BR112015022967B1 - HIGH AFFINITY 3F8 ANTI-GD2 ANTIBODY VARIANT OR A SINGLE-CHAIN VARIABLE FRAGMENT (SCFV) THEREOF, NUCLEIC ACID MOLECULE, COMPOSITION, AND METHOD FOR INCREASING ADCC OF A 3F8 ANTIBODY - Google Patents

Abstract

HIGH AFFINITY ANTI-GD2 ANTIBODIES OR A FRAGMENT THEREOF, NUCLEIC ACID MOLECULE, COMPOSITION, THEIR PRODUCTION METHODS, AND THEIR USES. In the present Application, high-affinity anti-GD2 antibody agents and various methods and reagents related thereto are described, including, for example, for the detection, prevention and/or therapeutic treatment of GD2-related diseases, in particular, neuroblastoma.

Description

REFERENCE TO RELATED ORDERS

[0001] This Application benefits from 35 USC § 119 (e) of United States Provisional Patent Application Serial No. 61/801,287 filed on March 15, 2013, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] This specification references a sequence listing sent in electronic format as an ascii .txt file named "2003080-0638_ST25" on March 14, 2014. The .txt file was generated on March 12, 2014 and it is 94 kb in size.

INTRODUCTION

[0003] Monoclonal antibody (MoAb) therapy is an accepted treatment modality for cancers, with five MoAbs having received FDA approval for solid tumors in adults, including colorectal and breast cancer, non-small cell lung cancer, squamous cell carcinoma and melanoma (Boyiadzis et al., 2008, Expert Opin Biol Ther 8, 1151-8; Yan et al., 2008, J Cancer 14, 178-83).

[0004] Among other things, the present invention provides the insight that MoAb therapy has remained inadequately explored for the treatment of pediatric cancers. Unlike chemotherapy or radiotherapy, MoAb therapy is nonmyelosuppressive and genotoxic and generally has few long-term toxicities. These are critical considerations for children. Most importantly, the MoAb is effective against metastatic cancer in the blood, bone marrow, and bone, typically found in high-risk neuroblastoma (NB). As a class of agents, the pharmacokinetics and toxicity of human or humanized IgG1 antibodies have been extensively studied. Furthermore, antibodies can carry cytotoxic payloads, whether immune-based, radioisotopes, toxins or enzymes, thereby increasing options for targeted therapy.

SUMMARY

[0005] The present invention provides antibody agents that bind to GD2 and are variants of a reference 3F8 antibody in that they contain one or more particular structural features that are not found in the reference 3F8 antibody and are described herein. In some embodiments, the antibody agents provided show improved stability and/or reduced immunogenicity relative to an otherwise identical 3F8 antibody that lacks one or more of the structural features described herein. In some embodiments, the antibody agents provided are useful in medicine, for example, in the diagnosis and/or treatment of NB. In some embodiments, the antibody agents provided are associated with, comprise and/or provide one or more payloads (e.g., a detectable payload and/or a therapeutic payload). A variety of methodologies for identifying, characterizing, preparing and/or using such antibody agents are also provided by the present invention.

[0006] In the present Application, high affinity anti-GD2 antibodies are specifically described with a new hu3F8 framework, hu3F8V5, which is designed to have reduced immunogenicity but greater stability. The hu3F8V5 antibodies were designed by optimizing the hu3F8V1 framework structure (described in WO 2011/160119 and Cheung et al., 2012, Oncoimmunology 1: 477-486) for reduced immunogenicity based on computational methods. First, the heavy chain and light chain sequences of hu3F8V1 were compared with the human germline sequences humIGHV199 and humIGKV025, respectively (EMBL database). Molecular simulations using CHARMm force fields (CHemistry at Harvard Molecular Mechanics) (B.R. Brooks et al., J. Comp. Chem. 30, 1545-1615 (2009)) were performed on each potential humanizing mutation based on the crystal structure of murine 3F8 (access to the 3VFG protein database) to determine whether the mutation was structurally permissive. Additionally, MHC class II T cell epitopes in hu3F8V1 were identified using the NN-alignment method in the Immune Epitope Database and minimized based on structurally permissive mutations. Based on a computational model of GD2 coupled to the 3F8 crystal structure (constructed using CDOCKER and Discovery software, Accelrys, San Diego, CA), CDR residues that were not modeled to interact directly with the GD2 antigen were considered for humanizing mutations.

[0007] Nine point mutations were made in hu3F8V1 to make hu3F8V5 (see Table 2) in an effort to reduce potential immunogenicity. All nine mutations were found to be structurally permissive for the computational model of 3F8 bound to its GD2 antigen. All mutations involve the change of murine residues left in the humanization of 3F8 to human germline sequences. (LC:K24R, LC:S56T, LC:V58I, HC:I20L, HC:M92V) involve framework residues. We additionally discovered 4 mutations in CDR H2 (HC: A62S, HC: F63V, HC: M64K, HC: S56G) that removed a strong T cell epitope as identified through in silico methods. We were surprised that our computational model of 3F8 bound to GD2 allowed for the suggested mutations in the CDR region, as it is unusual for those skilled in the art of humanizing antibodies via grafting methods to alter CDR residues.

[0008] To perform affinity maturation based on yeast display methods, we first synthesized a new biotinylated GD2 derivative to be used for selection. Previously, we have had no success using a standard biotinylated GD2 antigen (obtained from the Consortium for Functional Glycomics). A new synthetic GD2-azide derivative (Figure 2) was created by fusing a PEG spacer in order to observe GD2 in flow cytometry. Using this new analogue, we selected 2 mutations from a random library of yeast surface-displayed hu3F8 ScFvs which had increased binding to the synthetic GD2 analogue. The first was LC:D32H, which is located on the CDR L1, and the second was LC:E1K, which is a framework residue. The two mutations (LC:E1K and LC:D32H) were tested in recombinantly expressed ScFv hu3F8V1 and ScFv hu3F8V5 constructs and binding affinities to native GD2 were measured using Biacore analysis. Based on structural modeling, all hu3F8 ScFvs were made in the VL-VH format, as this allows less restricted access to the antigen-binding pocket. This is in contrast to most conventional ScFvs, which are constructed in the VH-VL format. Several variants were also tested in the full-length IgG1 format.

[0009] Therefore, the present invention provides new high-affinity anti-GD2 antibody agents, including intact antibodies, single-chain variable fragments (scFv) and other formats, containing specific structural features (mutations in relation to 3F8 and/or or hu3F8V1) which reduce immunogenicity and increase the affinity of the antibody for GD2.

[00010] In one embodiment, the present invention provides a new structure for the anti-GD2 antibody 3F8, namely hu3F8V5, having specific structural characteristics (mutations in relation to 3F8 and/or hu3F8V1) which reduce its immunogenicity and remove an epitope from T cell.

[00011] In another embodiment, the present invention provides anti-GD2 antibody agents with greater affinity for GD2, the antibody agents having specific structural characteristics in the light chain (LC), D32H located on the L1 CDR, and a residue of " framework" E1K.

[00012] Anti-GD2 antibody agents with a particular structural feature in the heavy chain (VH), G54I, are also provided.

[00013] The antibody agents of the present invention may have a single structural feature described above or two structural features in any combination, such as dual features, or more than two structural features in any combination, such as triple features. In some embodiments, these structural features can be introduced into any form of a 3F8 antibody, e.g., hu3F8V1, or in combination with other structural features already present in the 3F8 molecule for similar or alternative purposes, e.g., hu3F8V5. Although antibodies and single-chain variable fragments (scFv) that carry these structural features in both hu3F8V1 and hu3F8V5 are described here, it will be understood that mutations can be incorporated into any 3F8 antibody sequence in order to obtain increased affinity.

[00014] In one embodiment, the invention is directed to an anti-GD2 3F8 antibody having a light chain (LC) with a structural feature, either E1K or D32H, or two structural features, E1K and D32H, and/or a heavy chain (HC) with a G54I structural feature, as well as antibody compositions, antibody glycoforms, antibodies with increased stability, antibodies with increased binding to Fc receptors, antibodies with increased affinity for GD2, bispecific antibodies engineered to expressing a second distinct binding site or a bispecific T cell coupling agent or use of the Fv fragments of any of the antibodies of the present invention in constructing modular IgG for bispecific antibodies, random bispecific scFv couples to T cells (BiTE), trispecific or multispecific antibodies. Such antibodies and coding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants related thereto, and methods of producing and using the same, as described and empowered herein, in combination with that which is known in the art, form part of the present invention. Surprisingly, hu3F8 carrying any combination of the E1K, D32H and G54I structural features shows significantly more affinity for GD2 and significantly more PMN-ADCC and PBMC-ADCC activities than the parental hu3F8V1, with low Complement Mediated Cytotoxicity. CMC). Low CMC is desirable as it is believed to mediate the secondary effect of pain associated with anti-GD2 immunotherapy. This superiority was consistently observed in ADCC assays regardless of donor status or whether NK92 transfected with human CD16 or CD32 were used as killers. This was important, as ADCC is the proven mechanism for MoAb antitumor effects in general patients.

[00015] In one embodiment, the invention is directed to a 3F8 antibody agent comprising a light chain and a heavy chain based on hu3F8V1 or hu3F8V5, but differing in the presence of one or more structural features described herein, said antibody agent antibody binding with high affinity to GD2 and mediating a desired effect, for example, inhibition of cell growth in vitro, blocking the secondary effects of pain due to anti-GD2 antibody therapy, to name a few.

[00016] In one aspect, the antibody agent of the invention includes hu3F8V1 IgG with a single structural feature described herein, for example, hu3F8V1-E1K, hu3F8V1-D32H, hu3F8V1-G54I; hu3F8V1 IgGs with a dual structural feature as described here, for example, hu3F8V1-E1KD32H, hu3F8V1-E1KG54I and hu3F8V1-D32HG54I; hu3F8V1 IgGs with a triple structural feature as described here, for example, hu3F8V1-E1KD32HG54I; hu3F8V5 IgG; hu3F8V5 IgGs with a single structural feature as described here, e.g., hu3F8V5-E1K, hu3F8V5-D32H, hu3F8V5-G54I; hu3F8V5 IgGs with a dual structural feature as described here, for example, hu3F8V5-E1KD32H, hu3F8V5-E1KG54I and hu3F8V5-D32HG54I; and hu3F8V5 IgGs with a triple structural feature as described herein, for example, hu3F8V5-E1KD32HG54I.

[00017] The invention also includes fragments or a derivative of such an antibody, such as one or more portions of the antibody chain, such as the heavy chain constant, joining, diversity or variable regions or the constant, joining or variable regions light chain. The antibodies may be of any class, such as IgG, IgM, IgA or any subclass, such as IgG1, IgG2a, IgG4 and other subclasses known in the art. Antibodies useful in the present invention also include antigen-binding antibody fragments of the antibodies of the present invention including, but not limited to, Fab, Fab' and F(ab')2 fragments, Fd, single chain variable fragment (scFv) , single-chain antibodies, disulfide-linked or disulfide-stabilized Fvs (sdFv or dsFv).

[00018] Single chain variable fragments of the present invention include hu3F8V1-E1K scFv, hu3F8V1-D32H scFv, hu3F8V1-G54I scFv; hu3F8V1 scFv with a dual structural feature as described here, for example, hu3F8V1-E1KD32H scFv, hu3F8V1-E1KG54I scFv and hu3F8V1-D32HG54I scFv; hu3F8V1 scFv with a triple structural feature as described here, e.g., hu3F8V1-E1KD32HG54I scFv; hu3F8V5 scFv; hu3F8V5 scFv with a single structural feature as described here, e.g., hu3F8V5-E1K scFv, hu3F8V5-D32H scFv, hu3F8V5-G54I scFv; hu3F8V5 scFv with a dual structural feature as described here, for example, hu3F8V5-E1KD32H scFv, hu3F8V5-E1KG54I scFv and hu3F8V5-D32HG54I scFv; hu3F8V5 scFv with a triple structural feature as described herein, for example, hu3F8V5-E1KD32HG54I scFv and combinations thereof.

[00019] The invention also includes antibodies with a single domain comprising a VL or VH domain. Furthermore, antibodies can be produced by any method, such as phage display, or produced in any organism, egg, or cell line, including bacteria, insects, yeast (fungi), mammals, or other cell types or cell lines. cells that produce antibodies with desired characteristics, such as humanized antibodies. Antibodies can also be formed by combining a Fab moiety and an Fc region from different species or maintaining the complementarity determining regions and modifying the framework regions to that of another species.

[00020] Preferred anti-GD2 antibodies of the present invention comprise any of the following peptide sequences:

[00021] hu3F8V1 light chain with the structural characteristic D32H identified as SEQ ID NO: 1,

[00022] hu3F8V1 light chain with the structural characteristic E1K identified as SEQ ID NO: 2,

[00023] hu3F8V1 light chain with a double structural feature D32H and E1K identified as SEQ ID NO: 3,

[00024] hu3F8V1 heavy chain with the structural feature G54I identified as SEQ ID NO: 4,

[00025] hu3F8V5 gamma 1 heavy chain identified as SEQ ID NO: 5,

[00026] kappa light chain of hu3F8V5 identified as SEQ ID NO: 6,

[00027] hu3F8V5 light chain with the structural characteristic D32H identified as SEQ ID NO: 7,

[00028] hu3F8V5 light chain with the structural characteristic E1K identified as SEQ ID NO: 8,

[00029] hu3F8V5 light chain with a double structural feature E1K and D32H identified as SEQ ID NO: 9,

[00030] hu3F8V5 heavy chain with the structural feature G54I identified as SEQ ID NO: 10,

[00031] single chain variable fragment (scFv) of hu3F8V1 having a light chain variable region with the structural feature D32H identified in SEQ ID NO: 11,

[00032] scFv hu3F8V1 having a light chain variable region with the structural feature D32H and a heavy chain variable region with the structural feature G54I identified as SEQ ID NO: 12,

[00033] scFv hu3F8V1 having a light chain variable region with the structural feature E1K identified as SEQ ID NO: 13,

[00034] scFv hu3F8V1 having a light chain variable region with the structural feature E1K and a heavy chain variable region with the structural feature G54I identified as SEQ ID NO: 14,

[00035] scFv hu3F8V1 having a light chain variable region with the double structural feature E1K and D32H identified as SEQ ID NO: 15,

[00036] scFv hu3F8V1 having a light chain variable region with the double structural feature E1K and D32H and a heavy chain variable region with the structural feature G54I identified as SEQ ID NO: 16,

[00037] scFv hu3F8V1 having a heavy chain variable region with the structural feature G54I identified as SEQ ID NO: 17

[00038] scFv hu3F8V5 identified as SEQ ID NO: 18,

[00039] scFv hu3F8V5 having a light chain variable region with the structural feature D32H identified as SEQ ID NO: 19,

[00040] scFv hu3F8V5 having a heavy chain variable region with the structural feature G54I identified as SEQ ID NO: 20.

[00041] scFv hu3F8V5 having a light chain variable region with the structural feature D32H and a heavy chain variable region with the structural feature G54I identified as SEQ ID NO: 21,

[00042] scFv hu3F8V5 having a light chain variable region with the structural feature E1K identified as SEQ ID NO: 22,

[00043] scFv hu3F8V5 having a light chain variable region with the structural feature E1K and a heavy chain variable region with the structural feature G54I identified as SEQ ID NO: 23,

[00044] scFv hu3F8V5 having a light chain variable region with the double structural feature E1K and D32H identified as SEQ ID NO: 24 and

[00045] scFv hu3F8V5 having a light chain variable region with the double structural feature E1K and D32H and a heavy chain variable region with the structural feature G54I identified as SEQ ID NO: 25.

[00046] In another embodiment, the single-chain variable fragments described above can be linked, with or without linkers or spacers, to other scFvs with specificity for another antigen to produce bivalent or bispecific anti-GD2 antibodies. For example, the scFv sequence for huOKT3 with a linker and a spacer identified as SEQ ID NO: 26, or without a spacer identified as SEQ ID NO: 27, could follow any of the scFvs described in SEQ ID NOs: 11- 25. Alternatively, the scFv sequence for C825, anti-DOTA, identified as SEQ ID NO: 28 may follow any of the scFv sequences identified in SEQ ID NO: 11-25. Some examples of bispecific antibodies include:

[00047] scFv hu3F8V1-ligand-huOKT3 scFv with ADT-KGP spacer identified as SEQ ID NO: 29,

[00048] scFv hu3F8V1-ligand-huOKT3 scFv without spacer identified as SEQ ID NO: 30, and

[00049] scFv hu3F8V1-C825 scFv identified in SEQ ID NO: 31.

[00050] In some embodiments of the present invention, the provided antibody agents may be further modified with the carbohydrate composition, for example, to increase effector function, with a particular DEL triple residue characteristic (S239D/A330L/I332E ) in the hu3F8V1 heavy chain, one or more structural features in the heavy chain and/or in the VH-VL Ala43Ser interface for greater stability and/or combinations thereof.

[00051] The preferred antibody agents of the present invention are those that bind to human GD2 and perform the intended function, that is, effector function, pain blockage or cell growth inhibition. Some representative methods for determining the specificity and affinity of a monoclonal antibody through competitive inhibition can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988), incorporated herein by reference. At least one antibody of the present invention binds to at least one specified epitope specific to human GD2, subunit, fragment, portion or any combination thereof. The epitope may comprise at least one antibody binding region, which epitope is preferably comprised of at least 1 to 5 sugar or ceramide residues of at least a portion of GD2.

[00052] In one aspect, the present invention provides at least one anti-GD2 hu3F8 antibody isolated from the present invention that comprises either LC or HV bearing any of the structural features defined herein in any combination and the nucleic acid sequences encoding the same as:

[00053] the hu3F8V1 light chain with a single structural feature D32H is encoded by the polynucleotide identified in SEQ ID NO: 32,

[00054] the hu3F8V1 light chain with a double structural feature E1K and D32H is encoded by the polynucleotide identified in SEQ ID NO: 33,

[00055] the heavy chain of hu3F8V5 is encoded by the polynucleotide identified in SEQ ID NO: 34,

[00056] the hu3F8V5 light chain is encoded by the polynucleotide identified in SEQ ID NO: 35,

[00057] the hu3F8V5 light chain with a single structural feature D32H is encoded by the polynucleotide identified in SEQ ID NO: 36,

[00058] the hu3F8V5 light chain with a double structural feature E1K and D32H is encoded by the polynucleotide identified in SEQ ID NO: 37,

[00059] the hu3F8V5 heavy chain with a single structural feature G54I is encoded by the polynucleotide identified in SEQ ID NO: 38,

[00060] scFv hu3F8V1 with a single structural feature D32H in the light chain region is encoded by the polynucleotide identified in SEQ ID NO: 39,

[00061] scFv hu3F8V1 with a double structural feature E1K and D32H in the light chain region is encoded by the polynucleotide identified in SEQ ID NO: 40,

[00062] scFv hu3F8V1 with a triple structural feature E1K and D32H in the light chain region and G54I in the heavy chain region is encoded by the polynucleotide identified in SEQ ID NO: 41,

[00063] scFv hu3F8V5 is encoded by the polynucleotide identified in SEQ ID NO: 42,

[00064] scFv hu3F8V5 with a single structural feature D32H in the light chain region is encoded by the polynucleotide identified in SEQ ID NO: 43,

[00065] scFv hu3F8V5 with a double structural feature E1K and D32H in the light chain region is encoded by the polynucleotide identified in SEQ ID NO: 44, and

[00066] scFv hu3F8V5 with a triple structural feature E1K and D32H in the light chain region and G54I in the heavy chain region is encoded by the polynucleotide identified in SEQ ID NO: 45.

[00067] In some aspects, the present invention provides a diagnostic/detective or therapeutic immunoconjugate comprising an antibody component comprising any of the 3F8 MoAbs or fragments thereof of the present invention or an antibody fusion protein or fragment thereof comprising any of the 3F8 antibodies or fragments thereof of the present invention, wherein the antibody component is linked to at least one diagnostic/detection agent or at least one therapeutic agent.

[00068] In some aspects, the present invention provides a therapeutic immunoconjugate comprising a therapeutic agent, for example, selected from the group consisting of a radionuclide, boron, gadolinium or uranium atoms, an immunomodulator, such as a cytokine, a stem cell growth factor, a lymphotoxin, such as tumor necrosis factor (TNF), a hematopoietic factor, such as an interleukin (InterLeukin - IL), a colony stimulating factor (Colony Stimulating Factor - CSF), such as granulocyte-colony stimulating factor (Granulocyte-Colony Stimulating Factor - G-CSF) or granulocyte-macrophage colony-stimulating factor (Granulocyte Macrophage-Colony Stimulating Factor - GM-CSF), an interferon (InterFeroN - IFN), such as interferon-alpha, -beta or -gamma, and a stem cell growth factor, a hematopoietic factor, erythropoietin, thrombopoietin, an antibody, a hormone, a hormonal antagonist, an enzyme, an inhibitor enzyme, a photoactive therapeutic agent, a cytotoxic drug, such as antimitotic, alkylating, antimetabolite, angiogenesis inhibitors, apoptotics, alkaloid, COX-2 inhibitors and antibiotic agents, a cytotoxic toxin, such as plant, microbial and animal toxins and synthetic variations thereof, an angiogenesis inhibitor, a different antibody or a combination thereof.

[00069] In some aspects, the present invention also provides a multivalent, multispecific antibody or a fragment thereof that comprises one or more antigen binding sites with affinity for an antigen recognized by the 3F8 antibody and one or more hapten binding sites that have affinity for epitopes or haptens, in addition to GD2. In one embodiment, the multivalent or multispecific antibody or fragment thereof comprises a diagnostic/detection or therapeutic agent.

[00070] In some aspects, the present invention provides a method of delivering a diagnostic/detection agent, a therapeutic agent, or a combination thereof to a target comprising: (i) administering, to an individual, a multivalent antibody, multispecific or fragment thereof of the present invention; (ii) wait a sufficient period of time for a quantity of non-binding proteins to be eliminated from the individual's bloodstream; and (iii) administering to said individual a carrier molecule containing a diagnostic/detection agent, a therapeutic agent or a combination thereof that binds to a binding site of said antibody. In some embodiments, the diagnostic/detection agent or therapeutic agent is selected from the group consisting of isotopes, dyes, chromogens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormonal antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA and combinations thereof. The second specificity also includes hapten(s) conjugated to any of the group of agents described. These haptens include, but are not limited to, biotin and its derivatives, DOTA and its derivatives, DTPA and its derivatives, fluorescein and its derivatives, histamine and its derivatives, Deferoxamine and its derivatives.

[00071] In any of the methods of the present invention, the subject is preferably a mammal, such as a human or domestic animal.

[00072] In some embodiments, the present invention is a method for treating or identifying diseased tissues in an individual comprising: (a) administering, to said individual, a bispecific antibody or antibody fragment having at least one arm that specifically binds to a marker associated with diseased tissue and at least one other arm that specifically binds to an objectifiable conjugate, wherein said marker associated with diseased tissue is an antigen recognized by MoAb 3F8; (b) optionally administering to said individual a killing composition and allowing said composition to eliminate non-targeted antibodies or antibody fragments from circulation; and (c) administering to said subject a first objectifiable conjugate comprising a carrier moiety comprising or containing at least one epitope recognized by said at least one other arm of said bispecific antibody or antibody fragment and one or more agents therapeutic or combined diagnostics. Preferably, at least one arm that specifically binds to a target tissue is an anti-GD2 antibody or an anti-GD2 antibody fragment of the present invention.

[00073] In some aspects, the present invention provides a method for detecting or treating tumors expressing an antigen recognized by a 3F8 MoAb in a mammal comprising: (A) administering an effective amount of a bispecific antibody or an antibody fragment comprising at least one arm that specifically binds to a target tissue and at least one other arm that specifically binds to an objectifiable conjugate, wherein said arm that specifically binds to a target tissue is a 3F8 antibody of the present invention or a fragment thereof; and (B) administration of a conjugate capable of objectification. The conjugate subject to objectification can be selected from the group consisting of (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iii) DOTA -D-Asp-D- Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv) DOTA -D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v) DOTA -D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vi) DOTA -D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)- NH2; (vii) DOTA -D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2; (ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x) Ac-D-Phe-D-Lys(Bz-DTPA)- D-Tyr-D-Lys(Bz-DTPA)-NH2; (xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)- D-Lys(Tscg-Cys)-NH2; (xii) DOTA -D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)- D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv) Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2; (xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2; (xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2; fluorescein and its derivatives; desferrioxamine and its derivatives.

[00074] In some aspects, the present invention provides a method of objectification, wherein the method comprises: (A) injecting, into an individual undergoing such a procedure, a bispecific antibody, F(ab)2 fragment or F(ab')2 or single-chain Fv fragment, wherein the bispecific antibody or a fragment thereof has a first antibody binding site which specifically binds to an antigen recognized by a 3F8 MoAb of the present invention and has a second antibody binding site which specifically binds to a hapten, and allows the antibody fragment to aggregate at target sites; (B) optionally, eliminating non-targeted antibody fragments using a scavenging agent if the bispecific fragment is not largely eliminated from circulation within about 24 hours after injection and injection of a dextran modified with hapten or dendrimers or polymers, a which rapidly removes the antibody or non-targeted fragments in the liver for degradation (C) detection of the presence of the hapten by nuclear imaging or short-range detection of high levels of aggregated tracer at target sites using scanners or probe within hours of the first injection and carrying out said procedure, wherein said detection is carried out without the use of a contrast agent or subtraction agent. In a preferred embodiment, the hapten is labeled with a diagnostic/detection radioisotope, an image intensifying agent for MRI, a fluorescent label or a chemiluminescent label. Fluorescent markers may include rhodamine, fluorescein, renographin, fluorescein isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Chemiluminescent markers may include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester. Image intensifying agents for MRI include gadolinium and ferromagnetic substances. Antibody-hapten localization imaging detects intact tumor cells that carry GD2, which is critical for tumor evolution, measuring tumor response to treatment, detecting early relapse, and tumor surveillance. Intraoperative antibody-hapten localization detection provides precise tumor localization and uncovers hidden sites of disease to enable complete surgical resection as part of curative cancer therapy.

[00075] Also considered in the present invention is a multivalent, multispecific antibody or a fragment thereof that comprises one or more antigen binding sites with affinity for an antigen recognized by the 3F8 antibody and one or more hapten binding sites that have affinity for epitopes or haptens on cells (lymphocytes, natural killer cells, neutrophils, myeloid cells, neural stem cells, mesenchymal stem cells, leukemic cells, cytotoxic lymphocytes and B-lymphocytes). These bispecific antibodies or fragments can be administered via various routes, including intravenous, intrathecal and intratumoral, in mammals, including humans, to target endogenous cells or cells exogenously infused into sites or tissues or cells that bear the GD2 antigen. Alternatively, cells can be armed ex vivo using these bispecific antibodies or fragments prior to administration to mammals, including humans.

[00076] Also considered in the present invention is the use of 3F8 sequences or fragments thereof to create chimeric surface receptors specific for GD2 using genetic methods, to redirect cells (lymphocytes, natural killer cells, neutrophils, myeloid cells, stem cells neuronal cells, mesenchymal stem cells, leukemic cells, cytotoxic lymphocytes and B-lymphocytes) to tissues, organs or tumors carrying GD2, both for diagnostic purposes and for therapeutic applications.

[00077] The present invention provides, in one aspect, isolated nucleic acid molecules that comprise, are complementary to or hybridize with a polynucleotide encoding the aforementioned specific anti-GD2 antibodies comprising at least one specified sequence, domain, portion or variant of the same.

[00078] The present invention further provides recombinant vectors comprising said nucleic acid molecules encoding anti-GD2 antibody, host cells containing such nucleic acids and/or recombinant vectors, as well as methods of preparing and/or using such nucleic acids of antibodies, vectors and/or host cells. Thus, the invention comprises an isolated nucleic acid encoding at least one isolated mammalian anti-GD2 antibody or fragment thereof; an isolated nucleic acid vector comprising the isolated nucleic acid and/or a prokaryotic or eukaryotic host cell comprising the isolated nucleic acid. The host cell may optionally be at least one selected from COS-1, COS-7, HEK293, BHK21, CHO, CHO-S, DG44, BSC-1, Hep G2, 653, SP2/0, 293, HeLa cells. , myeloma cells or lymphoma cells or any cells derived, immortalized or transformed therefrom. Also provided is a method for producing at least one anti-GD2 antibody comprising translating the nucleic acid encoding the antibody under in vitro, in vivo or in situ conditions such that the antibody is expressed in detectable or recoverable amounts, including methods that use vectors which allow protein expression to be amplified using selection for growth and survival under the control of metabolic pathways or enzymes that include, but are not limited to, dihydrofolate reductase (DiHydroFolate Reductase - DHFR) or glutamine synthetase (Glutamine Synthase - GS ).

[00079] The present invention also provides at least one method for expressing at least one aforementioned anti-GD2 antibody in a host cell comprising culturing a host cell, as described herein, under conditions in which at least one anti-GD2 antibody is expressed in detectable and/or recoverable quantities.

[00080] The present invention also provides at least one composition comprising (a) an isolated nucleic acid encoding an isolated anti-GD2 antibody and/or an antibody as described herein; and (b) a suitable vehicle or diluent. The carrier or diluent may optionally be pharmaceutically acceptable in accordance with known carriers or diluents. The composition may optionally further comprise at least one other compound, protein or composition. In some of these compositions, the chimeric or humanized antibodies are conjugated to a cytotoxic agent (i.e., an agent that affects the viability and/or functions of a cell), such as a cytotoxic drug, a toxin, or a radionuclide.

[00081] The present invention further provides at least one anti-GD2 antibody or composition for administering a therapeutically effective amount to modulate or treat at least one GD2-related condition in a cell, tissue, organ, animal or patient and/or or before, subsequently to or during a related condition, as known in the art and/or as described herein. Thus, the invention provides a method for diagnosing or treating a GD2-related condition in a cell, tissue, organ or animal comprising contacting or administering a composition comprising an effective amount of at least one isolated anti-GD2 antibody or fragment. of the same of the present invention with or to the cell, tissue, organ or animal. The method may further comprise optionally using an effective amount of 0.001 to 50 mg/kg of an anti-GD2 antibody of the invention to the cells, tissue, organ or animal. The method may optionally further comprise contact or administration through at least one selected mode of administration parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intra-bronchial, intra-abdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrathecal, intra-Ommaya, intravitreal, intraocular, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal or transdermal. The method may optionally further comprise administration before, simultaneously or after contact with the antibody or administration of at least one composition comprising an effective amount of at least one compound or a protein or cell selected from at least one of a detectable marker. or a reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroidal anti-inflammatory drug (Non-Steroid Anti-Inflammatory Drug - NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic , a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an antibody or antibody-derived conjugates, an immunosuppressant, a growth hormone, a hormone replacement drug , a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or an analogue thereof, a cytotoxic agent or other anticancer agent, an antimetabolite agent, such as methotrexate, an antiproliferative agent, a cytokine, interleukin, growth factors, a cytokine antagonist and an anti-TNFα, white blood cells, T-cells, LAK cells, TIL cells, natural killer (NK) cells, monocytes, NKT cells, T cells or engineered NK cells or monocytes or granulocytes.

[00082] The present invention further provides at least one anti-GD2 antibody method for diagnosing at least one GD2-related condition in a cell, tissue, organ, animal or patient and/or before, subsequently to or during a condition related, as known in the art and/or as described herein.

[00083] The present invention also provides at least one composition, device and/or delivery method for diagnosing at least one condition with anti-GD2 antibody according to the present invention.

[00084] Also provided is a composition comprising at least one isolated humanized anti-GD2 antibody of the present invention and at least one pharmaceutically acceptable carrier or diluent. The composition may optionally further comprise an effective amount of at least one compound or protein selected from at least one of a detectable marker or reporter, a cytotoxic agent or other anticancer agent, an antimetabolite, such as methotrexate, a antiproliferative, a cytokine or cytokine antagonist, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroidal anti-inflammatory drug (NonSteroid Anti-Inflammatory Drug - NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement drug, a radiopharmaceutical; an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or similar.

[00085] Also provided is a medical device comprising at least one isolated mammalian anti-GD2 antibody of the invention, wherein the device is suitable for contacting or administering the at least one anti-GD2 antibody through at least one selected mode of parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal administration , intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrathecal, intra-Ommaya, intravitreal, intraocular, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual , intranasal or transdermal.

[00086] In a further aspect, the invention provides a kit comprising at least one chimeric or humanized anti-GD2 antibody or fragment of the description in lyophilized form in a first container and an optional second container comprising sterile water, sterile buffered water or at least at least one preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkylparaben, benzalkonium chloride, benzethonium chloride , sodium dehydroacetate and thimerosal, mannitol, sucrose, mannose, other sugars, Tween 80 or mixtures thereof in an aqueous diluent. In one aspect, in the kit, the concentration of the anti-GD2 antibody or portion or variant specified in the first container is reconstituted to a concentration of about 0.1 mg/ml to about 500 mg/ml with the contents of the second container. In another aspect, the second container further comprises an isotonicity agent. In another aspect, the second container further comprises a physiologically acceptable buffer. In one aspect, the disclosure provides a method of treating at least one condition related to GD2 comprising administering, to a patient in need thereof, a formulation provided in a kit and reconstituted prior to administration.

[00087] There is also provided an article of manufacture for pharmaceutical or diagnostic use in humans comprising packaging material and a container comprising a solution or a lyophilized form of at least one isolated humanized or chimeric anti-GD2 antibody of the present invention. The article of manufacture may optionally comprise a container with a component of an enteric, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intra-abdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebro-ventricular, delivery system device. intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrathecal, intra-Ommaya, intravitreal, intraocular, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual or intranasal.

BRIEF DESCRIPTION OF THE DRAWINGS

[00088] The drawings included here, which are comprised of the following Figures, are for illustrative, non-limiting purposes only.

[00089] Figure 1 shows the strength of staining intensity of different types of tumors with ScFv hu3F8V1.

[00090] Figure 2 shows the chemical structure of biotin-PEG-GD2 made from azide-GD2-oligosaccharide reacted with biotin (PEG) 4-alkyne using Click Chemistry.

[00091] Figure 3 shows BIAcore sensorgrams of dissociation rates for exemplary hu3F8V1 IgGs.

DETAILED DESCRIPTION OF CERTAIN MODALITIES Certain Settings

[00092] In the following description, a number of terms used in recombinant DNA and immunology are used extensively. In order to provide a clearer and more consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided.

[00093] Adult: As used herein, the term "adult" refers to a human being eighteen years of age or older. Body weights among adults can vary widely, with a typical range being 40 kg to 113 kg (90 pounds to 250 pounds).

[00094] Affinity: As known in the art, "affinity" is a measure of the tightness with which a particular ligand (e.g., an antibody) binds to its partner (e.g., an epitope). Affinities can be measured in different ways.

[00095] Amino acids: As used here, the term "amino acid", in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; In some embodiments, an amino acid is an L-amino acid. "Conventional amino acid" refers to any of twenty conventional L-amino acids commonly found in naturally occurring peptides. "Unconventional amino acids" refers to any amino acid other than conventional amino acids, regardless of whether they are prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids including, but not limited to, salts, amino acid derivatives (such as amides) and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified through methylation, amidation, acetylation, protecting groups and/or replacement with other chemical groups that can alter the circulating half-life of the peptide. tid without adversely affecting its activity. Amino acids can participate in a disulfide bond. Amino acids may comprise one or more post-translational modifications, such as association with one or more chemical entities (e.g., methyl groups, ethyl groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol, lipid portions, carbohydrate portions, biotin portions, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and/or an amino acid residue of a peptide. It will be evident from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[00096] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to human beings of either sex and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects and/or worms. In certain embodiments, the animal is susceptible to DV infection. In some embodiments, an animal may be a transgenic animal, genetically modified animal, and/or a clone.

[00097] Antibody: The term "antibody" is a terminology recognized in the art and is intended to include molecules or active fragments of molecules that bind to known antigens. Examples of active fragments of molecules that bind to known antigens include the Fab and F(ab')2 fragments. These active fragments can be derived from an antibody of the present invention through a variety of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For a further description of general techniques for the isolation of active antibody fragments see, for example, Khaw, B. A. et al., J. Nucl. Med. 23:1011-1019 (1982). The term "antibody" also includes chimeric and bispecific antibodies and other available formats.

[00098] In some embodiments, an antibody as described herein is or comprises a full-length immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binds) portion of a molecule. of immunoglobulin, such as an antibody fragment.

[00099] An antibody fragment is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, sFv and so on. Regardless of the structure, an antibody fragment binds to the same antigen that is recognized by the intact antibody. For example, a 3F8 monoclonal antibody fragment binds to an epitope recognized by 3F8. The term "antibody fragment" also includes any synthetic or genetically modified protein that includes antigen-binding structures and acts as an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of variable regions, such as "Fv" fragments consisting of heavy or light chain variable regions, recombinant single-chain polypeptide molecules in which light and heavy chain variable regions are linked by a peptide linker ("scFv proteins") and minimal recognition units consisting of amino acid residues that are or mimic the hypervariable region.

[000100] For example, in some embodiments, an antibody fragment comprises one or more, and in some embodiments all, the Complementary Determining Regions (CDRs) found in a heavy or light chain of the parent antibody. In some embodiments, an antibody fragment further includes a sequence adjacent to a CDR. In some embodiments, an antibody fragment includes a sequence identical to a portion of the intact parental antibody; in some embodiments, the portion includes 1, 2 or 3 CDRs; In some embodiments, the portion corresponds to a full-length chain. In some embodiments, the portion is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50 or more amino acids in length.

[000101] The expression "monoclonal antibody" is a recognized terminology in the art. Monoclonal antibodies are antibodies that are monospecific that are the same because they are made by one type of immune cells that are all clones of a single parental cell.

[000102] There are a variety of methods in the art for producing monoclonal antibodies. For example, monoclonal antibodies can be made by recombinant DNA methods, such as those described in United States Patent 4,816,567. In this context, the term "monoclonal antibody" refers to an antibody derived from a single eukaryotic phage or prokaryotic clone. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies, or such chains from human beings, humanized or from other sources). Once isolated, DNA can be placed into expression vectors, which are then transformed into host cells, such as NS0 cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, yeast cells , algal cells, eggs, or myeloma cells that otherwise do not produce immunoglobulin protein, to achieve the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by replacing the coding sequence with the constant domains of the human heavy and light chains of a desired species rather than the homologous human sequences (U.S. Patent No. 4,816,567; Morrison et al ., supra) or by covalently joining, to the immunoglobulin coding sequence, all or part of the coding sequence for a polypeptide other than immunoglobulin. Such non-immunoglobulin polypeptide may be replaced by the constant domains of an antibody of the invention or may be replaced by the variable domains of an antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

[000103] In some embodiments, an "antibody agent" is or comprises an antibody or a fragment thereof or an agent that comprises or consists of such an antibody or fragment thereof.

[000104] Comparable: The term "comparable" is used here to describe two (or more) sets of conditions or circumstances that are sufficiently similar to each other to permit comparison of results obtained or observed phenomena. In some embodiments, comparable sets of conditions or circumstances are characterized by a plurality of substantially identical features and one or a small number of varying features. Those skilled in the art will appreciate that sets of conditions are comparable to each other when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in the results obtained or phenomena observed with the different sets of conditions or circumstances are caused by or indicative of variation in those characteristics that are varied.

[000105] Corresponding to: As used here, the term "corresponding to" is often used to designate the position/identity of an amino acid residue in a polypeptide of interest. Those skilled in the art will appreciate that, for simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a related reference polypeptide, such that an amino acid "corresponding to" a residue in the position 190, for example, need not actually be amino acid 190 of a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; Those skilled in the art will readily appreciate how to identify "corresponding" amino acids.

[000106] Dosage Form: As used herein, the terms "dosage form" and "unit dosage form" refer to a physically discrete unit of a therapeutic protein (e.g., antibody) for the patient to be treated. Each unit contains a predetermined amount of active material calculated to produce the desired therapeutic effect. It will be understood, however, that the total dosage of the composition will be decided by the treating physician within the scope of medical judgment.

[000107] Dosage Regimen: A "dosage regimen" (or "therapeutic regimen"), as the term is used herein, is a set of unit doses (typically more than one) that are administered individually to an individual, typically separate for periods of time. In some embodiments, a given therapeutic agent has a recommended dosage regimen, which may involve one or more doses. In some embodiments, a dosage regimen comprises a plurality of doses, each of which is separated from each other by a period of time of the same duration; In some embodiments, a dosage regimen comprises a plurality of doses and at least two different time periods separate the individual doses. In some embodiments, all doses within a dosage regimen are the same unit dose amount. In some embodiments, different doses within a dosage regimen are of different amounts. In some embodiments, a dosage regimen comprises a first dose in a first dose amount, followed by one or more additional doses of a second dose amount different from the first dose amount. In some embodiments, a dosage regimen comprises a first dose of a first dose amount, followed by one or more additional doses in a second dose amount equal to the first dose amount.

[000108] Epitope: The term "epitope" is recognized in the art. It is generally understood by those skilled in the art to refer to the region of an antigen or antigens that interacts with an antibody. An epitope of a protein or peptide antigen or sugar may be linear or conformational or may be formed from contiguous or noncontiguous amino acids and/or sugar sequences of the antigen. The GD2 molecule, like many carbohydrates, contains many epitopes. Those skilled in the art will appreciate that, in some embodiments, the antibody agents provided may bind (e.g., cross-react) with variants of their target epitopes, e.g., may contain substitutions, modifications, additions, deletions, or mimetics. chemical properties of one or more amino acid or sugar residues. Such variant epitopes, and their use in accordance with the provided antibody agents, are within the scope of the present invention. Anti-idiotypic antibodies are an embodiment of the present invention. In some embodiments, amino acid or sugar epitopes or mimetic/chemical peptides or anti-idiotypic antibodies provide a convenient method, for example, by eluting GD2 from MoAb or GD2 MoAb over immunoaffinity columns. Furthermore, truncations of these epitopes may be possible.

[000109] Identity: As used herein, the term "identity" refers to the overall relationship between polymeric molecules, for example, between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two nucleic acid sequences, for example, may be accomplished by aligning the two sequences for purposes of optimal comparison (e.g., gaps may be introduced into one or both of a first and a second nucleic acid sequence). nucleic acid for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of an aligned sequence for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , at least 95% or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. Sequence comparison and determining the percentage of identity between two sequences can be performed using a mathematical algorithm. For example, the percentage of identity between two nucleotide sequences can be determined using the Meyers and Miller algorithm (CABIOS, 1989, 4: 11-17), which was incorporated into the ALIGN program (version 2.0), using a PAM120 residue weight table, a gap length penalty of 12, and a gap length penalty of 4. The percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package using an NWSgap-dna.CMP matrix.

[000110] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (either in nature and/ or in an experimental environment) and/or (2) produced, prepared and/or manufactured by the hand of man. Isolated substances and/or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of the other components with which they were initially associated. In some embodiments, the isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. As used here, a substance is "pure" if it is substantially free of other components. As used herein, the calculation of the percentage purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvents, water, etc.).

[000111] Naked: In some embodiments, an antibody agent can be said to be "naked" if it is not conjugated to a payload (e.g., a diagnostic or therapeutic agent). Such naked antibody agents are useful in a variety of contexts, including because the Fc portion of the antibody molecule confers effector functions such as complement fixation and Antibody-Dependent Cell Cytotoxicity (ADCC), as which establish mechanisms of action that can result in cell lysis. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric, humanized or human antibodies. However, it is possible that the Fc portion is not required for therapeutic function, rather that an antibody exerts its therapeutic effect through other mechanisms, such as inducing cell cycle arrest and apoptosis. In this case, naked antibodies also include the unconjugated antibody fragments defined above.

[000112] Chimeric: A "chimeric" antibody is a recombinant protein that contains the variable domains, including the complementarity determining regions (CDRs) of an antibody derived from a species, preferably a rodent antibody, whereas the constant domains of the antibody molecule are derived from those of a human antibody. For veterinary applications, the constant domains of the chimeric antibody can be derived from other species, such as a cat or dog.

[000113] Humanized: A "humanized" antibody is a recombinant protein in which the CDRs of an antibody from one species, for example, a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody to heavy and light variable domains. light humans. The constant domain of the antibody molecule is derived from those of a human antibody.

[000114] Human: The term "human" is often used to refer to an antibody obtained from transgenic mice that have been "manipulated" to produce specific human antibodies in response to antigenic challenge. In such a technique, elements of the human heavy and light chain locus are introduced into mouse types derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. Transgenic mice can synthesize human antibodies specific to human antigens, and mice can be used to produce hybridomas that secrete human antibodies. Methods for obtaining human antibodies from transgenic mice are described in Green et al., Nature Genet. 7: 13 (1994), Lonberg et al., Nature 368: 856 (1994) and Taylor et al., Int. Immun. 6:579 (1994). A fully human antibody can also be constructed using genetic methods or chromosomal transfection, as well as phage display technology, which are known in the art. See, for example, McCafferty et al., Nature 348: 552553 (1990) for the production of human antibodies and fragments thereof in vitro from immunoglobulin gene repertoires from non-immunized variable domain donors. In this technique, antibody variable domain genes are cloned in-frame into a major or minor envelope protein gene of a filamentous bacteriophage and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection for the gene encoding the antibody that exhibits these properties. In this way, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for its evaluation see, for example, Johnson and Chiswell, Current Opinion in Structural Biology 3: 5564-571 (1993).

[000115] Human antibodies can also be generated by B cells activated in vitro. See United States Patent Nos. 5,567,610 and 5,229,275, which are incorporated herein by reference in their entirety.

[000116] Therapeutic Agent: A therapeutic agent is an entity that, when administered according to a particular regimen, tends to achieve a desired therapeutic benefit. In some embodiments, a therapeutic agent can be administered separately, simultaneously or sequentially with an antibody moiety or conjugated to an antibody moiety, i.e., the antibody or antibody fragment or a subfragment, and is useful in treating a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, chelating agents, boron compounds, photoactive agents or dyes, and radioisotopes.

[000117] Diagnostic Agent: A diagnostic agent is an entity that is detectable when administered. In some embodiments, a diagnostic agent is administered conjugated to an antibody moiety, i.e., antibody or antibody fragment or subfragment, and is useful for diagnosing or detecting a disease by locating cells containing the antigen. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (e.g., biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, and enhancing agents (e.g., paramagnetic ions) for Magnetic Resonance imaging. Imaging - MRI). United States Patent No. 6,331,175 describes the MRI technique and the preparation of antibodies conjugated to an enhancing agent for MRI and is incorporated in its entirety by reference. Preferably, the diagnostic agents are selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. In some embodiments, a diagnostic agent may comprise a radioactive or non-radioactive tracer, a contrast agent (such as for MRI, CT, or ultrasound), and the radioactive tracer may be a gamma-, beta-, alpha-, Auger isotope. electron or positron emitter. In order to load an antibody component with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent that has a long tail to which is attached a plurality of chelating groups for binding the ions. Such tail may be a polymer, such as polylysine, polysaccharide or other derivatized or derivatizable chain having pendant groups to which chelating groups may be attached such as, for example, ethylenediaminetetraacetic acid (Ethylene Diamine Tetra Acetic - EDTA), diethylenetriaminepentaacetic acid (Diethylene Triamine Penta Acetic - DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes and similar groups known to be useful for this purpose. Chelates can be coupled to antibodies using standard chemistry. The chelate is typically linked to the antibody through a group that allows the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal internal aggregation and/or cross-linking. Other less common methods and reagents for conjugating chelates to antibodies are described in United States Patent No. 4,824,659 to Hawthorne, entitled "Antibody Conjugates", filed April 25, 1989, the disclosure of which is incorporated herein by reference in its entirety. Particularly useful metal chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs used as diagnostic isotopes for radioimaging. The same chelates, when in complex with non-radioactive metals, such as manganese, iron and gadolinium, are useful for MRI when used together with the antibodies of the invention. Macrocyclic chelates, such as NOTA, DOTA and TETA, are of use with a variety of metals and radiometals, more particularly, with radionuclides of gallium, yttrium and copper, respectively. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates, such as macrocyclic polyethers, which are of interest for stable binding nuclides, such as 223Ra for RAIT, are encompassed by the invention.

[000118] Immunoconjugate: An "immunoconjugate" is a conjugate (e.g., a covalent bond) of an antibody component with a payload (e.g., diagnostic or therapeutic agent).

[000119] Immunomodulator: An "immunomodulator" is an agent that, when present, typically stimulates cells of the immune system to proliferate or become activated in an immune response cascade, such as macrophages, B cells and/or T cells. An example of an immunomodulator, as described here, is a cytokine. As those skilled in the art will understand, certain interleukins and interferons are examples of cytokines that stimulate the proliferation of T cells or other immune cells.

[000120] Expression Vector: An "expression vector" is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operatively linked" to regulatory elements.

[000121] Host Cell: A recombinant "host cell" can be any prokaryotic or eukaryotic cell that contains a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells, as well as transgenic animals, which have been genetically manipulated to contain the cloned gene(s) into the chromosome or genome of the host cell or host cell cells.

[000122] Mutant: As used herein, the term "mutant" refers to an entity that shows significant structural identity with a reference entity, but differs structurally from the reference entity in the presence or level of one or more chemical moieties compared with the reference entity. In many embodiments, a mutant also differs functionally from its reference entity. Generally speaking, whether a given entity is properly considered to be a "mutant" of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any reference chemical or biological entity has certain characteristic structural elements. A mutant, by definition, is a distinct chemical entity that shares one or more of such characteristic structural elements. To give some examples, a small molecule may have a characteristic central structural element (e.g., a macrocyclic core) and/or one or more characteristic pendant moieties such that a mutant of the small molecule is one that divides the central structural element and the characteristic pendant portions, but differs in the other pendant portions and/or types of bonds present (single versus double, E versus Z, etc.) within the core, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to each other in linear or three-dimensional space and/or contribute to a particular biological function, a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to each other in space linear or three-dimensional. For example, a mutant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently. linked to the polypeptide backbone. In some embodiments, a mutant polypeptide shows an overall sequence identity to a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99%. Alternatively or additionally, in some embodiments, a mutant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a mutant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a mutant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a mutant polypeptide shows a reduced level of one or more biological activities compared to the reference polypeptide.

[000123] Multispecific: A "multispecific" antibody is an antibody that can simultaneously bind to at least two targets that are of different structure, for example, two different antigens, two different epitopes on the same antigen, or a hapten and an antigen or epitope . A specificity would be, for example, for a B cell, T cell, plasma or mast cell antigen or epitope. Another specificity could be for a different antigen on the same cell type, such as CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74 or CD22 on B cells. Multivalent, multispecific antibodies are constructs that have more than one binding site and binding sites are of different specificity. For example, a bispecific diabody, where one binding site reacts with one antigen and another with another antigen.

[000124] Bispecific: A "bispecific" antibody is an antibody that can simultaneously bind to two targets that are of different structure. Bispecific antibodies (BsAb) and bispecific antibody fragments (bsFab) have at least one arm that specifically binds, for example, GD2 and at least one other arm that specifically binds an objectifiable conjugate that carries a therapeutic agent or diagnosis. A variety of bispecific fusion proteins can be produced using molecular engineering techniques. In one form, the bispecific fusion protein is divalent consisting, for example, of an scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In another form, the bispecific fusion protein is tetravalent consisting, for example, of an IgG with two binding sites for one antigen and two identical scFvs for a second antigen.

[000125] Recent methods for producing bispecific MoAbs include engineered recombinant MoAbs that have additional cysteine residues so that they cross-bind more strongly than the more common immunoglobulin isotypes. See, for example, FitzGerald et al., Protein Eng. 10 (10): 1221-1225, 1997. Another approach is to engineer recombinant fusion proteins by linking two or more different antibody segments with a single chain or fragments of antibody with the required dual specificities. See, for example, Coloma et al., Nature Biotech. 15:159163, 1997. A variety of bispecific fusion proteins can be produced using molecular engineering techniques.

[000126] Bispecific fusion proteins by linking two or more different antibodies with a single chain or antibody fragments are produced in a similar way. Recombinant methods can be used to produce a variety of fusion proteins. In some embodiments, a flexible linker attaches the scFv to the heavy chain constant region of the 3F8 antibody. Alternatively, the scFv can be linked to the light chain constant region of another humanized antibody. Appropriate linker sequences required for in-frame binding of heavy chain Fd to scFv are introduced into the VL and Vkappa domains via PCR reactions. The DNA fragment encoding the scFv is then ligated into an assay vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and ligated into a vector containing a DNA sequence encoding the VH region of a hu3F8 antibody. The resulting vector can be used to transfect an appropriate host cell, such as a mammalian cell, for expression of the bispecific fusion protein.

[000127] In some embodiments, the hu3F8 antibodies and fragments thereof of the present invention can also be used to prepare functional single-chain bispecific antibodies (bscAb), also called diabodies, and can be produced in mammalian cells using recombinant methods. See, for example, Mack et al., Proc. Natl. Academic. Sci., 92: 7021-7025, 1995, incorporated herein by reference. For example, bscAb are produced by joining two single-chain Fv fragments via a glycine-serine linker using recombinant methods. The light chain V and heavy chain V domains of two antibodies of interest are isolated using conventional PCR methods known in the art. Single-chain bispecific antibodies and bispecific fusion proteins are included within the scope of the present invention.

[000128] In some embodiments, the ultimate use of the bispecific diabodies described here is for pre-targeting subsequent GD2-positive cells for specific delivery of diagnostic/detective or therapeutic agents. These diabodies selectively bind to target antigens, allowing increased affinity and longer residence time at the desired site. Furthermore, non-antigen-bound diabodies are eliminated from the body quickly and exposure of normal tissues is minimized. In certain particular embodiments, diabodies for use in the present invention may comprise or be conjugated with one or more diagnostic/detective and/or therapeutic agents such as, for example, isotopes, drugs, toxins, cytokines, hormones, growth factors. , conjugates, radionuclides and metals. For example, the metal gadolinium is used for magnetic resonance imaging (MRI). Radionuclides are also available as diagnostic and therapeutic agents (whether with diabodies or not), especially those in the energy range of 60 to 4000 keV.

[000129] The construct capable of objectification can be of varied structure, but is selected not only to avoid induction of an immune response, but also for rapid elimination in vivo when used within the scope of the bsAb objectification method. Hydrophobic agents are best at inducing strong immune responses, while hydrophilic agents are preferred for rapid in vivo elimination; thus, a balance between hydrophobic and hydrophilic needs is established. This is achieved, in part, based on the use of hydrophilic chelating agents to compensate for the inherent hydrophobicity of many organic moieties. Furthermore, subunits of the objectifiable construct can be chosen which have opposite solution properties, for example, peptides, which contain amino acids, some of which are hydrophobic and some of which are hydrophilic. In addition to peptides, carbohydrates can be used.

[000130] Peptide: Peptides that have as little as two amino acid residues can be used, preferably two to ten residues, in some embodiments, also coupled to other moieties, such as chelating agents.

[000131] Polypeptide: The term "polypeptide" is used here as a generic term to refer to the native protein, fragments or analogues of a polypeptide sequence. Consequently, native protein fragments and analogues are species of the polypeptide genus. The polypeptides according to the invention comprise the immunoglobulin heavy chain molecules represented in SEQ ID NOS: 4, 5, 10 and the light chain immunoglobulin molecules represented in SEQ ID NOS: 1, 2, 3, 6, 7, 8, 9, as well as antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules with immunoglobulin light chain molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogues.

[000132] Linker: In some embodiments, a "linker" used in a conjugate should have a low molecular weight compared to the conjugate, preferably having a molecular weight of less than 50,000 daltons and, advantageously, less than about 20,000 daltons , 10,000 daltons or 5,000 daltons, including metal ions that may be in chelates. In some embodiments, the presence of hydrophilic chelate moieties in the linker moieties can help ensure rapid elimination in vivo. In addition to hydrophilicity, chelating agents can be selected for their metal-binding properties and can be altered at will since, at least for those ligands whose BsAb epitope is part of the peptide or is a non-chelate chemical hapten, the Recognition of the metal-chelate complex is no longer a problem.

[000133] Mutant: As used herein, the term "mutant" refers to an entity that shows significant structural identity with a reference entity, but differs structurally from the reference entity in the presence or level of one or more chemical units compared to that of reference entity. In many embodiments, a mutant also differs functionally from its reference entity. Generally speaking, whether a given entity is properly considered to be a "mutant" of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any reference chemical or biological entity has certain characteristic structural elements. A mutant, by definition, is a distinct chemical entity that shares one or more of such characteristic structural elements. To provide some examples, a small molecule may have a characteristic central structural element (e.g., a macrocyclic core) and/or one or more characteristic pendant moieties such that a mutant of the small molecule is one that divides the central structural element and the characteristic pendant portions, but differs in the other pendant portions and/or types of bonds present (single versus double, E versus Z, etc.) within the core, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to each other in linear or three-dimensional space and/or contribute to a particular biological function, a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to each other in space linear or three-dimensional. For example, a mutant polypeptide may differ from a reference polypeptide as a result of one or more differences in the amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently linked to the polypeptide backbone. In some embodiments, a mutant polypeptide shows an overall sequence identity to a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99%. Alternatively or additionally, in some embodiments, a mutant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a mutant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a mutant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a mutant polypeptide shows a reduced level of one or more biological activities compared to the reference polypeptide.

[000134] Chelating Agent: A chelating agent, such as DTPA, DOTA, TETA or NOTA, can be used in any of a variety of circumstances, including in conjugates. The same chelating agents, when in complex with non-radioactive metals, such as Mn, Fe and Gd, can be used for MRI when used together with the bsAb of the invention. Macrocyclic chelating agents such as NOTA (1,4,7-triaza-cyclononane-N,N',N''-triacetic acid), DOTA and TETA (p-bromoacetamido-benzyl tetraethylaminetetraacetic acid) are of use with a variety of metals and radiometals, most particularly Ga, Y and Cu radionuclides, respectively.

[000135] Conjugate: In some embodiments, antibody agents are used in conjugates. In some particular embodiments, a chelating agent, such as DTPA, DOTA, TETA or NOTA, or a suitable peptide, to which a detectable label, such as a fluorescent molecule or cytotoxic agent, such as a heavy metal or radionuclide, can be attached. conjugated. For example, a therapeutically useful immunoconjugate can be obtained by conjugating a photoactive agent or dye to an antibody fusion protein. Fluorescent compositions, such as fluorochromes, and other chromogens or dyes, such as visible light-sensitive porphyrins, have been used to detect and treat lesions by directing appropriate light to the lesion. In therapy, this has been called Photoradiation, phototherapy or photodynamic therapy (Jori et al. (eds), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain 22: 430 (1986)) . Furthermore, monoclonal antibodies have been coupled with photoactivated dyes to obtain phototherapy. Mew et al., J. Immunol. 130: 1473 (1983); idem., Cancer Res. 45: 4380 (1985); Oseroff et al., Proc. Natl. Academic. Sci. USA 83: 8744 (1986); ditto., Photochem. Photobiol. 46: 83 (1987); Hasan et al., Prog. Clinic. Biol. Res. 288: 471 (1989); Tatsuta et al., Lasers Surg. Med. 9:422 (1989); Pelegrin et al., Cancer 67: 2529 (1991). However, these previous studies did not include the use of endoscopic therapy applications, especially with the use of antibody fragments or sub-fragments. Thus, the present invention considers the therapeutic use of immunoconjugates comprising photoactive agents or dyes.

[000136] Prevention: As used herein, the terms "prevent" and "prevention" refer to the prevention of the recurrence or appearance of one or more symptoms of a disease in an individual as a result of the administration of a prophylactic agent or therapeutic agent.

[000137] Combination: As used here, the term "in combination" refers to the use of one or more prophylactic and/or therapeutic agents. The use of the term "in combination" does not restrict the order in which prophylactic and/or therapeutic agents are administered to an individual with a disorder. A first prophylactic or therapeutic agent may be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours , 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes , 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks later) administration of a second prophylactic or therapeutic agent to an individual with a disorder.

[000138] Effector Function: "Effector function", as used herein, means a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or a ligand. Effector functions include, but are not limited to, Antibody-Dependent Cell Cytotoxicity, Antibody Dependent Cell Mediated Phagocytosis (ADCP), and Complement Mediated Cytotoxicity (CMC). ). Effector functions include both those that act upon antigen binding and those that act independently of antigen binding.

[000139] Effector Cell: "Effector cell", as used herein, means a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, natural killer cells, T lymphocytes, B lymphocytes and may be from any organism including, but not limited to, humans, mice, rats, rabbits and monkeys.

[000140] Fc Ligand: "Fc Ligand", as used herein, means a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc-ligand complex. Fc ligands include, but are not limited to, FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16A), FcyRIIIB (CD16B), FcyRI (CD64), FcεRII (CD23), FcRn, C1q, C3, staphylococcal protein A, protein Streptococcal G and viral FcyR. Fc ligands may include unknown molecules that bind to Fc.

[000141] Derivative: As used herein, the term "derivative", in the context of polypeptides or proteins, refers to a polypeptide or protein comprising an amino acid sequence that has been altered by introducing substitutions, deletions or additions of residues of amino acids. The term "derivative", as used herein, also refers to a polypeptide or protein that has been modified, that is, through the covalent attachment of any type of molecule to the polypeptide or protein. For example, but without limitation, an antibody may be modified, for example, through glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein. , etc.. A polypeptide or protein derivative can be produced through chemical modifications using methods known to those skilled in the art including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.. Furthermore, a polypeptide derivative or protein derivative has a similar or identical function to that of the polypeptide or protein from which it was derived.

[000142] Fragment: As used herein, the term "fragment" refers to a peptide or polypeptide that comprises an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 amino acid residues contiguous, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous At least 175 amino acid residues contiguous to 200 amino acid residues or at least 250 amino acid residues contiguous to the amino acid sequence of another polypeptide. In a specific embodiment, a fragment of a polypeptide retains at least one function of the polypeptide.

[000143] Effective Amount: As used herein, the term "effective amount" refers to an amount of a given compound, conjugate or composition that is necessary or sufficient to obtain a desired biological effect. An effective amount of a given compound, conjugate or composition in accordance with the methods of the present invention would be the amount that achieves this selected result and such amount can be determined as a matter of routine by those skilled in the art using assays that are known in the art. and/or which are described here without the need for undue experimentation. For example, an effective amount to treat or prevent cancer metastasis could be the amount necessary to prevent migration and invasion of a tumor cell across the basement membrane or across an endothelial layer in vivo. The term is also synonymous with "sufficient quantity". The effective amount for any particular application may vary depending on factors such as the disease, disorder or condition being treated, the particular composition to be administered, the route of administration, the size of the individual and/or the severity of the disease or condition. Those skilled in the art can empirically determine the effective amount of a particular compound, conjugate or composition of the present invention in accordance with the guidance provided herein without the need for excessive experimentation.

[000144] About: As used herein in relation to a given measured quantity, the term "about" refers to the normal variation in this measured quantity that would be expected by the specialist making the measurement and exercising a level of care commensurate with the measurement objective and the accuracy of the measuring equipment used. Unless otherwise noted, "about" refers to a variation of +/- 10% of the given value.

[000145] Isolated: By an "isolated" polypeptide or a fragment, variant or derivative thereof is meant a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide that can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of the invention, as are native or recombinant polypeptides that have been separated, fractionated or partially or substantially purified by any suitable technique.

[000146] Small Molecule: In general, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, the small molecule is less than about 4 kD, about 3 kD, about 2 kDa, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol . In some embodiments, the small molecules are non-polymeric. In some embodiments, according to the present invention, the small molecules are not proteins, polypeptides, oligopeptides, polynucleotides, peptides, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, etc.

[000147] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting full or nearly full extent or degree of a characteristic or property of interest. Those versed in biological techniques will understand that biological and chemical phenomena will rarely, if ever, terminate and/or proceed to completion or achieve or avoid an absolute result. The term "substantially" is therefore used here to capture the potential absence of integrity inherent in many biological and chemical phenomena.

[000148] Therapeutically Effective Amount: As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic protein that confers a therapeutic effect on the treated individual, with a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some assay or marker) or subjective (i.e., the individual provides an indication or feels an effect). In particular, "therapeutically effective amount" refers to an amount of a protein or composition effective to treat, ameliorate, or prevent a desired disease or condition or to exhibit a detectable therapeutic or preventive effect, such as by improving symptoms associated with the disease, preventing or delay the onset of the disease and/or also decrease the severity or frequency of disease symptoms. A therapeutically effective amount is typically administered in a dosage regimen that may comprise several unit doses. For any particular therapeutic protein, a therapeutically effective amount (and/or an appropriate dosage unit within an effective dosage regimen) may vary, for example, depending on the route of administration, in combination with other pharmaceutical agents. Furthermore, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition used; the patient's age, body weight, general health, sex, and diet; the timing of administration, route of administration and/or rate of excretion or metabolism of the specific fusion protein employed; the duration of treatment; and similar factors, as is well known in the medical art.

[000149] Treatment: As used herein, the terms "treatment", "treat" or "treated" refer to prophylaxis and/or therapy, in particular where the objective is to prevent or delay (lessen) an unwanted physiological change or disorder , such as the progression of multiple sclerosis. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction in extent of disease, stabilization (i.e., no worsening) of disease state, delay or delay of disease progression, improvement or palliation of disease state, and remission ( whether partial or total), whether detectable or not detectable. "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment. Those in need of treatment include those who already have the disorder or condition, as well as those with a propensity to have the disorder or condition or those in whom the condition or disorder must be avoided. By "individual" or "subject" or "animal" or "patient" or "mammal" is meant any individual, particularly a mammalian individual, for whom diagnosis, prognosis or therapy is desired. Mammalian individuals include humans and other primates, domestic animals, farm animals and zoo animals, sporting animals or companion animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows and so on.

[000150] Unit Dose: The expression "unit dose", as used herein, refers to an amount administered as a single dose and/or in a physically discrete unit of a pharmaceutical composition. In many embodiments, a unit dose contains a predetermined amount of an active agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some embodiments, more than one unit dose is administered to achieve a total single dose. In some embodiments, it is necessary, or expected to be necessary, to administer multiple unit doses to achieve a desired effect. A unit dose may be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained-release formulation or drug delivery device that contains a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a dosage unit may be present in a formulation that includes any of a variety of components in addition to the agent(s). therapeutic(s). For example, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc., may be included as described below. It will be appreciated by those skilled in the art, in many embodiments, that a total adequate daily dose of a particular therapeutic agent may comprise a portion or a plurality of unit doses, and may be decided, for example, by the treating physician within the scope of medical judgment. In some embodiments, the specific effective dose level for any particular patient or organism may depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active compound used; specific composition used; the individual's age, body weight, general health, sex, and diet; the timing of administration and excretion rate of the specific active compound used; duration of treatment; additional drugs and/or therapies used in combination or coincident with the specific compound(s) used and similar factors well known in the medical art.

Neuroblastoma

[000151] Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In ~50% of cases, curative strategies must address both soft tissue masses and bone marrow metastases (Bone Marrow - BM). Dose-intensive chemotherapy improves the possibility of tumor resection and postsurgical irradiation reduces the risk of recurrence at the primary site to <10% (Kushner et al., 2001, J Clin Oncol 19, 2821-8). However, disease in BM, as evidenced by histology or metaiodobenzylguanidine (MIBG) scan, often persists and is headed toward a lethal outcome (Matthay et al., 2003, J Clin Oncol 21, 2486-91; Schmidt et al., 2008, Eur J Cancer 44, 1552-8). Furthermore, osteomedullary relapse is common, despite achieving almost complete remission after induction therapy. Attempts to intensify treatment have resulted in both acute and long-term side effects, both of great concern for young patients. There is a paucity of promising new agents, and to date few, if any, target/pathway-specific small molecules have demonstrated major clinical benefit in patients with NB, although many promising leads continue to accumulate. With a cure rate of <30% at toxicity thresholds among stage 4 patients diagnosed at >18 months of age, there is plenty of room for improvement (Pearson et al., 2008, Lancet Oncol 9, 247-56).

[000152] The present invention comprises the recognition that several factors make NB well suited for MoAb-targeted immunotherapy. First, the MoAb highly efficiently mediates the Antibody-Dependent Cell Cytotoxicity of NB in the presence of human white blood cells. Second, the MoAb induces Complement Mediated Cytotoxicity (CMC) of NB cells, which lack the decline-accelerating factor CD55 (Cheung et al., 1988, J Clin Invest 81, 1122-8) and homologous restriction factor CD59 (Chen et al., 2000, Cancer Res 60, 3013-8). Complement deposition in NB cells increases ADCC through activation of the iC3b receptor on neutrophils (Kushner and Cheung, 1992, Blood 79, 1484-90, Metelitsa et al., 2002, Blood 99, 4166-73), available even after dose-intensive treatment or myeloablative chemotherapy plus stem cell transplantation, if colony-stimulating factors are provided (Mackall, CL, 2000, Stem Cells 18, 10-8). Third, the use of intensive chemotherapy (standard of care for NB) to achieve clinical remission causes lymphopenia and prolonged immunosuppression (Mackall et al., 2000, Blood 96, 754-762), so that patients are less likely to reject Murine, chimeric or humanized MoAbs (Kushner et al., 2007, Pediatr Blood Cancer 48, 430-4).

Reference Anti-GD2 Antibodies

[000153] GD2 is a di-sialoganglioside abundant in tumors of neuroectodermal origin, including neuroblastoma and melonoma, with highly restricted expression in normal tissues. At least two anti-GD2 antibody families have been clinically tested for the treatment of NB, i.e., 3F8 (Cheung et al., 1985, Cancer Res 45, 2642-9) and 14.18 (Mujoo et al., 1989, Cancer Res 49, 2857-61).

[000154] chimeric ch14.18 consists of the variable region of murine MoAb 14.18 and the constant regions of human IgG1-K (Gillies et al., 1989, J Immunol Methods 125, 191-202). It demonstrates ADCC and CMC of NB and melanoma cells in vivo (Barker et al., 1991, Cancer Res 51, 144-9; Barker and Reisfeld, 1993, Cancer Res 52, 362-7; Mueller et al., 1990, PNAS USA 87, 5702-05). Based on encouraging clinical responses in phase I studies, ch14.18 was tested in large phase II studies as consolidation therapy for stage 4 NB (German studies NB90 and NB97). For the 166 patients > 12 months at diagnosis, even though event-free survival (EFS) was similar in patients receiving ch14.18 when compared with patients receiving maintenance chemotherapy, overall survival (Overall Survival - OS) was improved and the BM relapse rate reduced in patients treated with ch14.18 (Simon et al., 2004, J Clin Oncol 22, 3549-57).

[000155] In 2001, the Children's Oncology Group (COG) initiated a randomized phase III trial to study the efficacy of combining ch14.18 with GMCSF and IL-2 in preventing NB relapse in patients in complete remission. - CR) after autologous stem cell transplantation (Autologous Stem-Cell Transplantation - ASCT) (ClinicalTrials.gov NCT00026312) (Gilman et al., J Clin Oncol. 27: 85-91, 2009), where a significant improvement in survival no progression (Progression Free Survival - PFS) and OS at 2 years was found (Yu et al., N Engl J Med 363: 1324-1334, 2010).

[000156] 3F8, a murine IgG3 MoAb specific for GD2, induces cell death and mediates efficient ADCC and CMC against NB in vitro (Cheung et al., 2007, supra). Among patients with chemotherapy-resistant marrow disease despite dose-intensive induction plus an aggressive rescue regimen, 80% achieved overall BM remission after 1 to 2 5-day cycles of antibody plus GM-CSF therapy. (Kushner et al., 2007, Proc Amer Soc Clin Oncol 25, 526s). Given the activity of m3F8 against chemotherapy-resistant marrow disease, the use of m3F8 has been expanded to patients in their first remission with encouraging results. These favorable clinical outcomes in children may be improved if m3F8 is provided as maintenance therapy during the first 3-5 years of highest recurrence risk. However, the Human Anti-Mouse Antibody (HAMA) response is a limiting factor when the immune system recovers when chemotherapy is finished. One strategy to reduce HAMA is to chimerize or humanize 3F8.

[000157] We previously described the engineering and isolation of humanized 3F8 (hu3F8-IgG1 H1L1, hereinafter hu3F8V1) (described in WO 2011/160119 and Cheung et al., 2012, Oncoimmunology 1: 477-486, both incorporated herein in full by reference). These antibodies were made using conventional recombinant methods and selected for high expression by CHO-DG44 cell lines in serum-free medium. Measured using surface plasmon resonance used by Biacore systems, the humanized 3F8 maintained a similar KD to that of the m3F8. In contrast to other anti-GD2 antibodies, hu3F8V1 had a substantially slower Koff, which translated into slower clearance in vitro. Like m3F8, humanized 3F8 inhibited cell growth in vitro, not typical for other anti-GD2 antibodies. Both Peripheral Blood Mononuclear Cells (PBMCs)-ADCC and neutrophil (PMN)-ADCC assays of hu3F8V1 were superior (10 to > 1000-fold) to those of m3F8, although the CMC was lower. This superiority was consistently observed in ADCC assays regardless of donors or whether NK92 cells transfected with human CD16 or CD32 were used as killers. Hu3F8V1 showed a superior antitumor effect against NB xenotransplantation when compared with m3F8.

Provided Antibody Agents

[000158] The present invention comprises the recognition that, to improve therapeutic efficiency, new humanized forms of the antibody with increased affinity are necessary. The present invention encompasses the recognition that it would be desirable to develop antibodies (or other antibody agents) that are variants of 3F8 and/or hu3F8V1. The present invention provides, in particular, such antibodies and antibody agents. That is, the present invention provides several antibody agents that show significant structural identity with 3F8 and/or hu3F8V1 and, in addition, show improved functional characteristics (e.g., stabilization and/or affinity or specificity) compared to those observed with 3F8 and /or hu3F8V1. In a specific preferred embodiment, the invention encompasses a molecule comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification with respect to a wild-type Fc region, such that said molecule has an affinity -nity altered by an FcyR, provided that said variant Fc region does not have a substitution at positions that make a direct contact with FcyR based on structural crystallographic analysis and Fc-FcyR interactions, such as those described by Sondermann et al. , 2000 (Nature, 406: 267-273, which is incorporated herein by reference in full). Examples of positions within the Fc region that make direct contact with FcyR are amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C'/E loop), and amino acids 327-332 (M/L loop). In some embodiments, the molecules of the invention comprise variant Fc regions that comprise modification of at least one residue that makes a direct contact with an FcyR based on structural and crystallographic analysis.

[000159] One aspect of the invention includes hu3F8 antibodies with altered affinities for activating receptors and/or inhibitors having variant Fc regions with one or more amino acid modifications, wherein said one or more amino acid modifications are a substitution at position 239 for aspartic acid, at position 330 by leucine and at position 332 by glutamic acid.

[000160] The invention encompasses molecules that comprise a variant Fc region with additions, deletions and/or substitutions of one or more amino acids in the Fc region of an antibody of the present invention in relation to a reference antibody (e.g., Fc region of a reference antibody 3F8), for example, in order to alter the effector function or increase or decrease the Fc affinity delivered to the FcR. It is within the ability of those skilled in the art, given the guidance provided herein, how to prepare and use such variant Fc regions. Therefore, the invention encompasses molecules comprising variant Fc regions that bind with a greater affinity to one or more FcyRs. Such molecules preferably mediate the effector function more effectively, as discussed below.

[000161] In some embodiments, the invention encompasses molecules that comprise a variant Fc region that binds with a weaker affinity to one or more than one FcyRs reference antibody (e.g., a reference 3F8 antibody) Fc region. The reduction or elimination of effector function is desirable in certain cases, for example, in the case of antibodies whose mechanism of action involves blocking or antagonism, but not killing, cells carrying a target antigen. The reduction or elimination of effector function would be desirable in cases of autoimmune disease where blocking FcyR would activate receptors on effector cells (This type of function would be present on host cells). In general, the increase in effector function would be directed towards tumor and foreign cells.

[000162] In certain embodiments, the Fc variants of the present invention can be combined with other Fc modifications, including, but not limited to, modifications that alter the effector function. The invention encompasses combining an Fc variant of the invention with other Fc modifications to provide additive, synergistic or novel antibody or Fc fusion properties. Preferably, the Fc variants of the invention improve the phenotype of the modification with which they are combined. For example, if an Fc variant of the invention is combined with a mutant known to bind FcyRIIIA with greater affinity than a comparable molecule comprising a wild-type Fc region; combination with a mutant of the invention results in a greater fold enhancement in FcyRIIIA affinity.

[000163] In some embodiments, the Fc variants of the present invention are incorporated into an antibody agent (e.g., an antibody or an Fc fusion) that comprises one or more modified glycoforms, i.e., a carbohydrate composition that is covalently linked to a molecule comprising an Fc region, wherein said carbohydrate composition differs chemically from a parent molecule comprising an Fc region.

[000164] The invention covers antibodies with modified glycosylation sites, preferably without altering the functionality of the antibody, for example, GD2 binding activity. As used herein, "glycosylation sites" include any specific amino acid sequence to which an antibody to an oligosaccharide (i.e., carbohydrates containing two or more simple sugars linked together) specifically and covalently binds. Oligosaccharide side chains are typically attached to the backbone of an antibody via either N-OR-O bond. N-glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxy amino acid, e.g., serine, threonine. An Fc glycoform, hu3F8-H1L1-IgG1n that lacked certain oligosaccharides including fucose and the N-terminal acetylglucosamine was produced in special CHO cells and showed enhanced ADCC effector function.

[000165] In some embodiments, the present invention encompasses methods for modifying the carbohydrate content of an antibody of the invention by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and fall within the scope of the invention, see, for example, United States Patent No. 6,218,149; document EP 0 359 096 B1; United States Publication No. 2002/0028486; document WO 03/035835; United States Publication No. 2003/0115614; United States Patent No. 6,218,149; United States Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety. In other embodiments, the present invention encompasses methods for modifying the carbohydrate content of an antibody of the invention by deleting one or more endogenous carbohydrate moieties of the antibody. In a specific embodiment, the invention encompasses the exclusion of the glycosylation site of the Fe region of an antibody, changing position 297 from alanine to asparagine.

[000166] Manipulated glycoforms can be useful for a variety of purposes, including, but not limited to, increasing or reducing effector function. Engineered glycoforms can be generated by any method known to one of skill in the art, for example, using modified strains or variant expression, by coexpression with one or more enzymes, for example, di-n-acetylglucosaminyltransferase III (GnTI11), by expression of a molecule comprising an Fc region in various organisms or cell lines of various organisms or by modification of carbohydrate(s) after the molecule comprising the Fc region has been expressed. Methods for generating modified glycoforms are known in the art and include, but are not limited to, those described in Umana et al., 1999, Nat. Biotechnol 17: 176-180; Davies et al., Biotechnol Bioeng 2001, 774: 288-294; Shields et al., 2002, J Biol Chem 277: 26733-26740; Shinkawa et al., 2003, J Biol Chem 278: 3466-3473) United States Patent No. 6,602,684; United States Publication No. 10/277,370; United States Publication No. 10/113,929; PCT document WO 00/61739A1; PCT document WO 01/292246A1; PCT document WO 02/311140A1; PCT document WO 02/30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton, NJ); the GLYCOMAB™ Glycosylation Engineering Technology (GLYCART Biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, for example, document WO 00061739; EA01229125; US 20030115614;. Okazaki et al., 2004, JMB, 336: 1239-1249, each of which is incorporated herein by reference in its entirety.

[000167] Also included as polypeptides of the present invention are derivatives, fragments, analogues or variants of the previous polypeptides, and any combination thereof. The terms "fragment", "variants", "derivative" and "analog" when referring to anti-GD2 antibodies or antibody polypeptides include any polypeptides that retain at least some of the antigen-binding properties of the corresponding native antibody or polypeptide, that is, those polypeptides that retain the ability to bind to one or more GD2 epitopes.

[000168] The polypeptide fragments of the present invention include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed herein elsewhere.

[000169] Variants of anti-GD2 antibodies and antibody polypeptides useful in accordance with the present invention include fragments, as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art or unnatural amino acids. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions.

[000170] Anti-GD2 antibody derivatives and antibody polypeptides useful in accordance with the present invention are polypeptides that have been altered to present additional characteristics not found in the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein, a "derivative" of an anti-GD2 antibody or polypeptide antibody refers to an object polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be replaced by proline; 5- hydroxylysine can be replaced by lysine; 3-methylhistidine can be replaced by histidine; homoserine can be replaced by serine; and ornithine can be replaced by lysine.

[000171] In some embodiments, the antibody agents provided show functional properties, as set forth in the Examples presented here. For example, in some embodiments, the agents provided show improved binding relative to an original 3F8 antibody and/or a humanized version thereof (e.g., hu3F8V1). Exemplary humanized versions include those that have one or more structural features as described herein. In some given embodiments, a structural feature includes one or more amino acid substitutions that correspond to a sequence appearing in a human framework region of an immunoglobulin variable region sequence. In some given embodiments, a structural feature includes one or more amino acid substitutions that correspond to a sequence appearing in a human CDR region of an immunoglobulin variable region sequence. In some determined embodiments, a structural feature includes one or more amino acid substitutions that reduce the immunogenicity of the provided agent relative to a parent antibody. In some given embodiments, a structural feature includes one or more amino acid substitutions, which reduces, enhances, or eliminates a T cell epitope of the provided agent relative to a parent antibody. In some embodiments, the agents provided have a structural characteristic that includes those set forth in Table 2.

[000172] In some embodiments, the antibody agents provided bind to GD2 with an affinity of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than the affinity of a different antibody that binds GD2. In some embodiments, the antibody agents provided bind GD2 with an affinity of at least 2-fold, at least 3-fold, at least four-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold. times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times or at least 20 times more than the affinity of a different antibody for GD2. In some embodiments, the antibody agents provided bind GD2 with an affinity greater than 20-fold, greater than 30-fold, greater than 40-fold, greater than 50-fold, greater than 60-fold, greater than 70-fold, greater than 80-fold, greater than 90-fold, or greater than 100-fold greater than that of a different antibody that binds GD2. In some embodiments, the antibody agents provided show binding affinities for different gangliosides such as, for example, GD1b, which are within 2, within 3, about 4, within 5, within 6, within 7 , within 8, to about 9 or about 10 times of each other's affinity.

[000173] In some embodiments, the antibody agents provided show a relative potency (e.g., ratio of EC50 of 3F8/EC50 of antibody or EC50 of hu3F8V1/EC50 of antibody) in an ADCC or CMC assay within a range as described and/or exemplified here. In some embodiments, the antibody agents provided show a relative potency of at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 10.5, at least 11.0, at least 11.5, at least 12, 0, at least 12.5, at least 13.0, at least 13.5, at least 14.0, at least 14.5, at least 15.0, at least 15.5, at least 16.0, at least 16.5, at least 17.0, at least 17.5, at least 18.0, at least 18.5, at least 19.0, at least 19.5, at least 20.0, at least 20.5, at least 21.0, at least 21.5, at least 22.0, at least 22.5, at least 23.0, at least 23.5, at least 24.0, at least 24, 5, at least 25.0, at least 25.5, at least 26.0, at least 26.5, at least 27.0, at least 27.5, at least 28.0, at least 28.5, at least 29.0, at least 29.5 or at least 30.0 when compared to a parental antibody that binds GD2.

[000174] In some embodiments, the antibody agents provided show binding to GD2 with a KD (nM) less than 100 nM, less than 90 nM, less than 80 M, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM or less than 10 nM. In some certain embodiments, the antibody agents provided show binding to GD2 with a KD (nM) less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM , less than 3 nM, less than 2 nM or less than 1 nM. In some certain embodiments, the antibody agents provided show binding to GD2 with a KD (nM) of about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM. , about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1.0 nM, about 1.1 nM, about 1, 2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0 nM, about 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM, about 2.8 nM, about 2.9 nM or about 3.0 nM. In some certain embodiments, the antibody agents provided show binding to GD2 with a KD (nM) of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, of about 6 nM, about 7 nM, about 8 nM or about 9 Nm.

[000175] In some embodiments, the antibody binding agents provided show GD2 with a Koff (S-1) whose lower limit is about 2.0x10-4 s-1 and upper limit is about 20.0x10- 4 s-1. In some embodiments, the provided antibody binding agents display GD2 with a Koff (S-1) whose lower limit is selected from the group consisting of 2x10-4 s-1, 3x10-4 s-1, 4x10-4 s -1, 5x10-4 s-1, 6x10-4 s-1, 7x10-4 s-1, 8x10-4 s-1, 9x10-4 s-1 or more, and whose upper limit is greater than the limit lower and is selected from the group consisting of 10x10-4 S-1, 11x10-4 S-1, 12x10-4 S-1, 13x10-4 S-1, 14x10-4 S-1, 15x10-4 S-1 , 16x10- 4 S-1, 17x10-4 S-1, 18x10-4 S-1, 19x10-4 S-1, 20x10-4 S-1 or more. In some determined embodiments, antibody agents provided show binding to GD2 with a Koff (S-1) of about 2.9x10-4 s-1, 5.1x10-4 s-1, 6.9x10-4 s-1 , 8.8x10-4 s-1 or 18.5x10-4 s-1.

Humanized Antibody Agents

[000176] In one embodiment, the antibodies provided by the present invention are monoclonal antibodies, which in a preferred embodiment are humanized versions of cognate anti-GD2 antibodies derived from other species. A humanized antibody is an antibody produced by recombinant DNA technology in which some or all of the amino acids of a human immunoglobulin light or heavy chain are not required for the framework regions of the antigen-binding variable domains (e.g., the constant regions e) are used to replace the corresponding amino acids of the light or heavy chain of the cognate, non-human antibody. By way of example, a humanized version of a murine antibody to a given antigen has in both its heavy and light chains (1) the constant regions of a human antibody; (2) framework regions of the variable domains of a human antibody; and (3) the murine CDR antibody. When necessary, one or more residues in the human framework regions can be changed to residues in corresponding positions in the murine antibody so as to preserve the binding affinity of the humanized antibody for the antigen. This change is sometimes called a "back mutation." Likewise, mutations can be made forward to return the murine sequence to a desired motif, for example, stability or affinity for the antigen. For example, for hu3F8-H1L1 (or hu3F8V1) back mutations were required at 19 positions in the heavy chain sequence and 17 positions in the light chain in order to maintain in vitro binding affinity. Humanized antibodies are generally less likely to provoke an immune response in humans compared to human chimeric antibodies because the former contains considerably fewer non-human components.

[000177] Suitable methods for preparing humanized antibodies of the present invention are described in, for example, EP 0 239 Winter 400; Jones et al, Nature 321:. 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988); Queen et al., Proc. Nat. Acad. Sci USA 86: 10029 (1989); United States Patent No. 6,180,370; and Orlandi et al., Proc. Natl. Academic. Sci. USA 86: 3833 (1989); the descriptions of all of which are incorporated herein by reference in their entirety. In general, transplantation of murine (or other non-human) cDNA to a human antibody is achieved as follows. cDNAs encoding the heavy and light chain variable domains are isolated from a hybridoma. The DNA sequences of the variable domains, including the CDRs, are determined by sequencing. The DNAs encoding the CDRs are inserted into the corresponding regions of a human heavy or light chain variable domain antibody coding sequence, linked to human constant region gene segments of a desired isotype (e.g., Y1 for CH and for CL), are synthesized genes. Humanized heavy and light chain genes are coexpressed in mammalian host cells (e.g., CHO or NSO cells) to produce soluble humanized antibody. To facilitate large-scale production of antibodies, it is often desirable to select for high expression using a DHFR gene or GS gene in the production line. These producer cell lines are grown in bioreactors or hollow fiber culture system or WAVE technology, to produce bulk cultures of soluble antibody or to produce transgenic animals (e.g., goats, cows or sheep) that express the antibody in the milk (see, for example, United States Patent No. 5,827,690).

[000178] Using the methods described above, humanized and chimeric versions of the 3F8 antibody were generated. The cDNAs encoding the murine 3F8 variable regions of the light and heavy chains were used to construct vectors for expression of murine-human chimeras in which the murine 3F8 variable regions were linked to human IgGl (for the heavy chain) and human cap (for light chain) constant regions as described previously. Furthermore, new forms of hu3F8 with the glycosylation variant were created in order to increase binding to the Fc receptor and improve antigen affinity.

[000179] In order to produce humanized 3F8 antibodies, the human acceptor structure domains were chosen by homology with the corresponding human germline sequences. Using these chosen human acceptor structures, light and heavy chain variable domains were designed and a series of variants/versions of each were generated and expressed, as described below in the Examples.

[000180] Fully human antibodies are particularly desirable for the therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art, including phage display methods described above, using libraries of antibodies derived from human immunoglobulin sequences. See also, United States Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/60433, WO 98/24893, WO 98/16664, WO 96/34096, WO 96/33735, WO 91/10741 and; each of which is incorporated herein by reference in its entirety. The techniques of Cole et al, and Boerder et al, are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); E Boerner et al., J Immunol, 147 (1): 86-95, (1991)).

[000181] Human antibodies produced using other techniques, but maintaining the variable regions of the anti-GD2 antibody of the present invention are part of this invention. Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous mouse immunoglobulins, but which can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The heavy and light chain mouse immunoglobulin genes can be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal way with a selected antigen, for example, all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from transgenic mice immunized using conventional hybridoma technology. The human immunoglobulin transgenes harbored by transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for the production of human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, for example, PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; Pat USA. Nos. 5,413,923.; 5,625,126; 5,633,425; 5,569,825; 5661016; 5,545,806; 5,814,318; 5,886,793; 5,916,771; and 5,939,598, which are incorporated herein by reference in their entirety. Additionally, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, NJ) can be docked to deliver human antibodies directed against a selected antigen using similar technology. to that described above.

[000182] Furthermore, human MoAbs can be made by immunizing mice transplanted with human peripheral blood leukocytes or bone marrow splenocytes (e.g., trioma XTL techniques). Fully human antibodies that recognize a selected epitope can be generated using a technique known as "targeted selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a fully human antibody recognizing the same epitope (Jespers et al., Bio/Technology 12: 899-903 (1988)). .

[000183] As used herein, an "anti-GD2 antibody", "anti-GD2 antibody portion" or "anti-GD2 antibody fragment" and/or "anti-GD2 antibody variant" and so on include any protein or peptide containing molecule comprising at least a portion of an immunoglobulin molecule, which contains at least one complementarity determining region (CDR) of a heavy or light chain or a corresponding ligand-binding portion derived from any of the antibodies monoclonals described herein, in combination with a heavy chain or light chain variable region, heavy chain or light chain constant region, a framework region or any part thereof, of non-murine origin, preferably of human origin, which may be incorporated into an antibody of the present invention. Alternatively, the term "anti-GD2 antibody" refers collectively or individually to hu3F8V1 IgG with a single mutation, e.g. hu3F8V1-E1K, hu3F8V1-D32H, hu3F8V1-G54I; hu3F8V1 IgG with a double mutation, for example hu3F8V1-E1KD32H, hu3F8V1-E1KG54I, and hu3F8V1-D32HG54I; hu3F8V1 IgG with a triple mutation, for example hu3F8V1-E1KD32HG54I; hu3F8V5 IgG; hu3F8V5 IgG with a single mutation, e.g. hu3F8V5-E1K, hu3F8V5-D32H, hu3F8V5-G54I; hu3F8V5 IgG with a double mutation, for example hu3F8V5-E1KD32H, hu3F8V5-E1KG54I, and hu3F8V5-D32HG54I; and hu3F8V5 IgG with a triple mutation, for example hu3F8V5-E1KD32HG54I antibodies, and combinations thereof, as well as fragments and variable regions thereof, such as single-chain fragments of the present invention, including hu3F8V1-E1K scFv, scFv hu3F8V1-D32H, hu3F8V1-G54I scFv; hu3F8V1 scFv with a double mutation, for example hu3F8V1-E1KD32H scFv, hu3F8V1-E1KG54I scFv, and hu3F8V1-D32HG54I scFv; scFv hu3F8V1 with a triple mutation, for example hu3F8V1- E1KD32HG54I scFv; ScFv hu3F8V5; ScFv hu3F8V5 with a single mutation, e.g. hu3F8V5-E1K scFv, scFv hu3F8V5-D32H, hu3F8V5-G54I scFv; hu3F8V5 scFv with a double mutation, for example hu3F8V5-E1KD32H scFv, hu3F8V5-E1KG54I scFv, and hu3F8V5-D32HG54I scFv; hu3F8V5 scFv with a triple mutation, for example hu3F8V5-E1KD32HG54I scFv, and combinations thereof. Such an antibody is capable of modulating, decreasing, antagonizing, mitigating, alleviating, blocking, inhibiting, abrogating and/or interfering with at least one cellular function in vitro, in situ and/or in vivo, wherein said cell expresses the GD2. As a non-limiting example, an appropriate anti-GD2 antibody, specified part or variant of the present invention can bind with high affinity to a human GD2 epitope.

[000184] The term "antibody" is further intended to encompass antibodies, digest fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or a portion thereof, including single-chain antibodies and fragments thereof, each containing at least one CDR derived from an anti-GD2 antibody. Functional fragments include antigen-binding fragments that bind to mammalian GD2. For example, antibody fragments capable of binding to GD2 or portions thereof, including but not limited to Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction), and F (ab')2 (e.g., by digestion with pepsin), FACB (e.g., by digestion with plasmin), pFc' (e.g., by digestion with pepsin or plasmin), Fd (e.g., by digestion of pepsin, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the invention (see, e.g., Colligan, Immunology, supra).

[000185] Antibody fragments can be produced by enzymatic cleavage, synthesis or recombinant techniques, as is known in the art and/or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more terminal codons have been introduced upstream of the natural termination site. For example, a gene encoding a combination of F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and/or the hinge region of the heavy chain. The various antibody portions can be chemically joined by conventional techniques or can be prepared as a contiguous protein using genetic engineering techniques.

[000186] As used herein, "chimeric" antibodies or "humanized" antibodies or include any combination of the described herein anti-GD2 Abs or any CDR derived, combined with one or more proteins or peptides derived from a non-"CDR-grafted" murine, preferably the human antibody. According to the invention, chimeric or humanized antibodies include those in which the CDR is derived from one or more of the anti-GD2 Abs described herein and at least a portion or the remainder of the antibody is derived from one or more human antibodies. . Thus, the human antibody part may include the framework, Cl, CH domains (e.g., CH1, CH2, CH3), hinge, (VL, VH)) regions that are substantially non-immunogenic in humans. Regions of the antibody that are derived from human antibodies need not have 100% identity with human antibodies. In a preferred embodiment, as many human amino acid residues as possible are retained so that immunogenicity is negligible, but the human residues can be modified as necessary to support the antigen binding site formed while maximizing the CDR. simultaneously humanizing the antibody. Such changes or variations and, optionally, preferably maintain or reduce immunogenicity in humans or other species relative to unmodified antibodies. It is noted that a humanized antibody can be produced by a non-human animal or a prokaryotic or eukaryotic cell that is capable of functionally expressing a rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Furthermore, when the antibody is a single-chain antibody, it may comprise a binding peptide that is not found in native human antibodies. For example, an Fv may comprise a linker peptide, such as two to about twenty glycines or other amino acids, preferably 8-15 glycine residues or other amino acid residues, that links the heavy chain variable region and the variable region of the light chain. Such binding peptides are considered to be of human origin.

[000187] Antibody humanization can be accomplished by, for example, the synthesis of a combinatorial library comprising the six CDRs of a non-human target monoclonal antibody fused in structure with a set of individual human structures. A human framework library that contains genes representative of all known human germline heavy and light chain genes can be used. The resulting combinatorial libraries can then be screened for binding to antigens of interest. This approach may allow selection of the most favorable combinations of fully human structures in terms of maintaining binding activity to the parental antibody. The humanized antibodies can then be further optimized by a variety of techniques.

[000188] Antibody humanization can be used to evolve mouse or other non-human antibodies into "fully human" antibodies. The resulting antibody contains only the human sequence and the mouse sequence or no non-human antibodies, while maintaining similar binding affinity and specificity as the priming antibody.

[000189] For full-length antibody molecules, immunoglobulin genes can be obtained from genomic DNA or mRNA from hybridoma cell lines. Heavy and light chains of the antibody are cloned into a mammalian vector system. This is documented with double chain sequence analysis. The antibody construct can be expressed in other human or mammalian host cell lines. The constructs can then be validated by transient transfection assays and Western blot analysis of the expressed antibody of interest. Stable cell lines with the highest productivity can be isolated and screened using rapid assay methods.

[000190] At least one anti-GD2 antibody of the present invention can be produced, optionally, by a cell line, a mixed cell line, an immortalized cell or a clonal population of immortalized cells, as well known in the art. See, for example, Ausubel et al., Ed, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, New York (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, New York (1989); Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor, New York (1989), Colligan et al., eds, Current Protocols in Immunology, John Wiley & Sons, Inc., New York (19942001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, New York, NY, (1997-2001), each incorporated herein in its entirety by reference.

[000191] In one approach, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line, such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO , NS1, NS2, AE -1, L.5, > 243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A) or similar or heteromyelomas, fusion products thereof or any cell or cell fusion derived therefrom or any other suitable cell line, as known in the art. See, for example, www.atcc.org, www.lifetech.com, and so on, with antibody-producing cells, such as, but not limited to, isolated or cloned from the spleen, peripheral blood, lymph, tonsils or other immune or B cells containing cells or any other cells expressing the constant or variable heavy or light chain or framework sequences or CDR, whether as endogenous or heterologous nucleic acid, such as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptile, fish, mammal, rodent, equine, sheep, goat, ovine, primate DNA, eukaryotic, genomic, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and similar or any combination thereof. See, for example, Ausubel, supra, and Colligan, Immunology, supra, chapter 2, incorporated herein in its entirety by reference.

[000192] Any other suitable host cell can also be used for expression of heterologous or endogenous nucleic acid encoding an antibody or specified fragment thereof, of the present invention. Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods and cloned by limiting dilution or cell separation or other known methods. Cells that produce antibodies with the desired specificity can be selected by a suitable assay (e.g. ELISA).

[000193] The antibodies of the present invention can also be prepared using at least one anti-GD2 nucleic acid encoding antibody to provide transgenic animals or mammals, such as goats, cows, horses, sheep and so on, which produce such antibodies in the your milk. Such animals can be provided using known methods. See, for example, but without limitation, United States Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5994616, 5565362; 5,304,489, and so on, each of which is incorporated herein in its entirety by reference.

[000194] The antibodies of the present invention can additionally be prepared using at least one anti-GD2 nucleic acid encoding antibody to provide transgenic plants and plant cells in culture (e.g., but not limited to tobacco and corn) that produce these antibodies, specified portions or variants in parts of the plants or in cultured cells thereof. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large quantities of recombinant proteins, for example, using an inducible promoter. See, for example, Cramer et al., Curr. Top. Microbol. Immunol. 240: 95-118 (1999) and references cited therein. Furthermore, transgenic corn has been used to express mammalian proteins at commercial production levels, with biological activities equivalent to those produced in other recombinant systems or purified from natural sources. See, for example, Hood et al., Adv. Exp. Med. Biol. 464: 127-147 (1999) and references cited therein. Antibodies have also been produced in large quantities from transgenic plant seeds, including antibody fragments such as single-chain antibodies (scFv), including tobacco seeds and potato tubers. See, for example, Conrad et al., Plant Mol. Biol. 38: 101-109 (1998) and references cited therein. Thus, the antibodies of the present invention can also be produced using transgenic plants, according to known methods. See also, for example, Fischer et al., Biotechnol. Appl. Biochem. 30: 99-108 (October, 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., Plant Physiol. 109: 341-6 (1995); Whitelam et al., Biochem Soc. Trans. 22: 940-944 (1994); and the references cited there. Each of the above references is incorporated herein in its entirety by reference.

[000195] An anti-GD2 antibody can be recovered and purified from recombinant cell cultures by well-known methods, including, but not limited to, protein A purification, protein G purification, ammonium sulfate or ethanol precipitation, extraction acid, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. High-performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in Immunology or current protocols in Science Protein, John Wiley & Sons, New York, NY, (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, each incorporated herein in its entirety by reference.

[000196] The antibodies of the present invention include naturally purified products, products of chemical synthetic procedures and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plants, insects and mammalian cells. Depending on the host employed in a recombinant production procedure, the antibody of the present invention may be glycosylated or may be non-glycosylated, with glycosylated preferred.

[000197] The antibodies of the present invention include naturally purified products, products of chemical synthetic procedures and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plants, insects and mammalian cells. Depending on the host employed in a recombinant production procedure, the antibody of the present invention may be glycosylated or may be non-glycosylated, with glycosylated preferred. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, in sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14, all herein incorporated in their entirety by reference.

[000198] Purified antibodies can be characterized, for example, ELISA, ELISPOT, flow cytometry, immunocytology, BIACORE™ analysis, Sapidyne KINEXA™ kinetic exclusion assay, SDS-PAGE and Western blotting or by HPLC analysis, as well as well as a number of other functional assays disclosed herein.

[000199] A typical mammalian expression vector contains at least one promoter element, which mediates the initiation of mRNA transcription, the antibody coding sequence, and the signals necessary for transcription termination and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and intervening sequences flanked by RNA splicing donor and acceptor sites. Highly efficient transcription can be achieved with the early and late promoters of SV40, the long terminal repeats (LTR) of retroviruses, e.g., RSV, HTLVI, HIVI, and the early promoter of cytomegalovirus (CMV). However, cellular elements can also be used (e.g. the human actin promoter). Suitable expression vectors for use in the practice of the present invention include, for example, vectors such as pIRES1neo, pRetro-Off, pRetro-On, pLXSN or pLNCX (Clonetech Labs, Palo Alto, Calif.), PcDNA3.1 ( +/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that can be used include human Hela 293, H9 and Jurkat cells, NIH3T3 and C127, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, rat L cells, and Chinese hamster ovary cells ( Chinese Hamster Ovary - CHO).

[000200] Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. Cotransfection with a selectable marker such as DHFR, GPT, neomycin or hygromycin allows identification and isolation of transfected cells.

[000201] The transfected gene can also be amplified to express large amounts of the encoded antibody. The DHFR (dihydrofolate reductase) marker is useful for developing cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthetase (GS) (Murphy et al., Biochem J. 227: 277-279 (1991); Bebbington et al., Bio/Technology 10: 169-175 (1992)). . Using these markers, mammalian cells are grown in selective medium and cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for antibody production.

[000202] According to the present invention, anti-GD2 antibodies comprise any of hu3F8V1-E1K, hu3F8V1-D32H, hu3F8V1-G54I; hu3F8V1 with a double mutation, e.g., hu3F8V1-E1KD32H, hu3F8V1-E1KG54I, and hu3F8V1-D32HG54I; hu3F8V1 with a triple mutation, for example, hu3F8V1- E1KD32HG54I; hu3F8V5 IgG; hu3F8V5 with a single mutation, e.g. hu3F8V5-E1K, hu3F8V5-D32H, hu3F8V5-G54I; hu3F8V5 with a double mutation, for example hu3F8V5-E1KD32H, hu3F8V5-E1KG54I, and hu3F8V5-D32HG54I; and hu3F8V5 IgG with a triple mutation, for example hu3F8V5-E1KD32HG54I antibodies or an antibody in which the variable region or CDR is derived from any of hu3F8V1-E1K, hu3F8V1-D32H, hu3F8V1-G54I; hu3F8V1 with a double mutation, for example hu3F8V1-E1KD32H, hu3F8V1-E1KG54I, and hu3F8V1-D32HG54I; hu3F8V1 with a triple mutation, for example hu3F8V1-E1KD32HG54I; hu3F8V5 IgG; hu3F8V5 with a single mutation, e.g. hu3F8V5-E1K, hu3F8V5-D32H, hu3F8V5-G54I; hu3F8V5 with a double mutation, for example hu3F8V5-E1KD32H, hu3F8V5-E1KG54I, and hu3F8V5-D32HG54I; and hu3F8V5 with a triple mutation, for example hu3F8V5-E1KD32HG54I antibody and the frame and constant regions of the antibody are derived from one or more human antibodies. The variable region or CDR derived from the antibody preferably has between about 90% to about 100% identity with the variable region or CDR of any of hu3F8V1-E1K, hu3F8V1-D32H, hu3F8V1-G54I; hu3F8V1 with a double mutation, for example hu3F8V1-E1KD32H, hu3F8V1-E1KG54I, and hu3F8V1-D32HG54I; hu3F8V1 with a triple mutation, for example hu3F8V1- E1KD32HG54I; hu3F8V5; hu3F8V5 with a single mutation, e.g. hu3F8V5-E1K, hu3F8V5-D32H, hu3F8V5-G54I; hu3F8V5 with a double mutation, for example hu3F8V5-E1KD32H, hu3F8V5-E1KG54I, and hu3F8V5-D32HG54I; and hu3F8V5 with a triple mutation, for example hu3F8V5-E1KD32HG54I although any and all modifications, including substitutions, insertions and deletions, whether natural mutation or human manipulation are contemplated, as long as the antibody maintains the ability to bind to GD2. The regions of chimeric, humanized or CDR-grafted antibodies that are derived from human antibodies need not have 100% identity with human antibodies. In a preferred embodiment, as many human amino acid residues as possible are retained so that immunogenicity is negligible, but human residues, in particular framework region residues, are substituted as necessary and as taught herein. follow in accordance with the present invention. Such modifications as disclosed herein are necessary to support the antigen binding site formed by the CDRs, while maximizing the humanization of the antibody.

[000203] Amino acid sequences that are substantially the same as the sequences described herein include sequences that comprise conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid with a second amino acid that has physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first chemical and/or amino acid. Conservative substitutions include the substitution of one amino acid for another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.

[000204] Naturally, the number of amino acid substitutions a person skilled in the art would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions or deletions for any given anti-GD2 antibody, fragment or variant will not be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value thereof, as specified herein.

[000205] Amino acids of an anti-GD2 antibody of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., Au-subel , supra, Chapters 8, 15; Cunningham and Wells, Science 244: 10811085 (1989)). This last procedure introduces unique alanine mutations in all residues of the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least binding to GD2. Sites that are critical for the antibody can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol Biol 224: 899-904 (1992) and Vos et al., Science 255: 306-312 (1992)).

[000206] An anti-GD2 antibody may further comprise, optionally, a polypeptide of at least one of 70 to 100% of the contiguous amino acids of the CDRs derived from at least one of the sequence described here.

[000207] In one embodiment, the amino acid sequence of an immunoglobulin chain or a portion thereof (e.g., variable region, CDR) has about 70 to 100% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value thereof) for the amino acid sequence of at least one sequence in Tables 1 to 4.

[000208] Heavy chain and light chain variable regions Example sequences are provided here. Antibodies of the present invention, or specified variants thereof, may comprise any number of contiguous amino acid residues from an antibody of the present invention, wherein the number is selected from the group consisting of the integers of from 10 to 100% of the number of contiguous residues. of an anti-GD2 antibody. Optionally, this contiguous amino acid subsequence is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in length or any range or value therein. Furthermore, the number of such subsequences may be any integer selected from the group consisting of 1 to 20, such as at least 2, 3, 4 or 5.

[000209] According to the present invention, the nucleic acid sequences set forth in SEQ ID NOs: 32-45 and the deduced amino acid sequences of anti-GD2 antibodies are set forth in SEQ ID NOs: 1-31. The heavy and light chain variable regions each contain three CDRs, which combine to form the antigen-binding site. The three CDRs are surrounded by four structural regions that essentially function to support the CDRs. The CDR sequences within the heavy and light chain variable region sequences can be identified by computer-assisted alignment, according to Kabat et al. (1987), in Sequences of Proteins of Immunological Interest, 4th ed., United States Department of Health and Human Services, U.S. Government Printing Office, Washington, D.C. or by molecular modeling of the variable regions, for example, using the ENCAD program as described by Levitt (1983) J. Mol. Biol. 168:595.

[000210] The human genes encoding the (C) constant regions of the humanized antibodies, fragments and regions of the present invention can be derived from a human fetal liver library, by known methods. Human C region genes can be derived from any human cell, including those that express and produce human immunoglobulins. The human CH region can be derived from any of the classes or isotypes of human H chains, including known gamma, mu, alpha, delta, epsilon, and subtypes thereof, such as G1, G2, G3 and G4. Since the H chain isotype is responsible for the various effector functions of an antibody, the choice of the CH region will be guided by the desired effector functions, such as complement fixation or activity in antibody-dependent cellular cytotoxicity (ADCC). Preferably, the CH region is derived from gamma 1 (IgG1) or gamma 4 (IgG4).

[000211] The human CL region can be derived from any isotype of the human L chain, kappa or lambda, preferably kappa.

[000212] The genes encoding C regions of human immunoglobulins are obtained from human cells through standard cloning techniques (Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989) and Ausubel et al., eds. Current Protocols in Molecular Biology (1987-1993)) genes of the human C region are readily available from known clones containing genes representing the two classes of L chains, the five classes of H chains and subclasses thereof.

[000213] The sequences of the variable regions of the antibody can be modified by insertions, substitutions and deletions as the chimeric antibody maintains the ability to bind to human GD2. The person skilled in the art can determine the maintenance of this activity by carrying out the functional tests described below. The variable regions may have, for example, from about 50% to about 100% homology to the variable regions identified below. In a preferred embodiment, the variable regions of the antibody have from about 80% to about 100% homology to the variable regions identified below. In a more preferred embodiment, the variable regions have from about 90% to about 100% homology to the variable regions identified below.

[000214] In a specific aspect, preferred anti-GD2 MAbs of the disclosure comprise light chain variable regions having 95%, 96%, 97%, 98% or 99% amino acid sequence homology to the sequences identified herein and further comprise variable heavy chain regions having 95%, 96%, 97%, 98% or 99% amino acid sequence homology with the sequences identified herein.

[000215] Preferably, the antibody or antigen-binding fragment of an antibody or specified portion or variant of the present invention binds human GD2 and thereby partially or substantially neutralizes a GD2 protein or fragment and thereby inhibits activities mediated through GD2. As used herein, the term "neutralizing antibody" refers to an antibody that can inhibit GD2-dependent activity by about 20-120%, preferably by at least about 10, 20, 30, 40, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay. The ability of an anti-GD2 antibody to inhibit a GD2-dependent activity is preferably assessed by at least one suitable assay as described herein and/or as known in the described art.

[000216] As indicated, the invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and CDRs comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein. Such anti-GD2 antibodies may include one or more amino acid substitutions, deletions or additions, either natural mutations or by human manipulation, as specified herein. Preferably, such antibodies or antigen-binding fragments and comprising such antibody chains or CDRs can bind to human GD2 with high affinity.

[000217] As those skilled in the art will appreciate, the present invention includes at least one biologically active antibody of the present invention. Biologically active antibodies have a specific activity of at least 20%, 30% or 40%, and preferably at least 50%, 60% or 70%, and more preferably at least 80%, 90% or 95% to 100% of that of native (non-synthetic), endogenous or related and known antibody. Assay methods and measures of quantifying enzyme activity and substrate specificity are well known to those skilled in the art.

[000218] In another aspect, the invention relates to human antibodies and antigen binding fragments, as described here, which are modified by covalent attachment of an organic moiety. Such modification can produce an antibody or antigen-binding fragment with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic radical may be a linear or branched, hydrophilic polymeric group, fatty acid group or a fatty acid ester group. In particular embodiments, the hydrophilic polymeric group may have a molecular weight of about 800 to about 120,000 Daltons and may be a polyalkylene glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, polymer amino acid or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group may comprise from about eight to about forty carbon atoms.

[000219] The modified antibodies and fragments of the invention may comprise one or more organic groups that are covalently linked, directly or indirectly, with the antigen-binding antibody. Each organic group that is attached to an antibody or antigen-binding fragment of the invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic acids and dicarboxylic acids. A "hydrophilic polymeric group," as the term is used herein, refers to an organic polymer that is more soluble in water than in octane, for example, polylysine. Thus, an antibody modified by the covalent linkage of polylysine is encompassed by the invention. Hydrophilic polymers suitable for modifying antibodies of the invention may be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, and so on), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and so on), hydrophilic amino acid polymers (for example, polylysine, polyarginine, polyaspartate and so on), polyalkane oxides (for example, polyethylene oxide, polypropylene oxide and so on on) and polyvinylpyrrolidone. Preferably, the hydrophilic antibody-modifying polymer of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. For example PEG5000 and PEG20,000, where the subscript is the average molecular weight of the polymer in daltons, can be used. The hydrophilic polymeric group can be substituted with one to about six alkyl groups, fatty acid groups or fatty acid esters. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared employing suitable methods. For example, a polymer comprising an amine group can be coupled to a fatty acid or fatty acid ester carboxylate, and an activated carboxylate (e.g., activated with N,N-carbonyl diimidazole) onto a fatty acid or fatty acid ester. Fatty acid can be coupled to a hydroxyl group in a polymer.

[000220] Fatty acids and fatty acid esters suitable for modifying antibodies of the invention may be saturated or may contain one or more unsaturation units. Fatty acids that are suitable for modifying antibodies of the invention include, for example, n-dodecanoate, n-tetradecanoate, n-octadecanoate, n-eicosanoate, docosanoate-N,N-triacontanoate, N-tetracontanoate, cis-.delta.9 - octadecanoate, all cis-.delta.5,8,11,14-eicosatetraenoate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and so on. Suitable fatty acid esters include monoesters of dicarboxylic acids comprising a straight or branched chain lower alkyl group. The lower alkyl group may comprise from one to about twelve, preferably one to about six, carbon atoms.

[000221] Modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. A "modifying agent" as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group. An "activating group" is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second group of chemicals. For example, amino-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chlorine, bromine, fluorine, iodine), N-hydroxysuccinimidyl (NHS) esters, and so on. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and so on. An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hernanson, GT, Bioconjugate Techniques, Academic Press: San Diego, California (1996)). An activating group can be linked directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) or through a linker moiety, e.g., a divalent C1-C12 group, in which one or more atoms carbon may be replaced by a heteroatom such as oxygen, nitrogen or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3--, -NH-, to name a few. Agents comprising a modifying linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid, in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described or it can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimide fatty acid derivative. (See, e.g., Thompson, et al., WO 92/16221, the entire teachings of which are incorporated herein by reference).

[000222] The modified antibodies of the invention can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, the organic moieties can be linked to the antibody in a non-site-specific manner by employing an amine-reactive modifying agent, e.g., a PEG NHS ester. Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intrachain disulfide bonds) of an antibody or antigen-binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified antibody of the invention. Modified human antibodies and antigen-binding fragments comprising an organic moiety that is bound to specific sites of an antibody of the present invention can be prepared using suitable methods such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147- 153 (1992); Werlen et al., Bioconjugate Chem, 5: 411-417 (1994); Kumaran et al., Protein Sci 6 (10): 2233-2241 (1997); Itoh et al., Bioorg. Chem, 24 (1): 59-68 (1996); Capella et al., Biotechnol Bioeng, 56 (4): 456-463 (1997)), and the methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego , California (1996).

[000223] The antibodies of the invention can bind to human GD2 with a wide range of affinities (KD), as shown below.

[000224] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method (see, for example, Berzofsky et al., "Antibody-Antigen Interactions" in Fundamental Immunology, Paul, W.E., Ed, Raven Press : New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company : New York, New York (1992); and ways described here). The measured affinity of a particular antibody-antigen interaction may vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen binding parameters are preferably made with standardized antibody and antigen solutions, and a standardized buffer, such as the buffers described herein.

[000225] Anti-GD2 antibodies useful in the methods and compositions of the present invention are characterized by binding to GD2 and, preferably, have low toxicity. In particular, a specified antibody, fragment or variant of the invention, wherein the individual components, such as the variable region, constant region and framework, individually and/or collectively, optionally and preferably have low immunogenicity, is useful. in the present invention. Antibodies that can be used in the present invention are optionally characterized by their ability to treat patients for prolonged periods with measurable attenuation of symptoms and low and/or acceptable toxicity. Low immunogenicity or acceptable and/or high affinity, as well as other suitable properties may contribute to achieving therapeutic results. "Low immunogenicity" is defined here as significantly increasing HAHA, HACA, or HAMA responses in less than about 75% or, preferably, less than about 50% of treated patients and/or increasing low titers in the treated patient ( Elliott et al., Lancet 344: 1125-1127 (1994), incorporated herein in its entirety by reference).

[000226] Bispecific, heterospecific, heteroconjugate or similar antibodies can also be used, which are monoclonal antibodies, humanized antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for at least one GD2 protein, the other is for any other antigen. Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two pairs of immunoglobulin heavy chain and light chain, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305: 537 (1983)). Because of the random arrangement of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, which is usually carried out by affinity chromatography steps, is quite laborious and product yields are low. Similar procedures are described, for example, in WO 93/08829, United States Patent Nos. 6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989. 530, 5,959,084, 5,959,083, 5,932,448, 5,833,985, 5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549, 4,676. 980, WO 91/00360 , WO 92/00373, EP 03089, Traunecker et al., EMBO J. 10: 3655 (1991), Suresh et al., Methods in Enzymology 121: 210 (1986); Chan and Carter, 2010, Nature Rev. 10, 301316; Weiner et al., 2010, Nature Rev. 10, 317-327; each incorporated herein in its entirety by reference.

[000227] In certain embodiments, antibodies that bind to GD2 can be used in unconjugated form. In other embodiments, antibodies that bind to GD2 can be conjugated, for example, to a detectable marker, a drug, a prodrug, or an isotope.

[000228] In certain methods of the invention described in greater detail below, such as methods of detecting GD2 expression in cells or tissues as a measure of the metastatic potential of tumor cells or as a form of identification in in situ carcinomas (e.g. DCIS or LCIS) in tissues, anti-GD2 antibodies are conjugated to one or more detectable markers. For such uses, antibodies may be detectably labeled through covalent or non-covalent attachment of a contrast agent or other enzymatic, radioisotope, isotope, fluorescent, toxic, chemiluminescent, or chromogenic agent for nuclear magnetic resonance.

[000229] Examples of suitable chromogenic markers include diaminobenzidine and 4-hydroxyazo benzene-2-carboxylic acid.

[000230] Examples of suitable enzyme markers include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, glycerol-α phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.

[000231] Examples of suitable radioisotope labels include 3H, 111In, 125I, 131I, 32P, 35S, 14C, 51Cr, 57To, 58Co, 59Fe, 75Se, 152Eu, 90Y, 67Cu, 217Ci, 211At, 212Pb, 47 Sc, 109Pd, etc .. 111In is a preferred isotope where in vivo imaging is used, as it avoids the problem of dehalogenation of antibodies binding to the GD2 marker with 125I or 131I by the liver. Furthermore, this radionucleotide has a gamma emission energy more favorable for imaging (Perkins et al., Eur J. Nucl Med 70: 296-301 (1985); Carasquillo et al., J. Nucl Med 25: 281287 (1987) ). For example, monoclonal antibodies coupled to 111In with 1-(β-isothiocyanatobenzyl)-DPTA have shown little uptake in non-tumor tissues, in particular the liver, and therefore increases tumor localization specificity (Esteban et al., J. Nucl Med. 28: 861-870 (1987)).

[000232] Examples of suitable non-radioactive isotopic labels include 157Gd, 55Mn, 162Dy, 52Tr and 56Fe.

[000233] Examples of suitable fluorescent tags include a 152Eu tag, a fluorescein tag, an isothiocyanate tag, a rhodamine tag, a phycoerythrin tag, a phycocyanin tag, an allophycocyanin tag, a Green Fluorescent Protein tag (Green Fluorescent Protein - GFP), an o-phthaldehyde marker and a fluorescamine marker.

[000234] Examples of suitable toxin markers include diphtheria toxin, ricin and cholera toxin.

[000235] Examples of chemiluminescent labels include a luminol label, an isoluminol label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase marker and an aequorin marker.

[000236] Examples of nuclear magnetic resonance contrast agents include heavy metal nuclei, such as Gd, Mn and iron.

[000237] Typical techniques for binding labels described above for anti-GD2 antibodies are provided by Kennedy et al., Clin. Chem. Acta 70: 1-31 (1976) and Schurs et al., Clin. Chem. Acta 81: 1-40 (1977). Coupling techniques mentioned herein are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxysuccinimide ester method, all of which are incorporated herein by reference.

[000238] For use in certain therapeutic methods of the invention, such as ablation of residual tumor cells after surgery or prevention of metastases, anti-GD2 antibodies can be conjugated with one or more drugs, prodrugs or isotopes. Such preferred conjugates comprise one or more linkers, for example, one or more antibodies or fragments, derivatives or variants thereof, that bind to GD2, conjugated to one or more cytotoxic agents; Such conjugates are useful in the methods of treating and preventing tumor metastasis provided by the invention. According to certain embodiments of the invention, the anti-GD2 antibody is conjugated to a cytotoxic agent. Cytotoxic agents, e.g., chemotherapeutics, useful for generating anti-GD2 antibody-cytotoxic agent conjugates are well known in the art and include, but are not limited to, cisplatin, carboplatin, oxaliplatin, paclitaxel, melphalan, doxorubicin, methotrexate, 5-fluorouracil, etoposide, mechlorethamine, cyclophosphamide, bleomycin, microtubule poisons, and acetogenin. Other chemotherapeutic agents suitable for use in accordance with this aspect of the invention are well known and will be familiar to those skilled in the art.

[000239] The use of conjugates of one or more anti-GD2 antibodies and one or more small molecule toxins, such as a calicheamycin, a maytansine (United States Patent No. 5,208,020), a trichothene and CC1065 are also considered here. In one embodiment of the invention, the anti-GD2 antibody is conjugated to one or more maytansine molecules (e.g., about 1 to about 10 maytansine molecules per anti-GD2 antibody). Maytansine can, for example, be converted to May-SS-Me, which can be reduced to May-SH3 and reacted with modified anti-GD2 antibody (Chari et al., Cancer Research. 52: 127-131 (1992) ) to generate a maytansinoid-anti-GD2 antibody conjugate.

[000240] Alternatively, the anti-GD2 antibody can be conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at subpicomolar concentrations. Structural analogues of calicheamicin can be used (Research Hinman et al., Cancer 53: 3336-3342 (1993) and Lode et al., Cancer Research. 58: 2925-2928 (1998)).

[000241] Enzymatically active toxins and fragments thereof that can be used to produce conjugates with one or more anti-GD2 antibodies include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa) , ricin A chain, Abrin, chain, modecin, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, inhibitor of Sapaonaria officinalis, gerarnin, mitogelin, restrictocin, fenomycin, enomycin and trichothecenes. See, for example, document WO 93/21232 published in the English language on October 28, 1993, the description of which is incorporated herein by reference in its entirety. Maitansinoids can also be conjugated with one or more anti-GD2 antibodies.

[000242] The present invention also considers anti-GD2 antibodies conjugated to a compound with nucleolytic activity (for example, a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNAase).

Exemplary Alternative Formats

[000243] In some particular embodiments, anti-GD2s antibody agents or sequences thereof are used in multispecific (e.g., bispecific) format. In some embodiments, bispecific MoAbs may be comprised of dual variable domains, with one domain being the anti-3F8 variable domain and the other domain chosen from a group consisting of anti-OKT3 to redirect T cells toward tumor cytotoxicity or DOTA-metal. , C8.2.5 for multi-step pretargeting or Clone 35, CD137, for ADCC with anti-41BB-scFv as agonist or with CD137, 41BBL for ADC with 41 BBL as agonist. An N297A mutation in the CH2 domain results in aglycosylation, leading to no binding to FcR or C1q. The amino acid sequence of (hu3F8V1-scFv) - (huOKT3-scFv) with linker and spacer is shown in SEQ ID NO: 29 and without spacer in SEQ ID NO: 30. The amino acid sequence of (scFv hu3F8V1)- ( C8.2.5-scFv) (based on Orcutt et al., 2010, Protein Eng Design and Selection 23, 221) is shown in SEQ ID NO: 31.

[000244] A bispecific antibody (anti-GD2 and anti-DOTA) can be used in a first multi-step pre-targeting step, followed by blood elimination using DOTA(metal)-dextran as the elimination agent, with a third stage of introduction of therapeutic products conjugated to DOTA(metal), such as DOTA(metal)- radioactive metal, DOTA(metal)-nanoparticles, DOTA(metal)-liposomes, DOTA(metal)-drugs, DOTA(metal) -DNA, DOTA(metal)-RNA and DOTA(metal)-toxins. Since C8.2.5 has different affinities for each type of DOTA-metal complex, the affinity of the pre-targeted C8.2.5 to the scavenging agent and the DOTA-ligand can be precisely controlled.

[000245] The amino acid sequence of hu3F8 and its variants presented here can be used to construct chimeric antigen receptors (CAR), as previously shown for other anti-GD2 antibodies (Krause et al., 1998, J Exp Med 188, 619 -626). The strategy of reorienting CAR immune effector cells is independent of the MHC-peptide-TCR interaction and allows cells to react against a wide variety of antigens on the cell surface (Davies and Maher, 2010, Achivum Immunologiae Et Therapiae Experimentalis 58, 165-178 ). Various methods have been used in designing vehicles, with most of them employing the antigen-binding domain of a monoclonal antibody in the form of a single-chain variable fragment (scFv) for antigen recognition. Early T cell activation receptors originated from studies allowed investigators to elucidate the role of the CD3Ç chain (Irving and Weiss, 1991, Cell 64, 891-901; Romeo et al., 1992, Cell 68, 889-897) . In subsequent studies, scFv of interest were fused to the CD3Ç chain (Eshhar et al., 1993, PNAS USA 90, 720-724) or FcεRIγ (Weijtens et al., 1996, J Immunol 157, 836843) and were found to both are sufficient for T cell activation. Although this characterized CAR construction, the incorporation of co-stimulatory molecules came about after it was discovered that first generation CARs were able to induce T cell proliferation only up to 2-3 cell divisions. , quickly followed by cell death (Gong et al., 1999, Neoplasia 1, 123-127). By expressing CD80 on the target tumor cell, researchers were able to show that CAR-expressing cells could be stimulated again, leading to further increases in the number of T cells. The first CARs that incorporated the costimulatory molecule CD28 alongside the CD3Ç chain showed large improvements over those expressing the CD3Ç chain alone (Krause et al., 1998, supra; Haynes et al., 2002, Blood 100, 3155-3163; Maher et al., 2002, Nature Biotech 20, 70-75); this includes an absolute increase in the number of T cells as well as an increase in IL-2 production. Since then, several other groups have begun to use other costimulatory molecules in combination with CD3Ç alone or with both CD3Ç and CD28. These additional signaling molecules include 4-1BB (Wang et al., 2007, Human Gene Ther 18, 712-725; Brentjens et al., 2007, Clin Res Cancer 13, 54265432; Imai et al., 2004, Leukemia 18, 676-684; Finney et al., 2004, J Immunol 172, 104-113), DAP10 (Brentjens et al., 2007, supra), OX40 (Brentjens et al., 2007, supra; Finney et al., 2004, supra; Wilkie et al., 2008, J Immunol 180, 4901-4909; Nguyen and Geiger, 2003, Gene Therapy 10, 594-604; Pule et al., 2005, Mol Ther 12, 933-941) and ICOS (Finney et al., 2004, supra), and have been applied in the context of T cells as well as NK cells (Daldrup-Link et al., 2005, European Radiology 15, 413; Imai and Campana, 2004, J Biol Reg Homeostatic Ag 18 , 62-71; Roberts et al., 1998, J. Immunol 375-384; Kruschinski et al., 2008, PNAS USA 105, 17481-17486; Pegram et al., 2008, J Immunol 181, 34493455). Although first-generation CARs are the only ones that have been tested in the clinic to this point, both in vitro and in vivo comparisons have demonstrated clear superiority with second- and third-generation CARs (Haynes et al., 2002, supra; Brentjens et al. , 2007, supra; Teng et al., 2004, Human Gene Ther 15, 699-708; Haynes et al., 2002, J Immunol 169, 5780-5786; Kowolik et al., 2006, Cancer Res 66, 10995-11004 ; Loskog et al., 2006, Leukemia 20, 1819-1928; Moeller et al., 2004, Cancer Gene Therapy 11, 371-379; Vera et al., 2006, Blood 108, 3890-3897).

[000246] Currently, most investigators use human peripheral T cells, however, others have recently begun the use of EBV-specific T cells (Rossig et al., 2002, Blood 99, 20092016), lymphoid progenitor cells (Zakrzewski et al ., 2006, Nature Med 12, 1039-1047; Zakrzewski et al., 2008, Nature Biotech 26, 453- 461) and unfractionated bone marrow cells (Papapetrou et al., 2009, J Clin Invest 119, 157-168 ; Wang et al., 1998, Nature Med 4, 168172). Leukemia killer cell lines (e.g., NK92, NK92MI, KHYG-1), which are cytolytic and easy to culture, can also provide a continuous supply of CAR-expressing effector cells for preclinical and clinical testing. NK92MI is a human NK cell line derived from non-Hodgkin's lymphoma and transduced with human IL-2 cDNA; previous studies have demonstrated its strong cytotoxic capabilities in mouse models (Tam et al., 1999, J Hematol 8, 281-290; Korbelik and Sun, 2001, Inter J Cancer 93, 269-274). Furthermore, NK92 cells have also been used in clinical practice and have been proven to be safe after a series of Phase I studies in patients with renal cell carcinoma and melanoma (Arai et al., 2008, Cytotherapy 10, 625-632). By virtue of their ease of maintenance in vitro and relatively short doubling times, these cells are ideal effectors for various cytotoxicity assays to test a variety of targeting approaches. Although studies using the original IL-2-dependent NK92 cell line showed minimal toxic effects in both mice and humans, IL-2-transduced NK92MI cells may have greater leukemogenic potential. One method by which researchers attempt to prevent leukemogenesis in SCID mice using NK92 cells is by irradiating the effectors with 3000 cGy prior to inoculation. In Phase I clinical trials, this is sufficient to prevent uncontrolled proliferation of NK92MI cells within the immunocompromised patient. An alternative safety mechanism is one that involves the use of suicide genes. A common example is the use of the herpes virus thymidine kinase gene, which functions by killing a cell expressing the gene through administration of acyclovir or ganciclovir (Helene et al., 1997, J. Immunol 5079-5082).

Nucleic acids

[000247] The nucleotide and amino acid sequence of the variable regions of the heavy and light chains of the MoAbs of the invention are described in the present application. The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize, under stringent hybridization conditions, to polynucleotides that encode an antibody of the present invention.

[000248] Polynucleotides can now be obtained by any method known in the art. For example, once the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)) which, in brief, involves the synthesis of overlapping oligonucleotides that contain portions of the sequence encoding the antibody, hybridization and ligation of oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[000249] Alternatively, a polynucleotide encoding an antibody can be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g. , an antibody cDNA library or a cDNA library generated from nucleic acid, preferably poly A + RNA, isolated from any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the present invention ) by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, for example, a cDNA clone from of a cDNA library encoding the antibody. The amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[000250] Once the nucleotide sequence and corresponding amino acid sequence of the antibody are determined, the nucleotide sequence of the antibody can be manipulated using processes well known in the art for manipulating nucleotide sequences, e.g., DNA techniques. recombinant, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons; NY, both of which are incorporated herein by reference in their entirety), to generate antibodies that have a different amino acid sequence, e.g., to create amino acid substitutions, deletions, and/or insertions .

[000251] The nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA including, but not limited to, cDNA and genomic DNA obtained through cloning or produced synthetically or any combinations thereof. DNA can be triple-stranded, double-stranded or single-stranded or any combination thereof. Any portion of at least one strand of DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also known as the antisense strand.

[000252] The isolated nucleic acid molecules of the present invention may include nucleic acid molecules comprising an Open Reading Frame (ORF), optionally with one or more introns, for example, but without limitation, at least a portion specific for at least one CDR, such as CDR1, CDR2 and/or CDR3 of at least one heavy chain or light chain; nucleic acid molecules comprising the coding sequence for an anti-GD2 antibody or variable region; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, by virtue of the degeneracy of the genetic code, still encode at least one anti-GD2 antibody, as described herein and/or as known in technique.

[000253] The present invention provides isolated nucleic acids that hybridize, under selective hybridization conditions, with a polynucleotide described herein. Thus, the polynucleotides of the present embodiment can be used to isolate, detect and/or quantify nucleic acids comprising such polynucleotides. For example, the polynucleotides of the present invention can be used to identify, isolate or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic sequences or cDNA isolated from or otherwise complementary to a cDNA from a human or mammalian nucleic acid library.

[000254] Nucleic acids may conveniently comprise sequences other than a polynucleotide of the present invention. For example, a multiple cloning site comprising one or more restriction endonuclease sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Additionally, translatable sequences can be inserted to assist in isolating the translated polynucleotide of the present invention. For example, a hexahistidine tag sequence provides a convenient means of purifying the proteins of the present invention. The nucleic acid of the present invention - excluding the coding sequence - is optionally a vector, adapter or linker for cloning and/or expressing a polynucleotide of the present invention.

[000255] Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, assist in isolating the polynucleotide, or improve the introduction of the polynucleotide into a cell. The use of cloning vectors, expression vectors, adapters and linkers is well known in the art (see, for example, Ausubel, supra; or Sambrook, supra).

[000256] A vector comprising any of the above-described isolated or purified nucleic acid molecules, or fragments thereof, is further provided by the present invention. Any of the above nucleic acid molecules or fragments thereof can be cloned into any suitable vector and can be used to transform or transfect any suitable host. The selection of vectors and methods for constructing them are commonly known to those skilled in the art and are described in general technical references (See, generally, "Recombinant DNA, Part D", Methods in Enzymology, Vol. 153, Wu and Grossman , eds., Academic Press (1987)). Desirably, the vector comprises regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host (e.g., bacteria, fungus, plant or animal) into which the vector is to be introduced, as appropriate and taking into account whether the vector is DNA or RNA. Preferably, the vector comprises regulatory sequences that are specific to the host gender. More preferably, the vector comprises regulatory sequences that are specific to the host species.

[000257] In addition to the replication system and the inserted nucleic acid, the construct may include one or more marker genes that allow the selection of transformed or transfected hosts. Marker genes include resistance to biocides, e.g., resistance to antibiotics, heavy metals, etc., auxotrophic complementation in a host to provide prototrophy, and so on.

[000258] Suitable vectors include those that are designed for propagation and expansion or for expression or both. For example, a cloning vector is selected from the group consisting of the pUC series, the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden) and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as ÀGT10, ÀGT11, ÀZapII (Stratagene), ÀEMBL4 and ÀNM1149, can also be used. Examples of plant expression vectors include pBI110, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Carlsbad, Calif.) can also be used according to the manufacturer's recommendations.

[000259] An expression vector may comprise a native or non-native promoter operatively linked to an isolated or purified nucleic acid molecule, as described above. The selection of promoters, e.g., strong, weak, inducible, and developmentally specific, tissue specific, is within the skill in the art. Similarly, the combination of a nucleic acid molecule, or fragment thereof, as described above with a promoter is also within the skill in the art.

[000260] Suitable viral vectors include, for example, retroviral vectors, parvovirus-based vectors, for example, adeno-associated virus (AAV), adenoviral-AAV chimeric vectors and adenovirus-based vectors and lentiviral vectors, such as vectors based on the Herpes simplex virus (Herpes Simplex Virus - HSV). These viral vectors can be prepared using conventional recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

[000261] A retroviral vector is derived from a retrovirus. Retrovirus is an RNA virus capable of infecting a wide variety of host cells. Upon infection, the retroviral genome integrates into the host cell genome and is replicated together with the host cell's DNA, thus constantly producing viral RNA and any nucleic acid sequence incorporated into the retroviral genome. As such, long-term expression of (a) therapeutic factor(s) is possible when using retroviruses. Retroviruses considered for use in gene therapy are relatively nonpathogenic, although pathogenic retroviruses exist. When employing pathogenic retroviruses, for example, the Human Immunodeficiency Virus (HIV) or Human T-Cell Lymphotrophic Virus (HTLV), care must be taken to alter the viral genome to eliminate toxicity. to the host. A retroviral vector can additionally be manipulated to render the virus replication-deficient. As such, retroviral vectors are considered particularly useful for stable gene transfer in vivo. Lentiviral vectors, such as HIV-based vectors, are exemplary retroviral vectors used for gene delivery. Unlike other retroviruses, HIV-based vectors are known to incorporate their passenger genes into non-dividing cells and therefore may be of use in treating persistent forms of the disease.

[000262] Optionally, the isolated or purified nucleic acid molecule or a fragment thereof, when bound to another nucleic acid molecule, can encode a fusion protein. The generation of fusion proteins is within the skill of those skilled in the art and may involve the use of restriction enzymes or recombinant cloning techniques (see, for example, GatewayTM (Invitrogen)). See also United States Patent No. 5,314,995.

[000263] Taking into account the foregoing, the present invention also provides a composition comprising an isolated or purified nucleic acid molecule described above, optionally in the form of a vector. The composition may comprise other components, as further described herein.

[000264] Isotopes are also available for the production of radioconjugated anti-GD2 antibodies for use in the therapeutic methods of the present invention. Examples include 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, and radioactive isotopes of Lu.

[000265] Conjugates of anti-GD2 antibody and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4-( N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis compounds -azide (such as bis(p-azidabenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). 14Carbon-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide with anti-GD2 antibody. See document WO 94/11026. The linker may be a "cleavable linker" which facilitates the release of the cytotoxic drug into the cell. For example, an acid-sensitive linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Research 52: 127-131 (1992)) can be used.

[000266] Alternatively, a fusion protein comprising the antibody and anti-GD2 ligand cytotoxic agent can be made, for example, by means of recombinant techniques or peptide synthesis.

Compositions

[000267] The anti-GD2 antibody compositions of the present invention include any suitable and effective amount of a composition or pharmaceutical composition comprising at least one anti-GD2 antibody agent for use in delivering the antibody agent provided to a cell, tissue , organ, animal or patient in need of such modulation, treatment or therapy.

[000268] The present invention also provides at least one anti-GD2 antibody composition comprising at least one, at least two, at least three, at least four, at least five, at least six or more anti-GD2 antibodies, as described herein and/or as known in the art, which are provided in a non-naturally occurring composition, mixture or form. Such compositions comprise compositions comprising at least one or two non-naturally occurring C- and/or N-terminal full-length specified variants, domains, fragments, or variants, the amino acid sequence of the anti-GD2 antibody selected from the group consisting of 70 to 100% of the contiguous amino acids of the CDR regions of the antibodies described herein or specified fragments, domains or variants thereof. Preferred anti-GD2 antibody compositions include at least one or two full-length fragments, variants or domains, such as at least one CDR or LBR containing portions of the anti-GD2 antibody sequences described herein. Other preferred compositions comprise 40 to 99% to 70 to 100% of at least one CDR region of an anti-GD2 Ab described herein. Such composition percentages are by weight, volume, concentration, molarity or molality as liquid or dry solutions, mixtures, suspensions, emulsions or colloids, as known in the art or as described herein.

[000269] Anti-GD2 compounds, compositions or combinations of the present invention may further comprise at least one of any suitable auxiliary such as, but without limitation, a diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. . Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known in the art such as, but without limitation, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990. Pharmaceutically Acceptable Carriers may be routinely selected which are suitable for the mode of administration, solubility and/or stability of the anti-GD2 antibody, fragment or variant composition, as well known in the art or as described herein.

[000270] Pharmaceutical excipients and additives useful in the present composition include, but are not limited to, proteins, peptides, amino acids, carbohydrates and lipids (for example, sugars, including monosaccharides, di-, tri-, tetra- and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, esterified sugars and so on; and polysaccharides or sugar polymers), which may be present alone or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein and so on. Representative amino acid/antibody components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame and so on. A preferred amino acid is glycine.

[000271] Suitable carbohydrate excipients for use in the invention include, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose and so on; disaccharides, such as lactose, sucrose, trehalose, cellobiose and so on; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches and so on; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol and so on. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose and raffinose.

[000272] Anti-GD2 antibody compositions may also include a buffer or an agent for pH adjustment; Typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include salts of organic acids, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; Tris, tromethamine hydrochloride or phosphate buffers. Preferred buffers for use in the present compositions are salts of organic acids, such as citrate.

[000273] Additionally, the anti-GD2 antibody compositions of the invention may include polymeric excipients/additives, such as polyvinylpyrrolidones, Ficoll (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin ), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "Tween 20" and "Tween 80"), lipids (e.g., phospholipids, acids fatty acids), steroids (e.g. cholesterol) and chelating agents (e.g. EDTA).

[000274] These and other known pharmaceutical excipients and/or additives suitable for use in the anti-GD2 antibody compositions, portion or variants according to the invention are known in the art, for example, as listed in "Remington: The Science & Practice of Pharmacy", 19th Ed., Williams & Williams, (1995) and in "Physician's Desk Reference", 51st Ed., Medical Economics, Montvale, NJ (1998), the descriptions of which are incorporated herein in their entirety by reference. Preferred carrier materials or excipients are carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or polymeric agents.

[000275] As mentioned above, the invention provides stable formulations which are preferably a phosphate buffer with saline or a chosen salt, as well as preserved formulations and solutions containing a preservative, as well as preserved formulations for multiple use suitable for pharmaceutical or veterinary use comprising at least one anti-GD2 antibody in a pharmaceutically acceptable formulation. Preserved formulations contain at least one known preservative or, optionally, selected from the group consisting of at least one of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g. hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and so on), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in a aqueous diluent.

[000276] Any suitable concentration or mixture may be used, as known in the art, such as 0.001-5% or any range or value thereof such as, but without limitation, 0.001, 0.003, 0.005, 0.009, 0.01, 0 .02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 , 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2 ,2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 , 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9 or any range or value therein. Non-limiting examples include, for preservatives, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g. 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001 to 0.5 % thimerosal (e.g., 0.005, 0.01), 0.001 to 2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0% ), 0.0005 to 1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05 , 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and so on.

[000277] As mentioned above, the invention provides an article of manufacture comprising packaging material and at least one vial comprising a solution of at least one anti-GD2 antibody with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, in that said packaging material includes a label indicating that such solution can be kept for a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48 , 54, 60, 66, 72 hours or more. The invention further comprises an article of manufacture comprising packaging material, a first vial comprising at least one lyophilized anti-GD2 antibody and a second vial comprising an aqueous buffer diluent or prescribed preservative, wherein said packaging material includes a label that instructs a patient to reconstitute at least one anti-GD2 antibody in the aqueous diluent to form a solution that can be maintained over a period of twenty-four or more hours.

[000278] The range of at least one anti-GD2 antibody in the product of the present invention includes amounts that provide, upon reconstitution, if in a wet/dry system, concentrations of about 1.0 micrograms/ml to about 1000 mg /ml, although lower and higher concentrations are possible and are dependent on the intended delivery vehicle, e.g., solution formulations will differ in transdermal, pulmonary, transmucosal, or mini osmotic pump patch methods.

[000279] Preferably, the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. Preferred preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl paraben (methyl, ethyl, propyl, butyl and so on), benzalkonium, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof. The concentration of preservative used in the formulation is a concentration sufficient to produce an antimicrobial effect. Such concentrations are dependent on the selected preservative and are easily determined by those skilled in the art.

[000280] Other excipients, for example, isotonicity agents, buffers, antioxidants, conservation enhancers, can optionally and preferably be added to the diluent. An isotonicity agent, such as glycerin, is often used in known concentrations. A physiologically tolerated buffer is preferably added to provide better pH control. The formulations may cover a wide range of pH values, such as from a pH of about 4 to a pH of about 10, and preferred ranges of a pH of about 5 to a pH of about 9, and a more preferred range of from about 6.0 to about 8.0. Preferably, the formulations of the present invention have a pH between about 6.8 and about 7.8. Preferred buffers include phosphate buffers, more preferably sodium phosphate, particularly phosphate buffered saline (PBS).

[000281] Other additives, such as pharmaceutically acceptable solubilizers, such as Tween 20 (polyoxyethylene sorbitan monolaurate (20)), Tween 40 (polyoxyethylene sorbitan monopalmitate (40)), Tween 80 (polyoxyethylene (20) sorbitan ), Pluronic F68 (block copolymers of polyoxyethylene/polyoxypropylene) and PEG (polyethylene glycol), or non-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, PLURONIC® polyols, other copolymers in block and chelating agents, such as EDTA and EGTA, may optionally be added to the formulations or compositions to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation. The presence of a pharmaceutically acceptable surfactant attenuates the aggregation propensity of the protein.

[000282] The formulations of the present invention can be prepared by means of a process that comprises mixing at least one anti-GD2 antibody and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl and so on), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous diluent. Mixing the at least one anti-GD2 antibody and preservative in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one anti-GD2 antibody in the buffered solution is combined with the desired preservative in a buffered solution in amounts sufficient to provide the protein and preservative in the desired concentrations. Variations of this process will be recognized by those skilled in the art. For example, the order in which components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.

[000283] The claimed formulations may be provided to patients as clear solutions or as double vials comprising a vial of lyophilisate of at least one anti-GD2 antibody which is reconstituted with a second vial containing water, a preservative and/or excipients, preferably a phosphate buffer and/or saline solution and a salt chosen in an aqueous diluent. A single solution vial or double vial that requires reconstitution can be reused multiple times and may be sufficient for a single or multiple patient treatment cycles and therefore may provide a more convenient treatment regimen than those currently available.

[000284] The present claimed articles of manufacture are useful for administration over a period of immediately to twenty-four hours or more. Consequently, the presently claimed articles of manufacture offer significant advantages to the patient. The formulations of the invention may optionally be safely stored at temperatures of about 2°C to about 40°C and retain the biological activity of the protein for extended periods of time, thus allowing for a label on the package indicating that the solution may be maintained and/or used for a period of 6, 12, 18, 24, 36, 48, 72 or 96 hours or more. If a preserved diluent is used, such a label may include use for up to 1 to 12 months, half a year, a year and a half, and/or two years.

[000285] Solutions of at least one anti-GD2 antibody of the invention can be prepared by means of a process that comprises mixing at least one antibody in an aqueous diluent. Mixing is carried out using conventional dissolving and mixing procedures. To prepare a suitable diluent, for example, a measured amount of at least one antibody in water or buffer is combined in amounts sufficient to provide the protein and, optionally, a preservative or buffer in desired concentrations. Variations of this process will be recognized by those skilled in the art. For example, the order in which components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.

[000286] The claimed products may be provided to patients as clear solutions or as double vials comprising a vial of lyophilisate of at least one anti-GD2 antibody that is reconstituted with a second vial containing the aqueous diluent. A single solution vial or double vial that requires reconstitution can be reused multiple times and may be sufficient for a single or multiple patient treatment cycles and thus provide a more convenient treatment regimen than those currently available.

[000287] The claimed products may be provided indirectly to patients by supplying to pharmacies, clinics or other such institutions and units, clear solutions or double vials comprising a vial of lyophilisate of at least one anti-GD2 antibody that is reconstituted with a second bottle containing the aqueous diluent. The clear solution, in this case, can be up to a liter or even more, constituting a large reservoir from which small portions of the solution of at least one antibody can be obtained once or multiple times for transfer to smaller vials and provided by the pharmacy or clinic to the patients. your clients and/or patients.

[000288] Recognized devices comprising these single-vial systems include pen-injector devices for dispensing a solution, such as BD Pens, BD AUTOJECTOR®, HUMAJECT®, for example, as made or developed by Becton Dickensen (Franklin Lakes, NJ,), Disetronic (Burgdorf, Switzerland; Biojet, Portland, Oreg.; National Medical Products, Weston Medical (Peterborough, United Kingdom), Medi-Jet Corp (Minneapolis, Minn.). Recognized devices comprising a dual vial system include pen-injector systems for reconstituting a lyophilized drug into a cartridge for dispensing reconstituted solution, such as HUMATROPEN®.

[000289] The presently claimed products include packaging material. The packaging material provides, in addition to the information required by regulatory bodies, the conditions under which the product can be used. The packaging material of the present invention provides instructions to the patient for reconstituting at least one anti-GD2 antibody in the aqueous diluent to form a solution and how to use the solution over a period of 2 to 24 hours or more for the product from the two vials wet/ dry. For the single bottle, solution product, the label indicates that such solution can be used over a period of 2 to 24 hours or more. The presently claimed products are useful for use as a pharmaceutical product for humans.

[000290] The formulations of the present invention can be prepared by means of a process that comprises mixing at least one anti-GD2 antibody and a selected buffer, preferably a phosphate buffer with saline or a chosen salt. Mixing the at least one antibody and the buffer in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one antibody in water or buffer is combined with the desired buffering agent in water in amounts sufficient to provide the protein and buffer in desired concentrations. Variations of this process will be recognized by those skilled in the art. For example, the order in which components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.

[000291] The claimed stable or preserved formulations may be provided to patients as clear solutions or as double vials comprising one vial of lyophilisate of at least one anti-GD2 antibody that is reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent medium. A single solution vial or double vial that requires reconstitution can be reused multiple times and may be sufficient for a single or multiple patient treatment cycles and thus provide a more convenient treatment regimen than those currently available.

[000292] At least one anti-GD2 antibody in the stable or preserved formulations or solutions described herein can be administered to a patient in accordance with the present invention through a variety of delivery methods, including SC or IM injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micropump or other well-known means appreciated by those skilled in the art.

[000293] In one embodiment of the present invention, pharmaceutical compositions comprising an anti-GD2 antibody of the description facilitate the administration of humanized antibodies to an organism, preferably an animal, more preferably a mammal. Particular mammals include cattle, canines, horses, felines, sheep, swine, and nonhuman primates and humans. Humans are particularly preferred.

[000294] Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient will normally be present in an amount of about 0.5 to 99.999% by weight based on the total weight of the composition.

[000295] For parenteral administration, the antibody can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or provided separately, with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1 to 10% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The lyophilized vehicle or powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized using known or suitable techniques.

[000296] Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences A. Osol, a standard reference text in this field.

[000297] Formulations for parenteral administration may contain, as common excipients, sterile water or saline solution, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and so on. Aqueous or oily injection suspensions can be prepared using a suitable emulsifier or humidifier and suspending agent in accordance with known methods. Agents for injection may be a non-toxic diluting agent not for oral administration, such as an aqueous solution or a sterile injectable solution or suspension in a solvent. As the usable vehicle or solvent, water, Ringer's solution, isotonic saline, etc. They are allowed; as normal solvent or suspending solvent, sterile non-volatile oil can be used. For this purpose, any type of fatty acid and non-volatile oil can be used, including natural or synthetic fatty oils or semi-synthetic fatty acids; natural or synthetic or semi-synthetic mono-, di- or tri-glycerides. Parental administration is known in the art and includes, but is not limited to, conventional means of injection, a pressurized needleless injection device as described in United States Patent No. 5,851,198, and a laser piercing device as described in U.S. Pat. United States No. 5,839,446, incorporated herein in its entirety by reference.

Combined Therapy

[000298] In some embodiments, the provided anti-GD2 antibodies are optionally administered further comprising at least one selected from at least one TNF antagonist (e.g., but without limitation, an antibody to TNF or a fragment of a soluble TNF receptor). or fragment, fusion proteins thereof or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, sodium gold thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine) , a muscle relaxant, a narcotic, a Non-Steroid Anti-Inflammatory Drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial (e.g. aminoglycosides, an antifungal agent, an antiparasitic, an antiviral, a carbapenem, a cephalosporin, a fluoroquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a corticosteroid, an anabolic steroid, a diabetes-related agent, a mineral, a nutraceutical, a thyroid agent, a vitamin, a calcium-related hormone, an erythropoietin (e.g., epoetin alfa), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressant (e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, an antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analogue, domase alfa (Pulmozyme), a cytokine or a cytokine antagonist. Suitable dosages are well known in the art. See, for example, Wells et al., eds, Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), each of which is incorporated herein in its entirety by reference.

[000299] Such anti-cancer or anti-infective agents may also include toxin molecules that are associated, linked, co-formulated or co-administered with at least one antibody of the present invention. The toxin may optionally act to selectively kill the pathological cell or tissue. The pathological cell may be a cancer or another cell. Such toxins may be, but are not limited to, a purified or recombinant toxin or toxin fragment comprising at least one cytotoxic functional domain of the toxin, for example, selected from at least one of ricin, diphtheria toxin, a poison toxin or a toxin. bacterial. The term toxin also includes endotoxins and exotoxins, both produced by any naturally occurring, mutant or recombinant bacteria or viruses that can cause any pathological condition in humans and other mammals, including toxic shock, which can result in death. Such toxins may include, but are not limited to, E. coli heat-sensitive enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome-1 toxin (TSST). -1), staphylococcal enterotoxin A (SEA), B (SEB) or C (SEC), streptococcal enterotoxins and so on. Such bacteria include, but are not limited to, strains of an enterotoxigenic E. coli species (ETEC), enterohemorrhagic E. coli (e.g., strains of serotype 0157:h7), Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcus pyogenes ), Shigella species (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Salmonella species (e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella enteritidis), Clostridium species (e.g., Clostridium perfringens, Clostridium difficile , Clostridium botulinum), Camphilobacter species (e.g., Camphlobacter jejuni, Camphlobacter fetus), Helicobacter species, (e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae, Vibrios parahemolyticus), Klebsiella species, Pseudomonas aeruginosa, and Streptococci. See, for example, INTERNAL MEDICINE, 3rd ed., pages 1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds., Bacterial Infections of Humans: Epidemiology and Control, 2nd Ed., pages 239-254, Plenum Medical Book Co., New York (1991); Mandell et al., Principles and Practice of Infectious Diseases, 3rd Ed., Churchill Livingstone, N.Y. (1990); Berkow et al., eds., The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Wood et al., FEMS Microbiology Immunology, 76: 121-134 (1991); Marrack et al., Science, 248: 705-711 (1990), the content of such citations being incorporated herein entirely by reference.

Production

[000300] At least one anti-GD2 antibody used in accordance with the present invention can be produced by recombinant means, including from mammalian cells or transgenic preparations, or can be purified from other biological sources, as described herein or as known in the art.

[000301] Furthermore, taking into account the foregoing, the present invention provides a host cell comprising an isolated or purified nucleic acid molecule described above, optionally in the form of a vector. It is more preferable that the cell of the present invention expresses the vector, so that the oligonucleotide or a fragment thereof is efficiently transcribed and translated by the cell. Examples of cells include, but are not limited to, a human cell, a human cell line, E. coli (e.g., E. coli TB-1, TG-2, DH5α, XL-Blue MRF' (Stratagene), SA2821 and Y1090), B. subtilis, P. aerugenosa, S. cerevisiae, N. crassa, insect cells (e.g., Sf9, EA4) and others presented here below. The host cell may be present in a host, which may be an animal, such as a mammal, in particular a human being.

[000302] In a specific embodiment, using routine recombinant DNA techniques, one or more of the CDRs identified here can be inserted within "framework" regions. The framework regions may be naturally occurring or consensus framework regions and, preferably, human framework regions (see, for example, Chothia et al., J. Mol Biol 278: 457-479 (1998) for a list of human framework regions). Preferably, the polynucleotide generated by combining the framework regions and CDRs encodes an antibody that specifically binds to GD2. One or more amino acid substitutions can be made within the framework regions, and preferably the amino acid substitutions improve binding of the antibody to its antigen. Furthermore, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues that participate in an intrachain disulfide bond to generate antibody molecules that lack one or more intrachain disulfide bonds. Other changes to the polynucleotide are encompassed by the present invention and are within the scope of skill in the art.

applications

[000303] A chimeric or human high-affinity GD2 neutralizing antibody would be desirable for use in diseases where GD2 is expressed, for example, GD2 is expressed in >50% in melanoma (Zhang et al., 1997, Int. J. Cancer 73, 42-49), 88% in osteosarcoma (Heiner et al., 1987, Cancer Res. 47, 5377-5388) and 93% in soft tissue sarcomas, including liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma and sarcoma of spindle cells (Chang et al., 1992, Cancer 70, 633-638), as well as brain tumors (Longee et al., 1991, Acta Neuropathol. 82, 45-54). Anti-GD2 antibodies have been tested in melanoma patients (Saleh et al., 1992, Hum Antibodies Hybridomas 3, 19-24; Cheung et al., 1987, J. Clin Oncol 5, 1430-1440; Choi et al., 2006, Cancer Immunol Immunother 55, 761-774), sarcomas (Choi et al., 2006, supra; Yeh et al., 1992, The fifth Asia and Oceania Congress of Nuclear Medicine and Biology Proceedings, page 104), lung cancer small cell tumors (Grant et al., 1996, Eur. J. Nucl. Med. 23, 145-149), brain tumors (Arbit et al., 1995, Eur. J. Nucl. Med. 22, 419 -426) , via IV injection, as well as compartmental therapy using Ommaya reservoirs (Kramer et al., 2007, J. Clin. Oncol 25, 5465-5470). GD2 is also a tumor target in retinoblastoma (Chantada et al., 2006, J. Pediatr. Hematol. Oncol. 28, 369-373) and HTLV-1-infected T cells in leukemia (Furukawa et al., 1993, PNAS USA 90, 1972-1976). In a preferred aspect, an anti-GD2 antibody of the disclosure can be used to treat neuroblastoma. Anti-GD2 antibodies or derivatives thereof can be used as a single agent or in combination with other therapeutic agents. Furthermore, these Mabs can be used as a chemical sensitizing agent, whereby their use can increase the therapeutic efficacy of cytotoxic agents. These antibodies can be used as a radiosensitizer, so their use can improve the effectiveness of radiation. They can also be used in combination with other immunotumor agents, such as IL-2, IL-12 and/or IFNalpha. Furthermore, anti-GD2 antibodies can be used in combination with other monoclonal antibodies, such as anti-TNF-alpha, IL-12/IL-23, IL-2, GPIIb/IIIa receptor, CD52, CD20, RSV, HER2/neu receptor and so on; as well as with commercially approved antibodies, including Rituxan, Herceptin, Mylotarg, Campath, Zevalin, Bexxar, Erbitux, Avastin and Vetibix.

[000304] Thus, the present invention also provides a method for modulating or treating at least one GD2-related disease in a cell, tissue, organ, animal or patient, as known in the art or as described herein, using at least one anti-GD2 antibody. -GD2 of the present invention.

[000305] Adoptive immunotherapy trials in 1986 using lymphokine-activated killer cells (LAK) and tumor-infiltrating lymphocytes (TIL) reported occasional tumor responses in patients. Donor lymphocyte infusion has shown even greater success in patients with chronic myeloid leukemia after allogeneic stem cell transplantation or patients with post-transplant EBV-associated lymphoproliferative disease (Post-Transplant Lymphoproliferative Disease - PTLD). In solid tumors, CTL has been successful in treating malignant melanoma during the lymphopenic phase obtained through high-dose chemotherapy. Bispecific antibodies are made by fusing two hybridomas to create hybrid immunoglobulin molecules with two binding sites. Antibodies don't just arrest T-cell tumors; they cross-link with CD3 on T cells and initiate the activation cascade. In this way, TCR-based cytotoxicity is redirected to desired tumor targets bypassing MHC restrictions. Arming of activated T cells (Activated T Cells - ATCs) polyclonally with anti-CD3 x anti-TAA (BsAb or BiTE antibody) combines the targeting specificity of MoAb (e.g., hu3F8, where TAA is GD2) with cytotoxicity perforin/Granzyme-mediated non-MHC restricted T cells. BsAb or BiTE can ex vivo arm expanded activated T cells prior to infusion into a patient. This strategy converts each ATC into a specific CTL (Thakur and Lum, 2010, Curr Opin Mol Ther 12, 340-349; Grabert et al., 2006, Clin Cancer Res 12, 569-576).

[000306] Tumors escape T cells through a number of mechanisms: low or no MHC expression (e.g., NB), dysfunction of T cell signaling, decreased presentation of tumor peptides on MHC, absence of co-stimulatory molecules and induction of regulatory T cells that inhibit CTL and humoral responses. Since killing effected by BsAb or BiTE-armed ATCs is not MHC restricted, this strategy should overcome some of these tumor escape mechanisms. Tumors secrete TGF-β by shifting the immune response from T cells to a Th2 type, downregulating the secretion of interleukin 2 (IL-2) and IFN-Y, while upregulating IL-10 and IL- 6, all leading to immune suppression. T cells redirected by BsAb or BiTE may bypass these negative effects of regulatory cytokines, as ATC armed with BiTE targets IL-2 independently. Patients treated with BsAb or BiTE-armed T cells targeting their tumors have increased levels of TNF-α and IFN-Y, which would bias T cells toward a Th1 response. Furthermore, cytotoxic T cells kill through their Fas ligand (FasL) that couples to Fas receptors (CD95) on tumor cells. Unfortunately, FasL in tumor cells can also induce T cell apoptosis. TCR stimulation through the CD3 cascade protects CD8+ cells from CD95-mediated suicide. Armed ATCs resist CD95-induced cell death by cross-linking the TCR with BsAb or BiTE. The ability of T cells to serially kill, that is, one T cell consecutively kills target tumors, proliferates in the process, and moves into lymphatics and soft tissues has increased the possibility of taking NB cells, as they metastasize, out. from the marrow space to form tumor masses. Recent studies using targeting human cancers with BsAb or BiTE have shown promise.

[000307] There is growing evidence, in particular from analyzes of patients who have received allogeneic hematopoietic cell transplants, that supports the potential of T cells to suppress or eradicate lymphomas and certain forms of leukemia (Reilly et al., 2010, Semin Immunol 22, 162-172). However, there are no convincing data supporting a role for T cells in controlling solid tumors in children. This is consistent with the fact that several of these tumors do not express inherited HLA class I or II alleles (e.g., neuroblastoma) (Raffaghello et al., 2005, Oncogene 24, 46344644; Wolfl et al., 2005, Cancer Immunol . Immunother 54, 400-406) or express only class I alleles and at low levels (e.g. rhabdomyosarcoma) (Prados et al., 2006, Neoplasma 53, 226-231). Furthermore, expression of important costimulatory molecules, such as B7.1 and ICAM-1, is often low or undetectable. As a result, the ability of these tumors to induce T cell responses is poor and the potential for effector T cells to engulf the tumors, through binding to T cell receptor tumor antigens presented by HLA alleles, is limited. Furthermore, the most effective therapies currently available for neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma, and desmoplastic small round cell tumors employ alkylating immunosuppressive agents, particularly cyclophosphamide, at doses that induce profound T-lymphopenia. Bifunctional antibodies enable targeted engagement of T cells and exploration of their effector functions through non-HLA-restricted CD3-mediated activation rather than their antigen-specific HLA-restricted TCRs. Studies of certain bifunctional monoclonal antibodies specific for CD3 and a tumor antigen, such as CD-19, HER-2 neu or CEA, have demonstrated the ability of these antibodies to connect cytotoxic T cells to tumor cells expressing the other target antigen. (Bargou et al., 2008, Science 321, 974-977; Topp et al., 2009, Blood (ASH Annual Meeting Abstracts) 114, 840; Kiewe et al., 2006, Clin Cancer Res 12, 3085-3091; Lutterbuese et al., 2009, J Immnother 32, 341352). Once both antibody receptors are involved, a cytotoxic T cell response is initiated against the tumor cells. The T cell response involves the formation of a cytotoxic synapse between the T cell receptor and the tumor cell, as well as induction of perforin- and granzyme-mediated apoptosis of the tumor cells (Offner et al., 2006, Mol Immunol 43, 763-771; Brischwein et al., 2006, Mol Immunol 43, 1129-1143). CD3 engagement also activates T cells, inducing proliferation and production of effector cytokines that enhance the antitumor effect (Brischwein et al., 2006, supra; Brischwein et al., 2007, J Immunother 30, 798-807). Surprisingly, activated T cells upregulate an anti-apoptotic protein, C-FLIP, which protects them against the cytotoxic effects of TNF and the Fas ligand generated during T cell activation (2002, Dreir et al., Int J Cancer 100, 690-697). As a result, the T cell response is amplified. As a consequence, picogram levels of the bifunctional antibody can exert significant antitumor effects in vitro (Lutterbuese et al., 2009, supra; Brandl et al., 2007, Cancer Immunol Immunother 56, 1551-1563) in vivo, as shown in models preclinical animals and particularly in the results of early clinical trials of bispecific CD3/CD19 in the treatment of B-cell lymphomas and ALL (Topp et al., 2009, supra; Kiewe et al., 2006, supra). It is postulated that induced T cell responses may also recruit naïve T cells and stimulate the generation of tumor-specific T cells at tumor sites (Koehne et al., 2002, Blood 99, 1730-1740). Bispecific antibodies can also be used to target effector cells other than T lymphocytes. These effector cells include NK cells, B lymphocytes, dendritic cells, macrophages, monocytes, neutrophils, mesenchymal stem cells, neural stem cells, and others. stem cells for cells, tissues or organs that express GD2. When the tissue is a tumor, effector cells can be exploited to kill or deposit proteins (e.g., cytokines, antibodies, enzymes, or toxins), radioactive isotopes for diagnosis or therapy. When the tissue is a normal organ, effector cells can be similarly exploited to deliver proteins or isotopes for diagnosis or therapy.

[000308] The present invention includes a method for modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient including, but not limited to, at least one of: multiple myeloma, leukemia, acute leukemia, acute lymphoblastic leukemia (Acute Lymphoblastic Leukemia - ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (Acute Myeloid Leukemia - AML), chronic myelocytic leukemia (Chronic Myelocytic Leukemia - CML), chronic lymphocytic leukemia (Chronic Lymphoctyic Leukaemia - CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, cell carcinoma renal, pancreatic carcinoma, prostate carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignant character, solid tumors, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, cancer related to bone resorption, bone pain related to cancer ; suppression of cancer metastasis; improvement of cachexia in cancer; and the treatment of inflammatory diseases, such as mesangial proliferative glomerulonephritis and so on. Such a method may optionally be used in combination with, through administration before, concurrently or after administration of such an antibody to GD2, radiotherapy, an antiangiogenic agent, a chemotherapeutic agent, a farnesyl transferase inhibitor or the like.

[000309] The present invention also provides a method for modulating or treating at least one GD2-related immune disease, in a cell, tissue, organ, animal or patient including, but not limited to, at least one of rheumatoid arthritis, juvenile rheumatoid arthritis , juvenile-onset systemic rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis/optic neuritis /uveitis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener's granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures, allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, pneumonitis hypersensitivity, transplants, rejection of transplanted organs, graft versus host disease, systemic inflammatory response syndrome, sepsis syndrome, Gram positive sepsis, Gram negative sepsis, culture negative sepsis, fungal sepsis, febrile neutropenia, urosepsis, meningococcemia, trauma /hemorrhage, burns, radiation exposure, acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis, alcohol ionizing hepatitis, chronic inflammatory pathologies, sarcoidosis, Crohn's disease, sickle cell anemia, diabetes, nephrosis, atopic diseases, hypersensitivity reactions , allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic disease, thrombocytopenia, organ or tissue graft rejection, kidney transplant rejection, rejection heart transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, bone marrow transplant rejection (BMT), skin graft rejection, cartilage transplant rejection, acute graft rejection, intestinal transplant rejection, fetal thymus implant rejection, parathyroid transplant rejection, xenotransplant rejection of an organ or tissue, graft rejection, antireceptor hypersensitivity reactions, Graves' disease, Raynoud's disease, type diabetes Insulin-resistant B, asthma, myasthenia gravis, antibody-mediated cytotoxicity, type III hypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome (polyneuropathy syndrome, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes), polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin change syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus, chronic active hepatitis, primary biliary cirrhosis, vitiligo, vasculitis, post-MI cardiotomy, type IV hypersensitivity, contact dermatitis, hypersensitivity pneumonitis, graft rejection, granulomas due to intracellular organisms, drug sensitivity, metabolic/idiopathic syndrome, Wilson's disease, hemachromatosis, alpha-deficiency 1-antitrypsin, diabetic retinopathy, Hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis assessment, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, chronic neonatal lung disease, chronic obstructive pulmonary disease (COPD), lymph -familial hematophagocytic histiocytosis, dermatological conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerular nephritis, acute renal failure, hemodialysis, uremia, toxicity, pre-eclampsia, OKT3 therapy, anti-CD3 therapy, cytokine therapy, chemotherapy, radiotherapy (e.g., including but not limited to asthenia, anemia, cachexia, and so on), chronic salicylate poisoning, sleep apnea, obesity, heart failure, sinusitis, inflammatory bowel disease, and so on. See, for example, Merck Manual, 12th-17th Editions, Merck & Company, Rahway, N.J. (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Hand-book, Wells et al., eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each fully incorporated by reference.

[000310] The present invention also provides a method for modulating or treating at least one infectious disease in a cell, tissue, organ, animal or patient including, but not limited to, at least one of: acute or chronic bacterial infection, parasitic processes or acute and chronic infectious diseases, including bacterial, viral and fungal infections, HIV infection/HIV neuropathy, meningitis, hepatitis (A, B or C or similar), septic arthritis, peritonitis, pneumonia, epiglottitis, E. coli 0157: h7, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, Mycobacterium tuberculosis, intracellular Mycobacterium avium, Pneumocystis carinii pneumonia, inflammatory disease pelvic, orchitis/epididymitis, Legionella, Lyme disease, influenza A, Epstein-Barr virus, hemaphagocytic syndrome, vital encephalitis/aseptic meningitis and so on.

[000311] Any of such methods may optionally comprise administering an effective amount of at least one composition or pharmaceutical composition comprising at least one anti-GD2 antibody to a cell, tissue, organ, animal or patient in need of such modulation. , treatment or therapy.

[000312] Any method of the present invention may comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-GD2 antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy . Such method may optionally further comprise co-administration or combined therapy for the treatment of such autoimmune diseases or malignancies, wherein administering said at least one anti-GD2 antibody, specified portion or variant thereof further comprises administering before, simultaneously and /or after at least one agent selected from a TNF antagonist (e.g., but without limitation, an antibody to TNF or fragment thereof, a soluble TNF receptor or fragment thereof, fusion proteins thereof or a TNF antagonist). small molecule TNF), an antibody to IL-18 or a fragment thereof, a small molecule antagonist of IL-18 or IL-18 receptor binding protein, an antagonist antibody to IL-1 (including IL-1 alpha and IL-1 beta) or a fragment thereof, a soluble IL-1 receptor antagonist, an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine , radiotherapy, an antiangiogenic agent, a chemotherapeutic agent, Thalidomide, a muscle relaxant, a narcotic, a non-steroidal anti-inflammatory drug (Non-Steroid Anti-Inflammatory Drug - NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial (e.g., aminoglycosides, an antifungal agent, an antiparasitic, an antiviral, a carbapenem, a cephalosporin, a fluoroquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial - biano), an antipsoriatic, a corticosteroid, an anabolic steroid, a diabetes-related agent, a mineral, a nutraceutical, a thyroid agent, a vitamin, a calcium-related hormone, an erythropoietin (e.g. epoetin alfa), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressant (e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone replacement, an estrogen receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, an antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analogue, domase alfa (Pulmozyme), a cytokine or an antagonist of cytokine. Suitable dosages are well known in the art. See, for example, Wells et al., eds, Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), each of which is incorporated herein in its entirety by reference.

[000313] TNF antagonists suitable for compositions, combined therapy, co-administration, devices and/or methods of the present invention (further comprising at least one antibody, specified portion and variant thereof, of the present invention) include, but are not limited to, anti-antibodies. -TNF, antigen-binding fragments thereof and receptor molecules that specifically bind to TNF; compounds that prevent and/or inhibit the synthesis of TNF, the release of TNF or its action on target cells, such as thalidomide, tenidape, phosphodiesterase inhibitors (e.g. pentoxifylline and rolipram), adenosine A2b receptor agonists and potentiators of adenosine A2b receptor; compounds that prevent and/or inhibit signaling to the TNF receptor, such as mitogen-activated protein kinase inhibitors (MAP); compounds which block and/or inhibit the cleavage of membrane TNF, such as metalloproteinase inhibitors; compounds that block and/or inhibit the activity of TNF, such as angiotensin converting enzyme (ACE) inhibitors (for example, captopril); and compounds that block and/or inhibit the production and/or synthesis of TNF, such as MAP kinase inhibitors.

[000314] Any method of the present invention may comprise a method for treating a GD2-mediated disorder or a disorder characterized by the expression of GD2 comprising administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-GD2 antibody. to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. Such method may optionally further comprise co-administration or combined therapy for the treatment of such immune diseases, wherein administration of said at least one anti-GD2 antibody, specified part or variant thereof, further comprises administering, before simultaneously and/or after , of at least one agent, as described above.

[000315] Typically, treatment of pathological conditions is accomplished by administering an effective amount or dosage of at least one anti-GD2 antibody composition whose total range, on average, will be at least about 0.01 to 500 milligrams of at least one anti-GD2 antibody per kilogram of patient per dose, and preferably at least about 0.1 to 100 milligrams of antibody/kilogram of patient per single or multiple administration, depending on the specific activity of the antibody contained in the composition. Alternatively, the effective serum concentration may comprise 0.1 to 5000 μg/ml serum concentration per single or multiple administration. Suitable dosages are known to those skilled in the art and will, of course, depend on the particular pathological state, the specific activity of the composition to be administered and the particular patient being treated. In some cases, to achieve the desired therapeutic amount, it may be necessary to provide repeated administration, that is, repeated individual administrations of a particular metered or monitored dose, where individual administrations are repeated until the desired daily dose or effect is achieved.

[000316] Preferred doses may optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100 to 500 mg/kg /administration or any range, value or fraction thereof to achieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2 ,0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0 , 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5 , 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5 ,0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0 , 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9 , 14, 14.5, 15, 15.5, 15.9, 1.6, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19 , 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1500, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 μg/ml of serum by single or multiple administrations or any range, value or fraction thereof.

[000317] Alternatively, the dosage administered may vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; age, health and weight of the recipient; nature and extent of symptoms, type of concomitant treatment, frequency of treatment and desired effect. Typically, an active ingredient dosage can be about 0.1 to 100 milligrams per kilogram of body weight. Usually 0.1 to 50 and preferably 0.1 to 10 milligrams per kilogram per administration or in extended release form is effective in obtaining the desired results.

[000318] As a non-limiting example, treatment of humans or animals can be provided as a one-time dosage or periodic dosage of at least one antibody of the present invention at 0.1 to 100 mg/kg, such as 0. 5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg per day in at least one on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or, alternatively or additionally, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 or, alternatively or additionally , at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 years old or any combination thereof , using a single or repeated doses of infusion.

[000319] The invention further relates to the administration of at least one anti-GD2 antibody by parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intra-abdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intrathecal, intra-Ommaya, intraocular, intravitreal, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal or transdermal. At least one anti-GD2 antibody composition may be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) administration or any other administration, particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration, in semisolid forms, in particular such as, but not limited to, creams and suppositories; for buccal or sublingual administration such as, but not limited to, in the form of tablets or capsules; or intranasally such as, but without limitation, in the form of powders, nasal drops or aerosols or certain agents; or transdermally such as, but not limited to, a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers, such as dimethyl sulfoxide, to modify the structure of the skin or increase the drug concentration in the transdermal patch (Junginger, et al. in "Drug Permeation Enhancement"; Hsieh, D. S., Eds., pages 59-90, Marcel Dekker, Inc. New York 1994, incorporated herein in its entirety by reference) or with oxidizing agents that allow the application of formulations containing proteins and peptides to the skin (WO 98/53847) or applications of electric fields to create transient transport pathways, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or application of ultrasound, such as sonophoresis (United States Patent Nos. 4,309,989 and 4,767,402) (the above publications and patents being incorporated herein in their entirety by reference).

[000320] For pulmonary administration, preferably, composition of at least one anti-GD2 antibody is distributed in a particle size effective to reach the lower respiratory tract of the lung or nasal sinuses. According to the invention, at least one anti-GD2 antibody can be delivered through any of a variety of inhalation or nasal devices known in the art for administering a therapeutic agent by inhalation. These devices capable of depositing aerosol formulations into a patient's sinus cavity or alveoli include metered dose inhalers, nebulizers, dry powder generators, sprayers, and so on. Other devices suitable for directing pulmonary or nasal administration of antibodies are also known in the art. All of these devices can use formulations suitable for administration to deliver antibodies in an aerosol. Such aerosols can be comprised of solutions (both aqueous and non-aqueous) or solid particles. Metered dose inhalers, such as the Ventolin® metered dose inhaler, typically use a propellant gas and require actuation during inspiration (see, for example, documents WO 94/16970, WO 98/35888). Dry powder inhalers, such as TURBUHALER™ (Astra), ROTAHALER® (Glaxo), Diskus® (Glaxo), devices marketed by Inhale Therapeutics, to name a few, use breath actuation of a mixed powder (U.S. Patent No. No. 4,668,218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, United States Patent No. 5,458,135 Inhale, WO 94/06498 Fisons, all incorporated herein in their entirety by reference) . Nebulizers, such as the ULTRAVENT® nebulizer (Mallinckrodt) and the ACORN II® nebulizer (Marquest Medical Products) (United States Patent No. 5,404,871 Aradigm, WO 97/22376), the above citations incorporated herein in their entirety by reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. generate aerosols of small particles. These specific examples of commercially available inhalation devices are intended to be representative of specific devices suitable for practicing the present invention and are not intended to limit the scope of the invention. Preferably, a composition comprising at least one anti-GD2 antibody is delivered by a dry powder inhaler or a spray. There are several desirable features of an inhalation device for administering at least one antibody of the present invention. For example, delivery by the inhalation device is advantageously reliable, reproducible and accurate. The inhalation device may optionally deliver small dry particles, e.g., of less than about 10 μm, preferably about 1-5 μm, for good breathability.

[000321] A spray including the anti-GD2 antibody protein composition can be produced by forcing a suspension or solution of at least one anti-GD2 antibody through a nozzle under pressure. Nozzle size and configuration, applied pressure, and liquid feed rate can be chosen to achieve the desired particle size. An electrospray can be produced, for example, by an electric field, in connection with a nozzle or capillary supply. Advantageously, particles of at least one anti-GD2 antibody protein composition delivered by a spray have a particle size of less than about 10 μm, preferably in the range of about 1 μm to about 5 μm, and more preferably from about 2 μm to about 3 μm.

[000322] Formulations of at least one anti-GD2 antibody protein composition suitable for use with a spray typically include an antibody protein composition in an aqueous solution at a concentration of about 0.1 mg to about 100 mg of at least one anti-GD2 antibody protein composition per ml of solution or mg/gm or any range or value thereof, for example, but without limitation, 0.1, 0.2, 0.3, 0, 4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/mL or mg/gm. The formulation may include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant and, preferably, zinc. The formulation may also include an excipient or agent for stabilizing the antibody protein composition, such as a buffer, a reducing agent, a dense protein or a carbohydrate. Dense proteins useful in formulating the antibody protein composition include albumin, protamine or the like. Typical carbohydrates useful in formulating the antibody protein composition include sucrose, mannitol, lactose, trehalose, glucose or the like. The formulation of the antibody protein composition may also include a surfactant, which may reduce or prevent aggregation induced on the surface of the at least one anti-GD2 antibody caused by atomization of the solution in the formation of an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene esters of fatty acids and alcohols, and polyoxyethylene sorbitol fatty acid esters. In general, amounts will vary between 0.001 and 4% by weight of the formulation. Especially preferred surfactants for purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20 or the like. Additional agents known in the art for formulating a protein, such as antibodies, or specified GD2 portions or variants may also be included in the formulation.

[000323] The antibody protein composition can be administered by a nebulizer, such as a jet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air source is used to create a high-velocity jet of air through an orifice. As the gas expands beyond the nozzle, a low pressure region is created which draws a solution of antibody protein composition through a capillary tube connected to a liquid reservoir. The liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol. A variety of configurations, flow rates and baffle types can be employed to achieve the desired performance characteristics of a particular jet nebulizer. In an ultrasonic nebulizer, high-frequency electrical energy is used to create vibrational mechanical energy, typically employing a piezoelectric transducer. This energy is transmitted to the antibody protein composition formulation, either directly or through a coupling fluid, creating an aerosol that includes the antibody protein composition. Advantageously, particles of the antibody protein composition delivered by a nebulizer have a particle size of less than about 10 μm, preferably in the range of about 1 μM to about 5 μm, and even more preferably about 2 μm to about 3 μm.

[000324] Formulations of at least one anti-GD2 antibody suitable for use with a nebulizer, whether jet or ultrasonic, typically include a concentration of about 0.1 mg to about 100 mg of at least one antibody protein anti-GD2 per ml of solution. The formulation may include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant and, preferably, zinc. The formulation may also include an excipient or agent for stabilizing at least one protein composition of the anti-GD2 antibody, such as a buffer, a reducing agent, a dense protein or a carbohydrate. Dense proteins useful in formulating at least one anti-GD2 antibody protein composition include albumin, protamine or the like. Typical carbohydrates useful in formulating at least one anti-GD2 antibody include sucrose, mannitol, lactose, trehalose, glucose or the like. The at least one anti-GD2 antibody formulation may also include a surfactant, which can reduce or prevent aggregation induced on the surface of the at least one anti-GD2 antibody caused by atomization of the solution in the formation of an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene esters of fatty acids and alcohols, and polyoxyethylene sorbitol fatty acid esters. In general, amounts will vary between 0.001 and 4% by weight of the formulation. Especially preferred surfactants for purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20 or the like. Additional agents known in the art for formulating a protein, such as antibody protein, may also be included in the formulation.

[000325] In a Metered Dose Inhaler (MDI), a propellant, at least one anti-GD2 antibody and any excipients or other additives are contained in a can as a mixture including a liquefied compressed gas. Actuation of the metering valve releases a mixture as an aerosol, preferably containing particles in the size range of less than about 10 μM, preferably about 1 μM to about 5 μm, and most preferably about 2 μm to about 3 μm. The desired aerosol particle size can be obtained by employing a protein formulation with the antibody composition produced by various methods known to those skilled in the art, including jet milling, spray drying, critical point condensation, or the like. Preferred metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant.

[000326] Formulations of at least one anti-GD2 antibody for use with a metered dose inhaler device will generally include a finely divided powder containing at least one anti-IL-6 antibody as a suspension in a non-aqueous medium, e.g. example, suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a ( hydrofluoroalkanes-134a), HFA-227 (hydrofluoroalkane-227) or similar. Preferably, the propellant is a hydrofluorocarbon. The surfactant may be chosen to stabilize at least one anti-GD2 antibody as a suspension in the propellant to protect the active agent against chemical degradation and so on. Suitable surfactants include sorbitan trioleate, soy lecithin, oleic acid or the like. In some cases, aerosols using solvent solutions such as ethanol are preferred. Additional agents known in the art for formulating a protein may also be included in the formulation.

[000327] Those skilled in the art will recognize that the methods of the present invention can be obtained by means of pulmonary administration of at least one anti-GD2 antibody composition by means of devices not described here.

[000328] Formulations for oral administration rely on the co-administration of adjuvants (for example, resorcinols and non-ionic surfactants, such as polyoxyethylene oleyl ether and polyethylene n-hexadecyl ether) to artificially increase the permeability of the intestinal walls , as well as coadministration of enzyme inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF), and trasylol) to inhibit enzymatic degradation. The active constituent compound of the solid-type dosage form for oral administration may be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers and glycerides. These dosage forms may also contain other type(s) of additives, for example, an inactive diluting agent, a lubricant such as magnesium stearate, paraben, a preservative agent such as sorbic acid, ascorbic acid , alpha-tocopherol, an antioxidant such as cysteine, a disintegrant, binder, thickener, buffering agent, sweetening agent, flavoring agent, flavoring agent, etc.

[000329] Tablets and pills can be further processed into enteric-coated preparations. Liquid preparations for oral administration include permitted emulsions, syrup, elixir, suspension and solution of preparations for medical use. These preparations may contain inactive diluting agents normally used in said field, for example, water. Liposomes have also been described as drug delivery systems for insulin and heparin (United States Patent No. 4,239,754). More recently, artificial polymer microspheres of mixed amino acids (proteinoids) have been used for drug delivery (United States Patent No. 4,925,673). Furthermore, carrier compounds described in United States Patent No. 5,879,681 and United States Patent No. 5,871,753 are used for delivery of orally biologically active agents known in the art.

[000330] For absorption through mucosal surfaces, compositions and methods of administering at least one anti-GD2 antibody include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide and a continuous aqueous phase, which promotes absorption across mucosal surfaces via mucoadhesion of emulsion particles (United States Patent No. 5,514,670). Suitable mucosal surfaces for application of the emulsions of the present invention may include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomach, intestinal and rectal routes of administration. Formulations for rectal or vaginal administration, e.g., suppositories, may contain, as excipients, e.g., polyalkylene glycols, petroleum jelly, cocoa butter, and so on. Formulations for intranasal administration may be solid and contain, for example, lactose as excipients, or may be aqueous or oily solutions of nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch and so on (United States Patent No. 5,849,695).

[000331] For transdermal administration, at least one anti-GD2 antibody is encapsulated in a delivery device, such as a liposome or polymeric nanoparticles, microparticle, microcapsule or microspheres (collectively referred to as microparticles, unless otherwise indicated). A number of suitable devices are known, including microparticles made from synthetic polymers, such as polyhydroxy acids, such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides and polyphosphazenes, and natural polymers, such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (United States Patent No. 5,814,599).

[000332] Sometimes, it may be desirable to administer the compounds of the present invention to the individual over extended periods of time, for example, for periods of a week to a year or more from a single administration. Various slow-release, depot, or implant dosage forms can be used. For example, a dosage form may contain a pharmaceutically acceptable non-toxic salt of the compounds that have a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid, such as acid phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acids, polygalacturonic acid and so on; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and so on or with an organic cation formed, for example, from N ,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), for example, a zinc tannate salt. Additionally, the compounds of the present invention or, preferably, a relatively insoluble salt, such as those just described, can be formulated in a gel, for example, an aluminum monostearate gel, for example, with sesame oil, suitable for injection. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts and so on. Another type of slow-release depot formulation for injection will contain the compound or dispersed salt encapsulated in a non-toxic, non-antigenic, slow-degrading polymer, such as a polylactic acid/polyglycolic acid polymer, for example, as described in the U.S. Pat. United States No. 3,773,919. Compounds or, preferably, relatively insoluble salts, such as those described above, can also be formulated into silastic cholesterol matrix pellets, particularly for use in animals. Additional slow-release, depot, or implant formulations, e.g., gaseous or liquid liposomes, are known in the literature (United States Patent No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems", J.R. Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

[000333] The contents of all references (including bibliographical references, granted patents, published patent applications and co-pending patent applications) cited throughout this application are expressly incorporated herein by reference.

[000334] Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are provided for illustration of the invention and are not intended to be limiting thereof.

EXAMPLES

[000335] The invention will be further illustrated by the following non-limiting examples. These examples are presented to assist in understanding the invention, but are not intended to, and should not be construed to, limit its scope in any way. The examples do not include detailed descriptions of conventional methods that are well known to those skilled in the art (molecular cloning techniques, etc.). Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is indicated in degrees Celsius, and pressure is or is close to atmospheric.

Purification of murine 3F8 antibodies and Fab fragment preparation

[000336] Murine anti-GD2 MoAb 3F8 (IgG3) was purified from concentrated hybridoma supernatant as previously described (Cheung et al., 1985, Cancer Res 45, 2642-2649). m3F8 Fab fragments were generated via papain digestion using a standard Fab preparation kit (Pierce Biotechnology, Rockford, IL).

Crystallization and data collection

[000337] The purified 3F8 Fab fragment was concentrated to 12 mg/ml in 20 mM HEPES, pH 6.5 and was crystallized in a suspension drop via vapor diffusion at 16 ° C against a reservoir containing reagent Hampton Index D7 containing 0.1 M BIS-TRIS, pH 6.5, 25% PEG 3350 (Hampton Research, Aliso Viejo, CA). The droplet was formed by mixing 1 μl of protein solution and 1 μl of reservoir solution. The crystals were protected by cryoprotectant containing 25% glycerol, 0.1 M BIS-TRIS, pH 6.5, 25% PEG 3350. Data was collected in the Argonne Advanced Photon Source's 24IDC beam. The crystals belonged to space group C2 and had diffraction at a resolution of 1.65 Å.

Structure determination and refinement

[000338] The structure of the Fab was solved through molecular replacement with the 2AJU entry of the PDB search model using Phaser (CCP4 Suite) (Mccoy et al., 2007, J. Appl. Crystallogr 40, 658-674). The best molecular replacement model was refined using Refmac5 (Murshudov et al., 1997, Acta Crystallogr D 53, 240-255), manual assembly was performed with O (Bailey, S., 1994, Acta Crystallogr D 50, 760763), addition of solvent with Arp-Warp (Lamzin and Wildon, 1993, Acta Crystallogr D 49, 129-147). The final model contained two m3F8 Fab polypeptide chains and 585 solvent molecules. The final model was deposited in the Protein Data Bank (access code 3VFG).

Molecular docking and in silico mutagenesis simulations

[000339] GLIDE anchoring was performed using the Schrodinger Suite 2009 platform (Schrodin, New York, NY). OPLS force fields were used to parameterize the proteins and ligands. Plots of top ligands were clustered within a root mean square deviation of 2.0 Å and scored by GlideScore. CDOC-KER docking and interaction energy measurements were performed using Discovery Studio 3.0 (Accelerys, San Diego, CA). CHARMm force fields were used to parameterize the proteins and ligands. Plots of top ligands were clustered within a root mean square deviation of 2.0 Å and scored by CDOCKER Interaction Energy. For all docking studies involving GD2, the ceramide tail was replaced with a methyl group (data not shown). Docking simulations were performed under rigid body conditions where ligand conformations were docked onto proteins/antibodies with rigid side chains. The energy of the final docked complexes was minimized with CHARMm using the Smart Minimizer algorithm in Discovery Studio 3.0 (Accelerys, San Diego, CA). In silico mutagenesis was performed by calculating the free energy of binding of the antibody:anchored antibody model using CHARMm force fields and the Mutation Energy Calculation protocol in Discovery Studio 3.0 (Accelerys, San Diego, CA).

Image rendering

[000340] Molecular structure images were rendered with Pymol (Schrodinger, New York, NY) for docking studies or with Discovery Studio 3.0 (Accelerys, San Diego, CA) for surface electrostatic potentials.

Modeling exposed hydrophobic surface area

[000341] The antigen binding site of MoAb 3F8 and MoAb 3F8 H:Gly54Ile was modeled in Discovery Studio 3.0 (Accelrys, San Diego, CA). Exposed hydrophobic surfaces were rendered using the Spatial Aggregation Propensity algorithm developed by Chennamsetty et al. (Chennamsetty et al., 2009, Proc Natl Acad Sci USA 106, 1193799842), where stretches of dynamically exposed hydrophobicity effective on the surface of a protein are quantified and colored red.

Cell culture

[000342] The human neuroblastoma cell line LAN-1 was provided by Dr. Robert Seeger (Children's Hospital of Los Angeles). M14 and OCM-1 melanoma cell lines were provided by Dr. David Cobrinik (Children’s Hospital of Los Angeles). All cell lines were cultured in F10 RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C. in an incubator with 5% CO2.

Construction of hu3F8 and variants

[000343] Humanized 3F8 genes were synthesized by CHO cells (Blue Heron Biotechnology or Genscript), as previously described (Cheung et al., 2012, Oncoimmunology 1, 477-486). Using the Bluescript vector (Eureka, CA), these hu3F8 heavy and light chain genes were transfected into DG44 cells and selected with G418 (Invitrogen, CA).

Antibody purification

[000344] Lines producing chimeric Hu3F8 and 3F8 were grown in Opticho serum-free medium (Invitrogen) and the mature supernatant collected, as previously described (Cheung et al., 2012, supra). The Protein A affinity column was pre-equilibrated with 25 mM sodium citrate buffer with 0.15 M NaCl, pH 8.2. Bound Hu3F8 was eluted with 0.1 M citric acid/sodium citrate buffer, pH 3.9 and alkalinized (1:10 v/v ratio) in 25 mM sodium citrate, pH 8.5. It was passed through a Sarto-bind-Q membrane and concentrated to 5 to 10 mg/ml in 25 mM sodium citrate, 0.15 M NaCl, pH 8.2.

Quantification of GD2 binding by ELISA and flow cytometry

[000345] ELISA was performed as previously described (Cheung et al., 2012, supra). Microtiter plates were coated with GD2 at 20 ng per well. 150 μl per well of 0.5% BSA in PBS (diluent) was added to each plate for at least 30 min at room temperature to block excess binding sites. 100 μl of standard and diluted samples (2-fold) were added to each well and incubated for 2.5 h at 37°C. After washing the plates with PBS, 100 μL of goat anti-human IgG antibody (H+L) (Jackson Research Laboratory) diluted 1:3500 in diluent was added to each well and incubated for 1 h at 4 °C. The colored reaction in the ELISA was developed with the OPD chromogen (Sigma) with the substrate hydrogen peroxide for 30 minutes at room temperature in the dark. The reaction was stopped with 5N H2SO4 and the optical density (OD) was read in an ELISA MRX plate reader (Dynex) at 490 nm.

[000346] To measure the binding retention of MoAbs to antigen-containing cells, the antibodies were incubated with M14 melanoma cells and washed successively. Cells were initially collected at 1x106 cells per round bottom tube, centrifuged and washed with PBS and resuspended in 100 μl of PBS per test tube. Cells were incubated with hu3F8 or hu3F8-Ile MoAbs ((1 μg MoAb/1x106 cells) for 30 minutes at 4°C. Cells were then subjected to successive cycles of washing using 5 ml of PBS with 3°C EDTA. mM, followed by pelleting, discarding supernatant, and resuspension.With each successive wash, samples were incubated with R-Phycoerythrin (R-PE)-conjugated anti-human IgG, Fcy fragment-specific secondary antibody (Jackson ImmunoResearch) for 30 minutes at 4°C in the dark, washed, and then analyzed by flow cytometry using a FACS Calibur BD instrument. Samples were prepared in triplicate.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by 51chromium release

[000347] ADCC assays were performed using NK-92MI cells stably transfected with the Fc receptor, human CD16, as previously described (Cheung et al., 2012, supra). Target cells LAN1-1, M14, OCM-1, U2OS, CRL1427, NCI-H345 were released with 2 mM EDTA in PBS without Ca2+/Mg2+ and washed in F10, before radiolabeling with 51Cr for ADCC assays.

Statistical Analysis

[000348] Curve fitting and statistical analyzes were performed using GraphPad Prism 5.0. Student's t-test was used for significance calculations.

EXAMPLE 1 In silico scanning mutagenesis of the 3F8GD2 model

[000349] In silico scanning mutagenesis was performed by taking the 12 residues that directly interacted with GD2 in the docked 3F8:GD2 model (L:Tyr37, L:Lys55, L:Val99, L:Leu102, H:Gly40, H:Tyr31 , H:Asn32, H:Asn34, H:Ser56, H:Ser58, H:Gly97 and H:Met98) and analyzing the effect of single point mutations for all possibilities at each site. The models were energy minimized using CHARMm force fields, then analyzed for changes in interaction energies (electrostatic, van der Waals, entropic). The best mutations are shown in Table 1. Only four mutations were found to increase the interaction energy of the bound complex by more than 1 kcal/mol (Table 1). Only one point mutation was found to have substantially higher interaction energy (H:Gly54Ile) by a weighted mutation energy of ~8 kcal/mol. Most of this increase in interaction energy was due to an increase in van der Waals contact with the antigen. The effects of double-point and triple-point mutations involving the 12 interacting residues were also calculated, but no combination of additional mutations was found to increase the interaction energy. Table 1 presents the results of in silico scanning mutagenesis of CDR residues that directly interact with the anchored GD2 antigen. Energies are shown in units of kcal/mol. TABLE 1

Antigen binding site analysis of 3F8 and 3F8-Ile (H:Gly54Ile)

[000350] The single point mutation derived from in silico scanning mutagenesis simulations (H:Gly54Ile, termed 3F8-Ile) was modeled for the antigen binding site of 3F8 (data not shown). Due to the hydrophobic nature of the H:Gly54Ile mutation, analysis of the hydrophobicity of the antigen binding site was performed using the Spatial Aggregation Propensity algorithm (Materials and Methods), which provides a measure of the stretches exposed to the hydrophobic solvent). MoAb 3F8 has a hydrophobic stretch in the GD2 binding site that is centered around H:Ile56 (data not shown). H:Ile56 protrudes out of the binding pocket and may help the antibody interact with the surface of the membrane surrounding the GD2 head group. The substitution of H:Gly54 for Ile increases the exposed hydrophobic surface area of the antigen-binding site and also increases the van der Waals contact with GD2 in the docked model (data not shown).

Tumor cell binding and killing properties of hu3F8 and hu3F8- Ile H:Gly54Ile

[000351] To test whether the H:Gly54Ile mutation increases the affinity for GD2 and ADCC of tumor cells, the mutation was engineered into the recently described humanized 3F8 (hu3F8) (Cheung et al., 2012, supra). Hu3F8 is less immunogenic than murine 3F8, retains the structural features of murine 3F8 found in this investigation, and is currently in phase I clinical trials. Hu3F8 and hu3F8 H:Gly54Ile (hu3F8-Ile) were constructed, expressed, purified, and tested regarding the connection to GD2 and ADCC. ELISA assays with GS2 showed that hu3F8-Ile had a negligible increase in binding relative to the efficiency of hu3F8 (EC50 of GD2 binding: hu3F8 48 ± 13 ng/mL, hu3F8-Ile 38 ± 11 ng/mL) (data not shown ). To test the avidity of these antibodies for binding to GD2 in their native environment on the surface of tumor cells, a washing experiment was performed in which antibodies bound to the surface of M14, a GD2(+) melanone cell line, were subjected to to consecutive washing cycles with PBS-EDTA (see Materials and Methods). Hu3F8-Ile showed a greater ability to resist washing of tumor cells (t1/2 of hu3F8-Ile = 3 washes, t1/2 of hu3F8 = 2 washes) (data not shown). No other mutations were found to increase antigen binding.

[000352] Hu3F8 and hu3F8-Ile were then tested for their efficiency in mediating LAN-1 neuroblastoma ADCC in the presence of the NK-92MI natural killer cell line transfected with the Fc receptor, human CD16 (data no. shown). Hu3F8-Ile consistently showed a ~9-fold increase in cytotoxicity potency compared to hu3F8 (IC50 of cell death: hu3F8 1.35 ± 0.15 ng/mL, hu3F8-Ile 0.15 ± 0.01 ng/mL) . A 6-7-fold increase in ADCC potency against M14 and OCM-1 melanoma cells was also observed (Cell death IC50 of M14 cells: hu3F8 25 ± 2.2 ng/mL, hu3F8-Ile 3.7 ± 1 .1 ng/mL; IC50 of cell death of OCM-1 cells: hu3F8 8.5 ± 0.8 ng/mL, hu3F8-Ile 1.5 ± 0.1 ng/mL). These increases in ADCC potency for hu3F8-Ile relative to hu3F8 were highly significant (p < 0.001).

[000353] To further optimize the potential clinical efficacy of humanized 3F8, we employed high-throughput in silico scanning mutagenesis at key interacting residues in the 3F8:GD2 docked model. We identified a single point mutation (H:Gly54Ile) that showed a modest increase in binding affinity in ELISA-GD2 assays and an increase in the ability to retain hu3F8-Ile binding to GD2 on a cell surface. Most surprisingly, we showed that hu3F8 had a ~6-9-fold increase in ADCC of GD2-positive tumor cell lines, including neuroblastoma, melanoma, osteosarcoma, and small cell lung cancer. The nature of the H:Gly54Ile mutation increases the hydrophobic surface area exposed at the antigen-binding site. Since GD2 is embedded on the membrane surface via a ceramide moiety, the addition of an Ile to the antigen-binding site can enhance ADCC by increasing the ability of MoAb 3F8 to remain bound to the membrane surface, as observed in cell washing experiments.

[000354] Carbohydrate antigens play an important role in several biological processes. The development of antibodies to target carbohydrates is important for the investigation of tumors, bacteria, blood groups, cell-cell adhesion interactions, hormone receptors, and viral toxins; and glycosylation of recombinant proteins (Heimburg-Molinaro and Rittenhouse-Olson, 2009, Methods Mol. Biol. 534,341-357). Since the immune response to saccharides is independent of T cells, antibodies generated against carbohydrate antigens are often produced as low-affinity IgM antibodies (Heimburg-Molinaro and Rittenhouse-Olson, 2009, supra). In order to generate higher affinity antibodies for therapeutic application, such as in the case of cancer immunotherapy, affinity maturation techniques often need to be employed to improve the therapeutic effect. Traditional antibody affinity maturation methods, such as yeast/phage/ribosomal display, rely on error-prone PCR that cannot provide the full range of diversity in each of the amino acids in the antibody CDR. In this investigation, we showed that in silico scanning mutagenesis could be employed even if a high-resolution cocomplex structure is not available. Additionally, we demonstrate that a modest increase in affinity can improve the functional properties of MoAb 3F8 for therapeutic targeting of GD2 tumor antigen. Although an increase in ADCC is expected to translate into better efficacy, this will have to be proven in a future clinical trial in patients. The use of these in silico techniques may provide a valuable addition to traditional experimental methods in developing the next generation of MoAb for the diagnosis or treatment of not only cancer, but other human disorders where carbohydrate epitopes are drug-targetable targets. .

Design of new hu3F8 ver5 framework

[000355] The hu3F8 V1 framework structure (document WO 2011/160119, Cheung et al., 2012, supra) was optimized for reduced immunogenicity based on computational methods. First, the hu3F8V1 heavy chain and light chain sequences were compared with the human germline sequences humIGHV199 and humIGKV025, respectively (EMBL database, www.vbase2.org). Molecular simulations using CHARMm force fields (CHemistry at Harvard Molecular Mechanics) (Brooks et al., 2009, J. Comp. Chem. 30, 1545-1615) were performed on each mutation for potential humanization based on the crystal structure of 3F8 (access to the 3VFG protein database, http://www.pdb.org) to determine whether the mutation was structurally permissive. Furthermore, MHC class II T cell epitopes on hu3F8 V1 were identified using the NN-alignment method in the Immune Epitope Database (http://www.iedb.org/) and minimized based on structurally permissive mutations. . Based on a computational model of GD2 anchored to the 3F8 crystal structure (constructed using CDOCKER and Discovery Studio software, Accelerys, San Diego, CA), CDR residues that were not modeled to interact directly with the GD2 antigen were considered. mutations for humanization.

Selection of hu3F8 mutants from yeast libraries

[000356] The methodology for generating and isolating higher affinity mutants was as described in the references (Zhao et al., Mol. Cancer Ther. 2011, 10, 1677-1685). Prior to FACS selection, yeast cells (1 x 109) were incubated with 10 μg of GD2-conjugated magnetic beads for 1 hour at room temperature in PBSA buffer (0.1% BSA in PBS), followed by separation. with a magnetic holder. The isolated spheres were washed 3 times with PBSA buffer, placed in 10 mL of SDCAA (casamino acids/synthetic dextrose) medium and grown overnight on a shaker at 30 °C at 250 rpm. Yeast cells recovered from the magnetic beads were induced in SG/RCAA (casamino acids/galactose/synthetic raffinose) medium for 18 hours at 20°C with shaking at 250 rpm. About 1 x 108 yeast cells were pelleted, washed twice with PBSA buffer, and resuspended in 1 ml of PBSA buffer with biotinylated GD2 and a 1:100 dilution of mouse anti-c-myc antibody (Invitrogen). After incubation, yeast cells were washed 3 times and then resuspended in 1 ml of PBSA buffer. Either a 1:100 dilution of goat anti-mouse IgG antibody conjugated to R-phycoerythrin (Invitrogen) or Alexa Fluor 488 (Invitrogen) was added to the yeast cells, incubated at 4°C for 30 min, and washed 3 times. with PBSA buffer again and then resuspended in PBSA buffer for classification. Selection gates were determined to select only the population with the highest antigen binding signals. The collected cells were cultured overnight in SDCAA medium at 30 °C and induced in SG/RCAA for the next round of selection. For the next three selections, approximately 1-2 x 107 yeast cells were used for staining with biotinylated IGF-1, respectively. Yeast plasmids were isolated using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research) according to the manufacturer's instructions and used for library construction templates. The 4th cycle plasmids were prepared, sequenced and characterized.

Expression of scFv hu3F8 and IgG1

[000357] scFvs were expressed and purified as previously described (Zhao et al., 2011, supra). HB2151 cells were transformed with plasmid containing pComb3x scFv sequences. Fresh individual colonies were inoculated into 2YT medium containing 100 μg/mL ampicillin and 0.2% glucose. The culture was induced by L-iso-propyl-thio-HD-galactopyranoside (0.5 mM final concentration). After overnight growth at 30°C, bacteria were centrifuged at 5,000 × g for 15 min. Soluble scFv was released from the periplasm by incubation at 30°C for 30 minutes. The clarified supernatant was recovered by Ni-NTA column purification. Recombinant ScFvs have FLAG and His tags. IgGs were expressed in CHO cells in suspension as described previously (Cheung et al., 2012, supra). Hu3F8 V5 IgGs were transiently expressed using HEK293 cells (Invitrogen Freestyle Expression system). IgGs were purified on a protein G column.

ELISA

[000358] For cross-reactivity with other gangliosides, GD2, GD1a and GD1b were coated on polyvinyl plates for microtitering at 20 ng per well in 90% ethanol. After air drying, the wells were blocked with 0.5% BSA in PBS at 150 μl per well for 1 h at room temperature. Antibodies were added in triplicate at 1 mg/ml (100 ml per well) in 0.5% BSA. After incubation for 1 h at room temperature and washing with PBS, goat anti-human IgG-HRP at a 1:5000 dilution for IgG antibodies or goat anti-Flag-HRP IgG at a 1:5000 dilution for antibodies scFv were added. After incubation for 1 h at 4°C and additional washing, color reaction was performed and the OD was read using an ELISA plate reader at 490 nm.

Affinity determination by surface plasmon resonance

[000359] Affinity was measured using a Biacore T100. In summary, gangliosides were directly immobilized on the CM5 sensor chip through hydrophobic interaction. The reference surface was immobilized with GM1. The active surface was immobilized with GD2 and GM1 in a 1:1 ratio or GD1b alone. The diluted mixture of GD2 and GM1 (50 μg/ml) or GD1b was injected (300 μl) at a flow rate of 15 μl/min over 20 min. Extensive washing was followed with 10 mM NaOH (typically five 20-μl washes at a flow rate of 5 μl/min) until a stable baseline was obtained.

Complement-mediated cytotoxicity (CMC) assay

[000360] Antibodies were tested for their direct effect on tumor cell growth and survival in the absence of human serum or human effector cells. Tumor targets were dissociated with 2 mM EDTA or trypsin-EDTA, washed, and plated in 96-well flat-bottom plates with human serum. After incubation for 24 hours in a 5% CO2 incubator at 37 °C, increasing concentrations of antibodies in F10 are added to each well. Control wells received F10 only. After incubation for 4 h at 37°C in 5% CO2, WST-8 reagent (Cayman Chemical Co.) was added to each well and incubated in the dark in a CO2 incubator at 37°C for 2-6 h. OD was read at 450 nm and 690 nm using an ELISA plate reader. The WST-8 assay was validated using direct cell counting using Trypan Blue (Sigma) or Beckman Coulter Counter (Beckman Coulter).

Antibody-dependent cell-mediated cytotoxicity (ADCC) by 51Chrome release

[000361] Target cells were released with 2 mM EDTA in PBS without Ca2+/Mg2+ and washed in F10. Antigen density was estimated using Quantum Simply Cellular anti-mouse IgG beads according to the manufacturer's instructions (Bangs Laboratories, Inc.). For cytotoxicity assays, 100 μCi of 51Cr was incubated with 106 target cells in a final volume of 250 μl and incubated for 1 h at 37 °C with gentle resuspension of the pellet at 15 min intervals. The cells were then washed and resuspended in 250 μl of F10 and incubated for 30 min at 37°C. After washing, the cells were counted and viability determined with trypan blue and quickly placed in 96-well U-bottom plates. Peripheral blood from normal volunteers was collected in heparinized tubes. Blood was mixed with 3% dextran/PBS and kept at room temperature for 20 min to sediment red cells. The white cells were then placed in Ficoll and separated into peripheral blood mononuclear cells (PBMCs) for PBMC-ADCC. Cells were washed in F10, counted and viability determined. PBMC-ADCC was performed in the presence of 10 U/ml IL-2. Antibodies were diluted in F10 starting at 1 μg/ml in 10-fold dilutions. The plates were incubated in a 37 °C, 5% CO2 incubator for 4 h. 51Cr released in ADCC supernatant was collected for gamma counting. Total release was determined using 10% sodium dodecyl sulfate (SDS) and spontaneous base release was determined with F10 only without effectors. An effector:target (E:T) ratio of 50:1 was generally used. Similarly, ADCC assays were performed using NK-92MI cells stably transfected with the Fc receptors, human CD16 or human CD32. Unlike PBMCs, no cytokines were required in the assay. The E:T ratio was maintained at 20:1.

Immunohistochemistry (ImmunoHistoChemistry - IHC)

[000362] Tumors and normal tissues were obtained from Memorial Sloan-Kettering Cancer Center with approval from the Institutional Review Board. Five- to seven-micrometer sections of instantly frozen tissue were fixed in acetone for 30 min at −20°C. Endogenous biotin binding activity was blocked by sequential treatment with avidin and biotin (Vector Avidin-Biotin Blocking kit; Invitrogen) for 20 min each. Sections were incubated with 3 μg/ml scFv-Flag at room temperature for 1 h. After washing, the sections were incubated with anti-FLAG HRP antibodies for 30 minutes at room temperature and subsequent incubation with 3,3-diaminobenzidine for 5 min. H&E staining was also performed.

EXAMPLE 2 Construction of hu3F8V5

[000363] Nine point mutations were made in hu3F8 V1 to make hu3F8 V5 (see Table 2), in an effort to reduce potential immunogenicity. All nine mutations were found to be structurally permissive for the computational model of 3F8 bound to its GD2 antigen. All mutations involve the change of murine residues left in the humanization of 3F8 to human germline sequences. Five of the mutations (LC:K24R, LC:S56T, LC:V58I, HC:I20L, HC:M92V) involved structural residues. Additionally, we found 4 mutations in CDR H2 (HC: A62S, HC: F63V, HC: M64K, HC: S56G) that removed a strong T cell epitope as identified by in silico methods. Although it is unusual for those skilled in the art of humanizing antibodies via grafting methods to alter CDR residues, our computational model of 3F8 bound to GD2 allowed us to manipulate these additional humanizing mutations.

Affinity maturation of hu3F8

[000364] To perform affinity maturation based on yeast display methods, we synthesized a new biotinylated derivative of GD2 to be used for selection. Previously, we were unsuccessful using a standard biotinylated GD2 antigen. Using a synthetic GD2-azide derivative (Figure 2), we fused it to a PEG spacer (see Example 7 below). Using this new GD2 analog, we selected 2 mutations from a random library of hu3F8 scFvs displayed on the surface of yeast, which had increased binding by the synthetic GD2 analog. The first was LC:D32H, which is located in CDR L1, and the second was LC:E1K, which is a framework residue.

[000365] Two mutations (LC:E1K and LC:D32H) were tested in the form of recombinantly expressed constructs ScFv hu3F8V1 and ScFv hu3F8V5 and binding affinities to native GD2 were measured using Biacore analysis. Based on structural modeling, all hu3F8 scFvs were made in the VL-VH format because this allows less restricted access to the antigen-binding pocket. This is in contrast to most conventional scFvs, which are constructed in the VH-VL format. Several variants were also tested in the full-length IgG1 format. Table 2 presents the design of hu3F8V5. TABLE 2

EXAMPLE 3 Binding affinities

[000366] The binding affinities of the hu3F8 variants tested in the scFv format (see Table 3) show a series of interesting conclusions. First, hu3F8V5, which was designed only because it is less immunogenic than hu3F8V1, had a slightly stronger binding affinity to GD2 than hu3F8V1. The two affinity maturation mutations (LC:E1K and LC:D32H), when expressed separately, show increased binding to GD2. When expressed together in the scFv hu3F8V1 or scFv hu3F8V5 formats, a more significant increase in binding affinity is observed (KD 7-12 times lower). When the double mutation (LC:E1K + LC:D32H) is combined with HC:G54I (based on in silico modeling, reference to the original patent), the binding affinity is not as strong as the double mutation individually, but still with higher affinity than hu3F8V1. hu3F8 G54I has been shown to have a 7-10-fold increase in ADCC for GD2-positive tumor cell lines.

[000367] Binding affinities for the hu3F8 variants in the full-length IgG1 format are shown in Table 4. Similar to the data for ScFvs, the double mutation LC:E1K + LC:D32H shows a higher affinity than the single mutation LC:D32H in both hu3F8 V1 and hu3F8 V5 formats. The overall increase of the LC:E1K + LC:D32H double mutation versus the parental is 8-10 fold in both the hu3F8 V1 and hu3F8 V5 formats. The contribution of LC:E1K to the increase in binding is unexpected since it is not a canonical CDR residue. Direct comparison of the binding affinities of hu3F8 V1 constructs with hu3F8 V5 IgG cannot be made, since the hu3F8 V5 constructs were transiently expressed in HEK 293 cells and may have altered structural properties that could affect binding. Table 3 presents the binding affinities of scFv hu3F8 for GD2, as measured by Biacore. Table 4 presents the binding affinities of hu3F8 IgGs to GD2 as measured by Biacore. TABLE 3 TABLE 4 *transiently expressed in HEK293 cells, may exhibit different glycosylation than when stably expressed in CHO cells

EXAMPLE 4 Cross-reactivity with other gangliosides

[000368] In cross-reactivity studies (Table 5 and data not shown), all hu3F8 variants had comparable cross-reactivity with GD1b (a ganglioside also present in neuroblastoma tumor cells) and no significant cross-reactivity with other gangliosides tested (GD1a , GD1b, GD3), which demonstrates that the hu3F8 variants retain the same specificity as the parental hu3F8V1. Table 5 presents the binding affinities of hu3F8 IgGs to GD1b, as measured by Biacore. TABLE 5

EXAMPLE 5 Antibody potency in ADCC and CMC

[000369] Anti-GD2 IgG1 antibodies were compared in ADCC assays using Peripheral Blood Mononuclear Cells (PBMCs) or NK92-CD16 (CD16-positive cultured NK cells) as effectors and LAN-1 neuroblastoma cells as target cells (data not shown). The ADCC potencies of these antibodies were calculated as the ratio (EC50 for 3F8)/(EC50 for MoAb). Relative to the parental hu3F8 V1 IgG, hu3F8 V1 LC:D32H and hu 3F8 V1 LC:E1K + LC:D32H were ~20 times stronger in PBMC-ADCC and 7 times stronger in NK92-CD16- ADCC (see Table 6) . Table 6 presents a summary of CCDA assays using hu3F8V1 IgG. TABLE 6 *Hu3F8 V1 is used as a reference

[000370] The same antibodies were tested for their ability to induce CMC using human sera as effectors and LAN-1 cells as targets (Table 7 and data not shown). hu3F8 V1 LC:D32H shows a small increase in CMC, whereas hu3F8 V1 LC:E1K + LC:D32H showed relatively the same amount of CMC as the parental hu3F8 V1. Relatively low complement activation was desirable, as complement activation is believed to mediate the secondary effect of pain associated with anti-GD2 immunotherapy. Table 7 presents a summary of CMC assays with hu3F8V1 IgG. TABLE 7

EXAMPLE 6 Immunohistochemistry in normal tissues and tumors

[000371] ScFv versions of hu3F8 V1, hu3F8 V1 LC: D32H and hu3F8 LC: E1K + LC: D32H were tested for tissue specificity by IHC in human neuroblastoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, desmoplastic small round cell tumors. normal human bodies and tissues (see Figure 1). Twelve normal tissues were also tested (see Table 8). Frontal lobe, pons, cerebellum, and spinal cord stained positive with both affinity-matured clones (hu3F8 V1 LC:D32H and hu3F8 LC:E1K + LC:D32H), but not the parental clone (hu3F8 V1) as expected, since GD2 is known to be present in neuronal tissues. When looking at IHC of different tumor samples, the affinity-matured clones (hu3F8 V1 LC:D32H and hu3F8 LC:E1K + LC:D32H) showed a higher level of staining in GD2-positive tumors relative to the parental antibody (hu3F8 V1). Table 8 presents the staining intensity of tissues with ScFvs hu3F8V1. TABLE 8 The concentration of scFv is 3 μg/ml; NB, neuroblastoma; intensity is defined as 0, 1 (weak, heterogeneous membrane staining), 2 (weak, homogeneous membrane staining), 3 (strong, heterogeneous membrane staining), and 4 (strong, homogeneous membrane staining).

EXAMPLE 7 Biotinylation of GD2

[000372] For the small-scale reaction, 100 μg of GD2-azide and 50 μg of DBCO-PEG4-biotin (Click Chemistry Tools) in 25 mL of water were reacted overnight at 4°C with slight rotation. The next day, excess DBCO-PEG4-biotin was inactivated by adding 30 μg of azide-PEG-azide (Click Chemistry Tools) and incubated for 1 h at room temperature. The product was diluted until reaching a concentration of 0.5 mg/ml and stored at -80°C.

FACS Analysis

[000373] Yeast cells displaying Hu3F8 scFvs were cultured and induced as for FACS analysis. Yeast cells (1 x 106) were incubated with 2 μg/ml of biotinylated GD2-azide-PEG4-biotin or GD2-Biotin in a 1:100 dilution of mouse anti-c-myc antibody for 30 min. in ice-cold PBS/0.1% BSA buffer. After washing once, cells were incubated with a 1:50 dilution of Alexa Fluor 488-conjugated goat anti-mouse antibody conjugated to streptavidin/R-phycoerythrin for 30 min on ice, then washed once more and resuspended at 0. .5 ml of PBSA buffer. Analysis was performed using a BD Bioscience FACS.

Results

[000374] Yeast cells displayed Hu3F8 scFv with cMyc tag on the cell surface, which were used to bind biotin-GD2 conjugates with or without a PEG spacer. In flow cytometry analysis, GD2 expression and Hu3F8 scFv binding were detected as X and Y axis, respectively. We found that the existence of a PEG spacer is necessary for observation of GD2 in flow cytometry analysis through comparison with spacer-free GD2 (data not shown). See Figure 2 for the chemical structure of biotin-PEG-GD2.

EXAMPLE 8 Measurement of MoAb dissociation rates via surface plasmon resonance

[000375] Dissociation rates of hu3F8 IgGs with affinity-increasing mutations were measured by surface plasmon resonance (BIAcore T100) using a high-density GD2 model.

[000376] Briefly, gangliosides were directly immobilized on a CM5 sensor chip through hydrophobic interaction. The reference surface was immobilized with GM1. The active surface was immobilized with pure GD2. GD2 (50 μg/ml) was injected (300 μl) at a flow rate of 15 μl/min over 20 min. Extensive washing was followed with 10 mM NaOH (typically five 20-μl washes at a flow rate of 5 μl/min) until a stable baseline was obtained. The results are shown in Table 9 and Figure 3.TABLE 9

[000377] As shown in Table 9 and Figure 3, hu3F8 double mutants (hu3F8V1 LC:D32H HC:G54I and hu3F8V1 LC:E1K LC:D32H) demonstrated the slowest dissociation rates, which were 3.6-6 .4 times slower than hu3F8V1. The single mutant (hu3F8 V1 LC:D32H) and the triple mutant (hu3F8V1 LC:E1K LC:D32H HC:G54I) demonstrated a 2.7- and 2.1-fold slower dissociation rate, respectively.

Antibody Potency in ADCC With Additional Tumor Cell Lines

[000378] Anti-GD2 IgG1 antibodies were compared in ADCC assays with NK92-CD16 (CD16-positive cultured NK cells) as effectors and IMR-32 neuroblastoma or M14 melanoma as targets (as described above). ADCC potencies were calculated as the ratio of EC50 of hu3F8V1/EC50 of antibody. The results are shown in Table 10.TABLE 10

[000379] The results show that, relative to the parental hu3F8 V1 IgG, the double mutants (hu3F8V1 LC:D32H HC:G54I and hu3F8V1 LC:E1K) demonstrated a 100- to 140-fold increase in ADCC of IMR-32 cells and a 22- to 25-fold increase in ADCC of M14 cells. The single mutant (hu3F8V1 LC:D32H) demonstrated a 25-fold increase in ADCC of IMR-32 cells and a 5-fold increase in ADCC of M14 cells. The triple mutant (hu3F8V1 LC:E1K LC:D32H HC:G54I) demonstrated a 15.6-fold increase in ADCC of IMR-32 cells and a 3.6-fold increase in ADCC of M14 cells.

Claims (14)

1. High-affinity 3F8 anti-GD2 antibody variant or a single-chain variable fragment (scFv) thereof, characterized by the fact that its structure is defined by a feature that is selected from the group consisting of: a mutation D32H in the light chain, an E1K mutation in the light chain, and a combination thereof, wherein the antibody or scFv comprises: (i) a light chain variable region sequence identified as SEQ ID NO: 1, 2 or 3 and a heavy chain variable region sequence identified as SEQ ID NO: 4; or (ii) a light chain variable region sequence identified as SEQ ID NO: 7, 8 or 9 and a heavy chain variable region sequence identified as SEQ ID NO: 5 or 10; or (iii) an scFv comprising a sequence identified by any of SEQ ID NOs: 11-16, 19 and 21-25. 2. Antibody or scFv variant according to claim 1, characterized by the fact that (1) the light chain variable region sequence identified as SEQ ID NO: 7, 8 or 9 and the heavy chain variable region comprises a G54I substitution and comprises a sequence identified as SEQ ID NO: 10. 3. Antibody variant or scFv, according to claim 1, characterized by the fact that the antibody comprises: (i) a light chain sequence identified as SEQ ID NO: 1 or 2 and a heavy chain sequence identified as SEQ ID N: 4, or (ii) a light chain sequence identified as SEQ ID NO: 7 or 8 and a heavy chain sequence identified as SEQ ID N: 5 or 10. 4. Antibody or scFv variant according to claim 1, characterized by the fact that: (i) the antibody comprises a light chain sequence identified as SEQ ID NO: 3 and a heavy chain sequence identified as SEQ ID N : 4, or (ii) the antibody comprises a light chain sequence identified as SEQ ID NO: 9 and a heavy chain sequence identified as SEQ ID NO: 5 or 10. 5. Antibody variant or scFv, according to claim 1, characterized by the fact that the antibody is a single-chain variable fragment (scFv), wherein the scFv comprises a sequence identified by any of the SEQ ID NOs: 11-16, 19 and 21-25. 6. Antibody variant according to claim 1, characterized by the fact that said antibody is a bispecific antibody comprising a first binding site comprising an scFv comprising a sequence identified as SEQ ID NOs: 11 to 16, 19, and 21 to 25, and a second binding site, wherein: (a) the second binding site comprises an anti-CD3 scFv identified as SEQ ID NO: 26 or 27; or (b) the second binding site comprises an anti-DOTA scFv identified as SEQ ID NO: 28. 7. Isolated nucleic acid molecule, characterized by the fact that it encodes an antibody or scFv variant, as defined in any one of claims 1 to 6, wherein said nucleic acid is selected from the group consisting of SEQ ID NOS: 32 to 34, and 36 to 41 and 43 to 45; or recombinant vector comprising said nucleic acid molecule. 8. Composition, characterized by the fact that it comprises the antibody or scFv variant, as defined in any one of claims 1 to 6; whereby: (i) the antibody or scFv is conjugated to a cytotoxic agent; and/or (ii) the composition further comprises a pharmaceutically acceptable carrier or diluent. 9. Antibody or scFv variant according to any one of claims 1 to 6, characterized by the fact that it is for use in the treatment or prevention of a medical condition or disease in an individual, wherein the medical condition or disease is determined by the expression of GD2, wherein: (a) said medical condition is neuroblastoma, melanoma, sarcoma, brain tumor or carcinoma; or (b) said disease is chosen from the group consisting of: osteosarcoma, liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leimyosarcoma, spindle cell sarcoma, brain tumor, small cell lung cancer, retinoblastoma, T-cell leukemia infected with HTLV-1 and other GD2-positive tumors. 10. Composition according to claim 8, characterized by the fact that it comprises a molecule comprising azide-GD2-oligosaccharide reacted with biotin-PEG-alkyne. 11. Method for increasing the ADCC of a 3F8 antibody, characterized by the fact that it comprises introducing a D32H substitution in the light chain of said antibody as defined in claim 1. 12. Therapeutic composition, characterized by the fact that it comprises a 3F8 antibody as defined in claim 1. 13. Antibody variant or scFv, as defined in any one of claims 1 to 6, characterized by the fact that it is radioactively labeled. 14. Diagnostic composition, characterized by the fact that it comprises an antibody or scFv variant as defined in any one of claims 1 to 6