NZ726501B2

NZ726501B2 - Human monoclonal antibodies to ganglioside gd2

Info

Publication number: NZ726501B2
Application number: NZ726501A
Authority: NZ
Inventors: Ritsuko Sawada; Wolfgang Scholz
Original assignee: Biontech Research And Development Inc
Priority date: 2014-06-04
Filing date: 2015-06-03
Publication date: 2021-08-31

Abstract

The present invention provides compositions for the production of an antibody or functional fragment thereof directed against disialoganglioside-GD2. The compositions of the invention include polynucleotides encoding a heavy chain and/or a light chain variable domain that binds to GD2. The invention also provides an isolated antibody or functional fragment thereof and methods of treating or preventing a disease, such as cancer or tumor formation, wherein the antibody or functional fragment includes a variable heavy chain domain and a variable light chain domain that has an amino acid sequence provided herein. The invention further provides a conjugate of an antibody or functional fragment thereof conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent, and methods of treating, preventing or diagnosing a disease in a subject in need thereof. also provides an isolated antibody or functional fragment thereof and methods of treating or preventing a disease, such as cancer or tumor formation, wherein the antibody or functional fragment includes a variable heavy chain domain and a variable light chain domain that has an amino acid sequence provided herein. The invention further provides a conjugate of an antibody or functional fragment thereof conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent, and methods of treating, preventing or diagnosing a disease in a subject in need thereof.

Description

Human Monoclonal Antibodies to Ganglioside GD2 CROSS-REFERENCE TO RELATED APPLICATION This application claims the beneﬁt ofU.S. Serial No. 62/007,874 ﬁled June 4, 2014, which is incorporated herein by reference in its entirety.

SE UENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on May 29, 2015, is named 12967228_SL.txt and is 69,238 bytes in size.

BACKGROUND OF INVENTION The present invention relates generally to antibodies directed t the disialoganglioside GD2. More speciﬁcally, this application relates to polynucleotides encoding human anti-GD2 antibodies and the corresponding d antibodies or fragment thereof, as well as to the use of such dies for diagnostic or therapeutic purposes. GD2 is attractive for speciﬁc ies such as antibody therapy because of the tumor selective expression pattern. Currently, l anti-GD2 antibodies have been developed, such as murine D2 antibodies, human-mouse chimeric anti-GD2 antibodies. However, these dies still have undesirable immune s. Thus, there remains a need to reduce the undesirable immune effects and enhance desirable antitumor effects of the anti-GD2 antibodies. This application discloses human monoclonal antibodies (mAbs) against GD2, which satisfy the need and provide related advantages.

SUMMARY OF INVENTION In accordance with the present invention, herein provided are compositions for ing antibodies or ﬁlnctional fragments f that bind GD2. The compositions include an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody includes a le heavy chain (VH) domain that has complementarity determining regions (VH CDRl, VH CDR2 and VH CDR3) provided herein. In one aspect, the isolated polynucleotide of the invention can also encode an antibody or a functional fragment thereof, n the antibody includes a VH domain that has an amino acid sequence provided herein. In another aspect, the isolated polynucleotide of the invention can also include a nucleic acid sequence provided , wherein the nucleic acid sequence encodes the VH domain of the antibody or ﬁanctional fragment thereof In another embodiment of the invention, the isolated polynucleotide can encode an antibody or a functional fragment thereof, wherein the dy includes a variable light chain (VL) domain that has mentarity determining s (VL CDRl, VL CDR2 and VL CDR3) provided herein. In one aspect, the isolated polynucleotide of the invention can also encode an antibody or a functional fragment thereof, wherein the dy includes a VL domain that has an amino acid sequence ed herein. In r aspect, the isolated polynucleotide of the invention can also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the VL domain of the antibody or ﬁanctional fragment f.

The compositions of the invention also include an isolated antibody or onal fragment f, n the antibody binds to GD2. In some embodiments, the invention es an isolated dy or functional fragment thereof that binds GD2, wherein the antibody or ﬁanctional fragment thereof includes a VH domain having VH CDRl , VH CDR2, and VH CDR3 regions provided herein. In other embodiments, the invention provides an isolated antibody or onal fragment thereof that binds GD2, wherein the antibody or functional fragment thereof includes a VH domain having an amino acid sequence provided herein.

In some embodiments, the invention provides an isolated antibody or ﬁanctional fragment thereof that binds GD2, wherein the antibody or functional fragment thereof includes a VL domain having VL CDRl, VL CDR2, and VL CDR3 regions provided herein. In other embodiments, the invention provides an ed antibody or functional fragment thereof that binds GD2, wherein the antibody or ﬁanctional fragment thereof includes a VL domain having an amino acid sequence provided herein. [0007a] In ular the t invention provides for isolated polynucleotides encoding an antibody or a functional fragment thereof that binds to GD2, said dy or functional fragment thereof comprising a variable heavy chain VH domain and a variable light chain VL domain, said VH domain having VH CDR1, VH CDR2 and VH CDR3 amino acid sequences and said VL domain having VL CDR1, VL CDR2 and VL CDR3 amino acid sequences, wherein said VH CDR1 amino acid sequence is residues 26-33 of SEQ ID NO: 2; said VH CDR2 amino acid sequence is residues 51-58 of SEQ ID NO: 2; said VH CDR3 amino acid sequence is residues 97-109 of SEQ ID NO: 2; said VL CDR1 amino acid sequence is residues 27-37 of SEQ ID NO: 4; said VL CDR2 amino acid ce is residues 55-57 of SEQ ID NO: 4; and said VL CDR3 amino acid sequence is residues 94-102 of SEQ ID NO: 4.

In some embodiments, the ion provides an isolated antibody or functional nt thereof that binds to GD2, wherein the antibody or functional fragment thereof includes both a VH domain and a VL domain, where the VH domain and the VL domain respectively include an amino acid sequence for the respective VH and VL domains of the clonal isolates provided herein, particularly a variable heavy chain VH domain having an amino acid sequence of SEQ ID NO: 2 and a variable light chain VL domain having an amino acid sequence of SEQ ID NO: 4.

In some embodiments, the invention es a conjugate having an antibody or functional nt provided herein that is conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In some aspects of the invention, a conjugate of the invention that includes a detectable agent can be used in a method for detecting and/or diagnosing tumor formation is a subject. Such methods can include administering an effective amount of the conjugate to a subject in need thereof. Further provided is the use of the conjugate, in the manufacture of a medicament for ing a tumor in a subject in need In some embodiments, the invention provides pharmaceutical itions having one or more antibody or functional fragment of the invention and a pharmaceutically acceptable carrier. In some aspects, the invention also provides a method for treating or preventing a disease in a subject in need thereof, by administering a eutically effective amount of a pharmaceutical composition of the ion. Further provided is the use of the pharmaceutical composition in the manufacture of a medicament, for the treating or preventing a disease in a subject in need thereof. In still another aspect, the invention (followed by page 3A) provides administering a second therapeutic agent concurrently or successively with an antibody or functional fragment of the invention. [0010a] These and other embodiments are described in r detail herein. Certain embodiments are included herein for completeness and may form the subject of one or more divisional applications.

BRIEF DESCRIPTION OF THE DRAWINGS shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 1B7. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 1 and amino acid sequence of SEQ ID NO: 2. The three mentarity determining regions (VH CDR1, VH CDR2 and VH CDR3) are also identified. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 1B7. The top n of the figure shows an alignment between the tide sequence of SEQ ID NO: 3 and amino acid sequence of SEQ ID NO: 4. The three complementarity determining s (VL CDR1, VL CDR2 and VL CDR3) are also identified. (followed by page 4) shows the nucleotide ce and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 2H12. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 5 and amino acid sequence of SEQ ID NO: 6. The three complementarity ining regions (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide sequence and the d amino acid sequence of the variable light (VL) chain domain of clone 2H12. The top portion of the ﬁgure shows an ent between the nucleotide sequence of SEQ ID NO: 7 and amino acid sequence of SEQ ID NO: 8. The three complementarity determining regions (VL CDRl, VL CDR2 and VL CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 1G2. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 9 and amino acid sequence of SEQ ID NO: 10. The three complementarity determining s (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 1G2. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: ll and amino acid sequence of SEQ ID NO: 12. The three complementarity determining regions (VL CDRl, VL CDR2 and VL CDR3) are also identiﬁed. shows the nucleotide ce and the encoded amino acid ce of the variable heavy (VH) chain domain of clone 1E9. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 13 and amino acid sequence of SEQ ID NO: 14. The three mentarity determining regions (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 1E9. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 15 and amino acid sequence of SEQ ID NO: 16. The three complementarity determining regions (VL CDRl, VL CDR2 and VL CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 1H3. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 17 and amino acid sequence of SEQ ID NO: 18. The three complementarity determining s (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide ce and the encoded amino acid sequence of the variable light (VL) chain domain of clone 1H3. The top portion of the ﬁgure shows an ent between the nucleotide sequence of SEQ ID NO: 19 and amino acid sequence of SEQ ID NO: 20. The three complementarity determining regions (VL CDRl, VL CDR2 and VL CDR3) are also identiﬁed.

FIG. ll shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 2F5. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 21 and amino acid ce of SEQ ID NO: 22. The three complementarity determining regions (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 2F5. The top portion of the ﬁgure shows an alignment between the nucleotide ce of SEQ ID NO: 23 and amino acid sequence of SEQ ID NO: 24. The three complementarity determining s (VL CDRl, VL CDR2 and VL CDR3) are also identiﬁed. shows the tide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 2F7. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 25 and amino acid sequence of SEQ ID NO: 26. The three complementarity ining regions (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 2F7. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 27 and amino acid sequence of SEQ ID NO: 28. The three complementarity determining regions (VL CDRl, VL CDR2 and VL CDR3) are also identiﬁed. shows the nucleotide sequence and the d amino acid sequence of the variable heavy (VH) chain domain of clone 2E12. The top portion ofthe ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 29 and amino acid sequence of SEQ ID NO: 30. The three complementarity determining s (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 2E12. The top portion of the ﬁgure shows an alignment between the nucleotide sequence of SEQ ID NO: 31 and amino acid sequence of SEQ ID NO: 32. The three complementarity determining regions (VL CDRl, VL CDR2 and VL CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 3 lF9. The top portion of the ﬁgure shows an alignment between the nucleotide ce of SEQ ID NO: 33 and amino acid sequence of SEQ ID NO: 34. The three complementarity determining regions (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid ce of the variable heavy (VH) chain domain of clone 3 lF9V2. The top portion of the ﬁgure shows an ent between the nucleotide sequence of SEQ ID NO: 35 and amino acid sequence of SEQ ID NO: 36. The three complementarity ining regions (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 3 lF9. The top portion of the ﬁgure shows an ent between the nucleotide sequence of SEQ ID NO: 37 and amino acid sequence of SEQ ID NO: 38. The three complementarity determining regions (VL CDRl, VL CDR2 and VL CDR3) are also identiﬁed. shows the nucleotide sequence and the d amino acid sequence of the variable heavy (VH) chain domain of clone 32E2. The top portion ofthe ﬁgure shows an alignment between the tide sequence of SEQ ID NO: 39 and amino acid sequence of SEQ ID NO: 40. The three complementarity determining regions (VH CDRl, VH CDR2 and VH CDR3) are also identiﬁed. shows the nucleotide sequence and the encoded amino acid ce of the le light (VL) chain domain of clone 32E2. The top portion of the ﬁgure shows an alignment between the nucleotide ce of SEQ ID NO: 41 and amino acid sequence of SEQ ID NO: 42. The three complementarity determining regions (VL CDRl, VL CDR2 and VL CDR3) are also identiﬁed. shows an alignment of the VH regions of human anti-GD2 monoclonal dies 1B7 (SEQ ID NO: 2), 2Hl2 (SEQ ID NO: 6), lG2 (SEQ ID NO: 10), 1E9 (SEQ ID NO: 14), lH3 (SEQ ID NO: 18), 2F5 (SEQ ID NO: 22), 2F7 (SEQ ID NO: 26), 2El2 (SEQ ID NO: 30), 3lF9 (SEQ ID NO: 34), 3lF9V2 (SEQ ID NO: 36), and 32E2 (SEQ ID NO: 40), with the consensus sequence (SEQ ID NO: 43) listed below. shows an alignment of the VL regions of human anti-GD2 onal antibodies 1B7 (SEQ ID NO: 4), 2Hl2 (SEQ ID NO: 8), lG2 (SEQ ID NO: 12), 1E9 (SEQ ID NO: 16), lH3 (SEQ ID NO: 20), 2F5 (SEQ ID NO: 24), 2F7 (SEQ ID NO: 28), 2El2 (SEQ ID NO: 32), 3 lF9 (SEQ ID NO: 38), and 32E2 (SEQ ID NO: 42), with the consensus sequence (SEQ ID NO: 44) listed below. shows antibody-dependent cell-mediated cytotoxicity (ADCC) of human anti-GD2 monoclonal antibodies 1B7, 3lF9, 3lF9V2, lG2, 2F7, 32E2 and 2Hl2. The ADCC activities were measured by Promega’s er assay using ered Jurket cells as effector cells. Target cells are various tumor cells, including SaOS2, H524, Hs578T, TC7l, and Lanl- luc. shows internalization of human anti-GD2 monoclonal antibodies into H524 cells. H524 cells were grown in the presence of 1B7, 3 lF9, or 3 lF9V2 complexed with Hum- ZAP, a saporin-conjugated anti-human IgG. The values were measured and ized to 100% growth in absence of Fab-ZAP. shows internalization of human anti-GD2 monoclonal antibodies into Lanl- luc cells. Lanl-luc cells were grown in the presence of 1B7, lG2, 2Hl2, 2F7, 3 lF9 or 32E2 complexed with Hum-ZAP, a saporin-conjugated anti-human IgG. The values were measured and normalized to 100% growth in absence of Fab-ZAP. shows the kinetics of internalization of anti-ganglioside antibodies into H524 (SCLC) tumor cells measured with a pH sensitive reporter by ﬂow cytometry. Cells that internalized the antibodies into the low pH environment of endosomes display a ﬂuorescence measured by ﬂow cytometry. shows the kinetics of internalization of anti-ganglioside antibodies into TC- 71 (sarcoma) tumor cells measured with a pH sensitive reporter by ﬂow cytometry. Cells that alized the antibodies into the low pH environment of endosomes display a ﬂuorescence measured by ﬂow cytometry. shows the survival of SCID mice engrafted with human SaOSZ (osteosarcoma) xenograft and treated with D2 antibodies or control. shows the growth of human TC-7l (sarcoma) xenograft tumors in SCID mice and treated with anti-GD2 antibodies or l.

DETAILED DESCRIPTION OF THE INVENTION Gangliosides expressed on the tumor cell surface can be targets for cancer immunotherapy. The itions ed herein are based, at least in part, on the identiﬁcation and terization of human antibodies that were generated from blood lymphocytes of individuals immunized with MabVax vaccines (MabVax Therapeutics, San Diego, CA) ning KLH-conjugated GD2L, GD3L and GM2 antigens as described, for e, in US. Patents Nos. 6,936,253; 7,001,601, and 6,916,476. At least 11 antibodies with high afﬁnity for GD2 (1B7, 2H12, 1G2, 1E9, 1H3, 2F5, 2F7, 2E12, 31F9, 31F9V2, and 32E2) were identiﬁed, expressed as recombinant antibodies, and further characterized in in vitro models. Of the eight antibodies tested, six (1B7, 2H12, 2F7, 2E12, 31F9V2, and 32E2) were potent in complement-dependent cytotoxicity (CDC) assays in at least one cancer cell line. All six antibodies tested show signiﬁcant activity in antibody-dependent cytotoxicity assays with ﬁve different cancer cell lines, albeit to ent degree. The two antibodies tested (1B7 and 31F9) also showed signiﬁcantl anti-tumor acitivty in vivo, in both a survival model and a subcutaneous tumor model. The translational relevance of the invention provided herein is twofold: First, GD2-KLH conjugate vaccine can elicit an D2 IgG and IgM antibody response in cancer ts and antibody producing cells can be recovered from patient blood samples. Second, the most potent antibodies that were generated in a al trial can be preserved and ultimately used as therapeutics, or in the generation of therapeutics, for a target cancer population. The high affinity of the antibodies provided herein and their high effector functions support this translational potential.

As used herein, the term “antibody” is intended to mean a polypeptide product of B cells within the immunoglobulin class of ptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids and each carboxy-terminal n of each chain includes a constant region (See Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford University Press; Kuby (1997) Immunology, Third Edition, W.H. Freeman and Company, New York). In the t of the present invention, the specific molecular antigen that can be bound by an antibody of the invention includes the target GD2.

The term “human” when used in reference to an antibody or a functional nt thereof refers an antibody or ﬁanctional fragment thereof that has a human variable region and/or a human constant region or a n thereof corresponding to human ne immunoglobulin sequences. Such human germline immunoglobulin sequences are described by Kabat et al. (1991) Sequences of Proteins of Immunological st, Fifth Edition, US. Department of Health and Human Services, NIH Publication No. 2. A human antibody, in the context of the present invention, can include an antibody that binds to GD2 and is encoded by a nucleic acid ce that is a lly ing somatic variant of the human germline immunoglobulin nucleic acid sequence. Exemplary methods of ing human antibodies are provided in Example I, but any method well known to those skilled in the art can be used.

The term lonal antibody” refers to an antibody that is the product of a single cell clone or hybridoma or a population of cells derived from a single cell. A monoclonal antibody also is intended to refer to an antibody produced by recombinant methods from heavy and light chain encoding immunoglobulin genes to produce a single molecular immunoglobulin species. Amino acid sequences for antibodies within a monoclonal antibody preparation are substantially homogeneous and the binding activity of antibodies within such a preparation exhibit substantially the same antigen binding activity. In contrast, onal antibodies are obtained from different B cells within a population, which are a ation of immunoglobulin WO 87811 molecules that bind a speciﬁc antigen. Each immunoglobulin of the polyclonal dies can bind a different epitope of the same antigen. Methods for producing both monoclonal antibodies and polyclonal antibodies are well known in the art (Harlow and Lane., Antibodies: A Laboratog Manual, Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (ed.), Antibody Engineering: A Practical Guide, W.H. Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

As used herein, the term “functional fragment” when used in reference to an antibody is ed to refer to a portion of the antibody including heavy or light chain polypeptides that retains some or all of the binding activity as the antibody from which the fragment was derived.

Such functional fragments can include, for example, an Fd, Fv, Fab, F(ab’), F(ab)2, 2, single chain Fv (scFv), diabody, triabody, tetrabody and minibody. Other functional nts can include, for example, heavy or light chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof so long as such ﬁanctional fragments retain binding activity.

Such antibody binding fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratog Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. y and hnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Pliickthun and Skerra, Meth.

Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistg, Second Ed., Wiley-Liss, Inc., New York, NY (1990).

The term “heavy chain” when used in nce to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal n includes a le region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The nt region can be one of five distinct types, referred to as alpha (or), delta (8), epsilon (a), gamma (y) and mu (u), based on the amino acid sequence of the heavy chain constant region. The ct heavy chains differ in size: or, 5 and y n approximately 450 amino acids, while u and 8 contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to ﬁve well known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant . The imate length of a light chain is 211 to 217 amino acids. There are two ct types, referred to as kappa (K) of lambda (9») based on the amino acid ce of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

The term “variable domain” or “variable region” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR). The CDRs of the light and heavy chains are ily responsible for the interaction of the antibody with n. Numbering of amino acid positions used herein is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (US. Department of Health and Human Services, Washington, DC.) 5th ed. A variable region can be a human variable A CDR refers to one of three ariable regions (H1, H2 or H3) within the non- framework region of the globulin (Ig or antibody) VH B-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL t framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prat.

Chem. 32: 1-75 (1978)). CDR region sequences also have been deﬁned structurally by Chothia as those residues that are not part of the conserved B-sheet framework, and thus are able to adapt different conformations ia and Lesk, J. M01. Biol. 196:901-917 (1987)). Both terminologies are well recognized in the art. The positions of CDRs within a canonical antibody WO 87811 2015/033954 le domain have been determined by comparison of numerous structures (Al-Lazikani et al., J. M01. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). e the number of residues within a ariable region varies in different dies, additional residues ve to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

For example, CDRs defined according to either the Kabat (hypervariable) or Chothia (structural) designations, are set forth in the Table 1 below.

Table 1: CDR ions Kabat1 Chothia2 Loop Location VH CDRl 31-35 26-32 linking B and C strands VH CDR2 50-65 53-55 linking C’ and C” strands VH CDR3 95-102 96-101 linking F and G strands VL CDRl 24-34 26-32 linking B and C strands VL CDR2 50-56 50-52 linking C’ and C” s VL CDR3 89-97 91-96 linking F and G strands 1 Residue numbering follows the lature of Kabat et al., supra 2 Residue numbering follows the nomenclature of Chothia et al., supra One or more CDRs also can be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to bind to a particular antigen of interest.

As used herein, the term “isolated” when used in nce to an antibody, antibody functional fragment or polynucleotide is intended to mean that the referenced molecule is free of at least one component as it is found in nature. The term includes an antibody, antibody functional fragment or polynucleotide that is removed from some or all other components as it is found in its natural environment. Components of an antibody’s natural environment include, for example, erythrocytes, leukocytes, ocytes, plasma, proteins, nucleic acids, salts and nutrients. Components of an dy functional fragment’s or polynucleotide’s natural environment include, for e, lipid membranes, cell organelles, proteins, nucleic acids, salts and nutrients. An antibody, antibody ﬁanctional fragment or polynucleotide of the invention can also be free or all the way to substantially free from all of these components or any other component of the cells from which it is ed or recombinantly produced.

As used herein, "isotype" refers to the antibody class that is d by heavy chain nt region genes. The heavy chains of a given antibody or ﬁanctional fragment determine the class of that antibody or ﬁanctional fragment: IgM, IgG, IgA, IgD or IgE. Each class can have either K or 9» light chains. The term “subclass” refers to the minor differences in amino acid sequences of the heavy chains that differentiate the subclasses. In humans there are two subclasses of IgA (subclasses IgAl and IgA2) and there are four sses of IgG (subclasses IgGl, IgG2, IgG3 and IgG4). Such classes and subclasses are well known to those skilled in art.

The terms “binds” or “binding” as used herein refer to an ction between molecules to form a complex. Interactions can be, for example, non-covalent ctions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. g of an antibody or functional fragment thereof can be detected using, for example, an enzyme-linked sorbant assay, a method provided in Example I or any one of a number of methods that are well known to those skilled in the art.

The strength of the total non-covalent interactions between a single antigen-binding site on an antibody or ﬁanctional fragment and a single epitope of a target molecule, such as GD2, is the affinity of the antibody or functional fragment for that epitope. The ratio of association (k1) to dissociation (k_1) of an antibody or ﬁanctional fragment thereof to a monovalent antigen (k1/ k_1) is the association constant K, which is a measure of affinity. The value of K varies for different complexes of antibody or ﬁanctional fragment and antigen and depends on both k; and k.1. The ation constant K for an dy or onal fragment of the invention can be determined using any method provided herein or any other method well known to those skilled in the art.

WO 87811 The affinity at one binding site does not always reﬂect the true strength of the interaction between an antibody or ﬁanctional fragment and an antigen. When complex ns containing multiple, ing antigenic determinants, such as a lent GD2, come in t with dies containing multiple binding sites, the interaction of dy or ﬁanctional fragment with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions n a multivalent antibody and antigen is called the avidity. The y of an antibody or functional fragment can be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity can compensate for low affinity as is sometimes found for pentameric IgM antibodies, which can have a lower affinity than IgG, but the high avidity of IgM, resulting from its multivalence, enables it to bind antigen effectively.

The specificity of an antibody or ﬁanctional fragment thereof refers to the ability of an individual antibody or ﬁanctional fragment f to react with only one antigen. An antibody or functional fragment can be considered specific when it can distinguish differences in the primary, secondary or tertiary structure of an antigen or isomeric forms of an antigen. The antibody can be cross-reactive if the binding epitope is present on other ns.

The term "polynucleotide" refers to a polymeric form of tides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof The sequence of a polynucleotide is composed of four tide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the terms “nucleotide sequence” or “nucleic acid sequence” is the alphabetical representation of a polynucleotide. A polynucleotide can include a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, mes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. cleotide also refers to both double- and single-stranded molecules. Unless otherwise speciﬁed or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. It is understood that the isolated polynucleotides and nucleic acids described herein are directed to non naturally WO 87811 occurring polynucleotides and nucleic acids. Non-naturally occurring polynucleotides and nucleic acids can include, but not limit to, cDNA and chemically synthesized molecules.

The term “encode” or tical equivalents thereof as it is used in reference to cleotides refers to a polynucleotide in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to e mRNA, which is then translated into a polypeptide and/or a fragment thereof. The antisense strand is the complement of such a polynucleotide, and the encoding sequence can be deduced therefrom.

The phrase “therapeutic agent” refers to any agent that can be used in the treatment, management or amelioration of a disease associated with expression of GD2 and/or a symptom d thereto. In certain embodiments, a therapeutic agent refers to an antibody or ﬁmctional fragment of the invention. In other embodiments, a therapeutic agent refers to an agent other than an antibody or functional fragment of the invention. A eutic agent can be an agent which is well known to be useful for, or has been or is currently being used for the ent, management or amelioration of a disease associated with expression of GD2 and/or one or more symptoms related thereto.

The phrase “diagnostic agent” refers to a substance administered to a subject that aids in the diagnosis of a disease. Such substances can be used to reveal, pinpoint, and/or define the localization of a disease causing process. In certain embodiments, a diagnostic agent includes a substance that is conjugated to an antibody or functional fragment of the invention, that when administered to a subject or contacted to a sample from a t aids in the diagnosis of cancer or tumor formation.

The phrase “detectable agent” refers to a substance that can be used to ascertain the nce or ce of a d molecule, such as an antibody or functional fragment of the invention, in a sample or subject. A detectable agent can be a substance that is capable of being ized or a substance that is otherwise able to be determined and/or measured (e.g., by quantitation).

An "effective amount" is an amount sufﬁcient to effect beneficial or desired results.

An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of les including the time period for which the individual dosage unit is to be used, the bioavailability of the agent, the route of administration, etc.

The phrase “therapeutically effective amount” as used herein refers to the amount of a therapeutic agent (e.g., an antibody or functional fragment provided herein or any other therapeutic agent provided herein) which is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease and/or a symptom related thereto. A therapeutically ive amount of a therapeutic agent can be an amount necessary for the reduction or amelioration of the advancement or progression of a given disease, reduction or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody or functional fragment provided herein).

The compound GD2, also known as GD2 ganglioside, ganglioside GD2, and ganglioside G2, is a disialoganglioside with a molecular formula of C74Hl34N4032 and a molar mass of 1591.86g/mol. Gangliosides are acidic glycosphingolipids found on the outer surface of most cell membranes. They can be targets for monoclonal antibodies (mAb) because of the high n density, lack of modulation, relative homogeneity in many tumors and the possibility of up-regulation by cytokines. Many tumors have al glycolipid composition and structure.

GD2 has been found in a wide um of human tumors, including those of neuroectodermal or lial origin, virtually all melanomas, and approximately 50% of tumor samples from osteosarcoma and soft-tissue sarcoma.

In some ments, the invention provides an isolated polynucleotide encoding an dy heavy or light chain or a functional fragment thereof, wherein the antibody heavy or light chain or functional fragment thereof encoded by the polynucleotide of the invention has one or more of the mentarity determining regions (CDRs) depicted in FIGS. 1-21 or listed in Table 2. An antibody or onal fragment thereof that includes one or more of the CDRs can cally bind to GD2 as described herein. Specific g to GD2 can include the speciﬁcity, affinity and/or avidity as ed in Example I for any of the dies provided herein. In some aspects, an antibody or ﬁanctional fragment thereof d by the polynucleotides of the invention can include the complement dependent cytotoxicity (CDC) activity and/or antibody-dependent cell-mediated cytotoxicity (ADCC) activity of any one of the 2015/033954 clonal es 1B7, 2H12, 1G2, 1E9, 1H3, 2F5, 2F7, 2E12, 3lF9, 3lF9V2 or 32E2 described . Methods for assessing the speciﬁcity, afﬁnity and/or avidity of an antibody or ﬁlnctional fragment thereof are well known in the art and exemplary methods are provided herein.

In some embodiments, the antibody or ﬁanctional fragment thereof of the invention includes less than six CDRs. In some embodiments, the antibody or ﬁlnctional fragment thereof includes one, two, three, four, or ﬁve CDRs ed from the group consisting ofVH CDRl, VH CDR2, VH CDR3, VL CDRl, VL CDR2, and/or VL CDR3. In speciﬁc embodiments, the antibody or ﬁanctional fragment thereof includes one, two, three, four, or ﬁve CDRs selected from the group consisting ofVH CDRl, VH CDR2, VH CDR3, VL CDRl, VL CDR2, and/or VL CDR3 of clonal isolates 1B7, 2H12, 1G2, 1E9, 1H3, 2F5, 2F7, 2E12, 31F9, 31F9V2 or 32E2 described herein.

In some ments, the present invention provides an isolated polynucleotide encoding an antibody heavy chain or a functional nt thereof, the antibody heavy chain or functional nt thereof including a variable heavy chain (VH) domain having VH CDRl VH CDR2 and VH CDR3 amino acid sequences, wherein the VH CDRl amino acid sequence is selected from the group consisting of residues 26-33 of SEQ ID NO: 2; residues 26-33 of SEQ ID NO: 6; residues 26-33 of SEQ ID NO: 10; residues 26-33 of SEQ ID NO: 14; residues 26-33 of SEQ ID NO: 18; residues 26-33 of SEQ ID NO: 22; es 26-33 of SEQ ID NO: 26; es 26-33 of SEQ ID NO: 30; residues 26-33 of SEQ ID NO: 34; residues 26-33 of SEQ ID NO: 36; and residues 26-33 of SEQ ID NO: 40; the VH CDR2 amino acid sequence is selected from the group consisting of residues 51-58 of SEQ ID NO: 2; residues 51-58 of SEQ ID NO: 6; residues 51-58 of SEQ ID NO: 10; residues 51-58 of SEQ ID NO: 14; residues 51-58 of SEQ ID NO: 18; residues 51-58 of SEQ ID NO: 22; residues 51-58 of SEQ ID NO: 26; residues 51-58 of SEQ ID NO: 30; residues 51-58 of SEQ ID NO: 34; residues 51-58 of SEQ ID NO: 36; and residues 51-58 of SEQ ID NO: 40; and the VH CDR3 amino acid sequence is selected from the group consisting of residues 97-109 of SEQ ID NO: 2; residues 97-109 of SEQ ID NO: 6; residues 97-108 of SEQ ID NO: 10; residues 97-108 of SEQ ID NO: 14; residues 97-108 of SEQ ID NO: 18; residues 97-108 of SEQ ID NO: 22; residues 97-109 of SEQ ID NO: 26; residues 97- 109 of SEQ ID NO: 30; residues 97-110 of SEQ ID NO: 34; residues 97-110 of SEQ ID NO: 36; and residues 97-108 of SEQ ID NO: 40.

In other ments, the present invention provides an isolated polynucleotide encoding an antibody heavy chain or a functional fragment f, the antibody heavy chain or functional fragment thereof including a variable heavy chain (VH) domain having VH CDRl VH CDR2 and VH CDR3 amino acid ces, wherein the VH CDRl amino acid sequence is encoded by the nucleic acid sequence selected from the group ting of residues 76-99 of SEQ ID NO: 1; residues 76-99 of SEQ ID NO: 5; residues 76-99 of SEQ ID NO: 9; residues 76- 99 of SEQ ID NO: 13; residues 76-99 of SEQ ID NO: 17; residues 76-99 of SEQ ID NO: 21; residues 76-99 of SEQ ID NO: 25; residues 76-99 of SEQ ID NO: 29; residues 76-99 of SEQ ID NO: 33; residues 76-99 of SEQ ID NO: 35; residues 76-99 of SEQ ID NO: 39; the VH CDR2 amino acid sequence is encoded by the nucleic acid sequence selected from the group consisting ofresidues 4 of SEQ ID NO: 1; residues 151-174 of SEQ ID NO: 5; residues 151-174 of SEQ ID NO: 9; residues 4 of SEQ ID NO: 13; residues 151-174 of SEQ ID NO: 17; residues 151-174 of SEQ ID NO: 21; residues 151-174 of SEQ ID NO: 25; es 151-174 of SEQ ID NO: 29; es 151-174 of SEQ ID NO: 33; residues 151-174 of SEQ ID NO: 35; residues 15 1-174 of SEQ ID NO: 39; and the VH CDR3 amino acid sequence is encoded by the nucleic acid sequence selected from the group consisting of residues 7 of SEQ ID NO: 1; residues 289-327 of SEQ ID NO: 5; residues 289-324 of SEQ ID NO: 9; residues 289-324 of SEQ ID NO: 13; residues 289-324 of SEQ ID NO: 17; residues 289-324 of SEQ ID NO: 21; residues 289-327 of SEQ ID NO: 25; residues 289-327 of SEQ ID NO: 29; residues 289-330 of SEQ ID NO: 33; residues 289-330 of SEQ ID NO: 35; es 289-324 of SEQ ID NO: 39.

In some embodiments, the present ion provides an isolated polynucleotide encoding an antibody heavy chain or a functional fragment thereof, wherein the antibody heavy chain or functional fragment includes a variable heavy (VH) chain domain having the VH CDRl, VH CDR2 and VH CDR3 amino acid sequence of the clonal isolate 1B7, 2Hl2, 1G2, 1E9, 1H3, 2F5, 2F7, 2El2, 3lF9, 31F9V2 or 32E2.

In some embodiments, the present invention provides an isolated polynucleotide encoding an dy heavy chain or a functional fragment thereof, the antibody heavy chain or functional fragment thereof including a variable heavy (VH) chain domain, wherein the VH domain has VH CDRl, VH CDR2, and VH CDR3 amino acid sequences selected from the group consisting of residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 2; residues 26-33, residues 51-58, and es 97-109 of SEQ ID NO: 6; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 10; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 14; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 18; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 22; residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 26; residues 26-33, es 51-58, and residues 97- 109 of SEQ ID NO: 30; es 26-33, residues 51-58, and residues 97-110 of SEQ ID NO: 34; residues 26-33, es 51-58, and residues 97-110 of SEQ ID NO: 36; and residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 40.

In other ments, the t invention provides an isolated polynucleotide encoding an antibody heavy chain or a functional fragment thereof, the antibody heavy chain or functional fragment thereof including a variable heavy (VH) chain domain, wherein the VH domain has VH CDR1, VH CDR2, and VH CDR3 amino acid sequences encoded by nucleic acid sequences selected from the group consisting of residues 76-99, residues 151-174, and residues 289-327 of SEQ ID NO: 1; residues 76-99, residues 151-174, and residues 289-327 of SEQ ID NO: 5; residues 76-99, residues 151-174, and residues 289-324 of SEQ ID NO: 9; residues 76-99, residues 151-174, and residues 4 of SEQ ID NO: 13; residues 76-99, residues 151-174, and residues 289-324 of SEQ ID NO: 17; residues 76-99, residues 151-174, and residues 4 of SEQ ID NO: 21; residues 76-99, residues 151-174, and residues 289- 327 of SEQ ID NO: 25; residues 76-99, residues 151-174, and residues 7 of SEQ ID NO: 29; residues 76-99, residues 151-174, and residues 289-330 of SEQ ID NO: 33; residues 76-99, residues 151-174, and residues 289-330 of SEQ ID NO: 35; es 76-99, residues 151-174, and residues 289-324 of SEQ ID NO: 39.

In another embodiment, the present invention provides an isolated polynucleotide encoding an antibody heavy chain or a functional nt thereof, the antibody heavy chain or functional fragment thereof including a le heavy (VH) chain , wherein the VH domain has an amino acid sequence is selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 6; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 18; SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 30; SEQ ID NO: 34; SEQ ID NO: 36; and SEQ ID NO: 40.

In yet another embodiment, the present invention provides an isolated polynucleotide encoding an antibody heavy chain or a functional fragment thereof, the antibody heavy chain or functional fragment thereof ing a variable heavy (VH) chain domain, wherein the VH domain amino acid sequence is encoded by the nucleic acid ce selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 5; SEQ ID NO: 9; SEQ ID NO: 13; SEQ ID NO: 17; SEQ ID NO: 21; SEQ ID NO: 25; SEQ ID NO: 29; SEQ ID NO: 33; SEQ ID NO: 35; and SEQ ID NO: 39.

In some ments, the t invention provides an isolated polynucleotide encoding an antibody light chain or a functional fragment thereof, the antibody light chain or functional fragment thereof including a variable light chain (VL) domain haVing VL CDRl VL CDR2 and VL CDR3 amino acid sequences, n the VL CDRl is selected from the group consisting of residues 27-37 of SEQ ID NO: 4; residues 27-37 of SEQ ID NO: 8; es 27-38 of SEQ ID NO: 12; residues 27-38 of SEQ ID NO: 16; residues 27-38 of SEQ ID NO: 20; residues 27-38 of SEQ ID NO: 24; residues 27-37 of SEQ ID NO: 28; residues 27-37 of SEQ ID NO: 32; residues 27-32 of SEQ ID NO: 38; and residues 27-38 of SEQ ID NO: 42; the VL CDR2 is selected from the group consisting of residues 55-57 of SEQ ID NO: 4; residues 55-57 of SEQ ID NO: 8; residues 56-58 of SEQ ID NO: 12; residues 56-58 of SEQ ID NO: 16; residues 56-58 of SEQ ID NO: 20; residues 56-58 of SEQ ID NO: 24; residues 55-57 of SEQ ID NO: 28; residues 55-57 of SEQ ID NO: 32; es 50-52 of SEQ ID NO: 38; and residues 56- 58 of SEQ ID NO: 42, and the VL CDR3 is selected from the group consisting of residues 94- 102 of SEQ ID NO: 4; residues 94-102 of SEQ ID NO: 8; residues 95-103 of SEQ ID NO: 12; residues 95-103 of SEQ ID NO: 16; residues 95-103 of SEQ ID NO: 20; residues 95-103 of SEQ ID NO: 24; residues 94-102 of SEQ ID NO: 28; residues 94-102 of SEQ ID NO: 32; residues 89- 97 of SEQ ID NO: 38; and residues 95-103 of SEQ ID NO: 42.

In some embodiments, the t invention provides an isolated polynucleotide encoding an antibody light chain or a functional fragment thereof, the antibody light chain or functional fragment thereof including a variable light chain (VL) domain haVing VL CDRl VL CDR2 and VL CDR3 amino acid sequences, n the VL CDRl is encoded by the nucleic acid sequence selected from the group consisting of residues 79-l 11 of SEQ ID NO: 3; residues 79-l 11 of SEQ ID NO: 7; residues 79-l 14 of SEQ ID NO: 11; residues 79-l 14 of SEQ ID NO: ; residues 79-l 14 of SEQ ID NO: 19; residues 79-l 14 of SEQ ID NO: 23; residues 79-l 11 of SEQ ID NO: 27; es 79-l 11 of SEQ ID NO: 31; residues 79-96 of SEQ ID NO: 37; and residues 79-114 of SEQ ID NO: 41; the VL CDR2 is encoded by the c acid sequence selected from the group consisting of residues 163-171 of SEQ ID NO: 3; 163-171 of SEQ ID NO: 7; 166-174 ofSEQ ID NO:11;166-174 ofSEQ ID NO: 15; 166-174 ofSEQ ID NO:19; 166-174 ofSEQ ID NO: 23; 163-171 ofSEQ ID NO: 27; 163-171 ofSEQ ID NO:31;148-156 of SEQ ID NO: 37; and 4 of SEQ ID NO: 41; and the VL CDR3 is encoded by the nucleic acid sequence selected from the group consisting of residues 6 of SEQ ID NO: 3; residues 6 of SEQ ID NO: 7; residues 283-309 of SEQ ID NO: 11; es 283-309 of SEQ ID NO: 15; residues 283-309 of SEQ ID NO: 19; residues 283-309 of SEQ ID NO: 23; residues 280-306 of SEQ ID NO: 27; residues 280-306 of SEQ ID NO: 31; residues 265-291 of SEQ ID NO: 37; residues 283-309 of SEQ ID NO: 41.

In some embodiments, the present invention provides an isolated polynucleotide encoding an antibody light chain or a functional fragment thereof, wherein the antibody light chain or functional fragment includes a le light (VL) chain domain having the VL CDRl VL CDR2 and VL CDR3 amino acid sequence of the clonal isolate 1B7, 2H12, 1G2, 1E9, 1H3, 2F5, 2F7, 2E12, 31F9, 31F9V2 or 32E2.

In some ments, the present invention provides an isolated polynucleotide encoding an antibody light chain or a functional fragment thereof, the antibody light chain or functional fragment f including a variable light chain (VL) domain, n the VL domain has VL CDRl, VL CDR2, and VL CDR3 amino acid sequences selected from the group ting of residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 4; residues 27- 37, residues 55-57, and residues 94-102 of SEQ ID NO: 8; residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 12; residues 27-38, residues 56-58, and es 95-103 of SEQ ID NO: 16; residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 20; residues 27- 38, residues 56-58, and residues 95-103 of SEQ ID NO: 24; residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 28; residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 32; residues 27-32, residues 50-52, and residues 89-97 of SEQ ID NO: 38; and residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 42.

In other embodiments, the present ion provides an isolated polynucleotide encoding an antibody light chain or a functional fragment thereof, the antibody light chain or functional nt thereof including a variable light chain (VL) domain, wherein the VL domain has VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are encoded by the nucleic acid sequence selected from the group consisting of es 79-111, residues 163-171, and es 280-306 of SEQ ID NO: 3; residues 79-111, residues 163-171, and residues 280- 306 of SEQ ID NO: 7; residues 79-114, residues 166-174, and residues 9 of SEQ ID NO: 11; residues 79-114, residues 166-174, and residues 283-309 of SEQ ID NO: 15; residues 79- 114, residues 166-174, and residues 283-309 of SEQ ID NO: 19; residues 79-114, residues 166- 174, and es 283-309 of SEQ ID NO: 23; residues 79-111, residues 163-171, and residues 280-306 of SEQ ID NO: 27; es 79-111, residues 163-171, and residues 280-306 of SEQ ID NO: 31; residues 79-96, residues 148-156, and residues 265-291 of SEQ ID NO: 37; residues 79- 114, residues 166-174, and residues 283-309 of SEQ ID NO: 41.

In another embodiment, the present invention provides an isolated polynucleotide encoding an dy light chain or a functional fragment thereof, the antibody light chain or functional fragment f including a variable light chain (VL) domain, wherein the VL domain has an amino acid sequence selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 38; and SEQ ID NO: 42.

In yet another ment, the present invention provides an isolated polynucleotide encoding an antibody light chain or a functional fragment thereof, the antibody light chain or onal fragment thereof including a variable light chain (VL) domain, wherein the VL domain amino acid sequence is encoded by the nucleic acid sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 7; SEQ ID NO: 11; SEQ ID NO: 15; SEQ ID NO: 19; SEQ ID NO: 23; SEQ ID NO: 27; SEQ ID NO: 31; SEQ ID NO: 37; and SEQ ID NO: 41.

In some embodiments, the present ion provides an isolated antibody or functional fragment thereof that binds to GD2. In some aspects, the antibody or ﬁanctional fragment f has one or more of the CDRs depicted in FIGS. 1-21 or listed in Table 2. An antibody or ﬁanctional fragment f that includes one or more of the CDRs, in particular CDR3, can speciﬁcally bind to GD2 as described herein. Specific binding to GD2 can e the speciﬁcity and ty as described in Example I for any of the antibodies provided herein.

In some aspects, an antibody or ﬁanctional fragment thereof of the invention can include the CDC activity and/or ADCC activity of any one of the clonal es 1B7, 2Hl2, 1G2, 1E9, lH3, 2F5, 2F7, 2El2, 31F9, 31F9V2 or 32E2 described herein.

In some embodiments, the present invention provides an isolated antibody or functional fragment thereof, wherein the antibody binds to GD2. Accordingly, in some s, the invention provides an isolated dy or functional fragment thereof that binds to GD2, the antibody or ﬁanctional fragment thereof including a variable heavy chain (VH) domain, the domain having VH CDRl, VH CDR2 and VH CDR3 amino acid sequences, wherein the VH CDRl amino acid sequence is selected from the group consisting of residues 26-33 of SEQ ID NO: 2; residues 26-33 of SEQ ID NO: 6; residues 26-33 of SEQ ID NO: 10; es 26-33 of SEQ ID NO: 14; residues 26-33 of SEQ ID NO: 18; residues 26-33 of SEQ ID NO: 22; residues 26-33 of SEQ ID NO: 26; residues 26-33 of SEQ ID NO: 30; residues 26-33 of SEQ ID NO: 34; residues 26-33 of SEQ ID NO: 36; and residues 26-33 of SEQ ID NO: 40; the VH CDR2 amino acid sequence is selected from the group consisting of es 51-58 of SEQ ID NO: 2; es 51-58 of SEQ ID NO: 6; residues 51-58 of SEQ ID NO: 10; es 51-58 of SEQ ID NO: 14; residues 51-58 of SEQ ID NO: 18; residues 51-58 of SEQ ID NO: 22; residues 51-58 of SEQ ID NO: 26; residues 51-58 of SEQ ID NO: 30; residues 51-58 of SEQ ID NO: 34; residues 51-58 of SEQ ID NO: 36; and residues 51-58 of SEQ ID NO: 40; and the VH CDR3 amino acid sequence is selected from the group consisting of residues 97-109 of SEQ ID NO: 2; residues 97-109 of SEQ ID NO: 6; residues 97-108 of SEQ ID NO: 10; residues 97-108 of SEQ ID NO: 14; residues 97-108 of SEQ ID NO: 18; residues 97-108 of SEQ ID NO: 22; residues 97-109 of SEQ ID NO: 26; residues 97-109 of SEQ ID NO: 30; residues 97-110 of SEQ ID NO: 34; residues 97-110 of SEQ ID NO: 36; and residues 97-108 of SEQ ID NO: 40.

In some other s, the invention provides an ed antibody or functional fragment f that binds to GD2, the antibody or functional fragment thereof including a variable heavy chain (VH) domain, wherein the VH domain has VH CDRl, VH CDR2, and VH CDR3 amino acid sequences ed from the group consisting of residues 26-33, residues 51- 58, and residues 97-109 of SEQ ID NO: 2; residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 6; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 10; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 14; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 18; residues 26-33, residues 51-58, and residues 97- 108 of SEQ ID NO: 22; residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 26; residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 30; residues 26-33, residues 51-58, and residues 97-110 of SEQ ID NO: 34; residues 26-33, residues 51-58, and es 97- 110 of SEQ ID NO: 36; and residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: In yet other aspects, the invention provides an isolated antibody or functional fragment thereof that binds to GD2, the antibody or onal fragment thereof including a variable heavy chain (VH) domain, wherein the VH domain has an amino acid sequence selected from the group ting of SEQ ID NO: 2; SEQ ID NO: 6; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 18; SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 30; SEQ ID NO: 34; SEQ ID NO: 36; and SEQ ID NO: 40.

In some ments, the present invention provides an isolated antibody or functional fragment thereof that binds to GD2, the antibody or ﬁanctional fragment thereof including a variable light chain (VL) domain, wherein the VL domain has VL CDRl , VL CDR2 and VL CDR3 amino acid sequences, wherein the VL CDRl is selected from the group consisting of residues 27-37 of SEQ ID NO: 4; residues 27-37 of SEQ ID NO: 8; residues 27-38 of SEQ ID NO: 12; residues 27-38 of SEQ ID NO: 16; residues 27-38 of SEQ ID NO: 20; residues 27-38 of SEQ ID NO: 24; residues 27-37 of SEQ ID NO: 28; residues 27-37 of SEQ ID NO: 32; residues 27-32 of SEQ ID NO: 38; and residues 27-38 of SEQ ID NO: 42; the VL CDR2 is selected from the group ting of residues 55-57 of SEQ ID NO: 4; residues 55-57 of SEQ ID NO: 8; residues 56-58 of SEQ ID NO: 12; es 56-58 of SEQ ID NO: 16; residues 56-58 of SEQ ID NO: 20; residues 56-58 of SEQ ID NO: 24; residues 55-57 of SEQ ID NO: 28; residues 55-57 of SEQ ID NO: 32; residues 50-52 of SEQ ID NO: 38; and residues 56- 58 of SEQ ID NO: 42, and the VL CDR3 is selected from the group ting of residues 94- 102 of SEQ ID NO: 4; residues 94-102 of SEQ ID NO: 8; residues 95-103 of SEQ ID NO: 12; residues 95-103 of SEQ ID NO: 16; residues 95-103 of SEQ ID NO: 20; residues 95-103 of SEQ ID NO: 24; residues 94-102 of SEQ ID NO: 28; residues 94-102 of SEQ ID NO: 32; residues 89- 97 of SEQ ID NO: 38; and residues 95-103 of SEQ ID NO: 42.

In some aspects, the t invention provides an isolated antibody or ﬁanctional fragment thereof that binds to GD2, the antibody or functional fragment thereof including a variable light chain (VL) domain, wherein the VL domain has VL CDRl, VL CDR2, and VL CDR3 amino acid sequences selected from the group consisting of es 27-37, residues 55- 57, and residues 94-102 of SEQ ID NO: 4; residues 27-37, es 55-57, and residues 94-102 of SEQ ID NO: 8; residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 12; residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 16; es 27-38, residues 56-58, and es 95-103 of SEQ ID NO: 20; residues 27-38, residues 56-58, and residues 95- 103 of SEQ ID NO: 24; residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 28; residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 32; residues 27-32, residues 50-52, and residues 89-97 of SEQ ID NO: 38; and residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 42.

In some other aspects, the present invention provides an isolated antibody or functional fragment thereof that binds to GD2, the antibody or ﬁanctional fragment f including a variable light chain (VL) , wherein the VL domain has an amino acid sequence selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 38; and SEQ ID NO: 42.

In some embodiments, the present ion provides an isolated antibody or functional fragment thereof that binds to GD2, the antibody or onal fragment thereof including a le heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain has an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 6; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 18; SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 30; SEQ ID NO: 34; SEQ ID NO: 36; and SEQ ID NO: 40; and the VL has an amino acid sequence selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 38; and SEQ ID NO: 42.

In some other embodiments, the present invention provides an isolated antibody or onal fragment thereof that binds to GD2, the antibody or ﬁanctional fragment thereof including a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain and the VL domain respectively include amino acid sequences from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; SEQ ID NO: 6 and SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 20; SEQ ID NO: 22 and SEQ ID NO: 24; SEQ ID NO: 26 and SEQ ID NO: 28; SEQ ID NO: 30 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 38; SEQ ID NO: 36 and SEQ ID NO: 38; and SEQ ID NO: 40 and SEQ ID NO: 42.

Table 2: CDRs of Clonal Isolates Variable Nucleic Acid Residues Amino Acid Sequence Domain 1B7 VH 151-174 289-327 2H12 VH 1G2 VH 76-99 151-174 4 26-33 51-58 97-108 1E9 VH 76-99 1-174 289-324 51 97-108 VH 76-99 151-174 289-324 26-33 51-58 97-108 2F7 VH 76-99 151-174 289-327 26-33 51-58 97-109 2E12 VH 76-99 151-174 289-327 26-33 51-58 97-109 31F9 VH 31F9 VL 79-96 148-156 1 27-32 50-52 89-97 32E2 VL 79-114 166-174 283-309 27-38 56-58 95-103 (NO: 41) (NO: 41) (NO: 41) (NO: 42) (NO: 42) (NO: 42) In another embodiment, the invention provides a variant of the polynucleotides provided herein. A t when used in reference to a polynucleotide includes a polynucleotide having one or more modiﬁed nucleotides, such as, but not d to, a methylated nucleotide or a nucleotide analog. Additionally, a variant polynucleotide can include a polynucleotide that is interrupted by non-nucleotide components. Modiﬁcations to a polynucleotide can be imparted before or after assembly of the polynucleotide using methods well known to those skilled in the art. For example, a polynucleotide can be modiﬁed after rization by conjugation with a labeling component using either enzymatic or chemical techniques (e.g., as described in Gottfried and Weinhold, 2011, m. Soc. Trans., 523-628; Paredes et al., 2011, Methods, 54(2):25 l-259).

The polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method well known in the art. Since the amino acid sequences of the variable heavy and light chain domains of 1B7, 2H12, 1G2, 1E9, 1H3, 2F5, 2F7, 2E12, 3lF9, 3lF9V2 and 32E2 are known (see, e.g., SEQ ID NOS: 2, 4, 6, 8, 10, l2, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42), nucleotide sequences encoding dies and modiﬁed versions of these antibodies can be determined using methods well known in the art, z'.e., nucleotide codons known to encode ular amino acids are assembled in such a way to generate a nucleic acid that s the antibody. Such a polynucleotide encoding the antibody can be assembled from chemically synthesized ucleotides (e.g., as described in Kutmeier et al., 1994, BioTechm'ques , which, brieﬂy, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, fragments, or variants thereof, annealing and ligating of those oligonucleotides, and then cation of the ligated oligonucleotides by PCR.

A polynucleotide encoding an antibody or a functional fragment thereof of the invention can be generated using the c acid sequence of the variable heavy and/or light chain s of isolates 1B7, 2H12, 1G2, 1E9, 1H3, 2F5, 2F7, 2E12, 3lF9, 3lF9V2 or 32E2 (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41). A nucleic acid encoding the antibody or functional fragment can be chemically synthesized or obtained from a suitable source (e.g., cDNA isolated from cells expressing the antibody or functional fragment thereof, such as hybridoma cells ed to express the antibody or functional fragment thereof) by PCR ampliﬁcation using synthetic primers hybridizable to the 3 ’ and 5’ ends of the sequence or by cloning using an oligonucleotide probe speciﬁc for the particular nucleic acid ce. Ampliﬁed nucleic acids generated by PCR can then be cloned into replicable g vectors using any method well known in the art.

In some aspects of the invention, the isolated antibody or functional fragment thereof is a monoclonal antibody. In some aspects of the invention, the isolated antibody or ﬁlnctional fragment f provided herein is an IgG or IgM isotype. In a further aspect of the invention, the antibody or function fragment thereof is an dy of the IgGl subclass.

In some embodiments, the present invention provides a method of producing an antibody or ﬁlnctional fragment thereof of the invention. The method of the invention can include introducing a polynucleotide of the invention into a host cell, culturing the host cell under conditions and for a sufﬁcient period of time to produce the encoded heavy and/or light chain of an antibody or functional fragment of the invention, and ing the heavy and/or light chain of an antibody or functional fragment. In other embodiments, the t invention provides a recombinant cell having a polynucleotide encoding an antibody or a functional fragment of the invention. In some aspects, the dy or function fragment f has the variable heavy chain domain and the variable light chain domain of the designated antibodies 1B7, 2H12, 1G2, 1E9, lH3,2F5,2F7,2E12,3lF9,3lF9V2 or 32E2.

Recombinant expression of an dy or functional fragment thereof of the ion that binds to a GD2 antigen can include construction of an expression vector containing a polynucleotide that encodes the heavy and/or light chain of an antibody or functional fragment of the invention. Once a polynucleotide encoding an antibody or ﬁlnctional fragment thereof (preferably, but not necessarily, containing the heavy and/or light chain le domain) of the invention has been obtained, the vector for the production of the dy or functional fragment can be ed by recombinant DNA technology using ques well known in the art. Methods for preparing a protein by expressing a polynucleotide containing an antibody or a functional fragment thereof encoding nucleotide sequence are described herein.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody or functional fragments thereof coding sequences and riate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in viva genetic recombination. The invention, thus, provides replicable vectors including a nucleotide sequence encoding an antibody or onal fragment thereof of the invention ly linked to a promoter. Such vectors can include the nucleotide sequence encoding the constant region of the dy molecule (see, e.g., International Publication Nos. WO 86/05 807 and WO 89/01036; and US. Patent No. 5,122,464) and the variable domain of the antibody can be cloned into such a vector for sion of the entire heavy, the entire light chain, or both the entire heavy and light chains.

The expression vector can be transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody or functional fragment thereof of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody or functional nt thereof of the invention operably linked to a heterologous promoter. In some embodiments for the expression of double-chained dies, vectors encoding both the heavy and light chains can be co-expressed in the host cell for sion of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems can be utilized to express the antibody or functional fragments f of the invention (see, e.g., US. Patent No. 5,807,715). Such host- expression systems represent vehicles by which the coding sequences of interest can be ed and subsequently puriﬁed, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the ion in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing dy coding sequences; insect cell s infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell s infected with recombinant virus expression vectors (e.g., cauliﬂower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g, Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g, metallothionein promoter) or from mammalian viruses (e.g, the adenovirus late promoter; the ia virus 7.5K er). In some s, ial cells such as Escherichia coli, or eukaryotic cells, especially for the expression of whole recombinant antibody, are used for the expression of a recombinant antibody or onal fragment. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major ediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, chnology 8:2). In some embodiments, antibodies or fragments thereof of the invention are ed in CHO cells. In one embodiment, the sion of nucleotide sequences encoding antibodies or functional fragments thereof of the invention which bind to GD2 is regulated by a constitutive er, inducible promoter or tissue speciﬁc promoter.

In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, s which direct the expression of high levels of fusion protein products that are readily puriﬁed can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody coding sequence can be d individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13 :3 101-3 109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with hione 5-transferase (GST). In general, such fusion ns are soluble and can easily be puriﬁed from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by n in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect , Autographa calz'form'ca r polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodopteraﬁugz’perda cells. The antibody or onal fragment coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems can be utilized.

In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in viva recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1355-359). Speciﬁc initiation signals can also be used for efficient translation of inserted antibody coding sequences. These signals e the ATG initiation codon and adjacent sequences. Furthermore, the tion codon must be in phase with the reading frame of the desired coding ce to ensure translation of the entire insert. These exogenous translational l signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate ription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153 :5 1-544).

] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.

Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the antibody or onal fragment. Different host cells have teristic and specific mechanisms for the post-translational processing and cation of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene t can be used. Such mammalian host cells include but are not limited to CH0, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not nously produce any immunoglobulin chains), O and HsS78Bst cells.

For long-term, high-yield production of inant proteins, stable expression is preferred. For example, cell lines which stably express the antibody or functional fragment of the invention can be engineered. Rather than using sion vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody molecule.

A number of selection systems can be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 7) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA. 77(6):3567-70; O’Hare et al., 1981, Proc. Natl. Acad. Sci.

USA 78: 1527); glutamine synthetase (GS), which is an enzyme responsible for the biosynthesis of ine using glutamate and ammonia (Bebbington et al., 1992, Biaotechnology 103169); gpt, which confers resistance to mycophenolic acid gan & Berg, 1981, Proc. Natl. Acad.

Sci. USA 78:2072); neo, which confers ance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3 :87-95; shev, 1993, Ann. Rev. Pharmacol. T0xic0l. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; May, 1993, TIB TECH 11(5): 155-215); and hygro, which confers resistance to hygromycin rre et al., 1984, Gene 30: 147). Methods well known in the art of recombinant DNA technology can be routinely applied to select the desired inant clone, and such methods are described, for e, in Ausubel et al. , Current Protocols in Molecular 2015/033954 Biology, John Wiley & Sons, NY ; Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY ; and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J.

Mol. Biol. 150: 1, which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector ampliﬁcation (for a review, see Bebbington and Hentschel, The use of vectors based on gene ampliﬁcation for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody or ﬁanctional fragment thereof is ampliﬁable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the ampliﬁed region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell can be co-transfected with two expression vectors of the invention, the ﬁrst vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two s can contain identical selectable markers which enable equal expression of heavy and light chain ptides. Alternatively, a single vector can be used which s, and is capable of sing, both heavy and light chain polypeptides.

In such situations, the light chain can be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci.

USA 77:2197-2199). The coding sequences for the heavy and light chains can include cDNA or c DNA.

Additionally, polynucleotides encoding the heavy and/or light chains of the antibody or onal fragment of the ion can be subjected to codon optimization using techniques well known in the art to achieve zed expression of an antibody or functional fragment of the invention in a desired host cell. For example, in one method of codon optimization, a native codon is substituted by the most frequent codon from a reference set of genes, n the rate of codon translation for each amino acid is designed to be high. Additional exemplary methods for generating codon optimized polynucleotides for expression of a desired protein, which can be applied to the heavy and/or light chains of the antibody or ﬁanctional fragment of the invention, are described in Kanaya et al., Gene, 238: 5 (1999), Wang et al., Mol. Biol. Evol., WO 87811 792-800 (2001), US. Patent 5,795,737, US. Publication 2008/0076161 and WO 2008/000632.

Once an antibody molecule of the invention has been produced by recombinant expression, it can be d by any method known in the art for puriﬁcation of an globulin molecule, for example, by chromatography (e.g., ion exchange, afﬁnity, particularly by afﬁnity for the c antigen after Protein A, and sizing column chromatography), centriﬁlgation, differential solubility, or by any other standard technique for the puriﬁcation of proteins. Further, the antibodies or functional fragments of the present invention can be fused to heterologous polypeptide sequences provided herein or otherwise known in the art to facilitate puriﬁcation. For example, an antibody or functional fragment of the invention can be puriﬁed through recombinantly adding a poly-histidine tag (His-tag), FLAG- tag, hemagglutinin tag (HA-tag) or myc-tag among others that are commercially available and utilizing puriﬁcation methods well known to those skilled in the art.

In some embodiments, the antibody ﬁlnctional fragment of the invention can be, but is not limited to, a Fab, a Fab’, a F(ab’)2, a Fabc, a scFV, a diabody, a triabody, minibody or a single-domain antibody (sdAB). With respect to antibodies and ﬁlnctional fragments thereof, various forms, alterations and modiﬁcations are well known in the art. The GD2 speciﬁc antibody fragments of the invention can include any of such various dy forms, alterations and modiﬁcations. Examples of such s forms and terms as they are known in the art are set forth below.

A Fab fragment refers to a lent fragment ting of the VL, VH, CL and CH1 s; a F(ab')2 fragment is a bivalent fragment including two Fab nts linked by a disulﬁde bridge at the hinge region; a Fd fragment consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH s of a single arm of an antibody; and a dAb fragment (Ward et al., Nature 341 :544-546, (1989)) consists of a VH domain.

An antibody can have one or more binding sites. If there is more than one binding site, the binding sites can be identical to one another or can be ent. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab nt has one binding site, while a “bispeciﬁc” or “biﬁlnctional” antibody has two different binding sites.

] A -chain antibody (scFv) refers to an antibody in which a VL and a VH region are joined via a linker (e. g., a synthetic sequence of amino acid residues) to form a continuous polypeptide chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., Science 242:423-26 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-83 (1988)). Diabodies refer to bivalent antibodies including two polypeptide chains, n each polypeptide chain includes VH and VL domains joined by a linker that is too short to allow for pairing between two s on the same chain, thus allowing each domain to pair with a mentary domain on another polypeptide chain (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993), and Poljak et al., Structure 2: 1 121-23 (1994)). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical n binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. rly, tribodies and tetrabodies are antibodies including three and four polypeptide , respectively, and forming three and four antigen binding sites, respectively, which can be the same or ent.

The present invention also provides an antibody or ﬁanctional fragment thereof derivative of 1B7, 2H12, 1G2, 1E9, 1H3, 2F5, 2F7, 2E12, 31F9, 31F9V2 and 32E2, wherein the antibody or ﬁanctional fragment binds to GD2. Standard techniques well known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody or functional fragment f of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. In some aspects, the derivative includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid tutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule.

In some embodiments, the invention provides an antibody or ﬁanctional fragment thereof having ed forms of naturally occurring amino acids, conservative substitutions, non-naturally occurring amino acids, amino acid analogues and mimetics so long as such the antibody or ﬁanctional fragment retains functional activity as defined herein. In one ment, the derivative has conservative amino acid substitutions that are made at one or more predicted non-essential amino acid residues. A conservative amino acid tution is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been deﬁned in the art. These families include amino acids with basic side chains (e.g., lysine, ne, histidine), acidic side chains (e.g., aspartic acid, ic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, cine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by tion nesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.

Following mutagenesis, the encoded dy or ﬁanctional fragment thereof can be expressed and the activity of the antibody or functional fragment can be determined.

In some embodiments, the invention provides an antibody or ﬁmctional fragment thereof having modified ﬁlcosylation, galactosylation and/or sialylation of an Fc fragment contained within an dy or functional fragment of the invention. Such cations of an Fc fragment can effect Fc receptor—mediated activity as discussed in Peipp et al., Blood, ll2(6):2390-2399 (2008). For example, glycoengineered therapeutic antibodies lacking core fucose residues from the Fc N—glycans exhibit strong ADCC at lower concentrations with much higher efficacy compared to ﬁJcosylated counterparts. Shields et al., J. Biol. Chem, 277(30):26733-40 (2002); Okazaki et al., JMol Bz'ol., 336:1239—1249 (2004); Natsume et al., J.

Immunol. Methods, 306:93—103 (2005). Methods for modifying the fucosylation, galactosylation and/or sialylation of an antibody for functional fragment thereof are well known in the art. For example, defucosylation approaches can be grouped into three methodologies (1) conversion of the N—glycosylation y of nonmammalian cells to the ‘humanized’ non- ﬁlcosylation pathway; (2) inactivation of the an lation pathway of mammalian cells and (3) in vitro chemical synthesis of cosylated N—glycoprotein or enzymatic modification ofN—glycans to non-fucosylated forms, as bed in Yamane-Ohnuki et al., MAbs., l(3):230- 236 (2009). It is understood that any one of these methods or any other method that is well known in the art can be used to produce an dy or functional fragment thereof having modiﬁed fucosylation, galactosylation and/or sialylation.

Antibodies or functional fragments thereof of the invention that bind to GD2 can be ed by any method known in the art for the synthesis of dies, in particular, by chemical synthesis or by recombinant sion techniques. The practice of the invention employs, unless otherwise indicated, tional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modiﬁcation, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g.,, Maniatis et al. (1982) Molecular Cloning: A Laboratogy , Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratogy Manual, Second Edition, Cold Spring Harbor tory Press; Sambrook et al. (2001) lar Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current ols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford sity Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press, Inc; each of which is incorporated herein by reference in its entirety.

] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of oma and recombinant technologies, or a combination thereof For example, onal dies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which is orated herein by reference in its entirety. A monoclonal antibody is not limited to antibodies produced through hybridoma technology. Other exemplary methods of producing monoclonal antibodies are known in the art. Additional exemplary methods of producing monoclonal antibodies are provided in Example I . dy functional fragments which bind GD2 can be ted by any technique well known to those of skill in the art. For example, Fab and F(ab’)2 fragments of the invention can be produced by proteolytic ge of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab’)2 fragments). F(ab’)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

The antibody functional fragments of the invention can also be generated using various phage y methods known in the art. For example, in phage display methods, functional antibody domains, such as the heavy and/or light chain variable regions having one, two, three, four, ﬁve or six CDRs provided , are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. The DNA encoding the VH and VL domains are ined er with an scFv linker by PCR and cloned into a id vector.

The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are lly filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to a particular n, such as GD2, can be selected or identified with antigen, e.g, using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibody functional fragments of the t invention include those disclosed in Brinkman et al., 1995, J.

Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184: 6; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57: 191-280; PCT Application No.

PCT/GB91/01134; International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO97/13844; and US. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, ,427,908, 5,516,637, 5,780,225, 5,658,727, 743 and 5,969,108; each ofwhich is incorporated herein by reference in its entirety.

As bed in the above nces, after phage selection, the antibody coding s from the phage can be isolated and used to generate whole antibodies, including human dies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described herein.

Techniques to inantly produce Fab, Fab’ and F(ab’)2 fragments can also be employed using s known in the art such as those disclosed in PCT ation No. WO 92/22324; Mullinax et al., 1992, BioTechm'ques l2(6):864-869; Sawai et al., 1995, AJRI 34:26- 34; and Better et al., 1988, Science 240: 1041-1043, each of which is incorporated by reference in its entirety.

To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a ﬂanking sequence to t the restriction site can be used to amplify the VH or VL ces in scFv clones. Utilizing cloning techniques well known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 1 constant region, and the PCR ampliﬁed VL domains can be cloned into vectors expressing a VL constant region, e.g, human kappa or lambda constant regions. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to te stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques well known to those of skill in the art.

In some embodiments, an antibody or ﬁanctional fragment of the invention is conjugated ent or non-covalent conjugations) or recombinantly fused to one or more diagnostic agent, detectable agent or therapeutic agent or any other desired molecule. The conjugated or recombinantly fused antibody or ﬁanctional fragment can be useful for monitoring or diagnosing the onset, development, progression and/or severity of a disease associated with the expression of GD2, such as cancer or tumor formation, as part of a clinical testing procedure, such as determining the cy of a particular therapy.

In some aspects, an antibody or ﬁanctional fragment thereof of the invention is ated with a detectable agent. Detection and diagnosis can be accomplished, for example, by coupling the antibody or functional nt of the invention to detectable substances including, but not limited to, radioactive materials, such as, but not limited to, ium (gng), iodine (1311, 1251, 1241, 1231, and 1211,), carbon( 14c, 11C), sulfur (355), tritium (3H), indium (“5111, 113In, 112In, and ), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), ﬂuorine (18F), 15O, 13N, 64Cu, 94mTc, 153 Sm, 177Lu, 159Gd, 149Pm, 140La, 175%, 166HO’ 86Y’ 90Y, 47Sc, 186Re’ 188Re’ 142Pr, IOSRh’ 97Ru, 68Ge’ 57Co, 65211, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Sn; and positron emitting metals using various positron emission aphies, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, alactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin and avidin/biotin; ﬂuorescent materials, such as, but not d to, umbelliferone, ﬂuorescein, cein isothiocynate, rhodamine, dichlorotriazinylamine ﬂuorescein, dansyl de or phycoerythrin; scent als, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin, and non-radioactive paramagnetic metal ions.

The present invention ﬁarther encompasses therapeutic uses of an antibody or functional fragment of the invention conjugated (covalent or non-covalent conjugations) or recombinantly fused to one or more therapeutic agent. In this context, for example, the dy can be conjugated or recombinantly fused to a therapeutic agent, such as a cytotoxin, e.g, a cytostatic or cytocidal agent, or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin or xic agent includes any agent that is detrimental to cells. A therapeutic agent can be a chemotherapeutic such as, but is not limited to, an anthracycline (e.g., doxorubicin and daunorubicin (formerly daunomycin)); a taxan (e.g., axel (Taxol) and docetaxel (Taxotere); an antimetabolite (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- ﬂuorouracil and decarbazine); or an alkylating agent (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU), lomustine , cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisdichlorodiamine platinum (II) (DDP) and cisplatin); an antibiotic (e.g., actinomycin D, bleomycin, mithramycin, and anthramycin ; an Auristatin molecule (e.g., atin PHE, bryostatin l, solastatin lO, monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF)); a hormone (e.g., glucocorticoids, progestins, androgens, and estrogens); a nucleoside analoge (e.g. Gemcitabine), a DNA-repair enzyme inhibitor (e.g., ide and topotecan), a kinase inhibitor (e.g., compound STlS7l, also known as Gleevec or imatinib mesylate); a cytotoxic agent (e.g., maytansine, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, stine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, ntrone, mithramycin, 1-dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof, and those compounds disclosed in US. Patent Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410, 372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872,223, ,863,904, 5,840,745, 5,728,868, 5,648,239, 5,587,459); a famesyl transferase inhibitor (e.g., R115777, BMS-2l4662, and those disclosed by, for example, US. Patent Nos: 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 387, 6,414,145, 6,410,541, 6,410,539, 581, 6,399,615, 905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218,406, 193, 6,187,786, 6,169,096, 6,159,984, 6,143,766, 6,133,303, 366, 6,124,465, 6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 574, and 6,040,305); a topoisomerase inhibitor (e.g., camptothecin, irinotecan, SN-38, topotecan, ocamptothecin, GG-211 (GI 147211), 1f, IST-622, rubitecan, pyrazoloacridine, XR-5000, saintopin, UCE6, UCE1022, TAN-1518A, TAN 1518B, KT6006, KT6528, ED-110, NB-506, ED-110, NB-506, fagaronine, coralyne, beta-lapachone and rebeccamycin); a DNA minor groove binder (e.g., Hoescht dye 33342 and Hoechst dye 33258); adenosine deaminase tors (e.g, Fludarabine phosphate and 2-Chlorodeoxyadenosine); or pharmaceutically acceptable salts, solvates, clathrates, or prodrugs thereof. A therapeutic agent can be a immunotherapeutic such as, but is not limited to, cetuximab, bevacizumab, heceptin, rituximab).

In addition, an antibody or functional fragment of the invention can be conjugated to a therapeutic agent such as a radioactive metal ion, such as emitters such as Bi or macrocyclic ors useful for conjugating radiometal ions, including but not limited to, 131In, 131 131 131 131 LU, Y, Ho, Sm, or a macrocycl1c or, such as l,4,7,l0-tetraazacyclododecane--_ N,N’,N’ ’,N’ ’ ’-tetraacetic acid (DOTA) which can be attached to the dy or ﬁmctional fragment via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50.

Further, an antibody or onal fragment of the invention can be conjugated (covalent or non-covalent conjugations) or recombinantly ﬁJsed to a therapeutic agent that modiﬁes a given biological response. Thus, therapeutic agents are not to be construed as limited to classical chemical therapeutic agents. For example, the therapeutic agent can be a protein, peptide, or polypeptide possessing a desired biological activity. Such proteins can include, for example, a toxin (e.g., abrin, ricin A, monas in, cholera toxin and diphtheria toxin); a protein such as tumor necrosis factor, y-interferon, (x-interferon, nerve growth factor, platelet d growth factor, tissue plasminogen activator, an apoptotic agent (e.g., TNF-y, AIM I, AIM II, Fas Ligand and VEGF), an anti-angiogenic agent (e.g., tatin, endostatin and a component of the coagulation pathway such as tissue factor); a biological response modifier (e.g. , a cytokine such as interferon gamma, interleukin-1, interleukin-2, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin- 1 0, eukin- 12, interleukin- 1 5 , interleukin-23, ocyte hage colony ating factor, and ocyte colony stimulating factor); a growth factor (e.g., growth e), or a coagulation agent (e.g., calcium, vitamin K, tissue factors, such as but not limited to, Hageman factor (factor XII), high- molecular-weight kininogen (HMWK), prekallikrein (PK), coagulation proteins-factors II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid, and ﬁbrin monomer).

The present invention encompasses antibodies or functional fragments of the invention recombinantly fused or chemically conjugated (covalent or non-covalent conjugations) to a heterologous protein or polypeptide to generate fusion proteins. In some aspects, such a polypeptide can be about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 amino acids in . In some aspects, the invention provides fusion proteins having a functional fragment of an antibody of the invention (e.g., a Fab fragment, Fd nt, Fv nt, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein or polypeptide. In one embodiment, the heterologous protein or polypeptide that the antibody or functional fragment is fused to is useful for targeting the antibody or ﬁanctional nt to a particular cell type, such as a cell that expresses GD2.

A ated or fusion protein of the invention includes any antibody or ﬁanctional fragment of the invention provided herein conjugated (covalent or non-covalent conjugations) or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In one embodiment, a conjugated or fusion protein of the invention includes a 1B7, 2H12, 1G2, 1E9, lH3, 2F5, 2F7, 2El2, 3lF9, 3lF9V2 or 32E2 antibody, and a stic agent, detectable agent or therapeutic agent. In another embodiment, a conjugated or fusion protein of the invention includes a functional fragment of 1B7, 2Hl2, 1G2, 1E9, lH3, 2F5, 2F7, 2El2, 3lF9, 3lF9V2 or 32E2 antibodies, and a diagnostic agent, detectable agent or therapeutic agent.

] In some embodiments, a conjugated or fusion protein of the present ion includes one or more VH CDRs having the amino acid sequence of any one of the VH CDRs depicted in SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, or 40 and a diagnostic agent, detectable agent or therapeutic agent. In r embodiment, a conjugated or fusion protein includes one or more VL CDRs having the amino acid sequence of any one of the VL CDRs depicted in SEQ ID NOS: 4, 8, l2, 16, 20, 24, 28, 32, 38, or 42 and a diagnostic agent, detectable agent or therapeutic agent.

] In some aspects, a conjugated or fusion protein of the invention includes a VH domain, the VH domain having VH CDRl, VH CDR2 and VH CDR3 amino acid sequences, wherein the VH CDRl amino acid sequence is selected from the group consisting of residues 26- 33 of SEQ ID NO: 2; residues 26-33 of SEQ ID NO: 6; residues 26-33 of SEQ ID NO: 10; residues 26-33 of SEQ ID NO: 14; residues 26-33 of SEQ ID NO: 18; residues 26-33 of SEQ ID NO: 22; residues 26-33 of SEQ ID NO: 26; es 26-33 of SEQ ID NO: 30; residues 26-33 of SEQ ID NO: 34; residues 26-33 of SEQ ID NO: 36; and residues 26-33 of SEQ ID NO: 40; the VH CDR2 amino acid sequence is selected from the group consisting of residues 51-58 of SEQ ID NO: 2; residues 51-58 of SEQ ID NO: 6; residues 51-58 of SEQ ID NO: 10; residues 51-58 of SEQ ID NO: 14; es 51-58 of SEQ ID NO: 18; residues 51-58 of SEQ ID NO: 22; residues 51-58 of SEQ ID NO: 26; residues 51-58 of SEQ ID NO: 30; residues 51-58 of SEQ ID NO: 34; residues 51-58 of SEQ ID NO: 36; and residues 51-58 of SEQ ID NO: 40; and the VH CDR3 amino acid ce is selected from the group consisting of residues 97-109 of SEQ ID NO: 2; residues 97-109 of SEQ ID NO: 6; residues 97-108 of SEQ ID NO: 10; residues 97-108 of SEQ ID NO: 14; residues 97-108 of SEQ ID NO: 18; residues 97-108 of SEQ ID NO: 22; residues 97-109 of SEQ ID NO: 26; residues 97-109 of SEQ ID NO: 30; residues 97-110 of SEQ ID NO: 34; residues 97-110 of SEQ ID NO: 36; and residues 97-108 of SEQ ID NO: 40, and a diagnostic agent, detectable agent or therapeutic agent.

In some other aspects, a conjugated or fusion n of the invention includes a VH domain, the VH domain having VH CDRl, VH CDR2, and VH CDR3 amino acid sequences ed from the group consisting of residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 2; residues 26-33, es 51-58, and residues 97-109 of SEQ ID NO: 6; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 10; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 14; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 18; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 22; residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 26; residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 30; es 26-33, residues 51-58, and residues 97- 110 of SEQ ID NO: 34; residues 26-33, residues 51-58, and residues 97-110 of SEQ ID NO: 36; and residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 40, and a stic agent, detectable agent or therapeutic agent.

In yet other aspects, a conjugated or fusion protein of the invention includes a VH domain, the VH domain having an amino acid sequence selected from the group ting of SEQ ID NO: 2; SEQ ID NO: 6; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 18; SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 30; SEQ ID NO: 34; SEQ ID NO: 36; and SEQ ID NO: 40, and a diagnostic agent, detectable agent or therapeutic agent.

In some embodiments, a conjugated or fusion n of the invention includes a VL domain, the VL domain having VL CDRl, VL CDR2 and VL CDR3 amino acid ces, wherein the VL CDRl is selected from the group consisting of residues 27-37 of SEQ ID NO: 4; residues 27-37 of SEQ ID NO: 8; es 27-38 of SEQ ID NO: 12; residues 27-38 of SEQ ID NO: 16; residues 27-38 of SEQ ID NO: 20; es 27-38 of SEQ ID NO: 24; residues 27-37 of SEQ ID NO: 28; residues 27-37 of SEQ ID NO: 32; residues 27-32 of SEQ ID NO: 38; and residues 27-38 of SEQ ID NO: 42; the VL CDR2 is selected from the group consisting of residues 55-57 of SEQ ID NO: 4; residues 55-57 of SEQ ID NO: 8; residues 56-58 of SEQ ID NO: 12; residues 56-58 of SEQ ID NO: 16; residues 56-58 of SEQ ID NO: 20; residues 56-58 of SEQ ID NO: 24; residues 55-57 of SEQ ID NO: 28; residues 55-57 of SEQ ID NO: 32; residues 50-52 of SEQ ID NO: 38; and residues 56-58 of SEQ ID NO: 42, and the VL CDR3 is selected from the group consisting of residues 94-102 of SEQ ID NO: 4; residues 94-102 of SEQ ID NO: 8; residues 95-103 of SEQ ID NO: 12; residues 95-103 of SEQ ID NO: 16; residues 95-103 of SEQ ID NO: 20; residues 95-103 of SEQ ID NO: 24; residues 94-102 of SEQ ID NO: 28; residues 94-102 of SEQ ID NO: 32; es 89-97 of SEQ ID NO: 38; and residues 95-103 of SEQ ID NO: 42, and a diagnostic agent, detectable agent or therapeutic agent.

In other embodiments, a conjugated or fusion protein of the ion includes a VL domain, the VL domain having VL CDRl, VL CDR2, and VL CDR3 amino acid sequences ed from the group consisting of residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 4; residues 27-37, residues 55-57, and es 94-102 of SEQ ID NO: 8; residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 12; es 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 16; residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 20; residues 27-38, residues 56-58, and es 95-103 of SEQ ID NO: 24; residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 28; residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 32; residues 27-32, residues 50-52, and residues 89- 97 of SEQ ID NO: 38; and residues 27-38, residues 56-58, and es 95-103 of SEQ ID NO: 42, and a diagnostic agent, detectable agent or eutic agent.

In some other aspects, a conjugated or fusion protein of the invention includes a VL domain, the VL domain an amino acid sequence selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 38; and SEQ ID NO: 42, and a diagnostic agent, detectable agent or therapeutic agent.

In some embodiments, a conjugated or fusion protein of the invention includes a VH domain and a VL domain, wherein the VH domain has an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 6; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 18; SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 30; SEQ ID NO: 34; SEQ ID NO: 36; and SEQ ID NO: 40; and the VL domain has an amino acid sequence selected from the group ting of SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 38; and SEQ ID NO: 42, and a diagnostic agent, detectable agent or therapeutic agent.

In some other embodiments, a conjugated or fusion protein of the ion includes a VH domain and a VL domain, wherein the VH domain and the VL domain respectively have an amino acid sequence from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; SEQ ID NO: 6 and SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 20; SEQ ID NO: 22 and SEQ ID NO: 24; SEQ ID NO: 26 and SEQ ID NO: 28; SEQ ID NO: 30 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 38; SEQ ID NO: 36 and SEQ ID NO: 38; and SEQ ID NO: 40 and SEQ ID NO: 42, and a diagnostic agent, detectable agent or therapeutic agent.

Methods for fusing or conjugating diagnostic agents, detectable agents or therapeutic agents (including polypeptides) to antibodies are well known, see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody rs Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And al Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future ctive Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer ion And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol. Rev. 62:119-58; US. Pat. Nos. ,336,603, 5,622,929, 5,359,046, 053, 5,447,851, 5,723,125, 181, 626, ,844,095, 5,112,946, 7,981,695, 8,039,273, 8,142,784; US. ations 2009/0202536, 2010/0034837, 2011/0137017, 2011/0280891, 2012/0003247; EP 307,434; EP 6; EP 394,827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991; Traunecker et al., Nature, 331 :84-86, 1988; Zheng et al., J. l., 90-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA, 89:11337-11341, 1992; and Senter, Current Opinion in Chemical Biology, 13:235-244 (2009), which are incorporated herein by reference in their entireties.

In another aspect, a stic agent, detectable agent or therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation.

WO 87811 2015/033954 Alternatively, such agents can be attached to the dy component using a heterobifunctional linker, such as N-succinyl yridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for such ation are well known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et a1., “Modiﬁcation of Antibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 -Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995).

Alternatively, a diagnostic agent, detectable agent or therapeutic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody. Methods for conjugating peptides to antibody components via an antibody carbohydrate moiety are well known to those of skill in the art. See, for example, Shih et al., Int. J. Cancer. 41 :832-839 (1988); Shih et al., Int. J.

Cancer. 46: 1 101-1 106 (1990); and Shih et al., US. Patent No. 5,057,313, all ofwhich are incorporated in their entirety by reference. The l method involves reacting an dy component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine ﬁanction and that is loaded with a plurality of peptide. This reaction s in an initial Schiff base (imine) linkage, which can be stabilized by ion to a secondary amine to form the ﬁnal conjugate.

However, if the Fc region is absent, for example, if an antibody ﬁanctional fragment as provided herein is ble, it is still possible to attach a diagnostic agent, a detectable agent or a therapeutic agent. A carbohydrate moiety can be introduced into the light chain variable region of a full-length antibody or dy fragment. See, for example, Leung et al., J.

Immunol., 154: 5919 (1995); US. Patent Nos. 5,443,953 and 6,254,868, all ofwhich are incorporated in their entirety by reference. The engineered carbohydrate moiety is used to attach the diagnostic agent, detectable agent or therapeutic agent.

The therapeutic agent conjugated or recombinantly fused to an antibody or functional fragment of the invention that binds to GD2 can be chosen to achieve the desired lactic or therapeutic effect(s). It is understood that it is within the skill level of a clinician or other WO 87811 medical personnel to consider the following when deciding which therapeutic agent to conjugate or recombinantly fuse to an antibody or functional fragment of the invention: the nature of the e, the severity of the disease, and the condition of the subject.

A conjugate or fusion antibody or functional fragment of the invention that is detectably labeled as provided herein and binds to GD2 can be used for diagnostic purposes to detect, diagnose, or r a disease, wherein the cells that cause or are associated with the disease express GD2. For example, as provided herein, cancer cells and tumors have been shown to s GD2, such as, but not limited to, neuroblastoma, osteosarcomas and other subsets of sarcomas, melanomas, gliomas, small cell lung cancer, breast cancer and breast cancer stem cells, medulloblastoma, and astrocytoma. Other types of sarcomas include, but are not limited be soft tissue sarcoma, osarcoma, liposarcoma, and leiomyosarcoma. Soft tissue sarcomas include, but are not limited to r soft part sarcoma, angiosarcoma, lioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, rcoma, gastrointestinal stromal tumor, liposarcoma, malignant peripheral nerve sheath tumor, Neuroflbrosarcoma, rhabdomyosarcoma.

Accordingly, the invention provides methods for detecting cancer or a tumor formation in a t by stering an effective amount of a conjugate or fusion antibody or functional fragment of the invention to a t in need thereof. In some aspects, the detection method can further include assaying the expression of a GD2 on the cells or a tissue sample of a subject using one or more conjugates or fusion antibodies or functional fragments of the invention that bind to GD2; and comparing the level of the GD2 with a l level, e.g., levels in normal tissue samples (e.g, from a subject not having a disease, or from the same subject before disease onset), whereby an increase in the assayed level of GD2 ed to the control level of the GD2 is indicative of the disease. Such diagnostic methods can allow health professionals to employ preventative es or aggressive treatment earlier than otherwise possible thereby preventing the development or further progression of the disease.

An antibody or functional fragment of the invention can also be used to assay GD2 antigen levels in a biological sample using classical immunohistological methods as provided herein or as well known to those of skill in the art (e.g., see en et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al., 1987, J. Cell . Biol. 105:3087-3096). Other antibody-based methods useful for detecting GD2 include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (mIn), and technetium (99Tc); luminescent labels, such as luminol; and ﬂuorescent labels, such as ﬂuorescein and rhodamine, and biotin.

In one aspect, the invention provides for the detection and diagnosis of disease in a human. In one ment, diagnosis includes: a) stering (for e, erally, subcutaneously, or intraperitoneally) to a subject an effective amount of a conjugate or ﬁJsion protein of the invention that binds to GD2; b) g for a time interval following the administering for permitting the conjugate or ﬁJsion protein to preferentially concentrate at sites in the subject where GD2 is expressed (and, in some aspects, for unbound conjugate or fusion protein to be d to background level); c) determining background level; and d) detecting the conjugate or fusion protein in the subject, such that detection of conjugate or fusion protein above the background level indicates that the subject has a disease. Background level can be ined by various methods including, comparing the amount of conjugate or fusion protein detected to a standard value previously determined for a particular .

It is understood that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images and can be readily determined by one of skill in the art. For example, in the case of a radioisotope conjugated to an antibody or ﬁanctional fragment of the invention, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 uries of 99Tc. The conjugate will then entially late at the location of cells which express GD2. In vivo tumor imaging is described in S.W. Burchiel et al. of Radiolabeled , opharmacokinetics Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of , S.W. el and BA. Rhodes, eds., Masson Publishing Inc. (1982).

] Depending on several variables, including the type of detectable agent used and the mode of administration, the time interval following the administration for permitting the conjugate to preferentially concentrate at sites in the subject and for unbound conjugate to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval following administration is 5 to 20 days or 5 to 10 days. In one embodiment, monitoring of a disease is carried out by repeating the method for diagnosing as provided , for e, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, or .

The presence of the conjugate or fusion protein can be detected in the subject using methods known in the art for in viva scanning. These methods depend upon the type of detectable agent used. A skilled artisan will be able to determine the appropriate method for ing a particular detectable agent. Methods and devices that can be used in the diagnostic methods of the ion include, but are not limited to, computed aphy (CT), whole body scan such as on emission tomography (PET), magnetic resonance imaging (MRI), and sonography. In one embodiment, an antibody or function fragment of the invention is conjugated to a radioisotope and is detected in the subject using a radiation responsive surgical ment. In another ment, an antibody or function fragment of the invention is ated to a ﬂuorescent compound and is detected in the subject using a ﬂuorescence sive scanning instrument. In another ment, an antibody or ﬁanction fragment of the invention is conjugated to a positron emitting metal, such as zirconium (89Zr) or any other positron emitting metal provided herein or that is well known in the art to be detectable by positron emission-tomography, and is detected in the subject using positron emission- tomography. In yet another embodiment, an antibody or function fragment of the invention is conjugated to a paramagnetic label and is detected in a subject using magnetic resonance g (MRI).

In one embodiment, the invention provides a pharmaceutical composition having an antibody or a functional fragment of the invention and a pharmaceutically able carrier. A pharmaceutically acceptable carrier that can be used in the pharmaceutical compositions of the invention include any of the standard pharmaceutical carriers known in the art, such as phosphate buffered saline solution, water and emulsions such as an oil and water emulsion, and various types of wetting agents. These pharmaceutical compositions can be prepared in liquid unit dose forms or any other dosing form that is sufficient for ry of the antibody or functional fragment of the invention to the target area of the subject in need of treatment. For example, the pharmaceutical itions can be prepared in any manner appropriate for the chosen mode of stration, e. g., intravascular, intramuscular, sub-cutaneous, intraperitoneal, etc. Other optional components, e.g., pharmaceutical grade stabilizers, buffers, preservatives, excipients and the like can be readily selected by one of skill in the art. The ation of a pharmaceutically composition, having due regard to pH, isotonicity, stability and the like, is within the level of skill in the art.

Pharmaceutical formulations containing one or more antibodies or ﬁanctional nts of the ion provided herein can be prepared for storage by mixing the antibody having the d degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other c acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; thonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 es) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, ne, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, ose or sorbitol; salt-forming counter-ions such as ; metal complexes (e.g., Zn- protein xes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).

Thus, in some embodiments, the invention provides a method for treating or preventing a disease in a subject in need thereof The methods of the invention can include administering a therapeutically effective amount of a ceutical composition provided herein to the subject. For example, the pharmaceutical composition can include one or more antibody or ﬁanctional fragment provided herein. Diseases that can be d or prevented using the methods of the invention include cancer, tumor formation and/or metastasis. In particular, the methods of the invention are useful for treating s or tumor formation wherein the cancer cells or tumor expresses GD2. Non-limiting examples of s or tumors that can be treated or prevented using the s of the invention include neuroblastoma, osteosarcomas and other subsets of sarcomas, melanomas, gliomas, small cell lung cancer, breast cancer, medulloblastoma, and astrocytoma. Other types of sarcomas include, but are not limited be soft tissue sarcoma, chondrosarcoma, liposarcoma, and leiomyosarcoma. Soft tissue sarcomas include, but are not limited to r soft part sarcoma, angiosarcoma, epithelioid sarcoma, extraskeletal chondrosarcoma, keletal osteosarcoma, f1brosarcoma, gastrointestinal l tumor, liposarcoma, malignant peripheral nerve sheath tumor, Neurofibrosarcoma, rhabdomyosarcoma. Breast cancer stem cells are also known to express GD2. Battulal VL et al., osz'de GD2 identiﬁes breast cancer stem cells motes tumorigenesis. J CLIN INVEST. l22(6):2066—2078 (2012).

Accordingly, in some aspects, the invention provides a method for treating cancer or a tumor formation in a subject in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or functional fragment f, wherein the antibody or ﬁanctional fragment binds to GD2 and has a variable heavy chain (VH) domain, the domain having VH CDRl, VH CDR2 and VH CDR3 amino acid sequences, wherein the VH CDRl amino acid sequence is selected from the group consisting of residues 26- 33 of SEQ ID NO: 2; residues 26-33 of SEQ ID NO: 6; residues 26-33 of SEQ ID NO: 10; residues 26-33 of SEQ ID NO: 14; residues 26-33 of SEQ ID NO: 18; es 26-33 of SEQ ID NO: 22; residues 26-33 of SEQ ID NO: 26; residues 26-33 of SEQ ID NO: 30; residues 26-33 of SEQ ID NO: 34; residues 26-33 of SEQ ID NO: 36; and residues 26-33 of SEQ ID NO: 40; the VH CDR2 amino acid sequence is selected from the group consisting of residues 51-58 of SEQ ID NO: 2; residues 51-58 of SEQ ID NO: 6; residues 51-58 of SEQ ID NO: 10; residues 51-58 of SEQ ID NO: 14; residues 51-58 of SEQ ID NO: 18; residues 51-58 of SEQ ID NO: 22; es 51-58 of SEQ ID NO: 26; residues 51-58 of SEQ ID NO: 30; residues 51-58 of SEQ ID NO: 34; residues 51-58 of SEQ ID NO: 36; and residues 51-58 of SEQ ID NO: 40; and the VH CDR3 amino acid sequence is selected from the group consisting of residues 97-109 of SEQ ID NO: 2; es 97-109 of SEQ ID NO: 6; residues 97-108 of SEQ ID NO: 10; residues 97-108 of SEQ ID NO: 14; residues 97-108 of SEQ ID NO: 18; residues 97-108 of SEQ ID NO: 22; residues 97-109 of SEQ ID NO: 26; residues 97-109 of SEQ ID NO: 30; residues 97-110 of SEQ ID NO: 34; residues 97-110 of SEQ ID NO: 36; and es 97-108 of SEQ ID NO: 40.

In some other aspects, the invention es a method for treating cancer or a tumor formation in a subject in need thereof by stering a therapeutically effective amount of a pharmaceutical composition having an antibody or ﬁanctional fragment thereof, wherein the antibody or ﬁanctional fragment binds to GD2 and has a variable heavy chain (VH) domain, the VH domain having VH CDRl, VH CDR2, and VH CDR3 amino acid sequences selected from the group consisting of residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 2; residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 6; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 10; residues 26-33, residues 51-58, and residues 97- 108 of SEQ ID NO: 14; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 18; residues 26-33, residues 51-58, and residues 97-108 of SEQ ID NO: 22; residues 26-33, residues 51-58, and residues 97-109 of SEQ ID NO: 26; residues 26-33, residues 51-58, and residues 97- 109 of SEQ ID NO: 30; es 26-33, residues 51-58, and residues 97-110 of SEQ ID NO: 34; residues 26-33, residues 51-58, and residues 97-110 of SEQ ID NO: 36; and residues 26-33, es 51-58, and residues 97-108 of SEQ ID NO: 40.

In yet other aspects, the invention provides a method for treating cancer or a tumor formation in a subject in need f by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or ﬁanctional fragment thereof, wherein the antibody or ﬁanctional fragment binds to GD2 and has a variable heavy chain (VH) domain, the VH domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 6; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 18; SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 30; SEQ ID NO: 34; SEQ ID NO: 36; and SEQ ID NO: 40.

] In some embodiments, the ion provides a method for ng cancer or a tumor ion in a t in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or ﬁanctional fragment thereof, wherein the antibody or ﬁanctional fragment binds to GD2 and has a variable light chain (VL) domain, the VL domain having VL CDRl, VL CDR2 and VL CDR3 amino acid sequences, wherein the VL CDRl is ed from the group consisting of residues 27-37 of SEQ ID NO: 4; residues 27-37 of SEQ ID NO: 8; residues 27-38 of SEQ ID NO: 12; residues 27-38 of SEQ ID NO: 16; residues 27-38 of SEQ ID NO: 20; residues 27-38 of SEQ ID NO: 24; residues 27-37 of SEQ ID NO: 28; residues 27-37 of SEQ ID NO: 32; residues 27-32 of SEQ ID NO: 38; and residues 27- 38 of SEQ ID NO: 42; the VL CDR2 is selected from the group ting of residues 55-57 of SEQ ID NO: 4; residues 55-57 of SEQ ID NO: 8; residues 56-58 of SEQ ID NO: 12; residues 56-58 of SEQ ID NO: 16; residues 56-58 of SEQ ID NO: 20; es 56-58 of SEQ ID NO: 24; residues 55-57 of SEQ ID NO: 28; residues 55-57 of SEQ ID NO: 32; residues 50-52 of SEQ ID NO: 38; and residues 56-58 of SEQ ID NO: 42, and the VL CDR3 is selected from the group consisting of residues 94-102 of SEQ ID NO: 4; residues 94-102 of SEQ ID NO: 8; residues 95- 103 of SEQ ID NO: 12; es 95-103 of SEQ ID NO: 16; residues 95-103 of SEQ ID NO: 20; residues 95-103 of SEQ ID NO: 24; es 94-102 of SEQ ID NO: 28; residues 94-102 of SEQ ID NO: 32; residues 89-97 of SEQ ID NO: 38; and residues 95-103 of SEQ ID NO: 42.

In some aspects, the present invention provides a method for treating cancer or a tumor formation in a subject in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or onal fragment thereof, wherein the antibody or ﬁanctional fragment binds to GD2 and has a variable light chain (VL) domain, the VL domain having VL CDRl, VL CDR2, and VL CDR3 amino acid sequences selected from the group consisting of residues 27-37, es 55-57, and residues 94-102 of SEQ ID NO: 4; residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 8; residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 12; residues 27-38, residues 56-58, and residues 95- 103 of SEQ ID NO: 16; residues 27-38, residues 56-58, and es 95-103 of SEQ ID NO: 20; residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 24; residues 27-37, residues 55-57, and residues 94-102 of SEQ ID NO: 28; es 27-37, residues 55-57, and residues 94- 102 of SEQ ID NO: 32; residues 27-32, residues 50-52, and residues 89-97 of SEQ ID NO: 38; and residues 27-38, residues 56-58, and residues 95-103 of SEQ ID NO: 42.

In some other aspects, the present invention provides a method for ng cancer or a tumor formation in a t in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or functional fragment thereof, wherein the antibody or ﬁanctional fragment binds to GD2 and has a variable light chain (VL) domain, the VL domain having an amino acid sequence selected from the group consisting of WO 87811 SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 38; and SEQ ID NO: 42.

In some embodiments, the present invention provides a method for treating cancer or a tumor formation in a subject in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or ﬁanctional fragment thereof that binds to GD2, the antibody or ﬁanctional fragment thereof including a le heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain has an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 6; SEQ ID NO: 10; SEQ ID NO: 14; SEQ ID NO: 18; SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 30; SEQ ID NO: 34; SEQ ID NO: 36; and SEQ ID NO: 40; and the VL domain has an amino acid sequence selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 38; and SEQ ID NO: 42.

In some other embodiments, the present invention provides a method for treating cancer or a tumor formation in a subject in need thereof by administering a therapeutically effective amount of a ceutical composition having an antibody or functional fragment thereof that binds to GD2, the antibody or functional fragment thereof ing a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain and the VL domain respectively have an amino acid sequence from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; SEQ ID NO: 6 and SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 20; SEQ ID NO: 22 and SEQ ID NO: 24; SEQ ID NO: 26 and SEQ ID NO: 28; SEQ ID NO: 30 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 38; SEQ ID NO: 36 and SEQ ID NO: 38; and SEQ ID NO: 40 and SEQ ID NO: 42.

Formulations, such as those described herein, can also contain more than one active compound as necessary for the particular disease being treated. In certain embodiments, formulations include an dy or ﬁanctional fragment of the invention and one or more active compounds with complementary ties that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are ive for the purpose intended. For e, an dy or functional fragment of the invention can be combined with one or more other therapeutic agents. Such combined therapy can be administered to the subject rently or successively.

Thus, in some embodiments, invention provides a method for treating or preventing a disease by administering a therapeutically effective amount of a pharmaceutical composition ed herein to a subject in need thereof, wherein the pharmaceutical composition includes an dy or onal fragment of the invention and a second eutic agent. The appropriate second eutic agent can be readily determined by one of ry skill in the art as discussed herein. In one aspect, the second therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent.

The pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more of the antibodies of the invention provided herein, and optionally one or more additional therapeutic agents, in a pharmaceutically acceptable carrier. Such ceutical compositions are useful in the prevention, treatment, management or amelioration of a disease, such as cancer or tumor formation, or one or more of the symptoms thereof.

The pharmaceutical compositions can contain one or more antibodies or onal fragments of the invention. In one embodiment, the antibodies or functional fragments are formulated into suitable pharmaceutical preparations, such as e solutions or suspensions for parenteral administration. In one embodiment, the antibodies or ﬁanctional fragments provided herein are formulated into pharmaceutical compositions using techniques and ures well known in the art (see, e.g., Ansel (1985) uction to Pharmaceutical Dosage Forms, 4th Ed., p. 126).

An antibody or functional fragment of the invention can be included in the pharmaceutical ition in a therapeutically effective amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. The therapeutically effective concentration can be determined empirically by testing the compounds in in vitro and in viva systems using routine methods and then extrapolated therefrom for dosages for humans. The tration of an antibody or functional fragment in the pharmaceutical composition will depend on, e.g., the physicochemical characteristics of the 2015/033954 antibody or ﬁanctional fragment, the dosage schedule, and amount administered as well as other factors well known to those of skill in the art.

In one embodiment, a therapeutically effective dosage produces a serum concentration of an antibody or functional fragment of from about 0.1 ng/ml to about 50-100 ug/ml. The pharmaceutical compositions, in another embodiment, provide a dosage of from about 0.001 mg to about 500 mg of antibody per kilogram of body weight per day.

Pharmaceutical dosage unit forms can be prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 30 mg, 100 mg or 500 mg, and in one embodiment from about 10 mg to about 500 mg of the antibody or functional fragment and/or a combination of other optional essential ingredients per dosage unit form.

The antibody or functional fragment of the invention can be stered at once, or can be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined cally using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values can also vary with the ty of the condition to be alleviated. It is to be further understood that for any particular subject, speciﬁc dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or ising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

] Upon mixing or addition of the antibody or ﬁanctional fragment of the ion, the resulting mixture can be a solution, suspension or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective tration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and can be cally determined.

The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as sterile parenteral ons or suspensions containing suitable quantities of the nds or pharmaceutically acceptable derivatives thereof. The antibody or ﬁanctional fragment can be, in one embodiment, ated and administered in unit- dosage forms or multiple-dosage forms. Unit-dose forms refers to physically te units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose ns a predetermined quantity of the dy or onal fragment of the invention sufficient to produce the desired eutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include es and syringes. Unit-dose forms can be administered in fractions or multiples thereof A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single ner to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials or bottles of pints or s. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.

In one embodiment, one or more antibody or ﬁanctional fragment of the invention is in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an antibody or functional fragment as ed herein and al pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, ol, glycols, ethanol, and the like, to thereby form a solution. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those d in this art; for example, see ton’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

Methods for administering a pharmaceutical composition of the invention are well known in the art. It is understood that the appropriate route of administration of a ceutical composition can be readily determined by a skilled clinician. ary routes of administration include intravenous injection, intramuscular ion, intradermal injection or subcutaneous injection. Moreover, it is understood that the formulation of the pharmaceutical composition can be readily adjusted to accommodate the route of administration. The invention also provides that following administration of a pharmaceutical composition of the invention, d, successive and/or repeated dosages of one or more pharmaceutical composition as provided herein can be administered to the subject.

The methods of the invention for treating a disease is intended to include (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a subject that can be predisposed to the disease but does not yet experience or y symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical ms; or (3) relieving the disease, i.e., g regression of the e or its clinical symptoms. The methods of the invention for preventing a e is ed to include forestalling of a clinical symptom indicative of cancer or tumor formation. Such forestalling includes, for example, the maintenance of normal physiological indicators in a subject.

Therefore, preventing can include the prophylactic treatment of a subject to guard them from the occurrence of tumor metastasis.

The therapeutically effective amount of the pharmaceutical composition used in the methods of the invention will vary depending on the pharmaceutical composition used, the disease and its severity and the age, weight, etc., of the subject to be treated, all of which is within the skill of the ing clinician. A subject that that can be d by the methods of the invention include a vertebrate, preferably a mammal, more preferably a human.

It is understood that modifications which do not substantially affect the activity of the s embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following es are intended to illustrate but not limit the present invention.

EXAMPLE I Human Monoclonal Antibodies to GD2 have Potent Antitumor ty The disialoganglioside GD2 has been found in a wide spectrum of human tumors, including neuroblastoma, osteo sarcomas and other subsets of sarcomas, melanomas, gliomas, small cell lung , breast cancer, medulloblastoma, and astrocytoma. Breast cancer stem cells are also known to express GD2. Battulal VL et al., Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis. J Clin Invest. 122(6):2066—2078 (2012).

Gangliosides are ideal s for onal antibodies (mAb) because of the high antigen density, lack of modulation, relative homogeneity in many tumors and the possibility of upregulation by cytokines. Accordingly, as described herein, fully human monoclonal antibodies (mAb) against GD2 were ted. Several mAbs were selected based on ELISA and FACS and ﬁlrther characterized. Ofthe tested antibodies, lB7, 2H12, 2F7, 2E12, and 3 lF9V2 showed high levels of complement-dependent cytotoxicity on some cancer cell lines; 1B7, 31F9, 31F9V2, and 2F7 showed high levels of antibody-dependent cell-mediated cytotoxicity on some cancer cell lines; and the antitumor activity was also conﬁrmed in in vivo models. Based on the potential of GD2 as a target for immune attack and their y, speciﬁcity, and effector functions, anti-GD2 mAb have clinical utility in the ent of .

Materials, cells, and antibodies Antigens: GD2-PAA-biotin (cat# 0832-BP), GM2-PAA-Biotin (cat#0835-BP) and A-biotin 898-BP) were purchased from ity (Moscow, ). Tn-PAA- biotin (cat#Ol-010), sTn-PAA-biotin (cat#0 l -05 9), TF-PAA-biotin (cat#0 l -023) and sLeA- PAA-biotin (cat#Ol-044) were purchased from Glycotech (Gaithersburg, MD). GD2-ceramide, GM2-ceramide, ramide, Fucosyl-GMl-ceramide and Globo-H-biotin were obtained from MSKCC (New York, NY). MUCl peptide-biotin (cat#353951) was purchased from American Peptide Company (Sunnyvale, CA). GM3-ceramide (cat#1503) was purchased from Matreya (Pleasant Gap, PA). Ceramide antigens were dissolved in Methanol and all others were resuspended in PBS at lmg/ml.

Cell Lines: H524, Lanl-luc, BXPC3, SK-MEL19, ST88, LSl4l, SaOS2 cell line were obtained from MSKCC (New York, NY). Capan2 (ATCC, HTB-80), DMS79 (ATCC, 49), Jurkat (ATCC, TIB-152), SK-MEL28 (ATCC, HTB72), HT29, (HTB-3 8) and MCF7 (ATCC, HTB22) were purchased from ATCC (Manassas, VA). TC-7l (AAC516) was purchased from Leibniz Institute DSMZ (Braunschweig, Germany).

Generation of D2 mAb-producing hybridomas and lymphoblastoid cell lines (LCL): Blood samples were obtained from patients in trials with GD2-/GD3-KLH conjugate vaccine in patients with melonama and GD2-/GD3-/GM2-KLH trivalent vaccine in ts with sarcoma. The melanoma Phase I trial was done at MSKCC while the sarcoma trial was a muticenter, blinded Phase II study. All samples were obtained under respective institutional and FDA approved IRB protocol and IND. Human B cells were isolated from approximately 80-90 ml of blood by eSep Human B Cell ment Cocktail (cat#15024, StemCell Technologies, Vancouver, BC, Canada) using gradient centrifugation on Histopaque- 1077 (cat#10771, Sigma, St Louis, MO). The B cells were cultured in RPMI-1640 medium (cat#10CV, Mediatech, Manassas, VA) supplemented with L-Glutamine (cat#25030081, Life Technologies, Carlsbad, CA), non-essential amino acids (cat#NE-01), sodium te (cat#SP-90), vitamin (cat#ME-30), penicillin/ streptomycin (PS-20), 10%FBS (cat#FB-01) from Omega Scientiﬁc, Tarzana, CA, and a mixture of stimulants and cytokines. Five to seven days later cells were fused by electrofusion to P3X63Ag8.653 myeloma cells (cat#PTA-8434, ATCC, Manassas, VA). LCLs were generated by infecting the B cell with EBV (B95-8 culture supernatant) in presence of IL2 and R848 or PS2006. Anti-GD2 producing hybridomas and LCLs were ﬁed using a GD2 speciﬁc ELISA.

ELISA: For the biotinylated Antigens: 50ul/well ofNeutrAvidin (cat#31000, Thermo Scientiﬁc, Rockford, IL) was coated on the ELISA plate (cat#655061, Greiner Bio-One, Monroe, NC) at 4ug/ml and incubated at RT for 2hrs. The plate was blocked with 1.25% human serum albumin (HuSA) (cat#HA25 S, Monobind, Lake Forest, CA) for 2hrs at RT or 4C, O/N. After g the plate with 0.05% Tween/PBS (PBS-0.05%T) twice, 50ul/well of biotinylated antigen (ﬁnal concentration at 2ug/ml in PBS) or PBS was added and incubated at RT for 30min or 4 0C, O/N. The plate was washed with PBS-T twice and ted with 100ul/well of puriﬁed mAbs diluted in 1.25% HuSA at 2ug/ml for 1hr at RT. After washing the plate with PBS- 0.05%T three times 100ul/well of secondary antibody, alkaline phosphatase conjugated-anti- human IgG or IgM (1 :3000 dilution in 1.25% HuSA, cat#075-1002 and cat#075-1003, tively, KPL, rsburg, nd) was added and ted at RT for 1hr. The plate was washed four times with PBS-0.05%T. A hundred ul/well pNPP ate (cat#PI34045, Thermo Scientiﬁc, Rockford, IL) was added and incubated at RT for 1hr and the reaction was stopped with 25 ul/well of 2N NaOH.

For the de antigens: 50ul/well of ceramide antigen (ﬁnal concentration at 5ug/ml in EtOH) or EtOH was coated on the 96 well plate (cat#269620, Thermo Scientiﬁc, Rockford, IL) and incubated at RT over 2hrs. The plate was washed with PBS once and blocked with 2.5% HuSA at RT for 2hrs or 4 0C, O/N. The plate was washed with PBS once and incubated with 100ul/well of puriﬁed mAbs diluted in 1.25% HuSA at 2ug/ml for 1hr at RT. The plate was washed with PBS twice before adding the 2Ild antibody as described above. The plate was washed with PBS-0.025%T three times before adding the substrate.

FACS: The cells were resuspended in 200ul/tube of PBS/1% BSA 3059, Sigma, St. Louis, M0) at 0.25xlO6 cells/ml. Puriﬁed mAb was added to the tube at 2mg/ml for IgG or 5mg/ml for IgM and incubated for 40min at 4 0C. After washing with PBS/1% BSA once 200ul of Fluorophore conjugated antibody, Alexa488-anti-human IgG (cat#H10120, Life Technologies, ad, CA) or Alexa488-anti-human IgM (cat#A21215, Life Technologies, Carlsbad, CA) was added to the tube and incubated for 40min at 4 0C. The cells were washed with PBS/1% BSA twice and analyzed with Guava ExpressPro software using the Guava Personal Cell Analysis-96 (PCA-96) System (Millipore, ica, MA).

Afﬁnity determination: Afﬁnity constants were determined using the principal of e Plasmon Resonance (SPR) with a BiaCore 3000 (GE Healthcare, Piscataway, NJ).

Custom synthesized biotin-labeled GD2-polyacrylamide (GD2-PAA-biotin) was obtained from Lectinity Holdings Inc (Moscow, ) and was coupled to a streptavidin coated biosensor chip (SA; Cat # BR100398) according to the manufacturer’s instructions. One ﬂow cell blocked with HSA and culture medium containing ﬁee biotin was used as a reference cell. The binding kinetic parameters were determined from several known concentrations of antibody diluted in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20) using the GD2-PAA-biotin coated ﬂow cell. The ﬁtting software provided by the BiaCore ment was used to calculate the ation and dissociation rates.

CDC Assay: The cells were washed with PBS twice, resuspended in 1ml of PBS at 106 cells/ml/tube and incubated with 12.5 ul/tube of Calcein AM (1mg/ml in DMSO) (cat#C3100MP, Invitrogen, Carlsbad, CA) at 37 0C for 30min. The d cells were washed with medium containing 10% FBS (complete medium) (cat#FB-12, Omega Scientiﬁc, Tarzana, CA) twice and resuspended in 1ml of complete medium. The labeled cells (50ul/well) were incubated with 100 ul well ofmAb diluted in te medium for 15min at 4C. 50 ul/well of human complements (cat#IPLA-CSER, Innovative Research, Novi, MI) diluted in complete medium were added to the wells and ted at 37 0C for 90min. The riate ﬁnal dilution for human complement was pre-determined for each cell line (between 1:5 and 1:16). After a centrifuge at 1600rpm for 8min supernatant (100ul/well) were transferred to a new ﬂuorescence 96 well plate (cat#7605, Thermo Scientiﬁc, Rockford, IL). Each sample was ed in triplicate. Control s that receive NP40 are used to determine maximal killing and samples receiving complement alone serve as baseline. The percentage of killed cells is determined by relative ﬂuorescent units and ated according to the following formula: % killed = (% sample — % complement alone) / (%NP40 - % complement alone)* 100.

Antibody-dependent ediated cytotoxicity Assay: ADCC Reporter Bioassay Core Kit (cat#G7010) was purchased from Promega (Madison, WI) and the assay was performed according to the instruction . Brieﬂy, effector cells (Jurkat from the core kit) and GD2 antigen expressing target cells were washed and resuspended in RPM11640 medium containing % low bovine IgG serum (core kit) at 3x106 cells/ml and 0.5x106 cells/ml, respectively. The target cells (12,500cells/25 ul/well) were incubated with 25 ul/well of anti-GD2 mAb (ﬁnal concentration at 5 ug/ml) or medium only, and the effector cells (75,000/25 ul/well) for 17hrs at 37 0C. 100 l of Bio-Glo luciferase assay substrate (core kit) was added and incubated for 10min. Relative light unit (RLU) was ed by Synergy 2 luminometer (BioTek, Winooski, VT). The effector cells and medium only wells served as baseline RLU. Each sample is tested in triplicate.

Intemalization Assay: Intemalization of anti-GD2 antibodies were evaluated by measuring the cytotoxic activity ofmAb and Hum-ZAP ary conjugate (cat#IT22, Advanced Targeting Systems, San Diego, CA) complex against GD2 expressing cell lines, H524 and Lan1-luc. Cells were plated into a 96 well plate (2,000 cells/ 90ul/well) and incubated overnight in duplicates. Anti-GD2 antibody was ted with Hum-ZAP secondary conjugates at RT ing to the manufacturer’s instruction. Next, 10 ul/well ofmAb and Hum-ZAP complex was added to the cells and incubated for 3 days. The ﬁnal concentration of the mAb was 10ug/ml. The assay was performed in cate. 25 ul of Thiazolyl Blue Tetrazolium Bromide (cat#M5655, Sigma, St Louis, MO) solution (5mg/ml in PBS) was added to each well and incubated at 37°C. After 2hrs incubation well of solubilization on (20% SDS/ 50% methylformamide, cat#D4551, Sigam, St Louis, MO) was added to each well and incubated for another 4 hrs at 37°C. The OD was measured at 570/690nm and values obtained with medium alone were used for plate background subtraction. Three parallel cultures t dy were used to normalize the sample values (Sample/Mean Untreated"< 100).

Internalization Assay using a pH sensitive Intracellular Fluorescent Probe: Goat anti-human lgG F(ab’)2 fragments (Jackson Research, cat#109006) were conjugated with pHAb Amine Reactive Dye ga, cat#G984l) according to the cturer’s protocol. pHAb is a pH sensor dye that shows ﬂuorescence only at acidic pH, which is encountered when the cells take up the antibodies into their lysosomes. In brief, primary antibody (1B7, 3 lF9, 5A7G3 or no mAb as reagent control) at 6ug/ml (200ul/tube) and anti-human lgG F(ab’)2-pHAb at 4.5 ug/ml (200 ul/tube) were incubated at RT for 20min. H524 or TC-7l cells were re-suspended in RPMIl640 +10% FBS + Glutamine +P/S medium at 1.5x106 cell/ml. Two hundred ul of cells were added to the primary plus secondary antibody mixture and incubated for 40min at 4°C. The cells were centrifuged and re-suspended in 0.5ml of culture medium, distributed into a 96 well tissue culture plate and incubated at 37°C, 5% C02.

Samples were taken at lhr, 2hrs, 4hrs and 24hrs for ﬂow cytometry analysis using Guava Express Pro software to ine the percentage of positive of cells. aft lantation model: Osteosarcoma SaOS2 cells were obtained from ATCC (Cat # HTB-85; Manassas, VA) and female CB17 SCID mice (5-8 weeks old) were purchased from Taconic (Germantown, NY). SaOS2 cells (1 x 106) in 0.1 ml complete growth media were injected via the tail vein on Day 0 using a BD insulin syringe with 28G needle (BD, Franklin Lakes, NJ) into 10 animals per group. 200ug mAb (1B7 or 3 lF9) was injected intraperitoneally on days 1, 4, 8, ll, 14, 21 and 28 post tumor cell ion. Survival was monitored daily and Kaplan-Meier survival curves were ted using GraphPad Prism 6.05 (GraphPad Software, San Diego, CA).

Ewing’s sarcoma TC-7l cells were obtained from the Leibniz-Institute DSMZ GmbH (Braunschweig, Germany) and ed as recommended. TC-7l cells (0.1x106) in 0.1 ml ofBD MatrigelTM Basement Membrane Matrix n Dickinson Bioscience) were injected subcutaneously into the right hind ﬂank of female CB17 SCID mice (5-8 weeks old) on day 0, 5 mice per group. 200ug mAb (1B7 or 31F9) was injected intraperitoneally on days 1, 4, 8, ll, 14, 21 and 28 post tumor cell injection. Animals in the control group received mock injections with PBS. Mice were monitored for tumor growth twice per week and tumor size was measured by caliper. Tumor volume (mm3) was calculated as length*width* width"< 0.5.

All procedures were performed under a protocol approved by the Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee.

Immunoglobin cDNA cloning and recombinant dy expression: Variable region of human mAb heavy and light chain cDNA was red by RT-PCR from the dual hybridoma or LCL cell line and ned into IgGl or IgM heavy chain, or IgK or IgL light chain expression vector as described before. Sawada-Hirai, R., et al. Human anti- anthrax protective antigen lizing monoclonal antibodies derivedfrom donors vaccinated with anthrax vaccine adsorbed. J IMMUNE BASED THER VACCINES 2(1): 5 (2004). Ig heavy chain or light chain sion vector were double ed with Not I and Sal I, and then both fragments were ligated to form a dual gene expression vector. CHO cells in 6 well-plate were transfected with the dual gene expression vector using Lipofectamine 2000 (cat#11668019, Life Technologies, Carlsbad, CA). After 24 hrs, transfected cells were transferred to 10 cm dish with selection medium [DMEM supplemented with 10% dialyzed FBS (cat#26400044, Life Technologies, Carlsbad, CA), 50 uM L-methionine sulphoximine (MSX, cat#M5379, Sigma, St Louis, MO), GS supplement (cat#58762C, Sigma, St Louis, MO) and penicillin/streptomycin (cat#PS-20, Omega Scientific, Tarzana, CA)]. Two weeks later MSX resistant transfectants were isolated and expanded. High anti-GD2 antibody producing clones were selected by measuring the antibody levels in tants in a GD2 specific ELISA assay and expanded for large scale mAb tions.

Results Generation of recombinant antibodies: The heavy and light chain variable regions from 11 selected antibodies were recovered by RT-PCR and cloned into full-length IgG or IgM heavy chain, or IgK or IgL light chain expression vectors. Molecular sequence analysis using IMGT/V-Quest (Brochet et al., Nucleic Acids Res., 36:W503—8 (2008)) revealed that the nine selected human anti-GD2 antibodies were derived from three ent VH families and all used kappa light chains. These IgG dies showed different CDR sequences with 5, 7, 8, 9, 10, or mutations deviating from the germ line, respectively (FIGS. 1-14, 17-21; Table 3). The IgM antibody (2E12) also utilizes the kappa light chain and has 3 heavy chain mutations (FIGS. 15-16; Table 3). Recombinant antibodies were produced in CHO cell lines in a wave bioreactor system and puriﬁed using n A or hydroxyapatite chromatography for IgG and IgM, tively.

The puriﬁed recombinant antibodies retained the properties of the original hybridoma-derived antibodies with respect to ELISA binding and speciﬁcity.

Table 3: cDNA Classiﬁcation of selected human anti-GD2 antibodies Mutations Mutations ironl ironl Clone ID germline DH (RF) JH VL germline 1B7 4*02 I(2-28*01 JI(3*01 2le “mm—.-mm 1G2 1-4801nun—n mm 1129 1-4801nun—n mm 1H3 1-46*01-m--_-— mm 2F5 mm m --m--—-—mm 2E12 1—3*01 2—15*01 3 8 8 13 K2-28*01 11,3 9 JK2*01 31F9 1-8*01 4—23*01 1 8,8 14 01 , , JK1*01 32E2 1-46*03 — 5—24*01 1 8,8 12 K4-1*01 9 K2*02 , 12,3, J Binding Speciﬁcity: In the antigen-speciﬁc ELISA assays, seven human anti-GD2 antibodies (1B7, 2H12, 1G2, 2F7, 2E12, 3lF9, and 32E2) showed strong reactivity against GD2- PAA conjugate, but not against GD3-PAA, Globo-H, MUCl, , sTn-PAA, TF-PAA, or SleA-PAA. Three antibodies (1B7, 2H12 and 2F7) also showed activity to A conjugates. Similarly, the seven antibodies also demonstrated strong reactivity with GD2- ceramide conjugate (GD2-cer), but not GD3-ceramide conjugate (GD3-cer), F-GMl-ceramide conjugate (F-GMl-cer), or GM3-ceramide conjugate (GM3-cer).

Table 4: Binding of anti-GD2 Antibodies Measured by ELISA Giﬂ- GDZ- GEE» Binding Speciﬁcity by EL Analysis of tumor cell binding: Cell e binding is crucial for cytotoxic activity and was ore tested next for anti-GD2 antibodies 1B7, 2H12, 1G2, 2F7, 2E12, 31F9, and 32E2. Flow cytometry showed strong binding of all seven antibodies to H524, a small cell cancer cell line, to Lanl-Luc, a neuroblastoma cell line, to Hs527T, a breast cancer cell line, and to TC71 and SaOSZ, two sarcoma cell lines. Positive binding was also detected between some of the seven antibodies and other cancer cell lines, including pancreatic cancer cells, acute T cell leukemia cells, ma cells, and other sarcoma cells. (Table 5).

Table 5: Binding of anti-GDZ-mAbs to Different Cell Lines ————————————m ““““““““““ S arcoma Mn mm 7 99.74 979.77 99.89 86.28 96.04 317.14 99.63 * IgM, 2 ug/nil Afﬁnity measurements: The relative afﬁnity/avidity of the binding to GD2 was probed by SPR using a streptaVidin-coated biosensor chip to capture biotinylated GD2-FAA (Table 6).

Table 6: Kinetic Parameters for anti-GD2 mAbs mAb KA(1IM) KD (M) ka(1lMs) kd(1ls)2 Species2 Isotype ‘ 70x101015x1062 ' 2 2 11x1010 CDC activity: To evaluate the onal activity of 1B7, 2H12, 1G2, 2F7, 2E12, 31F9, 31F9V2, and 32E2, we tested their cytotoxic activity with four different cells (H524, Lanl-Luc, Jurkat, and TC-71) in the presence of human serum as a source of complement. 1B7 showed close to 100% g actiVity at 10 ug/mL in three of the four cells tested. 2H12, 2F7, 31F9V2, and 32E2 all showed signiﬁcant levels of CDC actiVity toward some cell lines. 2E12, an IgM antibody, showed close to 100% killing actiVity at 5 ug/mL to H524, Lanl-Luc and TC- 71, which was expected because IgM antibodies were known to be more effective in complement-mediated cytotoxicity assays.

Table 7. ment Dependent Cytotoxicity -_—__ Final concentration: IgGs at l, IgM at 5ug/ml; * IgM Antibody-dependent ediated cytotoxicity: While 2E12 was more potent in the CDC assay, IgG antibodies were known to have antibody-dependent cell-mediated cytotoxicity (ADCC) actiVity, which is important for tumor killing in viva. Six D2 IgG antibodies (1B7, 31F9, 31F9V2, 1G2, 2F7, and 32E2) were tested with ﬁve different cell lines (SaOS2, H524, , TC71). Treatment with medium only was used as control. High levels of cytotoxicity were measured using 1B7, 3 lF9, 3 lF9V2 and 2F7 antibodies, especially with TC7l cell line (). 1G2 and 32E2 also showed some, albeit relatively low level activity.

Intemalization assay: lization of anti-GD2 antibodies were evaluated by measuring the cytotoxic activity ofmAb and Hum-ZAP secondary conjugate complex against GD2 expressing cell lines, H524 and Lanl-luc. Cells that internalize the complex die, while nonintemalized saporin leaves the cells unharmed. Treatment with medium only was used as control. As shown in , H524 cells were effectively killed in the presence of 1B7, 3 lF9, or 3 lF9V2. rly, the Lanl-Luc cells were effectively killed in the presence of 1B7, lG2, 2Hl2, 2F7, 3lF9, and 32E2 ().

Further Intemalization Assays were med using a pH sensitive intracellular ﬂuorescent probe, which directly measured the kinetics of internalization via a PH-sensitive ﬂuorescent tag. FIGS. 27 and 28 demonstrate the kinetics of intematilization of 1B7 and 3 lF9V2 into CLC) cells and TC-7l(Sarcoma) cells. As shown, a significant amount of D2 antibodies were internalized to both H524 (SCLC) tumor cells and TC-7l (sarcoma) tumor cells, as compared to 5A7G3(anti-GD3) and the (ab’)2-pHAb only.

In vivo Models: The mor effects of anti-GD2 dies were also evaluated in in vivo models. shows the results of a survival model, wherein the survival of SCID mice engrafted with human SaOS2 (osteosarcoma) xenograft was measured. As demonstrated in , the percent survival of engrafted mice was significantly increased by the treatment by either 3 lF9 or 1B7 compared to that in the control group, wherein the mice were injected with PBS only. shows the results of a subcutaneous tumor model, wherein the growth of human TC-7l ma) xenograft tumors in SCID mice was measured. As demonstrated in , the tumor volume in the engrafted mice was significantly reduced by the treatment by either 3 lF9 or 1B7 compared to that in the control group, where the mice were injected with PBS only.

Accoridngly, the above data demonstrate signiﬁcant potential to suppress or regress established tumors and e a survival benefit using human anti-GD2 mAb treatment. hout this application various publications have been referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully be the state of the art to which this invention pertains.

Although the invention has been described with reference to the examples provided above, it should be understood that various modiﬁcations can be made without departing from the spirit of the invention.

Claims

What we claim is:

1. An isolated polynucleotide encoding an antibody or a functional fragment thereof that binds to GD2, said antibody or functional fragment thereof comprising a variable heavy chain VH domain and a variable light chain VL domain, said VH domain having VH CDR1, VH CDR2 and VH CDR3 amino acid sequences and said VL domain having VL CDR1, VL CDR2 and VL CDR3 amino acid ces, wherein said VH CDR1 amino acid sequence is residues 26-33 of SEQ ID NO: 2; said VH CDR2 amino acid sequence is residues 51-58 of SEQ ID NO: 2; said VH CDR3 amino acid sequence is es 97-109 of SEQ ID NO: 2; said VL CDR1 amino acid sequence is residues 27-37 of SEQ ID NO: 4; said VL CDR2 amino acid sequence is residues 55-57 of SEQ ID NO: 4; and said VL CDR3 amino acid sequence is residues 94-102 of SEQ ID NO: 4.

2. An isolated polynucleotide ng an antibody or a functional nt thereof that binds to GD2, wherein said antibody or functional fragment thereof comprises a variable heavy chain VH domain having an amino acid sequence of SEQ ID NO: 2 and a le light chain VL domain having an amino acid sequence of SEQ ID NO: 4.

3. The isolated cleotide of claim 2, wherein said VH domain amino acid sequence is encoded by the c acid sequence of SEQ ID NO: 1; and said VL domain amino acid sequence is encoded by the nucleic acid sequence of SEQ ID NO: 3.

4. An isolated antibody or functional fragment thereof that binds to GD2, said antibody or functional fragment thereof comprising a variable heavy chain VH domain and a variable light chain VL domain, said VH domain having VH CDR1, VH CDR2 and VH CDR3 amino acid sequences and said VL domain having VL CDR1, VL CDR2 and VL CDR3 amino acid sequences, wherein said VH CDR1 amino acid sequence is residues 26-33 of SEQ ID NO: 2; said VH CDR2 amino acid sequence is es 51-58 of SEQ ID NO: 2; said VH CDR3 amino acid sequence is residues 97-109 of SEQ ID NO: 2 said VL CDR1 amino acid sequence is residues 27-37 of SEQ ID NO: 4; said VL CDR2 amino acid sequence is residues 55-57 of SEQ ID NO: 4; and said VL CDR3 amino acid sequence is residues 94-102 of SEQ ID NO: 4.

5. An isolated antibody or functional fragment thereof that binds to GD2, said antibody or functional fragment thereof comprising a variable heavy chain VH domain and a variable light chain VL domain, wherein said VH domain has an amino acid sequence of SEQ ID NO: 2; and said VL domain has an amino acid sequence of SEQ ID NO: 4.

6. The isolated antibody or functional fragment thereof in claim 4 or 5, wherein said antibody is a human antibody.

7. The isolated antibody or onal fragment thereof of claim 4 or 5, wherein said antibody functional fragment is ed from the group ting of a Fab, a Fab’, a 2, a scFV, a diabody, a triabody, and a minibody.

8. The ed antibody or functional fragment thereof of claim 4 or 5, wherein said antibody is a monoclonal antibody.

9. The isolated antibody or functional fragment thereof of claim 4 or 5, wherein said antibody is an IgG or IgM isotype.

10. The isolated antibody or functional fragment thereof of claim 9, n said IgG dy is an IgG1 subclass.

11. A conjugate comprising an isolated antibody or functional fragment thereof of claim 4 or 5 conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent.

12. The conjugate of claim 11, wherein said conjugate comprises a detectable agent.

13. The use of a conjugate according to claim 12, in the manufacture of a medicament for detecting a tumor in a subject in need thereof.

14. A pharmaceutical ition comprising the antibody or functional fragment thereof of claim 4 or 5 and a pharmaceutically acceptable carrier.

15. The use of the pharmaceutical composition of claim 14 in the manufacture of a ment for treating or preventing a disease in a t in need thereof.

16. The use according to claim 15, wherein said disease is cancer or a tumor formation, wherein the cells of said cancer or said tumor ses GD2.

17. The use according to claim 16, wherein said cancer or said tumor is selected from the group consisting of neuroblastoma, osteosarcomas and other subsets of sarcomas, mas, gliomas, small cell lung cancer, breast cancer, medulloblastoma, and astrocytoma.

18. The use according to claim 17, wherein said breast cancer comprises breast cancer stem cells.

19. The use according to claim 15, characterized in that the treating or preventing comprises administering a second therapeutic agent in combination with the pharmaceutical composition concurrently or successively.

20. The use of claim 19, wherein said second therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent.

21. An isolated polynucleotide according to claim 1, substantially as herein described or exemplified.

22. An isolated polynucleotide ing to claim 2, substantially as herein bed or exemplified.

23. An antibody or fragment thereof according to claim 4, substantially as herein described or exemplified.

24. An antibody or fragment f according to claim 5, substantially as herein described or exemplified.

25. A conjugate according to claim 11, ntially as herein described or exemplified.

26. A use according to claim 13, ntially as herein described or exemplified.

27. A pharmaceutical composition according to claim 14, substantially as herein described or exemplified.

28. A use according to claim 15, substantially as herein described or exemplified. VV()