% to approximately 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 that is 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 SEQ ID NOs: 18, 19, or 20.

One embodiment of the invention is an antibody, or an 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:21.

Another embodiment of the invention is 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:22.

Another embodiment of the present invention is an antibody, or an 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: 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.

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

Another exemplary embodiment provides an antibody, or an 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 approximately 80% to approximately 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 approximately 80% to approximately 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 that is 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 that is about 80% to about 100% homologous to an amino acid sequence selected from SEQ ID NOs: 26, 27, or 28.

The present invention also discloses an antibody, or an 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 shown in SEQ ID NO:29.

The present invention also discloses an antibody, or an 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 shown in SEQ ID NO:30.

The present invention also discloses an antibody, or an 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 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.

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

Another exemplary embodiment provides an antibody, or an 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 approximately 80% to approximately 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 approximately 80% to approximately 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 that is 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 SEQ ID NOs: 34, 35, or 36.

One embodiment of the invention is an antibody, or an 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.

Another embodiment of the present invention provides an antibody, or an 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 shown in SEQ ID NO:38.

In another embodiment of the present invention, an antibody, or an antigen-binding portion thereof, is provided, 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 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.

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

Another exemplary embodiment provides an antibody, or an 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 approximately 80% to approximately 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 approximately 80% to approximately 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 that is 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 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 present 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 the Globo H-expressing cancer cells are inhibited.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

Figures 3A to 3F are bar graphs showing the binding affinities of Globo H-2C2 mAb (Figure 3A), Globo H-7A11 mAb (Figure 3B), Globo H-3D7 mAb (Figure 3C), Globo H-2F8 mAb (Figure 3D), Globo H-1E1 mAb (Figure 3E), and Globo H-VK9 mAb (Figure 3F) to various carbohydrate antigens.

DETAILED DESCRIPTION

As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

As used herein, an "effective amount" refers to a dose of a vaccine or pharmaceutical composition sufficient to reduce the symptoms and signs of cancer, such as weight loss, pain, and a palpable mass, either clinically palpable or radiologically detectable by various imaging modalities. The terms "effective amount" and "therapeutically effective amount" are used interchangeably.

The term "subject" may refer to a vertebrate having cancer or a vertebrate believed to be in need of cancer treatment. 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 and dogs, livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and experimental animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary use and pharmaceutical preparations are contemplated herein.

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

The present invention provides pharmaceutical compositions and methods for treating or inhibiting cancer cells. The pharmaceutical compositions comprise antibodies that recognize carbohydrate antigens, including mouse monoclonal antibodies, humanized antibodies, chimeric antibodies, or antigen-binding portions of any of the foregoing. These antibodies (or antigen-binding portions thereof) can neutralize carbohydrate antigens and/or inhibit cancer cells. Therefore, the antibodies or antigen-binding portions thereof of the present invention can be used to treat or inhibit cancer cells.

Antibodies of the present invention include any protein or peptide comprising at least one complementarity determining region (CDR) of a heavy or light chain, or a ligand-binding portion thereof, derived from hybridomas designated 2C2 (deposited with the American Type Culture Collection as PTA-121138); 3D7 (deposited with the American Type Culture Collection as PTA-121310); 7A11 (deposited with the American Type Culture Collection as PTA-121311); 2F8 (deposited with the American Type Culture Collection as PTA-121137); or 1E1 (deposited with the American Type Culture Collection as PTA-121312), as described herein. Antibodies include antibody fragments, antibody variants, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies. Antibodies can be produced in mice, rabbits, or humans.

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

Thus, the anti-cancer antibodies of the invention include heavy or light chain variable regions, heavy or light chain constant regions, framework regions, or any combination of portions thereof of non-murine origin, preferably of human origin, which can be incorporated into the antibodies of the invention.

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

The term "antibody" is further intended to encompass antibodies, fragments, specific portions, and variants thereof, including antibody mimetics or portions of antibodies that mimic the structure and/or function of anti-cancer antibodies or specific fragments or portions 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 plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the present invention (see, e.g., Colligan, Immunology, supra).

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

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

The humanized antibodies of the present invention are antibodies from non-human species in which the amino acid sequences in the non-antigen binding region (and/or the antigen binding region) are altered so that the antibodies more closely resemble human antibodies while maintaining their original binding ability.

Humanized antibodies can be produced by replacing variable region sequences 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 sources of such nucleic acids are well known to those skilled in the art. Recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into a suitable expression vector.

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

According to one aspect of the invention, the positions of CDRs and framework residues are determined by the method disclosed in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. According to another aspect of the invention, the antibody or antigen-binding portion thereof may have the following structure:

Leader sequence-FW1-CDR1-FW2-CDR2-FW3-CDR3-,

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

The humanized antibodies of the present invention can be produced by methods well known in the art. For example, once a non-human (e.g., murine) antibody is obtained, the variable region can be sequenced and the positions of the CDRs and framework residues can be determined. Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242. Chothia, C. et al. (1987) J. Mol. Biol., 196: 901-917. DNA encoding the light and heavy chain variable regions can be optionally linked to the corresponding constant regions and then cloned into a suitable expression vector. CDR-grafted antibody molecules can be produced by CDR grafting or CDR substitution. One, two, or all CDRs of an immunoglobulin chain can be replaced. For example, all of the CDRs of a particular antibody may be derived from a non-human animal (e.g., a mouse, such as the CDRs shown in Table 1), or at least a portion of the CDRs may be replaced. Only the CDRs necessary for the antibody to bind to a predetermined carbohydrate antigen (e.g., Globo H) need be retained. Morrison, S.L., 1985, Science, 229:1202-1207. Oi et al., 1986, BioTechniques, 4:214. U.S. Patent Nos. 5,585,089; 5,225,539; 5,693,761; and 5,693,762. EP 519596. Jones et al., 1986, Nature, 321:552-525. Verhoeyan et al., 1988, Science, 239: 1534. Beidler et al., 1988, J. Immunol., 141: 4053-4060.

Also encompassed by the invention are antibodies, or antigen-binding portions thereof, comprising one or two variable regions as disclosed herein, with the other 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 a variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies can be produced using recombinant DNA technology. Morrison et al., Proc Natl Acad Sci, 81:6851-6855 (1984). For example, the gene encoding a murine (or other species) antibody molecule is cleaved with a restriction enzyme to remove the region encoding the murine Fc, and the equivalent portion of the gene encoding the human Fc constant region is then substituted into the recombinant DNA molecule. Chimeric antibodies can also be created using recombinant DNA technology, in which DNA encoding the murine V region is joined to DNA encoding the human constant region. Better et al., Science, 1988, 240:1041-1043. Liu et al., PNAS, 1987, 84:3439-3443. Liu et al., J. Immunol., 1987, 139:3521-3526. Sun et al., PNAS, 1987, 84:214-218. Nishimura et al., Canc. Res., 1987, 47:999-1005. Wood et al., Nature, 1985, 314:446-449. Shaw et al., J. Natl. Cancer Inst., 1988, 80:1553-1559. International Patent Publication Nos. WO1987002671 and WO 86/01533. European Patent Application Nos. 184,187; 171,496; 125,023; and 173,494. U.S. Patent No. 4,816,567.

The antibody may be full-length or may comprise a fragment (or fragments) of an antibody having an antigen-binding portion, including, 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, dimeric antibodies, trimeric antibodies, tetrameric antibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single-chain antibodies generated by attaching antibody fragments using recombinant methods or synthetic linkers are also encompassed by the present invention. Bird et al., Science, 1988, 242:423-426. Huston et al., Proc. Natl. Acad. Sci., USA, 1988, 85: 5879-5883.

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

All antibody isotypes are encompassed by the present invention, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, or IgE (all classes and subclasses are encompassed by the present invention). The antibody, or antigen-binding portion thereof, may 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 regions of the antibodies of the invention, or antigen-binding portions thereof, can be derived from non-human or human sources. The frameworks of the antibodies of the invention, or antigen-binding portions thereof, can be human, humanized, non-human (e.g., a murine framework modified to reduce antigenicity in humans), or synthetic (e.g., a consensus framework).

In one embodiment, an antibody of the invention, or an antigen-binding portion thereof, 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, specifically bind to Globo H with a dissociation constant (KD) of less than about 10E-7 M, less than about 10E-8 M, less than about 10E-9 M, less than about 10E-10 M, less than about 10E-11 M, or less than about 10E-12 M. In one embodiment, the antibody, or antigen-binding portion thereof, has a dissociation constant ( _KD ) of 1 to 10 _x 10E-9 or less. In another embodiment, KD is determined by surface plasmon resonance.

The antibodies have variable heavy and variable light chain 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 chain regions of the antibody produced by clone 2C2 and can also bind to carbohydrate antigens (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 the antibodies, or antigen-binding portions thereof, for example, antibodies produced by hybridoma 2C2, hybridoma 3D7, hybridoma 7A11, hybridoma 2F8, and hybridoma 1E1, are shown in Table 1.

In related embodiments, the antibodies or antigen-binding portions thereof include, for example, the variable heavy chain CDRs and/or variable light chain CDRs of antibodies produced by hybridoma 2C2, hybridoma 3D7, hybridoma 7A11, hybridoma 2F8, and hybridoma 1E1. The variable heavy chain and variable light chain CDRs and frameworks from these hybridoma clones are shown in Table 1.

Table 1: SEQ ID NO:1–90

The present invention also encompasses nucleic acids encoding an antibody of the present invention, or an antigen-binding portion thereof, that specifically binds to a carbohydrate antigen. 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 of the present invention, or an antigen-binding portion thereof.

In specific embodiments, an antibody of the invention, or antigen-binding portion thereof, comprises an antibody that is 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:

a) SEQ ID NO: 3 (hybridoma 2C2);

b) SEQ ID NO: 13 (hybridoma 3D7);

c) SEQ ID NO: 21 (hybridoma 7A11);

d) SEQ ID NO: 29 (hybridoma 2F8); or

e) a variable heavy chain region having the amino acid sequence of any one of SEQ ID NO: 37 (hybridoma 1E1).

In specific embodiments, an antibody of the invention, or antigen-binding portion thereof, comprises an antibody that is 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:

a) SEQ ID NO: 4 (hybridoma 2C2);

b) SEQ ID NO: 14 (hybridoma 3D7);

c) SEQ ID NO: 22 (hybridoma 7A11);

d) SEQ ID NO: 30 (hybridoma 2F8); or

e) a variable light chain region having the amino acid sequence of any one of SEQ ID NO: 38 (hybridoma 1E1).

In specific embodiments, the variable heavy chain region of the antibody, or antigen-binding portion thereof, comprises an amino acid sequence that is 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 SEQ ID NO: 3, and the variable light chain region of the 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% homologous to SEQ ID NO: 3. 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 amino acid sequence of SEQ ID NO:4.

In specific embodiments, the variable heavy chain region of the antibody, or antigen-binding portion thereof, comprises an amino acid sequence that is 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 SEQ ID NO: 13, and the variable light chain region of the 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% homologous to SEQ ID NO: 13. 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 amino acid sequence of SEQ ID NO: 14.

In specific embodiments, the variable heavy chain region of the antibody, or antigen-binding portion thereof, comprises an amino acid sequence that is 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 SEQ ID NO: 21, and the variable light chain region of the 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% homologous to SEQ ID NO: 21. 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 amino acid sequence of SEQ ID NO: 22.

In specific embodiments, the variable heavy chain region of the 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% homologous to the amino acid sequence of SEQ ID NO: 29, and the variable light chain region of the 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% homologous to the amino acid sequence of SEQ ID NO: 29. 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 amino acid sequence of SEQ ID NO: 30.

In specific embodiments, the variable heavy chain region of the antibody, or antigen-binding portion thereof, comprises an amino acid sequence that is 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 SEQ ID NO: 37, and the variable light chain region of the 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% homologous to SEQ ID NO: 37. 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 amino acid sequence of SEQ ID NO: 38.

The variable heavy chain region of the 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% homologous to:

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

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

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

d) 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 one of the CDRs (SEQ ID Nos: 39, 40, and 41) of the variable heavy chain region of the antibody produced by hybridoma 1E1.

The variable light chain region of the 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% homologous to:

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

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

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

d) 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 the antibody produced by hybridoma 1E1.

The variable heavy chain region of the 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% homologous to the CDRs of the variable heavy chain region of the antibody produced by hybridoma 2C2 (SEQ ID NOs: 5, 6, and 7) or the CDRs of the variable heavy chain region of the antibody produced by hybridoma 3D7 (SEQ ID NOs: 5, 6, and 7). 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the CDRs of the variable light chain region of the antibody produced by hybridoma 2C2 (SEQ ID NOs: 8, 9, and 10). Or the amino acid sequences of the CDRs (SEQ ID NOs: 18, 19, and 20) of the variable light chain region of the antibody produced by hybridoma 3D7.

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 2C2 antibody) or SEQ ID NO: 87 (heavy chain framework 1 of humanized 2C2 antibody, see Table 1), wherein the framework is a conjugated oligonucleotide sequence that is at least about 100% homologous to the leader sequence and the antibody produced by hybridoma 2C2. 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 SEQ ID NO: 88 (light chain framework 1 of humanized 2C2 antibody, see Table 1), and the framework is in the preceding between the guide sequence and CDR1 of the variable light chain region of the antibody produced by hybridoma 2C2.

In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a framework between CDR1 and CDR2 of the variable heavy 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% homologous to SEQ ID NO: 11 (heavy chain framework 2 in 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 positions of the amino acids of SEQ ID NO: 11 are depicted as follows:

*The amino acid at position 1 of FW2(W) is the residue adjacent to CDR1.

**The amino acid at position 14 of FW2(A) is the residue adjacent to CDR2.

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% homologous to SEQ ID NO: 12 (light chain framework 2 in Table 1). In an exemplary embodiment, the framework between CDR1 and CDR2 of the variable light chain region of the antibody produced by hybridoma 2C2 contains a proline at position 12. In another exemplary embodiment, the framework between 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 CDR1 and CDR2 of the variable light chain region of the antibody produced by hybridoma 2C2 contains a proline at position 12 and a tryptophan at position 13. The positions of the amino acids of SEQ ID NO: 12 are described as follows:

*The amino acid at position 1 of FW2(W) is the residue adjacent to CDR1.

**The amino acid at position 15 of FW2(Y) is the residue adjacent to CDR2.

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 SEQ 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 the antibody produced by hybridoma 2C2. 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: 86 (light chain framework 3) or SEQ ID NO: 90 (light chain framework 3 of a humanized antibody, shown in Table 1), and said framework is between CDR2 and CDR3 of the variable light chain region of the antibody produced by hybridoma 2C2.

The variable heavy chain region of the antibody, or antigen-binding portion thereof, can comprise one, two, or three or more CDRs comprising an amino acid sequence 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 the CDRs of the variable heavy chain region of the antibody produced by hybridoma 7A11 (SEQ ID NOs: 23, 24, and 25), and the variable light ... The antibody may comprise one, two, or three or more CDRs comprising an amino acid sequence 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 the CDRs of the variable light chain region of the antibody produced by hybridoma 7A11 (SEQ ID NOs: 26, 27, and 28).

The variable heavy chain region of the 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% homologous to the CDRs of the variable heavy chain region of the antibody produced by hybridoma 2F8 (SEQ ID NOs: 31, 32, and 33) or the CDRs of the variable heavy chain region of the antibody produced by hybridoma 1E1 (SEQ ID NOs: 31, 32, and 33). 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the CDRs of the variable light chain region of the antibody produced by hybridoma 2F8 (SEQ ID NOs: 34, 35, and 36) or by hybridoma 2F9. The amino acid sequences of the CDRs (SEQ ID NOs: 42, 43, and 44) of the variable light chain region of the antibody produced by the tumor 1E1 were obtained.

In certain embodiments, the variable regions corresponding to the variable regions in Table 1 have sequence variations. For example, a heavy chain variable region has 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 substituted or deleted, but retains substantially the same immunological properties, including, but not limited to, binding to carbohydrate antigens.

In certain embodiments, the CDRs have sequence variations corresponding to the CDRs in Table 1. 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 CDR are substituted or deleted, may be present in an antibody (or antigen-binding portion thereof) that binds to a carbohydrate antigen.

The antibody or its antigen-binding portion can be a peptide. Such peptides can include variants, analogs, heterologs, homologs, and derivatives of the peptide that exhibit biological activity, such as binding to carbohydrate antigens. The peptide can contain analogs of one or more amino acids (including, for example, non-naturally occurring amino acids, amino acids naturally occurring only in unrelated biological systems, amino acids modified from mammalian systems, etc.), peptides with substituted linkages, and other modifications known in the art.

Also within the scope of the present invention are antibodies or antigen-binding portions thereof in which specific amino acids have been substituted, deleted, or added. In exemplary embodiments, these substitutions have no 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 to enhance the binding affinity of the antibody for the antigen. In yet another exemplary embodiment, a small number of selected acceptor framework residues may be replaced by corresponding donor amino acids. The donor framework may be a mature or biocloned human antibody framework sequence or a corresponding sequence. Guidance for considering how to make phenotypically silent amino acid substitutions is provided by Bowie et al., Science, 247:1306-1310 (1990). Cunningham et al., Science, 244:1081-1085 (1989). Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994). T. Maniatis, E.F. Fritsch, and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). Pearson, Methods Mol. Biol., 243:307-31 (1994). Gonnet et al., Science, 256:1443-45 (1992).

An antibody, or an antigen-binding portion thereof, can 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 mediates association with another molecule (such as a streptavidin core region or a polyhistidine tag), an amino acid linker, a signaling sequence, an immunogenic carrier, or a ligand useful for protein purification, such as glutathione-S-transferase, a histidine tag, and Staphylococcal protein A. One type of derivatized protein is produced by cross-linking two or more proteins (of the same or different types). Suitable cross-linkers include those that are heterobifunctional, having two dissimilar reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, IL. Useful detectable agents with which proteins can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting exemplary fluorescent detectable agents include fluorescein, fluorescein thiocyanate, rose bengal, and phycoerythrin. Proteins or antibodies can also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, acetylcholinesterase, glucose oxidase, and the like. Proteins can also be derivatized with prosthetic groups (e.g., streptavidin/biotin and avidin/biotin).

Nucleic acids encoding functionally active variants of the antibodies or antigen-binding portions thereof of the present invention are also encompassed by the present invention. These nucleic acid molecules can be hybridized with nucleic acids encoding any of the antibodies or antigen-binding portions thereof of the present invention under conditions of moderate stringency, high stringency, or very high stringency. Guidelines for performing hybridization 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 x SSC at approximately 45°C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 60°C; 2) highly stringent hybridization conditions: 6 x SSC at approximately 45°C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65°C; and 3) very highly stringent hybridization conditions: 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2 x SSC, 1% SDS at 65°C.

Nucleic acids encoding the antibodies or antigen-binding portions thereof of the present invention can be introduced into expression vectors capable of expression in a suitable expression system, followed by isolation or purification of the expressed antibodies or antigen-binding portions thereof. Alternatively, nucleic acids encoding the antibodies or antigen-binding portions thereof of the present invention can be translated into a cell-free translation system. U.S. Patent No. 4,816,567. Queen et al., Proc Natl Acad Sci, USA, 86:10029-10033 (1989).

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

The nucleic acids of the present invention can be expressed in a variety of suitable cells, including prokaryotic and eukaryotic cells, such as bacterial cells (e.g., E. coli), yeast cells, plant cells, insect cells, and mammalian cells. Many mammalian cell strains are known in the art and include immortalized cell strains available from the American Type Culture Collection (ATCC). Non-limiting examples of cells include mammalian origin or all cell strains of similar mammalian characteristics, including but not limited to monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2/0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma, and lymphoma cells, parental cells, derivatives, and/or modified variants. Engineered variants include, for example, modifications of the glycan profile and/or position-specific integration of positional derivatives.

The invention also provides cells for containing the nucleic acids described herein. The cell can be a hybridoma or a transfectant, such as the hybridoma designated 2C2.

Alternatively, the antibodies of the present invention, or their antigen-binding portions, can be synthesized by solid-phase procedures known in the art. Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published IRL by Oxford University Press (1989). Methods in Molecular Biology, Volume 35: Peptide Synthesis Protocols, by M. W. Pennington and B. M. Dunn, eds., Chapter 7. Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Company, Rockford, IL (1984). G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, ed. E. Gross and J. Meienhofer, Vol. 1 and Vol. 2, Academic Press, New York (1980), pp. 3-254. M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984).

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

Any of a variety of carbohydrate antigens, particularly Globo H, can be used in the practice of the present 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 known as Gb5), stage-specific embryonic antigen 4 (SSEA-4), Gb-4, and Gb-3, Lewis antigens such as ^sLex , ^Lex , ^sLea , ^Lea , and ^Ley , polysaccharide antigens such as polysialic acid (PSA), sTn(c), and Tn(c), Thomsen-Friedenreich antigen (TF(c)), gangliosides such as GD1, GD2, GD3, fucosyl GM1, GM1, GM2, GM3, GD1α, and GM2, sulfitide antigens such as 6Gal-HSO3 _-SiaLex and 6GluNAc-HSO3 _- SiaLex. Other carbohydrate antigens include, but are not limited to, α-galactose, α-Man-6-phosphate, α-L-rhamnose, α-GalNAc(Tn), α-NeuAc-OCH ₂ C ₆ H ₄ -p-NHCOOCH ₂ , Fucα1-2Galβ1-4GalNAcβ(H type III), NeuAcα2-8NeuAcα, (NeuAcα2-8)2polysialic acid, NeuAca2-6Galb, NeuAcb2-6Gala(STn), Gala1-3Galb1-4GlaNAcb(NeuAca2-8)3, GalNAcαa-3(Fucα1-2)Galβ(blood type A), Galα1-3(Fucα1-2)Galβ(blood type B), 6Gal-HSO ₃ -SiaLex, 6GluNAc-HSO ₃ -SiaLex and α2-6 sialylated biantennary N-glycans.

In one embodiment, the anti-Globo H antibody or antigen-binding portion thereof can interact or bind to other highly selective carbohydrate antigens, such as those shown in Figures 3A and 3B. Non-limiting examples of carbohydrate antigens include SSEA-3, SSEA-4, and Lewis antigens.

In one embodiment, the present invention provides a method for producing hybridomas that express antibodies that specifically bind to a carbohydrate antigen (e.g., Globo H). The method comprises the steps of immunizing an animal with a composition comprising a carbohydrate antigen (e.g., Globo H); isolating spleen cells from the animal; generating hybridomas from the spleen cells; and selecting hybridomas that produce antibodies that specifically bind to Globo H. Kohler and Milstein, Nature, 256:495, 1975. Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N.Y., 1988.

In one embodiment, a carbohydrate antigen is used to immunize mice subcutaneously. One or more boosts may or may not be administered. Antibody titers in plasma can be monitored, for example, by ELISA (enzyme-linked immunosorbent assay) or flow cytometry. Mice with sufficient titers of anti-carbohydrate antigen antibodies are used for fusions. Mice may or may not be boosted with antigen three days before sacrifice and spleen removal. Mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line. The resulting hybridomas are then screened for antigen-specific antibodies. The cells are plated and then cultured in selective medium. Supernatants from individual wells are then screened by ELISA for human anti-carbohydrate antigen monoclonal antibodies. Antibody-secreting hybridomas are replated, screened again, and, if still positive for anti-carbohydrate antigen antibodies, can be cloned 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 aluminophosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan trioleate (Span 85)), CpG-containing nucleic acids, QS21 (saponin adjuvant), α-galactosylceramide or its synthetic analogs (e.g., C34, see US 8,268,969), MPL (Monophosphoryl Lipid A), 3DMPL (3-O-desacylated MPL), extracts from Aquila, ISCOMS (see, Sjolander et al., (1998), J. Leukocyte Biol., 64:713; WO 90/03184; WO 96/11711; WO 96/11720; WO 96/11731). 00/48630; WO98/36772; WO00/41720; WO06/134423 and WO07/026190), LT/CT mutations, poly (D, L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukin, Freund's adjuvant, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl)-ethylamine (CGP 19835A, called MTP-PE), and RIBI, which contains three components extracted from bacteria: monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

The immunized animal can be any animal capable of producing recoverable antibodies when administered with the immunogen, such as, but not limited to, rabbits, mice, rats, hamsters, goats, horses, monkeys, baboons, and humans. In one embodiment, the host is genetically modified to produce human antibodies, for example, a mouse expressing human immunoglobulin gene segments. U.S. Patent Nos. 8,236,311; 7,625,559 and 5,770,429, the disclosures of each of which are incorporated herein by reference in their entirety. Lonberg et al., Nature, 368(6474):856-859, 1994. Lonberg, N., Handbook of Experimental Pharmacology, 113:49-101, 1994. Lonberg, N. and Huszar, D., Intern. Rev. Immunol., 13: 65-93, 1995. Harding, F. and Lonberg, N., Ann. N.Y. Acad. Sci., 764: 536-546, 1995.

After the host is immunized and antibodies are generated, the antibodies are tested to determine whether they are specific for the target antigen and whether they exhibit any cross-reactions with other antigens. One method for performing these tests is a serum screening test, 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 known techniques. For example, in ELISA, a microtiter plate is coated with a toxin or toxoid antigen in PBS and then blocked with an unrelated protein, such as bovine serum albumin (BSA) diluted in PBS. A dilution of plasma from toxin-immunized mice is added to each well and cultured. The microtiter plate is washed and then cultured with a secondary antibody conjugated to an enzyme (e.g., alkaline phosphatase). After washing, the microtiter plate is developed with an enzyme substrate (e.g., ABTS) and analyzed at a specific OD. In other embodiments, to determine whether a selected monoclonal antibody binds to a target carbohydrate antigen or epitope, the antibody can be biotinylated, which can then be detected using a streptavidin-labeled probe. Anti-carbohydrate antigen antibodies can be tested for reactivity with carbohydrates by Western blotting.

Hybridomas that produce antibodies that bind to carbohydrate antigens, preferably with high affinity, can then be subcloned and further characterized. A single clone derived from each hybridoma that maintains the reactivity of the parental cell (by ELISA) can then be selected for cell bank preparation and antibody purification.

The antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention may also be described or specified in terms of their binding affinity for the antigen. The affinity of antibodies to carbohydrate antigens can be experimentally determined using any suitable method (see, e.g., Berzofsky et al., Antibody-Antigen Interactions, in Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company: New York, NY (1992); and the methods described herein). The measured affinity of a particular antibody-carbohydrate antigen interaction may vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., _KD , _Ka , _Kd ) can preferably be performed using standardized solutions of antibody and antigen, and standardized buffer.

The present antibodies or antigen-binding portions thereof have therapeutic, prophylactic, and/or diagnostic utility in vitro and in vivo. For example, these 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 recurrence, and/or diagnose cancer.

The antibodies or antigen-binding portions thereof can be administered to cells in culture, e.g., in vitro or ex vivo. For example, cells can be cultured in culture in vitro and contacted with 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 in vivo embodiments, the contacting step is effective within the subject and comprises administering the anti-toxin antibody or portion thereof to the subject under conditions effective to allow the antibody or portion thereof to bind to a carbohydrate antigen (e.g., Globo H) expressed on one or more cancer cells in the subject, e.g., breast cancer cells.

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

Methods for inhibiting cancer cells

The present invention also provides methods for inhibiting cell growth in vitro, ex vivo, or in vivo, wherein the cells, such as cancer cells, 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 present invention. The cells, such as cancer cells, can be primary cancer cells or cultured cells available from a tissue bank, such as the American Type Culture Collection (ATCC). The pathological cells can be carcinomas expressing Glolob H, gliomas, meningiomas, pituitary nematodes, or CNS metastases from systemic, lung, prostate, breast, hematopoietic, or ovarian cancers. The cells can be from a vertebrate, preferably a mammal, more preferably a human. U.S. Patent Publication No. 2004/0087651. Balassiano et al. (2002), Intern. J. Mol. Med., 10:785-788. Thorne et al. (2004), Neuroscience, 127:481-496. Fernandes et al. (2005), Oncology Reports, 13:943-947. Da Fonseca et al. (2008), Surgical Neurology, 70:259-267. Da Fonseca et al. (2008), Arch. Immunol. Ther. Exp., 56:267-276. Hashizume et al. (2008), Neuroncology, 10:112-120. In one embodiment, the cancer is a Globo H-expressing cancer. In another embodiment, the cancer is a SSEA-3-expressing cancer. In yet another embodiment, the cancer is a SSEA-4-expressing cancer. Cancers expressing Globo H, SSEA-3, and SSEA-4 include, but are not limited to, breast cancer, lung cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, and endometrial cancer, as well as colon cancer, liver cancer, nasopharyngeal cancer, skin cancer, oral cancer, kidney cancer, brain cancer, cervical cancer, and bladder cancer.

The in vitro efficacy of the antibodies of the present invention, or their antigen-binding portions, can be determined using methods known in the art. For example, the cytotoxicity of an antibody or its antigen-binding portion can be studied using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] cytotoxicity assay. The MTT assay is based on the principle that MTT, a tetrazolium salt, is taken up by metabolically active cells, where it is metabolized to a blue formazon product that can be detected spectroscopically. J. of Immunological Methods, 65:55-63, 1983. The cytotoxicity of an antibody or its antigen-binding portion can be studied using a colony formation assay. Functional testing for binding to the Globo H antigen can be performed using an ELISA. The cell cycle clock, based on antibodies or their antigen-binding portions, can be studied using standard propidium iodide (PI) staining and flow cytometry. Inhibition of invasion can be studied using a Boyden chamber. In this assay, a layer of reconstituted basement membrane, Matrigel, is coated onto a chemotaxis filter and serves as a barrier to cell migration within the Boyden chamber. Only cells with invasive capabilities can cross the Matrigel barrier. Other assays include, but are not limited to, cell viability assays, apoptosis assays, and morphology assays.

Testing can also be performed in vivo using murine models. See, e.g., B. Teicher, Tumor Models for Efficacy Determination, Mol Cancer Ther, 2006;5:2435-2443.

Pharmaceutical compositions

In one embodiment, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding portion thereof described herein and a pharmaceutically acceptable carrier. In another embodiment, the 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 for inhibiting 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 can be prepared as injectables, either as liquid solutions or suspensions, or in solid forms suitable for solution or suspension in a liquid carrier prior to injection. Pharmaceutical compositions can also be prepared as solid forms, emulsified, or encapsulated in liposome carriers or other particulate carriers used for sustained delivery of the active ingredient. For example, the pharmaceutical composition can be in the form of an oil emulsion, a water-in-oil emulsion, a water-in-oil-in-water emulsion, a site-specific emulsion, a stabilized emulsion, a visco-emulsion, a microemulsion, a nanoemulsion, a liposome, a microparticle, a microsphere, a nanosphere, a nanoparticle, and a variety of natural or synthetic polymers, such as non-absorbable impermeable polymers such as ethylene vinyl acetate copolymers and copolymers, swallowable polymers such as hydrogels, or absorbable polymers such as collagen and specific 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 present invention, or antigen-binding portions thereof, are formulated into pharmaceutical compositions for delivery to mammalian subjects. The pharmaceutical compositions are administered alone and/or mixed with a pharmaceutically acceptable carrier, excipient, or vehicle. Suitable carriers include, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Furthermore, the carrier may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers, or adjuvants. Pharmaceutically acceptable carriers may contain physiologically acceptable compounds that act, for example, to stabilize, increase, or decrease the absorption or clearance of the pharmaceutical compositions of the present invention. Physiologically acceptable compounds may include, for example, sugars such as glucose, sucrose, or polydextrose; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; detergents; liposomal carriers; or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives. See, for example, Remington's Pharmaceutical Science, 21st ed., Mack Publishing Company, Easton, Pa. ("Remington's"). The pharmaceutical compositions of the present invention may also include auxiliary substances, such as pharmaceutical agents, cytokines, or other biological response modifiers.

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

Actual methods for preparing such dosage forms are well known or will be apparent to those skilled in the art. See, for example, "Remington Pharmacological Sciences," Mack Publishing Company, Easton, Pennsylvania, 21st edition.

The pharmaceutical composition can be administered as a single dose treatment or in multiple doses according to 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, etc. For example, in one embodiment, the pharmaceutical composition according to the present invention is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

The administration period of an antibody according to the present invention, e.g., the time period over which the pharmaceutical composition is administered, can vary depending on any of a variety of variables, such as the subject's response, etc. For example, the pharmaceutical composition can 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 one year, from about one year to two years, or from about two years to about four years, or more.

For ease of administration and uniformity of dosage, oral or parenteral pharmaceutical compositions may be used in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each dosage contains a predetermined quantity of 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 dosage range for use in animals. In one embodiment, the dose of such a compound is within a range of circulating concentrations that includes the ED50 with minimal or no toxicity. The dose may vary within this range depending on the dosage form implemented and the route of administration utilized. In another embodiment, the therapeutically effective dose may be initially estimated from cell culture assays. In animal models, the dose may be formulated to achieve a circulating plasma concentration range that includes the _IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Sonderstrup, Springer, Sem. Immunopathol., 25:35-45, 2003. Nikula et al., Inhal. Toxicol. 4(12):123-53, 2000.

Exemplary, non-limiting ranges for a therapeutically or prophylactically effective amount of an antibody of the invention, or antigen-binding portion thereof, are from about 0.001 to about 60 mg/kg body weight, about 0.01 to about 30 mg/kg body weight, about 0.01 to about 25 mg/kg body weight, about 0.5 to about 25 mg/kg body weight, about 0.1 to about 20 mg/kg body weight, about 10 to about 20 mg/kg body weight, about 0.75 to about 10 mg/kg body weight, about 1 to about 10 mg/kg body weight, about 2 to about 9 mg/kg body weight, about 1 to about 2 mg/kg body weight, about 3 to about 8 mg/kg body weight, about 4 to about 7 mg/kg body weight, about 5 to about 6 mg/kg body weight, about 8 to about 13 mg/kg body weight, about 8.3 to about 12.5 mg/kg body weight, about 4 to about 6 mg/kg body weight, about 4.2 to about 6.3 mg/kg body weight, about 1.6 to about 1.75 ... About 2.5 mg/kg body weight, about 2 to about 3 mg/kg body weight, or about 10 mg/kg body weight.

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

The antibodies and antigen-binding portions thereof, pharmaceutical compositions, and methods of the present 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 implementing the invention are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Examples

Example 1: Hybridoma fusion and screening

Typical hybridoma fusions were performed. Mice received a primary immunization with Globo H-KLH (Keyhole Limpet Hemocyanin) conjugated to a saponin adjuvant, with subsequent boosters on days 7, 14, and 24. Bleeding tests were performed on days 10, 17, 21, and 24, and sera were tested for anti-Globo H antibody titers. 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 were fused with mouse spleen cells according to the procedure of Milstein (G. and Milstein C, 1975). Hybridoma supernatants were screened by affinity ELISA using 0.2 μg of Globo H-ceramide per well. Anti-Globo H Vk9 mAb served as a positive control. Hybridoma clones with an OD > twice the background value of the undiluted supernatant were selected. The first five hybridoma clones were 1E1, 2C2, 2F8, 3D7, and 7A11.

Example 2: Kinetic Analysis of Mouse Monoclonal Antibodies

Kinetic binding experiments were performed at 25°C using Biacore T100 (GE Healthcare) using single-cycle kinetics (SCK) and multi-cycle kinetics (MCK) methods.

Globo H was immobilized by amine coupling according to the manufacturer's instructions. Globo H-amine was diluted to 15 mg/ml in immobilization buffer (10 mM sodium acetate, pH 4.5) and immobilized at 25°C using a flow rate of 5 μl/min.

Anti-Globo H antibodies (Globo H-Vk9 mAb, Globo H-2C2 mAb, and Globo H-3D7 mAb) were diluted to 200 nM (50 nM) in running buffer. 200 μl of the 200 nM (50 nM) solution was mixed with 200 μl of running buffer to obtain a 100 nM (25 nM) solution. The following serial dilutions were performed: 200, 100, 50, 25, and 12.5 nM (assay concentrations: 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM). The diluted samples were placed in the rack positions and tested using the MCK and SCK methods. Samples used in the MCK and SCK sequences received a 420-second dissociation time. The surface was regenerated by a 40 sec injection of 10 mM glycine pH 2.0/1.5 (v/v = 1). SCK and MCK data were fitted to a 1:1 binding model using Biacore Evaluation software 2.0.

The results were analyzed for the dissociation constant ( _KD ), a measure used to describe the strength of binding between an antibody and an antigen; kon (1/Ms), the on-rate of formation of an antibody-antigen complex; koff (1/s), the off-rate of dissociation of the antibody-antigen complex; and Rmax, the maximum amount of analyte reaction. Table 2 shows the affinity and kinetic data for anti-Globo H antibodies derived from the following hybridomas: VK9, 2C2, and 3D7. The 3D7 and 2C2 antibodies had higher binding affinities than the VK9 antibody.

Table 2 Kinetic data for anti-Globo H antibodies

hybridoma 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 Analysis of Anti-Globo H Antibodies

The following anti-Globo H antibodies were tested for _EC50 and percentage of cell binding: Globo H-VK9 mAb, Globo H-1E1 mAb, Globo H-2C2 mAb, Globo H-2F8 mAb, Globo H-3D7 mAb, Globo H-7A11 mAb.

step:

ELISA affinity for _EC50 : Wells were coated with 0.2 μg of Globo H-ceramide per well on ice. After blocking, test antibody was added to the wells at concentrations ranging from 6.2 ng/ml to 51200 ng/ml. After incubation at room temperature for 1 hour, excess antibody was removed by washing three times. Goat anti-mouse IgG-HRP (1:533) was added. Color development was performed and quantified at 490 nm on a plate reader. _EC50 was determined using Prism 5.0 software.

FACS analysis of the percentage of cell-to-antibody binding: Cancer cell lines were prepared with a total of 200,000 cells per tube in 50 μl of FACS buffer. The designated antibodies were added to a final concentration of 1 μg/ml. After gentle shaking, the tubes were placed on ice and incubated for approximately 1 hour. After washing with FACS buffer, anti-mouse IgG-PE was added to a final concentration of 4 μg/ml in FACS buffer. After gentle shaking, the tubes were placed on ice and incubated for approximately 30 minutes. After washing with FACS buffer, the test cells were resuspended in 200 μl of FACS buffer. After performing flow cytometry, the percentage of cell binding was analyzed using WinMDI software. In the histogram, the secondary antibody was incubated only to define the background (M1) and binding (M2) regions. Based on the secondary antibody setting alone as background (M1), the percentage of the binding region (M2) for the designated antibody was determined.

Antibody binding to Globo H was assessed using fluorescence activated cell sorting (FACS) analysis in breast cancer cell lines (MCF-7), lung cancer cell lines (LLC1), and pancreatic cancer cell lines (HPAC).

Results: Table 3 summarizes the EC ₅₀ data for Globo H-2C2 mAb, Globo H-2F8 mAb, Globo H-3D7 mAb, Globo H-7A11 mAb, and Globo H-1E1 mAb. The results show that Globo H-2C2 mAb, Globo H-2F8 mAb, Globo H-3D7 mAb, and Globo H-7A11 mAb were more effective than Globo H-VK9 mAb in neutralizing Globo H antigen. FACS analysis showed that Globo H-2C2 mAb and Globo H-3D7 mAb had higher binding affinity for Globo H antigen than Globo H VK9 mAb in breast cancer cell lines. Furthermore, Globo H-2C2 mAb and Globo H-3D7 mAb had higher binding affinity for Globo H antigen than Globo H VK9 mAb in pancreatic cancer cell lines (HPACs). In addition, Globo H-1E1 mAb, Globo H-2C2 mAb, Globo H-3D7 mAb, and Globo H-7A11 mAb had higher binding affinity for Globo H antigen than Globo H VK9 mAb in lung cancer cell line (LLC1).

Table 3 _EC50 and binding affinity of anti-Globo H antibodies

The _EC50 value is the antibody concentration that neutralizes Globo H by 50%.

Example 4: In vivo anti-tumor evaluation of anti-Globo H antibodies

Nude mice weighing 33 g and bearing human pancreatic cancer (HPAC) xenografts were randomly divided into the following six experimental groups:

Mice were observed for tumor volume during 29 days, and the results were recorded and summarized in Figure 1.

Results: On day 29, tumor volume decreased in the following order: VK9 = 3D7 < 2C2. The tumor volume in the 2C2 group was significantly reduced compared with the control group (P < 0.05).

Example 5: In vivo anti-tumor evaluation of anti-Globo H antibodies

Nude mice weighing 27 g and bearing human breast cancer (MCF7) xenografts were randomly divided into the following five experimental groups:

Mice were observed for tumor volume over a period of 18 days, and the results are recorded and summarized in FIG2 .

Results: As shown in Figure 2, after day 15, tumor volume decreased in the following groups compared to the control group: 2C2 (4 mg/kg) > 2C2 (0.4 mg/kg). Starting from day 8, tumor volume in the 2C2 (4 mg/kg) group was significantly reduced compared to the control group (P < 0.05).

Example 6: Interaction of anti-Globo H antibodies

In vitro interaction assessment of anti-Globo H antibodies (Globo H 2C2, 7A11, 3D7, 2F8, 1E1 mAb and Globo H Vk9) was performed.

step:

GlycoDx cartridges were filled with 620 μL of wash buffer, 100 μL of diluted anti-Globo H antibody, 100 μL of blocking buffer, 120 μL of conjugation buffer, and 120 μL of matrix buffer. The cartridges were detected by a CCD analyzer, and the data were exported to an Excel spreadsheet for further analysis.

Results: As shown in Figure 3A, Globo H 2C2 mAb bound to Globo H and showed cross-reactivity to other carbohydrate antigens, such as Lewis antigens ( ^sLex and ^sLea ) and S15-S27 antigens (see Table 4 for a list of carbohydrate antigens). Globo H 7A11 mAb bound to Globo H and showed cross-reactivity to other carbohydrate antigens, such as Lewis antigens ( ^sLex and ^sLea ) and S15-S17, S19-S22 antigens (see Figure 3B). Globo H 3D7 mAb bound to Globo H and showed cross-reactivity to other carbohydrate antigens, such as Lewis antigens ( ^sLex , ^sLea , and ^Ley ) and S15-S22 antigens (see Figure 3C). Globo H 2F8 mAb binds to Globo H and shows cross-reactivity to other carbohydrate antigens, such as Lewis antigens (sLe ^x and sLe ^a ) and S15, S17, and S21 antigens (see Figure 3D). Globo H 1E1 mAb binds to Globo H and shows cross-reactivity to other carbohydrate antigens, such as Lewis antigens (sLe ^x ) and S16, S17, and S20-S22 antigens (see Figure 3E). In contrast, Globo H VK9 mAb binds only to Globo H and does not show cross-reactivity to other carbohydrate antigens (see Figure 3F).

Table 4. List of carbohydrate antigens in Figures 3A to 3C

Example 7: Binding affinity of anti-Globo H antibodies

The following in vitro evaluations of anti-Globo H humanized antibodies were performed. Table 5 lists the amino acid sequences of the heavy and light chain regions of the humanized antibodies derived from hybridoma 2C2.

Table 5. Amino acid sequence of 2C2 humanized antibody

The results of the binding affinity determination by ELISA are listed in Tables 6-9.

Table 6: Binding affinity of antibodies with chimeric light chain and humanized heavy chain

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

Table 7: Binding affinity of antibodies with humanized light chain and humanized heavy chain

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 chain and humanized heavy chain

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 chain and humanized heavy chain

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 substitution at position 12 of FW2 of the light chain (from P to L) reduces the binding affinity from 0.32-0.53 to 0.07-0.08, and amino acid substitution at position 13 of FW2 of the light chain (from W to L) reduces the binding affinity from 0.32-0.53 to 0.08-0.12.

While specific aspects of the present invention have been described and illustrated, such aspects should be considered as merely illustrative of the invention and not as limiting thereof, as construed in accordance with the appended claims. All publications and patent applications cited in this specification are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application was 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 apparent to those skilled in the art that changes and modifications may be made in light of the teachings of this invention without departing from the spirit or scope of the appended claims.

Sequence Listing: SEQ ID No. 1-90

Claims (16)

1. An antibody or its antigen-binding portion, comprising: The first heavy chain complementarity-determining region (HCDR1) is the amino acid sequence SEQ ID NO: 5. The second heavy chain complementarity-determining region (HCDR2) is the amino acid sequence SEQ ID NO: 6. The third chain complementarity-determining region (HCDR3) is the amino acid sequence SEQ ID NO: 7. The first light chain complementarity-determining region (LCDR1) is the amino acid sequence SEQ ID NO: 8. The second light chain complementarity-determining region (LCDR2) is the amino acid sequence SEQ ID NO: 9, and The third light chain complementarity-determining region (LCDR3) is the amino acid sequence SEQ ID NO: 10. The antibody or its antigen-binding portion binds to tumor-associated carbohydrate antigen (TACA). 2. An antibody or its antigen-binding portion, comprising: The first heavy chain complementarity-determining region (HCDR1) is the amino acid sequence SEQ ID NO: 5. The second heavy chain complementarity-determining region (HCDR2) is the amino acid sequence SEQ ID NO: 24. The third chain complementarity-determining region (HCDR3) is the amino acid sequence SEQ ID NO: 25. The first light chain complementarity-determining region (LCDR1) is the amino acid sequence SEQ ID NO: 8. The second light chain complementarity-determining region (LCDR2) is the amino acid sequence SEQ ID NO: 9, and The third light chain complementarity-determining region (LCDR3) is the amino acid sequence SEQ ID NO: 10. The antibody or its antigen-binding portion binds to tumor-associated carbohydrate antigen (TACA). 3. The antibody or its antigen-binding portion as described in claim 1, further comprising: A frame having 80% to 100% identity with the amino acid sequence of SEQ ID NO: 11, wherein the frame is between HCDR1 and HCDR2, and; A frame having 80% to 100% identity with the amino acid sequence of SEQ ID NO: 12, wherein the frame is between LCDR1 and LCDR2. 4. An antibody or its antigen-binding portion, including: The heavy chain variable domain is the amino acid sequence SEQ ID NO: 3, and the light chain variable domain is the amino acid sequence SEQ ID NO: 4. The antibody or its antigen-binding portion binds to TACA. 5. An antibody or its antigen-binding portion, comprising: The heavy chain variable domain is the amino acid sequence SEQ ID NO: 21, and the light chain variable domain is the amino acid sequence SEQ ID NO: 22. The antibody or its antigen-binding portion binds to TACA. 6. A humanized antibody or its antigen-binding portion, comprising: (a) Heavy chain region, wherein the heavy chain region contains three HCDRs, HCDR1, HCDR2 and HCDR3, whose amino acid sequences are shown in SEQ ID NO: 5, 6 and 7 respectively; The leader sequence and the framework between HCDR1, whose amino acid sequence has 80% to 100% identity with SEQ ID NO: 87; and The amino acid sequence within the framework between HCDR2 and HCDR3 has 80% to 100% identity with SEQ ID NO: 89, and (b) Light chain region, wherein the light chain region comprises three LCDRs, LCDR1, LCDR2 and LCDR3, whose amino acid sequences are shown in SEQ ID NO: 8, 9 and 10, respectively; The framework between the leader sequence and LCDR1, whose amino acid sequence has 80% to 100% identity with SEQ ID NO: 88; and The amino acid sequence within the frame between LCDR2 and LCDR3 has 80% to 100% identity with SEQ ID NO: 90. The antibody or its antigen-binding portion binds to TACA. 7. An antibody or its antigen-binding portion, comprising: (a) The heavy chain region, wherein the heavy chain region contains three HCDRs, namely HCDR1, HCDR2 and HCDR3, whose amino acid sequences are shown in SEQ ID NOs: 5, 6 and 7, respectively; and A frame containing an amino acid sequence between HCDR1 and HCDR2 that has 80% to 100% identity with SEQ ID NO: 11, wherein the frame contains glycine at position 9, and (b) A light chain region comprising three LCDRs, LCDR1, LCDR2, and LCDR3, whose amino acid sequences are shown in SEQ ID NO: 8, 9, and 10, respectively; and A frame between LCDR1 and LCDR2 having an amino acid sequence that is 80% to 100% identical to SEQ ID NO: 12, wherein the frame contains proline at position 12 and/or tryptophan at position 13. The antibody or its antigen-binding portion binds to TACA. 8. The antibody or its antigen-binding portion according to any one of claims 1 to 7, wherein the TACA is ^GloboH , sLex, sTn, Tn, ^sLea , α-NeuAc- _{OCH2C6H4 - p} -NHCOOCH2, Fucα1-2Galβ1-4GalNAcβ, NeuAca2-6Galb, _{Gala1-3Galb1-4GlaNAcb} , (NeuAca2-8)3, 6Gal-HSO3-SiaLex, 6GluNAc-HSO3-SiaLex, α2-6sialic acid biantennary N-glycan or polysialic acid. 9. The antibody or antigen-binding portion thereof of any one of claims 1-7, wherein the antibody or antigen-binding portion thereof is selected from: (a) an intact immunoglobulin molecule; and (b) scFv; (c) a Fab fragment; (d) F(ab')2 or (e) a disulfide-linked Fv. 10. A pharmaceutical composition comprising: The antibody or its antigen-binding moiety as described in any one of claims 1-9; and Pharmaceutically acceptable carrier. 11. The pharmaceutical composition of claim 10, further comprising at least one other therapeutic agent. 12. Use of the antibody or antigen-binding portion thereof according to any one of claims 1-9 in the preparation of a medicament for treating cancer. 13. The use of claim 12, wherein the cancer is breast cancer, lung cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, endometrial cancer, colon cancer, liver cancer, nasopharyngeal cancer, skin cancer, oral cancer, kidney cancer, brain cancer, cervical cancer, or bladder cancer. 14. An antibody or its antigen-binding moiety produced from a hybridoma named 2C2, deposited at ATCC number PTA-121138. 15. An antibody or its antigen-binding moiety produced from a hybridoma named 3D7, deposited at ATCC number PTA-121310. 16. An antibody or its antigen-binding moiety produced from a hybridoma named 7A11, deposited at ATCC number PTA-121311.