WO2018054353A1

WO2018054353A1 - Anti-globo h antibodies

Info

Publication number: WO2018054353A1
Application number: PCT/CN2017/102913
Authority: WO
Inventors: Gunasekaran Kannan; Dineli Wickramasinghe; Shouhua XIAO
Original assignee: Chang, Chih-Long
Priority date: 2016-09-23
Filing date: 2017-09-22
Publication date: 2018-03-29
Also published as: TW201825522A

Abstract

Provided herein are engineered antibodies that bind Globo H with high affinity and have increased stability against undesirable chemical modifications and large aggregate formation that can occur under high expression manufacturing conditions. Also provided are methods of manufacturing, pharmaceutical formulations, and uses of the engineered antibodies for the treatment of cancer.

Description

ANTI-GLOBO H ANTIBODIES

FIELD

This application relates to therapeutic antibodies that bind the carbohydrate antigen, Globo H. The subject antibodies have CDR sequences engineered for increased stability against undesirable modifications.

REFERENCE TO SEQUENCE LISTING

The official copy of the Sequence Listing is submitted concurrently with the specification as an ASCII formatted text file via EFS-Web, with a file name of “05384.005PV1. txt” , a creation date of September 23, 2016, and a size of 60.5 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.

BACKGROUND

Globo H is a hexasaccharide (formula: Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1→O-cer) that is one of a large group of tumor-associated carbohydrate antigens overexpressed on the surface of various epithelial cancer cells, including breast, colon, ovarian, pancreatic, lung, and prostate cancer cells. (See e.g.， Slovin et al.， “Carbohydrate Vaccines as Immunotherapy for Cancer, ” Immunology and Cell Biology (2005) 83: 418–428. ) The tumor-associated expression of Globo H makes it a strong candidate for the development of an immunotherapeutic and/or vaccine to treat such Globo H-associated cancers. Globo H, however, is often tolerated by the human immune system. Consequently, the immunogenicity induced by Globo H is limited. Attempts to immunize with Globo H have often resulted low titer of immunoglobulin M (IgM) and failure to class switch to immunoglobulin G (IgG) , as well as ineffective antibody affinity maturation.

WO 2015/143123 and WO 2015/143126 (Mackay Memorial Hospital) disclose an immunogenic Globo H compound and the resulting anti-Globo H antibodies of isotype IgG generated by this immunogen that exhibit high affinity for Globo H. Administration of these anti-Globo H antibodies resulted in significantly reduced tumor size in mouse xenograft models of ovarian, breast, and pancreatic tumors.

The above-described anti-Globo H antibodies, however, have structural features (e.g., CDR sequences) that can confer chemical instability, which makes them undesirable for further production scale-up and clinical study. Accordingly, there remains a need for anti-Globo H antibodies with high Globo H affinity, anti-tumor activity, and greater structural stability suitable for production scale-up, clinical studies, and ultimately, therapeutic treatment of cancer in humans.

SUMMARY

The present disclosure provides antibodies that specifically bind Globo H with high affinity and which have been engineered with increased stability against undesirable chemical modifications and large aggregate formation that can occur under high expression manufacturing conditions. In particular, replacement of an unpaired cysteine in CDR-H3 stabilizes the antibodies with little or no loss in Globo H binding affinity and ADCC activity.

Accordingly, in some embodiments, the present disclosure provides an anti-Globo H antibody comprising the complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, CDR-H1, CDR-H2, and CDR-H3, wherein:

(a) CDR-L1 comprises an amino acid sequence selected from SARSSVSYMH (SEQ ID NO: 1) , SASSSVSYMH (SEQ ID NO: 2) , SASSRVSYMH (SEQ ID NO: 3) , and RASSSVSYMH (SEQ ID NO: 4) ；

(b) CDR-L2 comprises an amino acid sequence selected from DTSKLAS (SEQ ID NO: 5) , ATSNLAS (SEQ ID NO: 6) , and WTSDRYS (SEQ ID NO: 7) ；

(c) CDR-L3 comprises an amino acid sequence selected from QQWSSNPLT (SEQ ID NO: 8) , QQWSSNPFT (SEQ ID NO: 9) , and QQHLHIPYT (SEQ ID NO: 10) ；

(d) CDR-H1 comprises an amino acid sequence selected from GFSLGTFDLGIG (SEQ ID NO: 11) , GFSLSTFDMGVG (SEQ ID NO: 12) , GSSLSTFDVGVG (SEQ ID NO: 13) , and GFSLSTFDLGIG (SEQ ID NO: 14) ；

(e) CDR-H2 comprises an amino acid sequence selected from HIWWDDDKYYNPALKS (SEQ ID NO: 15) , and HIWGDDDKYYNPALKS (SEQ ID NO: 16) ； and

(f) CDR-H3 comprises an amino acid sequence of formula selected from LSGNYLTSFYXDY (SEQ ID NO: 17) , LYGNYLTSFYXDY (SEQ ID NO: 18) , and LYGNYLRSYYXDY (SEQ ID NO: 19) , wherein X is an amino acid residue other than C, optionally, wherein X is an amino acid residue selected from the group consisting of A, S, T, and F.

In some embodiments of the anti-Globo H antibody, CDR-H3 comprises an amino acid sequence selected from LSGNYLTSFYADY (SEQ ID NO: 20) , LSGNYLTSFYSDY (SEQ ID NO: 21) , LSGNYLTSFYTDY (SEQ ID NO: 22) , LSGNYLTSFYFDY (SEQ ID NO: 23) , LYGNYLTSFYADY (SEQ ID NO: 24) , and LYGNYLRSYYADY (SEQ ID NO: 25) .

In some embodiments of the anti-Globo H antibody, CDR-L1 comprises the amino acid sequence SARSSVSYMH (SEQ ID NO: 1) ； CDR-L2 comprises the amino acid sequence selected from DTSKLAS (SEQ ID NO: 5) ； CDR-L3 comprises the amino acid sequence QQWSSNPLT (SEQ ID NO: 8) ； CDR-H1 comprises the amino acid sequence GFSLGTFDLGIG (SEQ ID NO: 11) ； and CDR-H2 comprises the amino acid sequence HIWWDDDKYYNPALKS (SEQ ID NO: 15) .

In some embodiments of the anti-Globo H antibody, the antibody comprises a light chain variable domain (V_L) having at least 90％identity to an amino acid sequence selected SEQ ID NO: 26, 27, 28, and 29, and a heavy chain variable domain (V_H) having at least 90％identity to an amino acid sequence selected from SEQ ID NO: 30, 31, and 32.

In some embodiments of the anti-Globo H antibody, the antibody comprises a light chain variable domain (V_L) having at least 90％identity to the amino acid sequence of SEQ ID NO: 26, and a heavy chain variable domain (V_H) having an amino acid sequence selected from SEQ ID NO: 33, 34, 35, and 36.

In some embodiments of the anti-Globo H antibody, the antibody comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 37, and the heavy chain comprises an amino acid sequence selected from SEQ ID NO: 39, 40, 41, and 42.

In any of the embodiments of the anti-Globo H antibody, the antibody binds to Globo H with a binding affinity of no more than 1 x 10^-7 M, optionally a binding affinity of no more than 1 x 10^-8 M. Further the present disclosure provides embodiments of the anti-Globo H antibody, wherein: (i) the antibody is a monoclonal antibody； (ii) the antibody is a human, humanized, or chimeric antibody； (iii) the antibody is a full length antibody of class IgG； (iv) wherein the antibody is an antibody fragment, optionally selected from the group consisting of F (ab') ₂, Fab', Fab, Fv, single domain antibody (VHH) , and scFv； (v) the antibody is an immunoconjugate, optionally, wherein the immunoconjugate comprises a chemotherapeutic agent； (vi) the antibody is a multispecific antibody, optionally a bispecific antibody； and (vii) the antibody is a synthetic antibody, wherein the CDRs are grafted onto a scaffold or framework other than an immunoglobulin scaffold or framework, optionally scaffold selected from an alternative protein scaffold and an artificial polymer scaffold.

In other embodiments, the present disclosure also provides isolated nucleic acids encoding the anti-Globo H antibodies disclosed herein. In some embodiments, the nucleic acid encodes a light chain and a heavy chain, wherein the nucleotide sequence encoding the light chain has at least 80％identity to SEQ ID NO: 45 and the nucleotide sequence encoding the heavy chain has at least 80％identity to SEQ ID NO: 46. In some embodiments, the nucleic acid encoding the heavy chain comprises a sequence selected from SEQ ID NO: 47, 48, 49, and 50.

In some embodiments of the nucleic acids, the nucleic acid further comprises a sequence encoding a signal peptide (SP) , optionally, wherein the signal peptide comprises the amino acid of SEQ ID NO: 43. In some embodiments, the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 44.

In some embodiments, the present disclosure also provides a host cell comprising a nucleic acid encoding an anti-Globo H antibody as disclosed herein.

The disclosure also provides a method of producing an anti-Globo H antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an anti-Globo H antibody so that an antibody is produced.

In some embodiments, the disclosure provides a pharmaceutical formulation comprising an anti-Globo H antibody as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises a chemotherapeutic agent.

The present disclosure also provides a method of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of an an anti-Globo H antibody as disclosed herein, or a therapeutically effective amount of a pharmaceutical formulation of an anti-Globo H antibody as disclosed herein. In some embodiments of the method of treatment, the cancer is a Globo H-positive expressing cancer. In some embodiments, the cancer is ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, or lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a plot of the SEC main peak area, which corresponds to the amount of purified, intact anti-Globo H antibody, over time (21 days) at 40 C, for the hMZ-2lw parental antibody ( “WT” ) and each of the following variant anti-Globo H antibodies: C100A, C100F, C100S, and C100T.

Figure 2 depicts a plot of the SEC main peak area, which corresponds to the amount of purified, intact anti-Globo H antibody, over time (21 days) at 4 C, for the hMZ-2lw parental antibody ( “WT” ) and each of the following variant anti-Globo H antibodies: C100A, and C100S.

Figure 3 depicts non-reducing capillary electrophoresis with SDS ( “nrCE-SDS” ) profiles of the parental anti-Globo H antibody, hMZ-2lw, and four variants, C100S, C100F, C100A, and C100T. The antibodies were purified as in Example 1, and nrCE-SDS conditions are described in Example 2. The areas of the intact antibody peaks at ～15 minutes indicates a purity of 94-95％for all antibodies.

Figure 4 depicts the differential scanning calorimetry ( “DSC” ) profile of the anti-Globo H variant, C100A, along with fitted peaks used to estimate the melting temperatures of the CH2, CH3, and Fab regions of the intact antibody. DSC conditions are described in Example 2.

Figure 5 depicts a plot of the ratio of dynamic light scattering ( “DLS” ) peak areas indicative of antibody large aggregate formation, versus the number of freeze-thaw cycles. The plots are for the purified parental anti-Globo H antibody hMZ-2lw ( “WT” ) , and the variants C100A and C100S. The antibodies were purified as in Example 1, and DLS described in Example 2. The plots indicate a linear increase in large aggregate formation for the parental antibody with increasing freeze-thaw cycles, whereas no large aggregate formation was observed for the variants after two cycles, and even after three cycles for C100S.

Figure 6 depicts plots of average optical density indicative of ADCC activity versus concentration for the parental anti-Globo H antibody, and the variants C100A and C100S. ADCC assay conditions were as described in Example 2, and plots were fitted with the 4-parameter curve fitting equation shown below the plot. The EC₅₀ values determined for each of the antibodies are shown below the plot and in Table 11 of Example 2.

Figure 7 depicts plots of average optical density indicative of ADCC activity versus concentration for the parental anti-Globo H antibody, and the variants C100F and C100T. ADCC assay conditions were as described in Example 2, and plots were fitted with the 4-parameter curve fitting equation shown below the plot. The EC₅₀ values determined for each of the antibodies are shown below the plot and in Table 11 of Example 2.

DETAILED DESCRIPTION

Overview of Various Embodiments

The present disclosure provides antibodies that bind Globo H with high affinity and which have been engineered for increased stability against undesirable chemical modifications and large aggregate formation that can occur under high expression manufacturing conditions. In particular, the unpaired cysteine residue in CDR-H3 of a parental anti-Globo H antibody has been substituted with other residues (e.g.， C100A, C100S, C100T, and C100F) to provide variant anti-Globo H antibodies with little or no loss in Globo H binding affinity and ADCC activity and increased stability. The parental antibody from which the variants of the present disclosure are derived is denoted “hMZ-2lw, ” and was disclosed in WO2015/143123. hMZ-2lw is a humanized anti-Globo H antibody of isotype IgG which exhibits high affinity for Globo H and results in significantly reduced tumor size in mouse xenograft models of ovarian, breast, and pancreatic tumors (see e.g.， WO2015/143123) . The present disclosure provides structures of the anti-Globo H antibody variants in terms on the amino acid and encoding nucleotide sequences of the various antibody sequence features (e.g.， CDRs, HVRs, V_H, and V_L, light and heavy chains) as disclosed in Table 1 and the accompanying Sequence Listing, and functional characteristics (e.g.， binding affinity, protein aggregate formation) as disclosed in the Examples. The disclosure also provides methods of manufacturing the antibody variants, pharmaceutical compositions and formulations comprising the antibody variants, and methods of treatments using the antibody variants.

For the descriptions herein and the appended claims, the singular forms “a” , and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. The use of “comprise, ” “comprises, ” “comprising” “include, ” “includes, ” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising, ” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of. ”

Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50, ” includes “2 to 25, ” “5 to 20, ” “25 to 50, ” “1 to 10, ” etc.

All publications, patents, patent applications, and other documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes.

It is to be understood that both the foregoing general description, including the drawings, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure.

Definitions

The technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

“Globo H, ” as used herein, refers to a hexasaccharide of formula, Fucα1→2Galβ1→3GalNAcβ1→3Galα1→ 4Galβ1→4Glcβ1→O-cer, having the structure:

Globo H is a member of a family of tumor-associated antigenic carbohydrates expressed on a variety of cell types, including cancer cells, especially cancer cells associated with breast, prostate and lung cancers (see e.g.， Dube DH, Bertozzi CR, (2005) “Glycans in cancer and inflammation. Potential for therapeutics and diagnostics, ” Nat Rev Drug Discov 4: 477–488) .

“Globo H-positive cell” refers to a cell that expresses Globo H on its surface. Generally, expression of Globo H on a cell surface can be determined using anti-Globo H antibodies in a method such as e.g.， immunohistochemistry, FACS, etc.

“Globo H-positive cancer” refers to a cancer comprising Globo H-positive cells.

“Antibody, ” as used herein, refers to a molecule comprising one or more polypeptide chains that specifically binds to, or is immunologically reactive with, a particular antigen. Exemplary antibodies of the present disclosure include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific (or heteroconjugate) antibodies (e.g.， bispecific antibodies) , monovalent antibodies, multivalent antibodies, antigen-binding antibody fragments (e.g.， Fab′, F (ab′) ₂, Fab, Fv, rIgG, and scFv fragments) , antibody fusions, and synthetic antibodies (or antibody mimetics) .

“Anti-Globo H antibody” or “antibody that binds Globo H” refers to an antibody that binds Globo H with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting Globo H. In some embodiments, the extent of binding of an anti-Globo H antibody to an unrelated, non-Globo H antigen is less than about 10％of the binding of the antibody to Globo H as measured, e.g.， by a radioimmunoassay (RIA) . In some embodiments, an antibody that binds to Globo H has a dissociation constant (Kd) of < 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.01 nM (e.g.， 10^-8 M or less, e.g.， from 10^-8 M to 10^-13 M, e.g.， from 10^-9 M to 10^-13 M) .

“Full-length antibody, ” “intact antibody, ” or “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

“Antibody fragment” refers to a portion of a full-length antibody which is capable of binding the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F (ab') ₂； diabodies； linear antibodies； single-chain antibody molecules (e.g.， scFv) ； and multispecific antibodies formed from antibody fragments.

“Class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes) , e.g.， IgGl , IgG2, IgG3, IgG4, IgA1 , and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

“Variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g.， Kindt et al.， Kuby Immunology, 6^th ed.， W. H. Freeman and Co.， page 91) . A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, e.g.， Portolano et al.， J. Immunol. 150: 880-887 (1993) ； Clarkson et al.， Nature 352: 624-628 (1991) ) .

“Hypervariable region” or “HVR, ” as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ( "hypervariable loops" ) . Generally, native antibodies comprise four chains with six HVRs； three in the heavy chain variable domains, VH (H1, H2, H3) , and three in the light chain variable domains, VL (L1, L2, L3) . The HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs) . Exemplary hypervariable loops occur at amino acid residues 26-32 (L1) , 50-52 (L2) , 91-96 (L3) , 26-32 (H1) , 53-55 (H2) , and 96-101 (H3) . (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) ) . Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al.， Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) .

“Complementarity determining region, ” or “CDR, ” as used herein, refers to the regions within the hypervariable regions of the variable domain which have the highest sequence variability and/or are involved in antigen recognition. Generally, native antibodies comprise four chains with six CDRs； three in the heavy chain variable domains, VH (H1, H2, H3) , and three in the light chain variable domains, VL (L1, L2, L3) . Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35 of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al.， supra) . With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops.

“Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL) : FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

“Native antibody” refers to a naturally occurring immunoglobulin molecule. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-to C-terminus, each heavy chain has a variable region (VH) , also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3) . Similarly, from N-to C-terminus, each light chain has a variable region (VL) , also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ) , based on the amino acid sequence of its constant domain.

“Monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e.， the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g.， variant antibodies contain mutations that occur naturally or arise during production of a monoclonal antibody, and generally are present in minor amounts) . In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes) , each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the term “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

“Chimeric antibody” refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

“Humanized antibody” refers to a chimeric antibody comprising amino acid sequences from non-human HVRs and amino acid sequences from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the FTVRs (e.g.， CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

“Human antibody” refers to an antibody which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

“Human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al.， Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991) , vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al.， supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

“Acceptor human framework” as used herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

“Fc region, ” refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc sequence is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl-terminus of the Fc sequence. However, the C-terminal lysine (Lys447) of the Fc sequence may or may not be present. The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain.

“Fc receptor” or “FcR, ” refers to a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some embodiments, an FcR is one which binds an IgG antibody (agamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcyRII receptors include FcyRIIA (an “activating receptor” ) and FcyRIIB (an “inhibiting receptor” ) , which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see, e.g.， Daeron, Annu. Rev. Immunol. 15: 203-234 (1997) ) . FcR, as used herein, also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 1 17: 587 (1976) and Kim et al, J. Immunol. 24: 249 (1994) ) and regulation of homeostasis of immunoglobulins. FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) ； Capel et al, Immunomethods 4: 25-34 (1994) ； and de Haas et al, J. Lab. Clin. Med. 126: 330-41 (1995) .

“Multivalent antibody, ” as used herein, is an antibody comprising three or more antigen binding sites. The multivalent antibody is preferably engineered to have the three or more antigen binding sites and is generally not a native sequence IgM or IgA antibody.

“Multispecific antibody” is an antibody having at least two different binding sites, each site with a different binding specificity. A multispecific antibody can be a full length antibody or an antibody fragment, and the different binding sites may bind each to a different antigen or the different binding sites may bind to two different epitopes of the same antigen.

“Fv fragment” refers to an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three HVRs of each variable domain interact to define an antigen binding site on the surface of the V_H-V_L dimer. Collectively, the six HVRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

“Fab fragment’ refers to an antibody fragment that contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. “F (ab') ₂ fragments” comprise a pair of Fab fragments which are generally covalently linked near their carboxy terminii by hinge cysteines between them. Other chemical couplings of antibody fragments also are known in the art.

“Antigen binding arm, ” as used herein, refers to a component part of an antibody fragment that has an ability to specifically bind a target molecule of interest. Typically the antigen binding arm is a complex of immunoglobulin polypeptide sequences, e.g.， HVR and/or variable domain sequences of an immunoglobulin light and heavy chain.

“Single-chain Fv” or “scFv” refer to antibody fragments comprising the V_H and V_L domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, an Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired antigen binding structure.

“Diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

“Linear antibodies" refers to the antibodies described in Zapata et al.， Protein Eng., 8 (10) : 1057-1062 (1995) . Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

“Naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g.， a cytotoxic moiety) or radiolabel.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g.， an antibody) and its binding partner (e.g.， an antigen) . “Binding affinity” refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g.， antibody and antigen) . The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd) . Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

“Binds specifically” or “specific binding” refers to binding of an antibody to an antigen with an affinity value of no more than about 1 x 10^-7 M.

“Affinity matured” antibody refers to an antibody with one or more alterations in one or more HVRs, compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

“Functional antigen binding site” of an antibody is one which is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen.

“Isolated antibody” refers to an antibody which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95％or 99％purity as determined by, for example, electrophoretic (e.g.， SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis) or chromatographic (e.g.， ion exchange or reverse phase HPLC) . For review of methods for assessment of antibody purity, see, e.g.， Flatman et al., J. Chromatogr. B 848: 79-87.

“Substantially similar” or “substantially the same, ” as used herein, refers to a sufficiently high degree of similarity between two numeric values (for example, one associated with a test antibody and the other associated with a reference antibody) , such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g.， Kd values) .

“Substantially different, ” as used herein, refers to a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g.， Kd values) .

“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC) ； Fc receptor binding； antibody-dependent cell-mediated cytotoxicity (ADCC) ； phagocytosis； down regulation of cell surface receptors (e.g.， B cell receptor) ； and B cell activation.

“Immunoconjugate” refers to an antibody conjugated to one or more heterologous molecule (s) , including but not limited to a cytotoxic agent.

“Cytotoxic agent, ” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes； chemotherapeutic agents or drugs； growth inhibitory agents； enzymes and fragments thereof such as nucleolytic enzymes； antibiotics； toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof； and the various antitumor or anticancer agents disclosed below.

“Disorder” is any condition that would benefit from treatment with a substance/molecule or method described herein.

“Cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation, such as cancer.

“Cancer” and “cancerous” refer to, or describe a physiological condition in mammals that is typically characterized by a cell proliferative disorder. Cancer generally can include, but is not limited to, carcinoma, lymphoma (e.g.， Hodgkin's and non-Hodgkin's lymphoma) , blastoma, sarcoma, and leukemia. More specific examples of cancer can include, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

“Tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer, ” “cancerous, ” “cell proliferative disorder, ” “proliferative disorder, ” and “tumor” are not mutually exclusive as referred to herein.

“Metastasis” refers to the spread of cancer and/or tumor from its primary site to other places in the body of an individual.

“Treatment, ” “treat” or “treating” refers to clinical intervention in an attempt to alter the natural course of a disorder in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desired results of treatment can include, but are not limited to, preventing occurrence or recurrence of the disorder, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disorder, preventing metastasis, decreasing the rate of progression, amelioration or palliation of a disease state, and remission or improved prognosis. For example, treatment can include administration of a therapeutically effective amount of pharmaceutical formulation comprising an anti-Globo H antibody to a subject to delay development or slow progression of a Globo H-positive cancer.

“Pharmaceutical formulation” refers to a preparation in a form that allows the biological activity of the active ingredient (s) to be effective, and which contain no additional components which are toxic to the subjects to which the formulation is administered.

“Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to the subject to whom it is administered. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

“Therapeutically effective amount” refers to the amount of an active ingredient or agent (e.g.， a pharmaceutical formulation) to achieve a desired therapeutic or prophylactic result, e.g., to treat or prevent a disease or disorder in a subject. In the case of a cancer, the therapeutically effective amount of the therapeutic agent is an amount that reduces the number of cancer cells； reduces the primary tumor size； inhibits (i.e.， slow to some extent and preferably stop) cancer cell infiltration into peripheral organs； inhibits (i.e.， slow to some extent and preferably stop) tumor metastasis； inhibits, to some extent, tumor growth； and/or relieves to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP) , the response rates (RR) , duration of response, and/or quality of life.

“Concurrently, ” as used herein to, refers to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent (s) continues after discontinuing the administration of one or more other agent (s) .

“Individual” or “subject” refers to a mammal, including but not limited to, domesticated animals (e.g.， cows, sheep, cats, dogs, and horses) , primates (e.g.， humans and non-human primates such as monkeys) , rabbits, and rodents (e.g.， mice and rats) .

“Anti-cancer therapeutic” refers to an agent useful for treating cancer. Exemplary anti-cancer therapeutics include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, anti-CD20 antibodies, platelet derived growth factor inhibitors (e.g.， Gleevec^TM (Imatinib Mesylate) ) , a COX-2 inhibitor (e.g., celecoxib) , interferons, cytokines, antagonists (e.g.， neutralizing antibodies) that bind to one or more of the following targets PDGFR-beta, BlyS, APRIL, BCMA receptor (s) , TRAIL/Apo2, other bioactive and organic chemical agents, and combinations thereof.

“Chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide

alkyl sulfonates such as busulfan, improsulfan and piposulfan； aziridines such as benzodopa, carboquone, meturedopa, and uredopa； ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine； acetogenins (especially bullatacin and bullatacinone) ； delta-9-tetrahydrocannabinol (dronabinol,

)； beta-lapachone； lapachol； colchicines； betulinic acid； a camptothecin (including the synthetic analogue topotecan (HYC

) , CPT-11 (irinotecan,

) , acetylcamptothecin, scopolectin, and 9-aminocamptothecin) ； bryostatin； callystatin； CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues) ； podophyllotoxin； podophyllinic acid； teniposide； cryptophycins (particularly cryptophycin 1 and cryptophycin 8) ； dolastatin； duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1) ； eleutherobin； pancratistatin； a sarcodictyin； spongistatin； nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard； nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine； antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma II and calicheamicin omegall (see, e.g., Nicolaou et ah, Angew. Chem Intl. Ed. Engl, 33: 183-186 (1994) ) ； CDP323, an oral alpha-4 integrin inhibitor； dynemicin, including dynemicin A； an esperamicin； as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores) , aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including

morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection

liposomal doxorubicin TLC D-99

pegylated liposomal doxorubicin

and deoxydoxorubicin) , epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin； anti-metabolites such as methotrexate, gemcitabine

tegafur

capecitabine

an epothilone, and 5-fluorouracil (5-FU) ； folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate； purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine； pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine； androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone； anti-adrenals such as aminoglutethimide, mitotane, trilostane； folic acid replenisher such as frolinic acid； aceglatone； aldophosphamide glycoside； aminolevulinic acid； eniluracil； amsacrine； bestrabucil； bisantrene； edatraxate； defofamine； demecolcine； diaziquone； elfornithine； elliptinium acetate； an epothilone； etoglucid； gallium nitrate； hydroxyurea； lentinan； lonidainine； maytansinoids such as maytansine and ansamitocins； mitoguazone； mitoxantrone； mopidanmol； nitraerine； pentostatin； phenamet； pirarubicin； losoxantrone； 2-ethylhydrazide； procarbazine；

polysaccharide complex (JHS Natural Products, Eugene, OR) ； razoxane； rhizoxin； sizofiran； spirogermanium； tenuazonic acid； triaziquone； 2, 2', 2'-trichlorotriethylamine； trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) ； urethan； vindesine

dacarbazine； mannomustine； mitobronitol； mitolactol； pipobroman； gacytosine； arabinoside ( "Ara-C" ) ； thiotepa； taxoid, e.g.， paclitaxel

albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE^TM) , and docetaxel

chloranbucil； 6-thioguanine； mercaptopurine； methotrexate； platinum agents such as cisplatin, oxaliplatin (e.g.,

), and carboplatin； vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine

vincristine

vindesine

and vinorelbine

etoposide (VP-16) ； ifosfamide； mitoxantrone； leucovorin； novantrone； edatrexate； daunomycin； aminopterin； ibandronate； topoisomerase inhibitor RFS 2 difluoromethylornithine (DMFO) ； retinoids such as retinoic acid, including bexarotene

bisphosphonates such as clodronate (for example,

or

) , etidronateNE-58095, zoledronic acid/zoledronate

alendronate

pamidronate

tiludronate

or risedronate

troxacitabine (a1, 3-dioxolane nucleoside cytosine analog) ； antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) ； vaccines such as

vaccine and gene therapy vaccines, for example,

vaccine,

vaccine, and

vaccine； topoisomerase 1 inhibitor (e.g.，

) ； rmRH (e.g.,

) ； BAY439 (sorafenib； Bayer) ； SU-1 1248 (sunitinib,

Pfizer) ； perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib) , proteosome inhibitor (e.g.， PS341) ； bortezomib

CCI-779； tipifarnib (Rl 1577) ； orafenib, ABT510； Bcl-2 inhibitor such as oblimersen sodium

pixantrone； EGFR inhibitors (see definition below) ； tyrosine kinase inhibitors (see definition below) ； serine-threonine kinase inhibitors such as rapamycin (sirolimus,

)； farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR^TM) ； and pharmaceutically acceptable salts, acids or derivatives of any of the above； as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone； and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN^TM) combined with 5-FU and leucovorin.

Chemotherapeutic agents also can include anti-hormonal agents or endocrine therapeutics which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. Such therapeutics may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen

4-hydroxytamoxifen, toremifene

idoxifene, droloxifene, raloxifene

trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3； pure anti-estrogens without agonist properties, such as fulvestrant

and EM8such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels) ； aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane

and nonsteroidal aromatase inhibitors such as anastrazole

letrozole

and aminoglutethimide, and other aromatase inhibitors include vorozole

megestrol acetate

fadrozole, and 4 (5) -imidazoles； lutenizing hormone-releaseing hormone agonists, including leuprolide (

and

) , goserelin, buserelin, and tripterelin； sex steroids, including progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide； onapristone； anti-progesterones； estrogen receptor down-regulators (ERDs) ； anti-androgens such as flutamide, nilutamide and bicalutamide； and pharmaceutically acceptable salts, acids or derivatives of any of the above； as well as combinations of two or more of the above.

Detailed Description of Various Embodiments

I. Anti-Globo H Antibodies

In some embodiments, the present disclosure provides structures of anti-Globo H antibody variants with increased stability in terms on the amino acid and encoding nucleotide sequences of the various well-known immunoglobulin features (e.g.， CDRs, HVRs, V_H, and V_L, light and heavy chains) . Table 1 below provides a summary description of the anti-Globo H antibody sequences of the present disclosure, and their sequence identifiers (SEQ ID NO: ) . The sequences are included in the accompanying Sequence Listing.

TABLE 1

1. Affinity of Anti-Globo H Antibody Variants

In some embodiments, the anti-Globo H antibody provided herein has a dissociation constant (Kd) of < 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10^-8 M or less, from 10^-8 M to 10^-13 M, e.g.， from 10^-9 M to 10^-13 M) . Binding affinity of a ligand to its receptor can be determined using any of a variety of assays, and expressed in terms of a variety of quantitative values. Specific Globo H binding assays useful in determining affinity of the antibodies are disclosed in the Examples herein. Additionally, antigen binding assays are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, enzyme-linked immunoabsorbent assay (ELISA) , “sandwich” immunoassays, surface plasmon resonance based assay (such as the BIAcore assay as described in WO2005/012359) , immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays.

Accordingly, in some embodiments, the binding affinity is expressed as Kd values and reflects intrinsic binding affinity (e.g.， with minimized avidity effects) . The anti-Globo H antibody variants of the present disclosure will normally have a sufficiently strong binding affinity for Globo H, for example, the antibody may bind Globo H with a Kd value of between 100 nM and 1 pM.

2. Antibody Fragments

In some embodiments, the anti-Globo H antibody of the present disclosure can be an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F (ab') 2, Fv, one-armed antibodies, scFv fragments, and other fragments described herein and known in the art. For a review of certain antibody fragments, see e.g.， Hudson et al. Nat. Med. 9: 129-134 (2003) . For a review of scFv fragments, see, e.g.， Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.， (Springer-Verlag, New York) , pp. 269-315 (1994) ； see also WO93/16185； and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a description of Fab and F (ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Other monovalent antibody forms are described in, e.g.， WO2007/048037, WO2008/145137, WO2008/145138, and WO2007/059782. One-armed antibodies are described, e.g.， in WO2005/063816. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific (see e.g.， EP0404097； WO93/01161； Hudson et al.， Nat. Med. 9: 129-134 (2003) ； and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) ) .

In some embodiments, the antibody fragments are single-domain antibodies which comprise all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc.， Waltham, MA； see, e.g.， US Pat. No. 6,248,516) .

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g.， E. coli or phage) , as described herein.

3. Chimeric and Humanized Antibodies

In some embodiments, the anti-Globo H antibody of the present disclosure can be a chimeric antibody. (See e.g.， chimeric antibodies as described in U.S. Pat. No. 4,816,567； and Morrison et al.， Proc. Natl. Acad. Sci. USA, 81 : 6851-6855 (1984) ) . In one embodiment, a chimeric antibody comprises a non-human variable region (e.g.， a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. It is contemplated that chimeric antibodies can include antigen-binding fragments thereof.

In some embodiments, the anti-Globo H antibody of the present disclosure is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g.， the antibody from which the CDR residues are derived) to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g.， in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008) , and are further described, e.g.， in Riechmann et al., Nature 332: 323-329 (1988) ； Queen et al.， Proc. Nat 1 Acad. Sci. USA 86: 10029-10033 (1989) ； US Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409； Kashmiri et al, Methods 36:25-34 (2005) (describing SDR (a-HVR) grafting) ； Padlan, Mol. Immunol. 28: 489-498 (1991) (describing "resurfacing" ) ； Dall'A cqua et al.， Methods 36: 43-60 (2005) (describing "FR shuffling" ) ； and Osbourn et al.， Methods 36: 61 -68 (2005) and Klimka et al.， Br. J. Cancer, 83 :252-260 (2000) (describing the "guided selection" approach to FR shuffling) .

Human framework regions that may be used for humanization include but are not limited to:framework regions selected using the "best-fit" method (see, e.g.， Sims et al. J. Immunol. 151: 2296 (1993) ) ； framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g.， Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992) ； and Presta et al. J. Immunol, 151 : 2623 (1993) ) ； human mature (somatically mutated) framework regions or human germline framework regions (see, e.g.， Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008) ) ； and framework regions derived from screening FR libraries (see, e.g.， Baca et al.， J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al.， J. Biol. Chem. 271: 2261 1-22618 (1996) ) .

4. Human Antibodies

In some embodiments, the anti-Globo H antibody of the present disclosure can be a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008) . Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005) . See also, e.g.， XENOMOUSE^TM technology in U.S. Pat. Nos. 6,075,181 and 6,150,584；

technology in U.S. Pat. No. 5,770,429； K-M

technology in U.S. Pat. No. 7,041,870； and

technology in U.S. Pat. Appl. Pub. No. US 2007/0061900) . Human variable regions from intact antibodies generated by such animals may be further modified, e.g.， by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. See, e.g.， Kozbor J. Immunol, 133 : 3001 (1984) ； Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987) ； and Boerner et al.， J. Immunol.， 147: 86 (1991) . Human antibodies generated via human B-cell hybridoma technology are also described in Li et al.， Proc. Natl. Acad. Sci. USA, 103 : 3557-3562 (2006) . Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) . Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20 (3) : 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology , 27 (3) : 185-91 (2005) .

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-Derived Antibodies

In some embodiments, the anti-Globo H antibody of the present disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Methods for producing such library-derived antibodies can be found in e.g.， Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al.， ed.， Human Press, Totowa, NJ, 2001) ； McCafferty et al.， Nature 348: 552-554； Clackson et al.， Nature 352: 624-628 (1991) ； Marks et al., J. Mol. Biol. 222: 581-597 (1992) ； Marks and Bradbury, m Methods in Molecular Biology 248: 161-175 (Lo, ed.， Human Press, Totowa, NJ, 2003) ； Sidhu et al.， J. Mol. Biol. 338 (2) : 299-310 (2004) ； Lee et al.， J. Mol. Biol. 340 (5) : 1073-1093 (2004) ； Fellouse, Proc. Natl. Acad. Sci. USA 101 (34) : 12467-12472 (2004) ； and Lee et al.， J. Immunol. Methods 284 (1-2) : 1 19-132 (2004) .

6. Multispecific Antibodies

In some embodiments, the anti-Globo H antibody of the present disclosure is a multispecific antibody, e.g.， a bispecific antibody. In some embodiments, the multispecific antibody is a monoclonal antibody having at least two different binding sites, each with a binding specificity for a different antigen, at least one of which specifically binds Globo H. In some embodiments, at least one of binding sites specifically binds a cytotoxic agent. In exemplary embodiments, an anti-Globo H antibody of the present disclosure is a bispecific antibody and can be used to localize a cytotoxic agent to cells which express Globo H.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see e.g.， Milstein and Cuello, Nature 305: 537 (1983) , WO 93/08829, and Traunecker et al.， EMBOJ. 10: 3655 (1991) ) . “Knob-in-hole" engineering can also be used (see, e.g.， U.S. Patent No. 5,731,168) .

Multispecific antibodies can also be made by engineering “electrostatic steering” effects that favor formation of Fc-heterodimeric antibody molecules rather than homodimers (WO 2009/089004A1) ； cross-linking two or more antibodies or fragments (see, e.g.， US Pat. No. 4,676,980, and Brennan et al.， Science, 229: 81 (1985) ) ； using leucine zippers to produce bispecific antibodies (see, e.g.， Kostelny et al.， J. Immunol, 148 (5) : 1547-1553 (1992)) ； using “diabody” technology for making bispecific antibody fragments (see, e.g.， Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) ) ； using single-chain Fv (scFv) dimers (see, e.g. Gruber et al.， J. Immunol, 152: 5368 (1994) ) ； or tri-specific antibodies (see e.g.， Tutt et al., J. Immunol. 147: 60 (1991) .

7. Antibody Variants

In some embodiments, variants of the anti-Globo H antibody of the present disclosure are also contemplated. For example, antibodies with improved binding affinity and/or other biological properties of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristic of Globo H antigen binding.

A. Substitution, Insertion, and Deletion Variants

In some embodiments, anti-Globo H antibody variants having one or more amino acid substitutions in addition to those described herein are provided. Sites for mutagenesis can include the HVRs and FRs. Typical “conservative” amino acid substitutions and/or substitutions based on common side-chain class or properties are well-known in the art and can be used in the embodiments of the present disclosure. The present disclosure also contemplates variants based on non-conservative amino acid substitutions in which a member of one of amino acid side chain class is exchanged for an amino acid from another class.

Amino acid side chains are typically grouped according to the following classes or common properties: (1) hydrophobic: Met, Ala, Val, Leu, Ile, Norleucine； (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln； (3) acidic: Asp, Glu； (4) basic: His, Lys, Arg； (5) chain orientation influencing: Gly, Pro； and (6) aromatic: Trp, Tyr, Phe.

Techniques are well-known in the art for amino acid substitution into an antibody and subsequent screening for desired function, e.g.， retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

Amino acid substitution variants can include substituting one or more hypervariable region residues of a parent antibody (e.g.， a humanized or human antibody) . Generally, the resulting variant (s) selected for further study will have modifications in certain biological properties (e.g.， increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g.， using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g.， binding affinity) .

A useful method for identifying residues or regions of an antibody that may be targeted for mutagenesis is “alanine scanning mutagenesis” (see e.g.， Cunningham and Wells (1989) Science, 244: 1081-1085) . In this method, a residue or group of target residues (e.g.， charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g.， Ala or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N-or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

Substitutions can be made in HVRs to improve antibody affinity. Such alterations may be made in “hotspots, ” i.e.， residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g.， Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) ) with the resulting variant V_H or V_L being tested for binding affinity. In one embodiment, affinity maturation can be carried out by constructing and reselecting from secondary libraries (see e.g.， in Hoogenboom et al.， Methods in Molecular Biology 178: 1-37 (O'Brien et al.， ed.， Human Press, Totowa, NJ, (2001) . ) Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g.， 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g.， conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots. ” In some embodiments of the variant V_H and V_L sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

B. Glycosylation Variants

In some embodiments, the anti-Globo H antibody of the present disclosure is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be carried by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

In embodiments where the antibody comprises an Fc region, the carbohydrate attached to the Fc region can be altered. Typically, native antibodies produced by mammalian cells comprise a branched, biantennary oligosaccharide attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (see, e.g.， Wright et al. TIBTECH 15: 26-32 (1997)) . The oligosaccharide may include various carbohydrates, such as mannose, N-acetyl glucosamine (GlcNAc) , galactose, and sialic acid, as well as, a fucose attached to a GlcNAc in the “stem” of the bi-antennary oligosaccharide structure. In some embodiments, the modifications of the oligosaccharide of an Fc region of an antibody can create a variant with certain improved properties.

In some embodiments, the anti-Globo H antibody of the present disclosure can be a variant of a parent antibody, wherein the variant comprises a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from about 1％to about 80％, from about 1％to about 65％, from about 5％to about 65％, or from about 20％to about 40％. The amount of fucose can be determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glyco-structures attached to Asn 297 (e.g.， complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry (see e.g.， WO 2008/077546) . Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues) ； however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e.， between positions 294 and 300, due to minor sequence variations in antibodies.

In some embodiments, the fucosylation variants can have improved ADCC function. See, e.g.， US Patent Publication Nos. US 2003/0157108, or US 2004/0093621. Examples of “defucosylated” or “fucose-deficient” antibodies and associated methods for preparing them are are disclosed in e.g.， US2003/0157108； US2003/0115614； US2002/0164328； US2004/0093621； US2004/0132140； US2004/0110704； US2004/0110282； US2004/0109865； WO2000/61739； WO2001/29246； WO2003/085119； WO2003/084570； WO2005/035586； WO2005/035778； WO2005/053742； WO2002/031140； Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004) ； Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) .

Cell lines useful for producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (see e.g.， Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986) ； US2003/0157108, and WO2004/056312) , and knockout cell lines, such as alpha-1 , 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.， Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) ； Kanda, Y. et al.， Biotechnol. Bioeng.， 94 (4) : 680-688 (2006) ； and WO2003/085107) .

C. Fc Region Variants

In some embodiments, an anti-Globo H antibody of the present disclosure can comprise one or more amino acid modifications in the Fc region (i.e.， an Fc region variant) . The Fc region variant may comprise a human Fc region sequence (e.g.， a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid substitution at one or more amino acid residue positions.

In some embodiments, the anti-Globo H antibody which is a Fc region variant can possess some, but not all of, the effector functions of the parent antibody, thereby making it a more desirable candidate for applications in vivo half-life of the antibody is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.

Fc region variant antibodies having reduced effector function can include an amino acid substitution at one or more of the following Fc region positions: 238, 265, 269, 270, 297, 327 and 329. (see, e.g.， U.S. Patent No. 6,737,056) . Such Fc region variants can include amino acid substitutions at two or more of positions 265, 269, 270, 297 and 327. Such Fc region variants can also include substitutions of both residues 265 and 297 to alanine (see e.g., US Pat. No. 7,332,581) . Fc region variants having improved or diminished binding to FcRs are disclosed in e.g.， U.S. Pat. No. 6,737,056； WO 2004/056312； and Shields et al.， J. Biol. Chem. 9 (2) : 6591-6604 (2001) . Fc region variants having improved ADCC can comprise one or more amino acid substitutions at e.g.， positions 298, 333, and/or 334 of the Fc region (based on EU numbering) . Fc region variants having altered (i.e.， either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC) , as described in e.g.， US Pat. No. 6,194,551, WO99/51642, and Idusogie et al.， J. Immunol. 164: 4178-4184 (2000) . Fc region variants with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) are disclosed in e.g.， US2005/0014934A1 (Hinton et al. ) . Such Fc region variants comprise amino acid substitutions at one or more of positions: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, and 434. Other examples of Fc region variants can be found in e.g.， U.S. Pat. Nos. 5,648,260 and 5,624,821； and WO94/29351.

Generally, in vitro and/or in vivo cytotoxicity assays can be carried out to confirm the reduction/depletion of CDC and/or ADCC activities in an Fc region variant. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity) , but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, et al.， Proc. Nat 'l Acad. Sci. USA 83: 7059-7063 (1986) ) and Hellstrom, et al.， Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985) ； 5, 821, 337 (see Bruggemann, M. et al.， J. Exp. Med. 166: 1351-1361 (1987) ) . Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI^TM nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA； and

non-radioactive cytotoxicity assay (Promega, Madison, WI) . Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g.， in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998) . Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g.， Clq and C3c binding ELISA in WO2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may be performed (see, e.g.， Gazzano-Santoro et al.， J. Immunol. Methods 202: 163 (1996) ； Cragg, M. S. et al.， Blood 101 : 1045-1052 (2003) ； and Cragg, M. S. and M. J. Glennie, SW 103: 2738-2743 (2004) ) . FcRn binding and in vivo clearance/half-life determinations can be performed using methods known in the art (see, e.g.， Petkova, et al.， Intl. Immunol. 18 (12) : 1759-1769 (2006) ) .

D. Cysteine Engineered Antibody Variants

In some embodiments, it is contemplated that the anti-Globo H antibody described herein can be substituted at specific non-CDR positions with cysteine residues so as to create reactive thiol groups. Such engineered “thioMAbs” can be used to conjugate the antibody to e.g.， drug moieties or linker-drug moieties and thereby create immunoconjugates, as described elsewhere herein. Cysteine engineered antibodies can be generated as described in e.g., U.S. Pat. No. 7,521,541. In some embodiments, any one or more of the following antibody residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain； A118 (EU numbering) of the heavy chain； and S400 (EU numbering) of the heavy chain Fc region.

E. Antibody Derivatives

In some embodiments, the anti-Globo H antibody of the present disclosure may be further modified (i.e.， derivatized) with non-proteinaceous moieties. Non-proteinaceous moieties suitable for derivatization of the antibody include, but are not limited to, water soluble polymers, such as: polyethylene glycol (PEG) , copolymers of ethylene glycol and propylene glycol, carboxy-methylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, poly-amino acid homo-polymers or random co-polymers, and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homo-polymers, polypropylene oxide/ethylene oxide co-polymers, polyoxy-ethylated polyols (e.g.， glycerol) , polyvinyl alcohol, and mixtures thereof. In some embodiments, modification of the antibody can be carried out using methoxy-polyethylene glycol propionaldehyde. The polymers may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody, e.g.， whether the antibody derivative will be used in a therapy under defined conditions.

8. Immunoconjugates

In some embodiments, the anti-Globo H antibody of the present disclosure can also be an immunoconjugate, wherein the immunoconjugate comprises an anti-Globo H antibody conjugated to one or more cytotoxic agents. Suitable cytotoxic agents contemplated by the present disclosure include chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g.， protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) , or radioactive isotopes.

In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) in which an anti-Globo H antibody, as described herein, is conjugated to one or more drugs. Drugs useful in immunoconjugates of the present disclosure can include an auristatin (see e.g., U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298) ； a dolastatin； a calicheamicin or derivative thereof (see e.g.， U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,710, 5,773,001 , and 5,877,296) ； an anthracycline, such as daunomycin or doxorubicin (see e.g.， U.S. Pat. No. 6,630,579； Kratz et al.， Current Med. Chem. 13: 477-523 (2006) ； Jeffrey et al.， Bioorganic &Med. Chem. Letters 16: 358-362 (2006) ； Torgov et al.， Bioconj. Chem. 16: 717-721 (2005) ； Nagy et al.， Proc. Natl. Acad. Sci. USA 97: 829-834 (2000) ； Dubowchik et al., Bioorg. &Med. Chem. Letters 12: 1529-1532 (2002) ； King et al.， J. Med. Chem. 45: 4336-4343 (2002) ) ； a maytansinoid (see e.g.， U.S. Pat. Nos. 5,208,020, 5,416,064) ； methotrexate； vindesine； a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel； a trichothecene； and CC1065.

In some embodiments, an immunoconjugate of the present disclosure comprises an anti-Globo H antibody as described herein conjugated to an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa) , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In some embodiments, an immunoconjugate of the present disclosure comprises an anti-Globo H antibody as described herein conjugated to a radioactive isotope (i.e., a radioconjugate) . A variety of radioactive isotopes are available for the production of such radioconjugates. Examples include ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ²¹²Pb, and radioactive isotopes of Lu. In some embodiments, the immunoconjugate may comprise a radioisotope for scintigraphic detection, or a spin label for NMR detection or MRI. Suitable radioisotopes or spin labels can include, as ¹²³I, ¹³¹ I, ¹¹¹In, ¹³C, ¹⁹F, ¹⁵N, ¹⁷O, various isotopes of Gd, Mn, and Fe.

Immunoconjugates of an anti-Globo H antibody and a cytotoxic agent, can be made using a variety of well-known bifunctional reagents and chemistries suitable for conjugating to proteins. Such reagents include but are not limited to: N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) , succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) , iminothiolane (IT) , bifunctional derivatives of imidoesters (e.g.， dimethyl adipimidate HQ) , active esters (e.g.， disuccinimidyl suberate) , aldehydes (e.g.， glutaraldehyde) , bis-azido compounds (e.g.， bis- (p-azidobenzoyl) -hexanediamine) , bis-diazonium derivatives (e.g.， bis- (p-diazoniumbenzoyl) -ethylenediamine) , diisocyanates (e.g.， toluene-2, 6-diisocyanate) , and bis-active fluorine compounds (e.g.， 1, 5-difluoro-2, 4-dinitrobenzene) .

Reagents for preparing immunoconjugates of the present disclosure can also include commercially available “cross-linking” reagents such as: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate) (see e.g.， Pierce Biotechnology, Inc.， Rockford, IL.， U.S.A) .

9. Synthetic Antibodies

In some embodiments, the anti-Globo H antibody of the present disclosure can be a synthetic antibody comprising a set of CDRs from an anti-Globo H immunoglobulin (e.g., CDR-L1, etc. ) grafted onto a scaffold or framework other than an immunoglobulin scaffold or framework, such as an alternative protein scaffold, or an artificial polymer scaffold.

Exemplary alternative protein scaffolds contemplated for preparation of synthetic antibodies of the present disclosure can include, but are not limited to: fibronectin, neocarzinostatin CBM4-2, lipocalins, T-cell receptor, protein-Adomain (protein Z) , Im9, TPR proteins, zinc finger domains, pVIII, avian pancreatic polypeptide, GCN4, WW domain Src homology domain 3, PDZ domains, TEM-1 beta-lactamase, thioredoxin, staphylococcal nuclease, PHD-fmger domains, CL-2, BPTI, APPI, HPSTI, ecotin, LACI-D1, LDTI, MTI-II, scorpion toxins, insect defensin-Apeptide, EETI-II, Min-23, CBD, PBP, cytochrome b-562, Ldl receptor domains, gamma-crystallin, ubiquitin, transferrin, and/or C-type lectin-like domains.

Exemplary artificial polymer (non-protein) scaffolds useful for synthetic antibodies are described in e.g.， Fiedler et al.， (2014) “Non-Antibody Scaffolds as Alternative Therapeutic Agents, ” in Handbook of Therapeutic Antibodies (eds S. Dübel and J. M. Reichert) , Wiley-VCH Verlag GmbH &Co. ； Gebauer et al.， Curr. Opin. Chem. Biol, 13: 245-255 (2009) ； Binz et al, Nat. Biotech.， 23 (10) : 1257-1268 (2005) .

II. Recombinant Methods and Compositions

The anti-Globo H antibody of the present disclosure can be produced using recombinant methods and materials well-known in the art of antibody production. In some embodiments, the present disclosure provides an isolated nucleic acid encoding an anti-Globo H antibody. The nucleic acid can encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g.， the light and/or heavy chains of the antibody) . In some embodiments, one or more vectors (e.g.， expression vectors) comprising nucleic acid sequences encoding an anti-Globo H antibody of the present disclosure are provided. In some embodiments, a host cell comprising nucleic acid sequences encoding an anti-Globo H antibody of the present disclosure are provided. In one embodiment, the host cell has been transformed with a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody. In another embodiment, the host cell has been transformed with a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.

In some embodiments of the recombinant methods, the host cell used is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, or a lymphoid cell (e.g.， Y0, NS0, Sp20) . In one embodiment, a method of making an anti-Globo H antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium) .

Briefly, recombinant production of an anti-Globo H antibody is carried out by isolating a nucleic acid encoding an antibody (e.g.， as described herein) and inserting this nucleic acid into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures well-known in the art (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the desired antibody) . Suitable host cells and culturing methods for cloning or expressing the antibody-encoding vectors are well-known in the art and include prokaryotic or eukaryotic cells. Typically, after expression, the antibody may be isolated from cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized, ” resulting in the production of an antibody with a partially or fully human glycosylation pattern (see e.g.， Gerngross, Nat. Biotech. 22: 1409-1414 (2004) , and Li et al., Nat. Biotech. 24: 210-215 (2006) ) .

Suitable host cells for the expression of glycosylated anti-Globo H antibodies of the present disclosure can also be derived from multicellular organisms (invertebrates and vertebrates) . Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts (see, e.g.， U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, and 7,125,978.

Examples of mammalian host cell lines useful for the production of the anti-Globo H antibodies of the present disclosure include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (see e.g.， Urlaub et al.， Proc. Natl. Acad. Sci. USA 77: 4216 (1980) ) ； myeloma cell lines such as Y0, NS0 and Sp2/0； monkey kidney CVl line transformed by SV40 (COS-7) ； human embryonic kidney line (293 or 293 cells as described, e.g.， in Graham et al., J. Gen Virol. 36: 59 (1977) ) ； baby hamster kidney cells (BHK) ； mouse Sertoli cells (TM4 cells as described, e.g.， in Mather, Biol. Reprod. 23: 243-251 (1980) ) ； monkey kidney cells (CVl) ； African green monkey kidney cells (VERO-76) ； human cervical carcinoma cells (HELA) ； canine kidney cells (MDCK； buffalo rat liver cells (BRL 3A) ； human lung cells (W138) ； human liver cells (Hep G2)； mouse mammary tumor (MMT 060562) ； TRI cells (see e.g.， in Mather et al.， Annals N Y. Acad. Sci. 383: 44-68 (1982) ) ； MRC 5 cells； and FS4 cells. For a general review of useful mammalian host cell lines suitable for antibody production, see, e.g.， Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.， Humana Press, Totowa, NJ) , pp. 255-268 (2003) .

III. Pharmaceutical Compositions and Formulations of Anti-Globo H Antibodies

The present disclosure also provides pharmaceutical compositions and pharmaceutical formulations comprising an anti-Globo H antibody. In some embodiments, the present disclosure provides a pharmaceutical formulation comprising an anti-Globo H antibody as described herein and a pharmaceutically acceptable carrier. Such pharmaceutical formulations can be prepared by mixing an anti-Globo H antibody, having the desired degree of purity, with one or more pharmaceutically acceptable carriers. Typically, such antibody formulations can be prepared as an aqueous solution (see e.g.， US Pat. No. 6,171,586, and WO2006/044908) or as a lyophilized formulation (see e.g.， US Pat. No. 6,267,958) .

Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. A wide range of such pharmaceutically acceptable carriers are well-known in the art (see e.g.， Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) . Exemplary pharmaceutically acceptable carriers useful in the formulations of the present disclosure can include, but are not limited to: buffers such as phosphate, citrate, and other organic acids； antioxidants including ascorbic acid and methionine； preservatives (such as octadecyldimethylbenzyl ammonium chloride； hexamethonium chloride； benzalkonium chloride； benzethonium chloride； phenol, butyl or benzyl alcohol； alkyl parabens such as methyl or propyl paraben； catechol； resorcinol； cyclohexanol； 3-pentanol； and m-cresol) ； low molecular weight (less than about 10 residues) polypeptides； proteins, such as serum albumin, gelatin, or immunoglobulins； hydrophilic polymers such as polyvinylpyrrolidone； amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine； monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins； chelating agents such as EDTA； sugars such as sucrose, mannitol, trehalose or sorbitol； salt-forming counter-ions such as sodium； metal complexes (e.g.， Zn-protein complexes) ； and/or non-ionic surfactants such as polyethylene glycol (PEG) .

Pharmaceutically acceptable carriers useful in the formulations of the present disclosure can also include insterstitial drug dispersion agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP) (see e.g.， US Pat. Publ. Nos. 2005/0260186 and 2006/0104968) , such as human soluble PH-20 hyaluronidase glycoproteins (e.g.， rHuPH20 or

Baxter International, Inc. ) .

It is also contemplated that the formulations disclosed herein may contain active ingredients in addition to the anti-Globo H, as necessary for the particular indication being treated in the subject to whom the formulation is administered. Preferably, any additional active ingredient has activity complementary to that of the anti-Globo H antibody activity and the activities do not adversely affect each other.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) .

In some embodiments, the formulation can be a sustained-release preparation of the antibody and/or other active ingredients. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

Typically, the formulations of the present disclosure to be administered to a subject are sterile. Sterile formulations may be readily prepared using well-known techniques, e.g., by filtration through sterile filtration membranes.

IV. Uses and Methods of Treatment

Any of the compositions or formulations comprising an anti-Globo H antibody of the present disclosure can be used in therapeutic methods as disclosed herein.

In some embodiments, the present disclosure provides a method of treating and/or preventing a cancer, comprising administering to a subject in need thereof, a therapeutically effective amount of an anti-Globo H antibody, or a composition or pharmaceutical formulation comprising an anti-Globo H antibody as described herein.

Administration of the antibody, composition, or pharmaceutical formulation in accordance with the method of treatment provides an antibody-induced therapeutic effect that protects the subject from a cancer, and/or treats the progression of a cancer in a subject, particularly a Globo H-positive cancer, or a similar carbohydrate-expressing cancer. In some embodiments of the method of treatment, the cancer is selected from breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, colorectal cancer and lung cancer. In some embodiments, the method of treatment can further comprise administration of one or more additional therapeutic agent or treatment, such as angiogenic inhibitors, chemotherapy, radiation, surgery, or other treatments known to those of skill in the art to prevent and/or treat cancer.

Such methods comprising administration of one or more additional agents can encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations) , and separate administration, in which case, administration of the antibody composition or formulation can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.

In some embodiments of the method of treatment of the present disclosure, the anti-Globo H antibody or pharmaceutical formulation comprising an anti-Globo H antibody is administered to a subject by any mode of administration that delivers the agent systemically, or to a desired target tissue. Systemic administration generally refers to any mode of administration of the antibody into a subject at a site other than directly into the desired target site, tissue, or organ, such that the antibody or formulation thereof enters the subject's circulatory system and, thus, is subject to metabolism and other like processes.

Accordingly, modes of administration useful in the methods of treatment of the present disclosure can include, but are not limited to, injection, infusion, instillation, and inhalation. Administration by injection can include intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.

In some embodiments, a pharmaceutical formulation of the anti-Globo H antibody is formulated such that the antibody is protected from inactivation in the gut. Accordingly, the method of treatments can comprise oral administration of the formulation.

In some embodiments, use of the compositions or formulations comprising an anti-Globo H antibody of the present disclosure as a medicament are also provided. Additionally, in some embodiments, the present disclosure also provides for the use of a composition or a formulation comprising an anti-Globo H antibody in the manufacture or preparation of a medicament, particularly a medicament for treating, preventing or inhibiting a cancer. In a further embodiment, the medicament is for use in a method for treating, preventing or inhibiting a cancer comprising administering to an individual having a cancer an effective amount of the medicament. In certain embodiments, the medicament further comprises an effective amount of at least one additional therapeutic agent, or treatment.

In a further embodiment, the medicament is for use in treating, inhibiting or preventing a cancer in a subject comprising administering to the subject an amount effective of the medicament to treat, inhibit or prevent the cancer.

For the prevention or treatment of a cancer, the appropriate dosage of the anti-Globo H antibody contained in the compositions and formulations of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of cancer to be treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The anti-Globo H antibody included in the compositions and formulations described herein, can be suitably administered to the patient at one time, or over a series of treatments. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

Depending on the type and severity of the disease, about 1 μg/kg to 150 mg/kg (e.g., 0.1-20 mg/kg) of anti-Globo H antibody in a formulation of the present disclosure is an initial candidate dosage for administration to a human subject, whether, for example, by one or more separate administrations, or by continuous infusion. In some embodiments, the administration of the anti-Globo H antibody comprises a daily dosage from about 1 mg/kg to about 100 mg/kg. In some embodiments, the dosage of anti-Globo H antibody comprises a daily dosage of at least about 1 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, at least about 20 mg/kg, or at least about 30 mg/kg.

Dosage administration can be maintained over several days or longer, depending on the condition of the subject, for example, administration can continue until the cancer is sufficiently treated, as determined by methods known in the art.

EXAMPLES

Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.

Example 1: Preparation, purification, and characterization of anti-Globo H antibody variants

This example illustrates the design of the recombinant constructs, cell-based production methods, and analytical characterization of a series of variants of the humanized anti-Globo H antibody, hMZ-2lw, wherein the amino acid residue cysteine located in CDR-H3 (C100 of the mature heavy chain) has been replaced by the amino acid residues, A, S, T, and F.

Preparation and purification of variant antibodies

The nucleotide sequences of SEQ ID NO: 45 and 46 encode the hMZ-2lw light chain (LC) and heavy chain (HC) amino acid sequences of SEQ ID NO: 37 and 38, respectively. Each of these light and heavy chain encoding nucleotide sequences are modified at the 5’-end with the nucleotide sequence of SEQ ID NO: 44, which encodes the human signal peptide (SP) sequence of SEQ ID NO: 43.

The nucleotide sequences of the variant nucleotide constructs are

The nucleotide sequence constructs encoding the precursor SP-LC and SP-HC amino acid sequences of hMZ-2lw and the C100 variants were introduced into a cell-based expression system using the following materials and methods.

Transfection: CHO-K1-C6 cells were prepared and maintained in exponential culture with HyCell TransFx-C medium (HyClone, Cat#SH30941.02) . Twenty-four hours prior to transfection, the CHO-K1-C6 cells were seeded at 7x10⁵ cells/mL with 30 mL culture medium in 125 mL shake flasks. On the day of transfection, the cell culture was at a cell density of ～1.2x10⁶ cells/mL, 97％viability. To prepare the transfection mixtures, 50 μg of the linearized expression plasmids (12.5 μg of pJH201-JHL2111-HC and 37.5 μg of pJH202-JHL2111-LC) was diluted in 0.6 mL OptiPRO SFM (Life Technologies, Cat#12309-050) ； meanwhile, 50 μL of FreeStyle MAX (Life Technologies, Cat#94764) was diluted in 0.6 mL OptiPRO SFM in parallel. The FreeStyle MAX solution was mixed with the DNA solution and incubated for 10 minutes. After incubation, the transfection mixtures were added to the desired CHO-K1-C6 cells.

Pool selection: forty-eight hours after transfection, cells were advanced to pool selection. In pool selection, four pools were generated by separating a bulk transfected pool into four different selection media, HT-200, HT-400, HT-800, and HT-1000 (shown below in Table 2) .

TABLE 2: Selection Media

HT	HyCell TransFx-C + 4 mM L-glutamine + 0.1％Pluronic F-68
HT-200	HyCell TransFx-C + 4 mM L-glutamine + 0.1％Pluronic F-68 + 200nM MTX
HT-400	HyCell TransFx-C + 4 mM L-glutamine + 0.1％Pluronic F-68 + 400nM MTX
HT-800	HyCell TransFx-C + 4 mM L-glutamine + 0.1％Pluronic F-68 + 800nM MTX
HT-1000	HyCell TransFx-C + 4 mM L-glutamine + 0.1％Pluronic F-68 + 1000nM MTX

When viability of selection media pools returned to greater than 85％, each pool was considered as cell recovery from pool selection and cryo-preserved as a research cell bank ( “RCB” ) .

Antibody expression: antibody pool which had recovered from, for example, HT-200 selection medium was expanded then inoculated in two 3 L shake flasks (Corning) . Initial volume was 800 mL HyCell-CHO medium (GE Healthcare, Piscataway, NJ) with 6 mM L-glutamine and 0.1％Pluronic-F68. Seeding density was 5 x 10⁵ cells/mL, pH was controlled by addition of base (Na₂CO₃) if pH was less than 6.8. Temperature was maintained in 37℃, agitation rate was 130 rpm and CO₂ was kept at 5％. Starting from Day 3, cells were fed 2.0％ActiCHO Feed A (GE Healthcare, Piscataway, NJ) and 0.2％ActiCHO Feed B (GE Healthcare, Piscataway, NJ) daily until Day 12. If glucose concentration was less than 3 g/L, the culture was supplemented with 5 g/L glucose.

Cell extracts containing the mature antibodies were further purified using the following materials and methods.

Affinity Chromatography: Antibodies were affinity captured by MabSelect SuRe chromatography (GE Healthcare, Piscataway, NJ) run with a flow rate of 20 CV/hr. The column was equilibrated with 25 mM Tris and 25 mM NaCl at pH 7.2. The protein was eluted by 200 mM acetate buffer at pH 2.8. The eluted protein was neutralized by adding 1 M Tris to pH ～5.2 and then filtered using a 0.2 μm PES filter.

Cation Exchange Chromatography (Poros 50HS) : The neutralized material from the affinity chromatography step was taken through the second column which is POROS 50HS (Life Technologies, Carlsbad, CA) . The column was equilibrated with buffer A (50 mM MES, pH 5.5) prior to loading the sample. The protein was gradient eluted using buffer B (50 mM MES, 0.5 M NaCl, pH 5.5) .

Buffer Exchange: UF/DF process was carried using a Pellicon 3 Ultracel 10 kDa ultrafiltration cassette (EMD Millipore, Billerica, MA) , mini-TFF system, TMP 1.2 bar. The protein was concentrated to 20 mg/mL in 10 mM sodium acetate pH 5.2, 9％sucrose.

The results of the above-described purification steps are shown in Table 3.

TABLE 3

Analytical characterization of antibody variants

Mass spectrometric analysis: Ultra-Performance Liquid Chromatography (UPLC) combined with MS analysis of the purified antibodies was carried out using a Acquity UPLC (Protein BEH C4 column) combined with Synapt G2/Si MS system (Waters Corp.， Milford, MA) . Buffer exchanged samples of purified material at 1 mg/mL concentration were reduced in 10mM DTT at 37℃ for 30 min and diluted using 5％ACN/0.1％formic acid. The raw data from the mass spectrometer was transferred to a processing computer in order to calculate deconvoluted molecular weights.

The MS results shown in Table 4 confirmed that the expected antibody variants were the primary molecular species present in the purified material.

TABLE 4

Size-exclusion chromatography (SEC) : A TOSOH column at 30℃ in a Waters Alliance 2695 instrument (Waters Corp.， Milford, MA) was utilized for SEC. The purified material samples were maintained at 2-8℃ temperature. In order to elute the protein, 20 mM sodium phosphate, 0.3 M sodium chloride at pH 6.8 was used. Elution profiles were captured at 280 nm detection at a flow rate 0.5 mL/min.

The SEC profiles for hMZ-2lw and each of the variants had a small leading peak at ～13.5-13.7 retention comprising 0.3-0.7％of the total peak area, a large main peak at ～16.2 –16.5 retention comprising 98-99％of the total peak areas, and a small shoulder at ～17.3 retention comprising 0.9％or less of the total peak areas. These SEC results indicated that the hMZ2-lw and the similarly purified antibody variants was > 98％.

SEC (using the same methods and materials described above) also was carried out to determine the stability of the purified variant to high-temperature stress (40 ℃) over an extended time period (10-21 days) . As shown in FIG. 1, the C100A variant showed stability to heat stress over the period of 21 days that was comparable to the parental hMZ-2lw antibody, whereas the C100S was slightly less stable to this stress.

Similarly, SEC measurements (using the same methods and materials described above) were made to determine the stability of the hMZ-2lw, C100A, and C100S variants at a constant storage temperature of 4 ℃ for a period of 21 days. As shown in FIG. 2, the parental antibody showed significant loss in the main SEC peak indicating loss of antibody over the 3 week period, whereas the C100A variant showed no loss of antibody under the same condition for the same time period. The C100S variant showed some slight loss of antibody but less than the parental antibody over the 3 week period at 4 ℃.

Non-reduced capillary electrophoresis with sodium dodecyl-sulfate (nrCE-SDS) : nrCE-SDS of the purified antibodies was carried out using materials and parameters described in Table 5.

TABLE 5

FIG. 3 shows the nrCE-SDS elution profiles for parent antibody, hMZ-2lw and the variants C100A, C100T, C100F, and C100S. All show a large main peak corresponding to the intact antibody that comprises 94-95％of the total peak areas. The nrCE-SDS results indicated that the purity of all of the antibody variants was 94-95％.

Differential scanning calorimetry (DSC) : DSC of the purified antibodies was carried out using the following instrument and parameters. Instrument: Nano DSC (TA Instruments, New Castle, Delaware, USA) ； scan range: 30-105 ℃； scan rate: 1 ℃/min； sample conc. : 2 mg/mL； buffer； 10 mM sodium acetate, pH 5.2, 9％sucrose. FIG. 4 shows a typical DSC profile of the C100A variant along with the fitted peaks used to estimate the melting temperatures of the CH2, CH3, and Fab regions of the intact antibody. The DSC results shown in Table 6 indicate that the antibody variants have thermal stability in their Fab region that is comparable or higher (e.g., C100F) than the parent hMZ-2lw antibody.

TABLE 6

Dynamic light scattering (DLS) : DLS of the purified antibodies was carried out using the following instrument and parameters. Instrument: Malvern Zetasizer Nano ZS (Malvern Instruments, Ltd.， Malvern, UK) ； sample concentration: 20 mg/mL. The DLS results shown in Table 7 show that the purified variants are very similar in size to the parental antibody hMZ-2lw.

TABLE 7

DLS also was performed on the parental antibody and variants C100A and C100S to determine if large aggregates (> 1000nm) formed after storage at 40 ℃ for 21 days. Consistent with the SEC measurements carried out at 40 ℃ for 21 days, no large aggregate formation was observed for the C100A and C100S variants following 21 days at 40 ℃.

Additionally, the stability of the variant antibodies to repeated freeze-thaw cycles was characterized with DLS using the same instrument and parameters as described above for detecting large aggregate formation. Samples of the parent antibody and the C100A and C100S variants were frozen and thawed for at least three cycles as follows. As shown in FIG. 5, significant formation of large aggregates was observed for the parental hMZ2-lw antibody after the first freeze-thaw, and formation continued to increase with each repeated freeze-thaw cycle. In contrast, the variants C100A and C100S exhibited no large aggregate formation after two freeze-thaw cycles, and the C100S variant exhibited no large aggregate formation even after three freeze-thaw cycles.

Example 2: Binding affinity and functional characterization of anti-Globo H antibody variants

This example illustrates the measurement of Globo H binding affinity and other functional characteristics of the anti-Globo H antibody variants.

ELISA: ELISA assay of the purified antibodies was carried out to determine Globo H binding using the following protocol. 1) . Streptavidin (Jackson ImmunoResearch, Cat#016-000-14) was diluted to 10 μg/mL in 1x PBS (Gibco, Cat#1715681) . 2) . 100 μL aliquots of the diluted streptavidin were added to each well of ELISA plates (NUNC, MaxiSorplates, #460124) . 3) . The ELISA plates were sealed with plastic film and incubated overnight at 4℃. 4) . Fluid was discarded and a 350 μL aliquot of blocking reagent in 1x PBS was added to each streptavidin coated well. 5) . The plates were incubated at room temperature for one hour. 6) . Fluid was discarded and the plates were washed 3 times with 1xPBS. 7) . Globo H-biotin was diluted to a final concentration of 0.02 μg/mL in 1x PBS, a 100μL aliquot of diluted Globo H-biotin was added to each streptavidin-coated well, and incubated for one hour at room temperature. 8) . Fluid was discarded and the plates were washed 6 times with wash buffer. 9) . hMZ-2Lw antibody sample was diluted to 3μg/mL in blocking solution and a 2-fold serial dilution was performed to generate 12 dilutions ranging from 3 μg/mL to 0.001 μg/mL. 100μL aliquot of serially diluted antibody was added to each well and incubated one hour at room temperature. Human IgG is used as an isotype control. 10) . Fluid was discarded and the plates washed 6 times with wash buffer. 11) . HRP-conjugated anti-human IgG-Fc antibody (Sigma#A0170) was diluted 1: 5000 in blocking solution. 100μL aliquot of diluted antibody was added to each well and incubated one hour at room temperature. 12) . Fluid was discarded and the plate washed 6 times with wash buffer. 13) . 100μL aliquot of TMB peroxidase substrate (Sigma #T0440-100ML) was added to each well and incubated at room temperature for 3 min to develop color. 14) . The reaction was stopped by adding 50μL of 2N H₂SO₄ to each well. The absorption is detected by an ELISA reader (Molecular Device, M2 spectrophotometer) at 450 nm with 540 nm as reference. Wash buffer: 0.05％Tween-20 in 1x PBS. Blocking solution: 1％BSA (Calbiochem, Cat#8170721000) in 1x PBS.

Relative binding affinities of the purified antibodies based on ELISA are shown in Table 8 below.

TABLE 8

Biacore: Biacore assay of the purified antibodies was carried out to determine globo H binding using a Biacore T200 instrument according to the following general protocol. Biotin-globo-H (30RU) was immobilized on a streptavidin (SA) chip. hMZ2 antibody material was flown at five different concentrations (7.5 to 120 nM) at 2 min injection for each concentration. For dissociation, 15 min injection was followed. In order to regenerate the immobilized biotin-globo-H, 10mM Glycine-HCl (pH 1.5) for 10 seconds was used. Data analysis was carried out assuming 1: 1 binding mode. Various binding parameters measured for the purified antibodies in two sets of assays based on Biacore are shown in Table 9 below.

TABLE 9

Relative binding affinities of the purified antibodies based on the Biacore assays are shown in Table 10 below.

TABLE 10

ADCC: ADCC assay of the purified antibodies was carried out to determine antibody activity was carried out using Promega Reporter Bioassay kit following the standard protocol (Promega Corp.， Madison WI, USA) . MCF7 cells at 0.6x10⁶ cells/mL density was used as target cells. Effector to target cells ratio was maintained at 2.5: 1.50 μL of cell suspension per well was incubated at 37℃ overnight. Antibody solution was prepared to 20-to 150 μg/ml. 1: 3 dilution was followed to prepare a total of 10 concentrations. 10 μL aliquots of diluted antibody were then transferred to each well which has the incubated cell suspension. 25 μL of effector cells were then transferred to each well, followed by incubation at 37℃ for 6 hours. Bio-Glo Luciferase assay reagent was added to each well to enable luminescence measurement using a plate reader. FIGS. 6 and 7 depict plots of the results of the ADCC assays. The EC₅₀ values derived from the plots are shown in Table 11.

TABLE 11

Antibody	EC₅₀
hMZ-2lw	27.8
C100A	66.5

C100S	118
C100F	3500
C100T	21,200

Results

The relative EC₅₀ values show that the C100A variant exhibits the highest ADCC activity of the variants, and only 2.4-fold lower activity than that of the parental antibody, MZ-2lw. The C100A variant also exhibits the highest Globo H binding affinity of the variant antibodies, only 1.6-fold lower affinity than hMZ-2lw.

While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention (s) .

Claims

An anti-Globo H antibody comprising complementarity determining regions CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, wherein:

(a) CDR-L1 comprises an amino acid sequence selected from SARSSVSYMH (SEQ ID NO: 1) , SASSSVSYMH (SEQ ID NO: 2) , SASSRVSYMH (SEQ ID NO: 3) , and RASSSVSYMH (SEQ ID NO: 4) ；

(b) CDR-L2 comprises an amino acid sequence selected from DTSKLAS (SEQ ID NO: 5) , ATSNLAS (SEQ ID NO: 6) , and WTSDRYS (SEQ ID NO: 7) ；

(c) CDR-L3 comprises an amino acid sequence selected from QQWSSNPLT (SEQ ID NO: 8) , QQWSSNPFT (SEQ ID NO: 9) , and QQHLHIPYT (SEQ ID NO: 10) ；

(d) CDR-H1 comprises an amino acid sequence selected from GFSLGTFDLGIG (SEQ ID NO: 11) , GFSLSTFDMGVG (SEQ ID NO: 12) , GSSLSTFDVGVG (SEQ ID NO: 13) , and GFSLSTFDLGIG (SEQ ID NO: 14) ；

(e) CDR-H2 comprises an amino acid sequence selected from HIWWDDDKYYNPALKS (SEQ ID NO: 15) , and HIWGDDDKYYNPALKS (SEQ ID NO: 16) ； and

(f) CDR-H3 comprises an amino acid sequence of formula selected from LSGNYLTSFYXDY (SEQ ID NO: 17) , LYGNYLTSFYXDY (SEQ ID NO: 18) , and LYGNYLRSYYXDY (SEQ ID NO: 19) , wherein X is an amino acid residue other than C.
The antibody of claim 1, wherein X is an amino acid residue selected from the group consisting of A, S, T, and F.
The antibody of any one of claims 1-2, wherein CDR-H3 comprises an amino acid sequence selected from LSGNYLTSFYADY (SEQ ID NO: 20) , LSGNYLTSFYSDY (SEQ ID NO: 21) , LSGNYLTSFYTDY (SEQ ID NO: 22) , and LSGNYLTSFYFDY (SEQ ID NO: 23) .
The antibody of any one of claims 1-3, wherein CDR-H3 comprises an amino acid sequence selected from LSGNYLTSFYADY (SEQ ID NO: 20) , LYGNYLTSFYADY (SEQ ID NO: 24) , and LYGNYLRSYYADY (SEQ ID NO: 25) .
The antibody of any one of claims 1-4, wherein CDR-H3 comprises the amino acid sequence LSGNYLTSFYADY (SEQ ID NO: 20) .
The antibody of any one of claims 1-5, wherein CDR-L1 comprises the amino acid sequence SARSSVSYMH (SEQ ID NO: 1) ； CDR-L2 comprises the amino acid sequence selected from DTSKLAS (SEQ ID NO: 5) ； CDR-L3 comprises the amino acid sequence QQWSSNPLT (SEQ ID NO: 8) ； CDR-H1 comprises the amino acid sequence GFSLGTFDLGIG (SEQ ID NO: 11) ； and CDR-H2 comprises the amino acid sequence HIWWDDDKYYNPALKS (SEQ ID NO: 15) .
The antibody of any one of claims 1-6, wherein the antibody comprises a light chain variable domain (V_L) having at least 90％identity to an amino acid sequence selected SEQ ID NO: 26, 27, 28, and 29, and a heavy chain variable domain (V_H) having at least 90％identity to an amino acid sequence selected from SEQ ID NO: 30, 31, and 32.
The antibody of any one of claims 1-7, wherein the antibody comprises a light chain variable domain (V_L) having at least 90％identity to the amino acid sequence of SEQ ID NO: 26, and a heavy chain variable domain (V_H) having at least 90％identity to the amino acid sequence of SEQ ID NO: 30.
The antibody of any one of claims 1-8, wherein the antibody comprises a light chain variable domain (V_L) having at least 90％identity to the amino acid sequence of SEQ ID NO: 26, and a heavy chain variable domain (V_H) having an amino acid sequence selected from SEQ ID NO: 33, 34, 35, and 36.
The antibody of claim 1, wherein the antibody comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 37, and the heavy chain comprises an amino acid sequence selected from SEQ ID NO: 39, 40, 41, and 42.
The antibody of claim 10, wherein the antibody comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 37, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 39.
The antibody of any one of claims 1-11, wherein the antibody binds to Globo H with a binding affinity of no more than 1 x 10^-7 M, optionally a binding affinity of no more than 1 x 10^-8 M.
The antibody of any one of claims 1-12, wherein the antibody is a monoclonal antibody.
The antibody of any one of claims 1-13, wherein the antibody is a human, humanized, or chimeric antibody.
The antibody of any one of claims 1-14, wherein the antibody is a full length antibody of class IgG.
The antibody of any one of claims 1-9, wherein the antibody is an antibody fragment, optionally selected from the group consisting of F (ab') ₂, Fab', Fab, Fv, single domain antibody (VHH) , and scFv.
The antibody of any one of claims 1-9, wherein the antibody is an immunoconjugate, optionally, wherein the immunoconjugate comprises a chemotherapeutic agent.
The antibody of any one of claims 1-9, wherein the antibody is a multispecific antibody, optionally a bispecific antibody.
The antibody of any one of claims 1-6, wherein the antibody is a synthetic antibody comprising the CDRs grafted onto a scaffold other than an immunoglobulin scaffold or immunoglobulin framework, optionally a scaffold selected from an alternative protein scaffold, and an artificial polymer scaffold.
An isolated nucleic acid encoding the antibody of any one of claims 1-19.
The nucleic acid of claim 20, further comprising a nucleic acid sequence encoding a signal peptide (SP) .
The nucleic acid of claim 21, wherein the signal peptide comprises the amino acid of SEQ ID NO: 43.
The nucleic acid of claim 22, wherein the signal peptide is encoded by the nucleotide sequence of SEQ ID NO: 44.
The nucleic acid of claim 20, wherein the nucleic acid encodes a light chain and a heavy chain, wherein the nucleotide sequence encoding the light chain has at least 80％identity to SEQ ID NO: 45 and the nucleotide sequence encoding the heavy chain has at least 80％identity to SEQ ID NO: 46.
The nucleic acid of claim 24, wherein the nucleic acid encoding the heavy chain comprises a sequence selected from SEQ ID NO: 47, 48, 49, and 50.
A host cell comprising a nucleic acid of any one of claims 20-25.
A method of producing an antibody comprising culturing the host cell of claim 26 so that an antibody is produced.
A pharmaceutical formulation comprising an antibody of any one of claims 1-19 and a pharmaceutically acceptable carrier.
The pharmaceutical formulation of claim 28 further comprising a chemotherapeutic agent.
A method of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody of any one of claims 1-19 or a therapeutically effective amount of a pharmaceutical formulation of any one of claims 28-29.
The method of claim 30, wherein the cancer is a Globo H-positive cancer.
The method of any one of claims 30-31, wherein the cancer is ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, or lung cancer.
The method of any one of claims 30-32, which further comprises administering to the subject one or more additional treatments selected from chemotherapeutic agent, radiation treatment, or surgery.