PROGNOSTIC AND THERAPEUTIC METHODS INCLUDING ANTI-GD2 IGA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/377,409, filed September 28, 2022, the contents of which are incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to methods for predicting prognosis of cancer patients treated with a GD2/GD3 vaccine comprising detecting the levels of anti-GD2-IgA and/or anti-GD2-IgG3 antibodies in biological samples obtained from the cancer patients. Also disclosed herein are methods for treating cancer (e.g., neuroblastoma) in a patient in need thereof comprising administering an effective amount of anti-GD2 antibodies of various isotypes (e.g., anti-GD2-IgAl or anti-GD2-IgA2) to the patient.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in .XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 27, 2023, is named 115872-2185_Sequence Listing.xml and is 61,440 bytes in size.

BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Neuroblastoma (NB) is the most common extracranial solid tumor of childhood; 50-60% of patients present with an unresectable primary tumor and metastases in the bone marrow (BM). ¹ Intensive induction chemotherapy and aggressive surgery have improved remission rates in young patients ² ; results have been less promising in adolescents and adults. ^3,4 Despite the use of myeloablative therapy with stem-cell support to eradicate minimal residual disease in distant sites, outcome of high-risk NB (HR-NB) remains unsatisfactory. Except in group-wide clinical trials, ⁵ ' ⁹ the long-term event-free survival (EFS) and overall survival (OS) rates are still <50%. After relapse, the prognosis is even worse with 5-10% long term survival. ⁹ These results, plus the potentially severe toxicities of chemotherapy and radiotherapy, are compelling reasons for pursuing novel immunotherapeutic approaches, including anti-GD2 immunotherapy and ganglioside GD2/GD3 cancer vaccine.

SUMMARY OF THE PRESENT TECHNOLOGY

[0006] In one aspect, the present disclosure provides a method for identifying a cancer patient that will show positive prognosis after receiving a GD2 vaccine comprising: detecting levels of anti-GD2-IgA and/or anti-GD2-IgG3 antibodies in a biological sample obtained from the cancer patient that are elevated compared to a predetermined threshold or a reference sample. In some embodiments, the method further comprises administering the GD2 vaccine to the cancer patient one or more times after the anti-GD2-IgA and/or anti- GD2-IgG3 antibodies are detected in the biological sample. The levels of anti-GD2-IgA, anti-GD2-IgG3 and/or anti-GD2-IgGl antibodies may be detected via ELISA, radioimmunoassay, scintillation proximity assays, fluorescence energy transfer (FRET), liquid chromatography, membrane filtration assays, nuclear magnetic resonance, surface plasmon resonance, immunoprecipitation, or mass spectroscopy. Additionally or alternatively, in some embodiments, the method further comprises detecting levels of anti- GD2-IgGl antibodies in a biological sample obtained from the cancer patient that are elevated compared to a predetermined threshold or a reference sample. The biological sample may comprise peripheral blood mononuclear cells, whole blood, serum or plasma.

[0007] Additionally or alternatively, in some embodiments, the GD2 vaccine comprises GD2 conjugated to a carrier, such as keyhole limpet hemocyanin (KLH). In certain embodiments, the GD2 vaccine further comprises GD3 conjugated to a carrier, e.g., KLH. In any of the above embodiments of the methods disclosed herein, the GD2 vaccine has been separately, sequentially or simultaneously co-administered with an effective amount of a yeast beta-glucan. In some embodiments, the yeast beta-glucan comprises a plurality of P- (1,3) side chains linked to a [3-( 1 ,3) backbone via [3-( 1 ,6) linkages, and has a range of average molecular weights from about 6 kDa to about 30 kDa. In certain embodiments, the yeast beta-glucan is formulated for oral administration and/or the GD2 vaccine has been formulated for parenteral administration. In any of the preceding embodiments of the methods described herein, the GD2 vaccine has been separately, sequentially or simultaneously co-administered with an adjuvant, such as QS21.

[0008] Additionally or alternatively, in some embodiments, the cancer patient shows increased progression free survival relative to a control cancer patient that has anti-GD2-IgA and/or anti-GD2-IgG3 antibody levels that are at or below the predetermined threshold.

[0009] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of an anti-GD2 IgA antibody. The anti-GD2 IgA antibody is an anti-GD2 IgAl antibody or anti- GD2 IgA2 antibody. In some embodiments, the anti-GD2 IgAl antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the anti-GD2 IgA2 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 12. Additionally or alternatively, in some embodiments, the anti-GD2 IgA antibody is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.

[0010] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of an anti-GD2 IgG3 antibody. In some embodiments, the anti-GD2 IgG3 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 4. Additionally or alternatively, in some embodiments, the anti-GD2 IgG3 antibody is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.

[0011] In any and all embodiments of the methods disclosed herein, the methods further comprise sequentially, separately or simultaneously administering to the subject an effective amount of an anti-GD2 IgGl antibody. In some embodiments, the anti-GD2 IgGl antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 15 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 16. Additionally or alternatively, in some embodiments, the anti-GD2 IgGl antibody is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.

[0012] In yet another aspect, the present disclosure provides a method for treating or preventing cancer in a subject in need thereof comprising: administering an effective amount of a GD2 vaccine to a donor to induce production of anti-GD2 IgA antibodies in the donor; isolating the anti-GD2 IgA antibodies from the donor; and administering an effective amount of the anti-GD2 IgA antibodies to a recipient subject. In some embodiments, the method further comprises expanding the anti-GD2 IgA antibodies ex vivo prior to step (c). The donor and the recipient subject may be the same or distinct.

[0013] In any of the above embodiments of the methods disclosed herein, the cancer expresses GD2 and/or GD3. In some embodiments, the cancer is selected from the group consisting of breast cancer, melanoma, small cell lung cancer, neuroblastoma, osteosarcoma, retinoblastoma, brain tumor, soft tissue sarcoma, Ewing’s sarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, and spindle cell sarcoma. In certain embodiments, the cancer is neuroblastoma.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Fig. 1 shows an exemplary patient vaccine regimen, where oral glucan was given late during the vaccine boost phase, total of 7 injections of GD2/GD3 with QS21 as subcutaneous adjuvant over one year.

[0015] Fig. 2A shows anti-GD2 IgGl seroconversion correlated strongly with progression-free survival following GD2/GD3 vaccine in high-risk neuroblastoma with a history of prior progression.

[0016] Fig. 2B shows anti-GD2 IgGl seroconversion correlated strongly with overall survival following GD2/GD3 vaccine in high-risk neuroblastoma with a history of prior progression. [0017] Fig. 3A shows anti-tumor hu3F8 class switch IgA mediated efficient polymorphonuclear neutrophils antibody-dependent cellular cytotoxicity (PMN-ADCC) when compared to IgGl (the x-axis is loglO scale).

[0018] Fig. 3B shows anti-tumor hu3F8 class switch IgA mediated efficient peripheral blood mononuclear cell antibody-dependent cellular cytotoxicity (PBMC-ADCC) when compared to IgGl (the x-axis is loglO scale).

[0019] Fig. 4A shows PMN-ADCC or neutrophil mediated by hu3F8-IgG subclasses (the x-axis is loglO scale).

[0020] Fig. 4B shows PBMC (peripheral blood mononuclear cells) mediated by hu3F8- IgG subclasses (the x-axis is loglO scale).

[0021] Fig. 5A shows Hu3F8-IgA synergizes with hu3F8-IgGl in PMN-ADCC. This synergism of anti-GD2 IgA with IgG was shown among patients following vaccination with GD2/GD3 ganglioside vaccine (the x-axis is loglO scale).

[0022] Fig. 5B shows Hu3F8-IgA synergizes with hu3F8-IgGl in PBMC (peripheral blood mononuclear cells). This synergism of anti-GD2 IgA with IgG was shown among patients following vaccination with GD2/GD3 ganglioside vaccine (the x-axis is loglO scale).

[0023] Fig. 6A shows Progression-free Survival based on anti-GD2 IgA seroconversion (>150 ng/ml at week 8) when combined with IgGl seroconversion (>150 ng/ml at week 8) among 102 patients treated in >2nd remission.

[0024] Fig. 6B shows Progression-free Survival based on anti-GD2 IgG3 seroconversion (>150 ng/ml at week 8) when combined with IgGl seroconversion (>150 ng/ml at week 8) among 102 patients treated in >2nd remission.

[0025] Fig. 7 shows a model for superior IgA-mediated tumor cell killing by neutrophils. FcaRI is able to interact with IgA in a 1 : 1 or a 2: 1 (FcaRI: IgA) stoichiometry. Thus, IgA can bind FcaRI bivalently resulting in more stable binding and recruitment of in total 4 ITAMs.

This scenario would initiate a robust IT AM signaling necessary for activating effector functions.

[0026] Fig. 8 shows immunoglobulin class switching. [0027] Fig. 9A shows the nucleic acid sequences of the Hu3F8 IgG3 light chain (SEQ ID NO: 1) and heavy chain (SEQ ID NO: 2). Fig. 9B shows the amino acid sequences of the Hu3F8 IgG3 light chain (SEQ ID NO: 3) and heavy chain (SEQ ID NO: 4). The VH and VL domain sequences are italicized and the leader sequences are underlined.

[0028] Fig. 10A shows the nucleic acid sequences of the Hu3F8 IgAl light chain (SEQ ID NO: 5) and heavy chain (SEQ ID NO: 6). Fig. 10B shows the amino acid sequences of the Hu3F8 IgAl light chain (SEQ ID NO: 7) and heavy chain (SEQ ID NO: 8). The VH and VL domain sequences are italicized and the leader sequences are underlined.

[0029] Fig. 11A shows the nucleic acid sequences of the Hu3F8 IgA2 light chain (SEQ ID NO: 9) and heavy chain (SEQ ID NO: 10). Fig. 11B shows the amino acid sequences of the Hu3F8 IgA2 light chain (SEQ ID NO: 11) and heavy chain (SEQ ID NO: 12). The VH and VL domain sequences are italicized and the leader sequences are underlined.

[0030] Fig. 12A shows the nucleic acid sequences of the Hu3F8 IgGl light chain (SEQ ID NO: 13) and heavy chain (SEQ ID NO: 14). Fig. 12B shows the amino acid sequences of the Hu3F8 IgGl light chain (SEQ ID NO: 15) and heavy chain (SEQ ID NO: 16). The VH and VL domain sequences are italicized and the leader sequences are underlined.

[0031] Fig. 13A shows the nucleic acid sequences of the Hu3F8 IgG4 light chain (SEQ ID NO: 17) and heavy chain (SEQ ID NO: 18). Fig. 13B shows the amino acid sequences of the Hu3F8 IgG4 light chain (SEQ ID NO: 19) and heavy chain (SEQ ID NO: 20). The VH and VL domain sequences are italicized and the leader sequences are underlined.

[0032] Fig. 14A shows the nucleic acid sequences of the Hu3F8 IgG2 light chain (SEQ ID NO: 21) and heavy chain (SEQ ID NO: 22). Fig. 14B shows the amino acid sequences of the Hu3F8 IgG2 light chain (SEQ ID NO: 23) and heavy chain (SEQ ID NO: 24).

DETAILED DESCRIPTION

[0033] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0034] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N. Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0035] Target occupancy, i.e. the percent of targets occupied by antibody or drug, is a function of (1) serum antibody or drug concentration at equilibrium at time t (Ct) and (2) their avidity for the target (apparent KD). For 150 ng/ml of IgGl (or 1 nM) and KD of 5 nM, the % GD2 occupancy can be estimated as Ct/(Ct+KD) = 16%. This provides an indirect measure of how much of the antigen GD2 (on tumor cells or normal cells) in this patient is occupied by each antibody class. Since GD2 occupancy determines efficiency of tumor killing by anti-GD2 antibodies, GD2 occupancy should have prognostic impact on outcome survival. No less important than antibody titer is the avidity of the induced antibodies well known to influence seroprotection following vaccines for infectious disease. ⁵¹ ' ⁵³

[0036] As disclosed in the Examples herein, cancer patients receiving a GD2/GD3 bivalent vaccine induced IgGl, IgG3 and IgA antibodies, whereas the IgG2 and the IgG4 blocking subclasses were low or absent. The discovery of anti-GD2 IgA and IgG3 among patients receiving GD2/GD3 vaccine is an unexpected finding with major therapeutic implications. This observation was surprising because increased IgG and/or IgM titers observed in vaccinated patient is not predictive/ cannot be equated with increased IgA titers. Additionally, increases in IgA titer is not always equated with therapeutic anti-tumor response.

[0037] Moreover, it was surprising that anti-GD2 IgA was effective to inducing antitumor responses against the carbohydrate antigen GD2, alone or in combination with IgGl . IgA has short serum half-life and hitherto was never believed to be important in determining cancer outcome. Until now, in order to study the properties of IgA, people have to either use genetically modified IgA or genetically modified mice. Hence so far no monoclonals are in clinical development or have been approved for human use. The methods of the present technology leverage GD2 vaccines as an approach to bypass the short half-life concern for anti-GD2 IgA.

Definitions

[0038] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0039] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0040] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.

[0041] An “adjuvant” refers to one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to one or more vaccine antigens. An adjuvant may be administered to a subject before, in combination with, or after administration of the vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponins), bacterial and fungal cell wall components (e.g., lipopolysaccarides, lipoproteins, and glycoproteins), hormones, cytokines, and co-stimulatory factors.

[0042] As used herein, an “antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen may be a polypeptide. An antigen may also be administered to an animal to generate an immune response in the animal.

[0043] As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0044] The terms “cancer” or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term “cancer” includes premalignant, as well as malignant cancers.

Examples of cancers include multiple myeloma, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, renal cell carcinoma, pancreatic carcinoma, prostatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, cancer related bone resorption, cancer related bone pain; the suppression of cancer metastasis; the amelioration of cancer cachexia.

[0045] As used herein, a “carrier” is an exogenous protein to which small, non- immunogenic or poorly immunogenic antigens e.g., haptens) can be conjugated to so as to enhance the immunogenicity of the antigens. Examples of such carriers include keyhole limpet hemocyanin (KLH), serum globulins, serum albumins, ovalbumins, and the like.

[0046] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0047] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0048] As used herein, the term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope.

[0049] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.

[0050] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0051] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0052] As used herein, the terms “identical” or percent “identity”, when used in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein)), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (e.g., NCBI web site). Such sequences are then said to be “substantially identical.” This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and/or additions, as well as those that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length.

[0053] As used herein, “immune response” refers to the action of one or more of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the aforementioned cells or the liver or spleen (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, infectious pathogens etc. An immune response may include a cellular response, such as a T-cell response that is an alteration (modulation, e.g., significant enhancement, stimulation, activation, impairment, or inhibition) of cellular, i.e., T-cell function. An immune response may also include humoral (antibody) response.

[0054] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

[0055] As used herein, the term “overall survival” or “OS” means the observed length of life from the start of treatment to death or the date of last contact.

[0056] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 ^th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0057]

[0058] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0059] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

[0060] As used herein, “prevention” or “preventing” of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0061] As used herein, "progression free survival" or “PFS” is the time from treatment to the date of the first confirmed disease progression per RECIST 1.1 criteria.

[0062] “RECIST” shall mean an acronym that stands for “Response Evaluation Criteria in Solid Tumors” and is a set of published rules that define when cancer patients improve (“respond”), stay the same (“stable”) or worsen (“progression”) during treatments. Response as defined by RECIST criteria have been published, for example, a Journal of the National Cancer Institute , Vol. 92, No. 3, Feb. 2, 2000 and RECIST criteria can include other similar published definitions and rule sets. One skilled in the art would understand definitions that go with RECIST criteria, as used herein, such as “Partial Response (PR),” “Complete Response (CR),” “Stable Disease (SD)” and “Progressive Disease (PD) .” As used herein, “CR2” refers to a patient that has achieved CR, relapsed and achieved CR again. [0063] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0064] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0065] As used herein, the term “seroconversion” refers to the development of detectable antibodies that are directed against a specific target antigen in the blood or serum of a subject as a result of infection or immunization.

[0066] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0067] As used herein, “specifically binds” refers to a molecule (e.g., an antibody or antigen binding fragment thereof) which recognizes and binds another molecule (e.g., an antigen), but that does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, can be exhibited, for example, by a molecule having a KD for the molecule to which it binds to of about IO ^-4 M, 10“ ⁵ M, 10’ ⁶ M, 10’ ⁷ M, 1( ⁸ M, 10’ ⁹ M, 10’ ¹⁰ M, 10 ^-11 M, or 10’ ¹² M. The term “specifically binds” may also refer to binding where a molecule (e.g., an antibody or antigen binding fragment thereof) binds to a particular polypeptide, or an epitope on a particular polypeptide, without substantially binding to any other polypeptide, or polypeptide epitope.

[0068] As used herein, "survival" refers to the subject remaining alive, and includes overall survival as well as progression free survival. [0069] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0070] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0071] It is also to be appreciated that the various modes of treatment or prevention of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0072] The term “vaccine” as used herein is a preparation used to enhance protective immunity against cancer, or infectious agents such as viruses, fungi, bacteria and other pathogens. A vaccine may be useful as a prophylactic agent or a therapeutic agent. Vaccines contain cells or antigens which, when administered to the body, induce an immune response with the production of antibodies and immune lymphocytes (T-cells and B-cells).

IgA in Cancer Therapy

[0073] Human antibodies are known to switch classes during the progress of an immune response, where IgM is typically seen as the early response, later other subclasses and isotypes such as IgG, IgGl, IgG2, IgG2, IgA, IgD and IgE are seen (Fig. 8). Human antibody class switches are irreversible and proceed from upstream classes to downstream classes, according to the order of the IGH constant region loci on the chromosome. ⁷⁰ The dominant class switch pathway leads from IgM/IgD to IgGl or IgAl. IgG3 subclass comes early, with nearly equal cytotoxic functions as IgGl, although only one-third of the serum half-life. IgA is well known to play an important role in dampening mucosal infections, as well as inflammatory or autoimmune diseases. ⁴⁷ [0074] For many therapeutic and diagnostic purposes, such as immunotherapy, IgG antibodies are favored because they have an attractive long plasma half-life, in contrast to IgA that naturally has a much shorter plasma half-life in comparison with IgG antibodies. In vivo tumor targeting using IgA anti-tumor mAbs has been difficult as mice do not express a homolog of the human FcaRI, and human IgA has a short half-life in mice. ⁶³ Specially engineered anti-HER.2 IgA mAbs to extend serum half-life enhanced in vivo BT-474 tumor response. ⁴⁸ ' ⁵⁰ However, the short half-life of IgA in serum is a major drawback for using IgA as a therapeutics.

Limitations of Conventional Anti-GD2 mAb Therapy in Neuroblastoma

[0075] NB expresses near equal levels of GD2 and GD3 (44 and 31 pg per gram of tumor, respectively). ¹⁰ These gangliosides are also found, albeit at low levels in neurons, peripheral pain fibers, epidermis and retina. Anti-GD2 monoclonal antibody (mAb) is a proven therapy for high risk NB, ¹¹ demonstrating efficacy world-wide in single arm studies ¹² ' ¹⁴ and randomized trials. ^{11 15} One major limitation is the typical pain and neuropathic side effects pervasive with anti-GD2 antibody immunotherapy, including sensory neuropathy, ophthalmoplegia and transverse myelitis, ¹⁶ in addition to the >40% risk of disease relapse with long follow-up.

[0076] In contrast to protein antigens, carbohydrate antigens like GD2 and GD3 are poorly immunogenic, thus requiring strong and safe immune adjuvants. ¹⁸ Most adjuvants for cancer vaccines currently in use or in development are all parenteral, ^19,20 and not all of them fulfill the benchmarks for efficacy: high seroconversion rates, robust and rapid IgG response, impact on disease outcome, and sustained immunological memory. ²¹ To boost helper T cells, vaccines can be conjugated to highly immunogenic protein scaffolds like keyhole limpet hemocyanin (KLH). ²² and then combined with subcutaneous (sc) immune adjuvants like QS- 21. ^{23, 24}

[0077] Despite conjugating GD2 to KLH, no anti-GD2 response was induced using adjuvant monophosphoryl lipid A. ²⁵ QS-21 plus ganglioside-KLH in patients with sarcoma induced mostly IgM response ²⁶ without clinical benefit (NCT01141491). Other vaccines targeting GM2 in melanoma, ²⁷ Globo H in breast cancer, ^28,29 and MUC1 in ovarian cancer ³⁰ have also failed to meet expectations. A common denominator was insufficient quality or quantity of antibody response. Anti-GD2 Antibodies of the Present Technology

[0078] Disclosed herein are GD2 binding antibodies of various isotypes, and their use in the treatment of cancer.

[0079] In some embodiments, the GD2 binding antibody is of the IgG3 isotype. One example of a GD2 binding antibody of the IgG3 isotype is the antibody comprising the light chain disclosed in SEQ ID NO: 3 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 1, and the heavy chain disclosed in SEQ ID NO: 4 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 2.

[0080] In certain embodiments, the GD2 binding antibody is of the IgAl isotype. One example of a GD2 binding antibody of the IgAl isotype is the antibody comprising the light chain disclosed in SEQ ID NO: 7 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 5, and the heavy chain disclosed in SEQ ID NO: 8 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 6.

[0081] In some embodiments, the GD2 binding antibody is of the IgA2 isotype. One example of a GD2 binding antibody of the IgA2 isotype is the antibody comprising the light chain disclosed in SEQ ID NO: 11 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 9, and the heavy chain disclosed in SEQ ID NO: 12 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 10.

[0082] In other embodiments, the GD2 binding antibody is of the IgGl isotype. One example of a GD2 binding antibody of the IgGl isotype is the antibody comprising the light chain disclosed in SEQ ID NO: 15 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 13, and the heavy chain disclosed in SEQ ID NO: 16 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 14.

[0083] In certain embodiments, the GD2 binding antibody is of the IgG4 isotype. One example of a GD2 binding antibody of the IgG4 isotype is the antibody comprising the light chain disclosed in SEQ ID NO: 19 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 17, and the heavy chain disclosed in SEQ ID NO: 20 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 18.

[0084] In some embodiments, the GD2 binding antibody is of the IgG2 isotype. One example of a GD2 binding antibody of the IgG2 isotype is the antibody comprising the light chain disclosed in SEQ ID NO: 23 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 21, and the heavy chain disclosed in SEQ ID NO: 24 and/or is encoded by the nucleic acid sequence disclosed in SEQ ID NO: 22.

[0085] Methods of Preparing Anti-GD2 Antibodies of the Present Technology. Initially, a target antigen is chosen to which an antibody of the present technology can be raised. For example, an antibody may be raised against the entire GD2 disialoganglioside, or to a portion of the GD2 disialoganglioside. Techniques for generating antibodies directed to such target antigens are well known to those skilled in the art. Examples of such techniques include, for example, but are not limited to, those involving display libraries, xeno or human mice, hybridomas, and the like. In some embodiments, the GD2 epitope may comprise at least 1-5 sugar residues or ceramide of at least a portion of GD2. In some embodiments, the GD2 epitope may comprise a penta-saccharide head group linked to a ceramide lipid tail.

[0086] It should be understood that recombinantly engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, which are directed to GD2 disial ogangliosides and fragments thereof are suitable for use in accordance with the present disclosure.

[0087] Anti-GD2 antibodies that can be subjected to the techniques set forth herein include monoclonal and polyclonal antibodies, and antibody fragments such as Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains and/or antibody fragments. Methods useful for the high yield production of antibody Fv-containing polypeptides, e.g., Fab' and F(ab')2 antibody fragments have been described. See U.S. Pat. No. 5,648,237.

[0088] Generally, an antibody is obtained from an originating species. More particularly, the nucleic acid or amino acid sequence of the variable portion of the light chain, heavy chain or both, of an originating species antibody having specificity for a target antigen is obtained. An originating species is any species which was useful to generate the antibody of the present technology or library of antibodies, e.g., rat, mouse, rabbit, chicken, monkey, human, and the like.

[0089] Phage or phagemid display technologies are useful techniques to derive the antibodies of the present technology. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding antibodies of the present technology, can be carried out in E. coli.

[0090] Due to the degeneracy of nucleic acid coding sequences, other sequences which encode substantially the same amino acid sequences as those of the naturally occurring proteins may be used in the practice of the present technology These include, but are not limited to, nucleic acid sequences including all or portions of the nucleic acid sequences encoding the above polypeptides, which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. It is appreciated that the nucleotide sequence of an immunoglobulin according to the present technology tolerates sequence homology variations of up to 25% as calculated by standard methods (“Current Methods in Sequence Comparison and Analysis,” Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.) so long as such a variant forms an operative antibody which recognizes GD2 antigens. For example, one or more amino acid residues within a polypeptide sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligands, etc. Additionally, an immunoglobulin encoding nucleic acid sequence can be mutated in vitro or in vivo to create and/or destroy translation, initiation, and/or termination sequences or to create variations in coding regions and/or form new restriction endonuclease sites or destroy pre-existing ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to in vitro site directed mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers (Pharmacia), and the like. [0091] Preparation of Polyclonal Antisera and Immunogens. Methods of generating antibodies or antibody fragments of the present technology typically include immunizing a subject (generally a non-human subject such as a mouse or rabbit) with a purified GD2 antigen or fragment thereof or with a cell expressing the GD2 antigen or fragment thereof. An appropriate immunogenic preparation can contain, e.g., a recombinantly-expressed GD2 antigen or a chemically-synthesized GD2 antigen. The GD2 disialoganglioside, or a portion or fragment thereof, can be used as an immunogen to generate an anti-GD2 antibody that binds to the GD2 disialoganglioside, or a portion or fragment thereof using standard techniques for polyclonal and monoclonal antibody preparation. In some embodiments, the GD2 epitope may comprise at least 1-5 sugar residues or ceramide of at least a portion of GD2. In some embodiments, the GD2 epitope may comprise a penta-saccharide head group linked to a ceramide lipid tail. The full-length GD2 disialoganglioside or fragments thereof, are useful as fragments as immunogens. In some embodiments, an antibody raised against the disialoganglioside forms a specific immune complex with the GD2 disialoganglioside. Multimers of a given epitope are sometimes more effective than a monomer.

[0092] If needed, the immunogenicity of the GD2 antigen can be increased by fusion or conjugation to a carrier protein such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such carrier proteins are known in the art. One can also combine the GD2 antigen with a conventional adjuvant such as Freund’s complete or incomplete adjuvant to increase the subject’s immune reaction to the antigen. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etcf human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0093] In describing the present technology, immune responses may be described as either “primary” or “secondary” immune responses. A primary immune response, which is also described as a “protective” immune response, refers to an immune response produced in an individual as a result of some initial exposure (e.g., the initial “immunization”) to a particular antigen, e.g., GD2 antigen. In some embodiments, the immunization can occur as a result of vaccinating the individual with a vaccine containing the antigen. For example, the vaccine can be a GD2 vaccine comprising one or more GD2-derived antigens. A primary immune response can become weakened or attenuated over time and can even disappear or at least become so attenuated that it cannot be detected. Accordingly, the present technology also relates to a “secondary” immune response, which is also described here as a “memory immune response.” The term secondary immune response refers to an immune response elicited in an individual after a primary immune response has already been produced.

[0094] Thus, a secondary immune response can be elicited, e.g., to enhance an existing immune response that has become weakened or attenuated, or to recreate a previous immune response that has either disappeared or can no longer be detected. The secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs upon stimulation of memory B cells that were generated at the first presentation of the antigen. Delayed type hypersensitivity (DTH) reactions are a type of cellular secondary or memory immune response that are mediated by CD4 ⁺ T cells. A first exposure to an antigen primes the immune system and additional exposure(s) results in a DTH.

[0095] Following appropriate immunization, the anti-GD2 antibody can be prepared from the subject’s serum. If desired, the antibody molecules directed against the GD2 antigen can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography to obtain the IgG fraction.

[0096] Monoclonal Antibody. In one embodiment of the present technology, the antibody is an anti-GD2 monoclonal antibody. For example, in some embodiments, the anti-GD2 monoclonal antibody may be a human or a mouse anti-GD2 monoclonal antibody. For preparation of monoclonal antibodies directed towards the GD2 antigen, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (See, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (See, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (See, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be utilized in the practice of the present technology and can be produced by using human hybridomas (See, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (See, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids that encode regions of antibodies can be isolated. PCR utilizing primers derived from sequences encoding conserved regions of antibodies is used to amplify sequences encoding portions of antibodies from the population and then DNAs encoding antibodies or fragments thereof, such as variable domains, are reconstructed from the amplified sequences. Such amplified sequences also can be fused to DNAs encoding other proteins - e.g., a bacteriophage coat, or a bacterial cell surface protein - for expression and display of the fusion polypeptides on phage or bacteria. Amplified sequences can then be expressed and further selected or isolated based, e.g., on the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on GD2. Alternatively, hybridomas expressing anti-GD2 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject’s spleen using routine methods. See, e.g., Milstein el al., (Galfire and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using standard methods will produce monoclonal antibodies of varying specificity (i.e., for different epitopes) and affinity. A selected monoclonal antibody with the desired properties, e.g., GD2 binding, can be used as expressed by the hybridoma, it can be bound to a molecule such as polyethylene glycol (PEG) to alter its properties, or a cDNA encoding it can be isolated, sequenced and manipulated in various ways. Synthetic dendromeric trees can be added to reactive amino acid side chains, e.g., lysine, to enhance the immunogenic properties of GD2. Also, CPG- dinucleotide techniques can be used to enhance the immunogenic properties of GD2. Other manipulations include substituting or deleting particular amino acyl residues that contribute to instability of the antibody during storage or after administration to a subject, and affinity maturation techniques to improve affinity of the anti-GD2 antibody.

[0097] Hybridoma Technique. In some embodiments, the antibody of the present technology is an anti-GD2 monoclonal antibody produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma techniques include those known in the art and taught in Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al. , Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art.

[0098] Phage Display Technique. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA and phage display technology. For example, anti-GD2 antibodies, can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them. Phages with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g., human or murine) by selecting directly with an antigen, typically an antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains that are recombinantly fused to either the phage gene III or gene VIII protein. In addition, methods can be adapted for the construction of Fab expression libraries (See, e.g., Huse, et al., Science 246: 1275-1281, 1989) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a GD2 antigen, e.g., derivatives, fragments, analogs or homologs thereof. Other examples of phage display methods that can be used to make the antibodies of the present technology include those disclosed in Huston et al., Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al. , Gene 187: 9-18, 1997; Burton et al. , Advances in Immunology 57: 191-280, 1994; PCT/GB91/01134; WO 90/02809;

WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; WO 96/06213; WO 92/01047 (Medical Research Council et a!. }, WO 97/08320 (Morphosys); WO 92/01047 (CAT/MRC); WO 91/17271 (Affymax); and U.S. Pat. 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, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. Methods useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides via disulfide bonds have been described by Lohning, U.S. Pat. No. 6,753,136. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0099] Generally, hybrid antibodies or hybrid antibody fragments that are cloned into a display vector can be selected against the appropriate antigen in order to identify variants that maintain good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). However, other vector formats could be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and/or screening.

[00100] Expression of Recombinant Anti-GD2 Antibodies. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA technology. Recombinant polynucleotide constructs encoding an anti-GD2 antibody of the present technology typically include an expression control sequence operably-linked to the coding sequences of anti-GD2 antibody chains, including naturally-associated or heterologous promoter regions. As such, another aspect of the technology includes vectors containing one or more nucleic acid sequences encoding an anti-GD2 antibody of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, the nucleic acid containing all or a portion of the nucleotide sequence encoding the anti-GD2 antibody is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below. Methods for producing diverse populations of vectors have been described by Lerner et al., U.S. Pat. Nos. 6,291,160 and 6,680,192. [00101] In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present disclosure, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present technology is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Such viral vectors permit infection of a subject and expression of a construct in that subject. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences encoding the anti-GD2 antibody, and the collection and purification of the anti-GD2 antibody, e.g., cross-reacting anti-GD2 antibodies. See generally, U.S. 2002/0199213. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin-resi stance or hygromycin-resi stance, to permit detection of those cells transformed with the desired DNA sequences. Vectors can also encode signal peptide, e.g., pectate lyase, useful to direct the secretion of extracellular antibody fragments. See U.S. Pat. No. 5,576,195.

[00102] The recombinant expression vectors of the present technology comprise a nucleic acid encoding a protein with GD2 binding properties in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operably-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, e.g., in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. Typical regulatory sequences useful as promoters of recombinant polypeptide expression (e.g., anti-GD2 antibody), include, e.g., but are not limited to, promoters of 3 -phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. In one embodiment, a polynucleotide encoding an anti-GD2 antibody of the present technology is operably-linked to an araB promoter and expressible in a host cell. See U.S. Pat. 5,028,530. The expression vectors of the present technology can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., anti-GD2 antibody, etc.).

[00103] Another aspect of the present technology pertains to anti-GD2 antibodyexpressing host cells, which contain a nucleic acid encoding one or more anti-GD2 antibodies. The recombinant expression vectors of the present technology can be designed for expression of an anti-GD2 antibody in prokaryotic or eukaryotic cells. For example, an anti-GD2 antibody can be expressed in bacterial cells such as Escherichia coh, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase. Methods useful for the preparation and screening of polypeptides having a predetermined property, e.g, anti- GD2 antibody, via expression of stochastically generated polynucleotide sequences has been previously described. See U.S. Pat. Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6,569,641.

[00104] Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a

- l- polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[00105] Examples of suitable inducible non-fusion E. coll expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET l id (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of distinct active peptide or protein domains to yield multifunctional polypeptides via polypeptide fusion has been described by Pack et al., U.S. Pat. Nos. 6,294,353; 6,692,935. One strategy to maximize recombinant polypeptide expression, e.g., an anti-GD2 antibody, in E. coll is to express the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli (See, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the present technology can be carried out by standard DNA synthesis techniques.

[00106] In another embodiment, the anti-GD2 antibody expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell G. 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, an anti-GD2 antibody can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides, e.g., anti-GD2 antibody, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, etal., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[00107] In yet another embodiment, a nucleic acid encoding an anti-GD2 antibody of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, e.g., but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that are useful for expression of the anti- GD2 antibody of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[00108] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1 : 268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji, et al. , 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477,

1989), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, Science 249: 374-379,

1990) and the a-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989). [00109] Another aspect of the present methods pertains to host cells into which a recombinant expression vector of the present technology has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[00110] A host cell can be any prokaryotic or eukaryotic cell. For example, an anti-GD2 antibody can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. Mammalian cells are a suitable host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. Co et al, J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[00111] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (See generally, Sambrook et al. , Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[00112] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the anti-GD2 antibody or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[00113] A host cell that includes an anti-GD2 antibody of the present technology, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) recombinant anti-GD2 antibody. In one embodiment, the method comprises culturing the host cell (into which a recombinant expression vector encoding the anti-GD2 antibody has been introduced) in a suitable medium such that the anti-GD2 antibody is produced. In another embodiment, the method further comprises the step of isolating the anti-GD2 antibody from the medium or the host cell. Once expressed, collections of the anti-GD2 antibody, e.g., the anti-GD2 antibodies or the anti-GD2 antibody-related polypeptides are purified from culture media and host cells. The anti-GD2 antibody can be purified according to standard procedures of the art, including HPLC purification, column chromatography, gel electrophoresis and the like. In one embodiment, the anti-GD2 antibody is produced in a host organism by the method of Boss et al., U.S. Pat. No. 4,816,397. Usually, anti-GD2 antibody chains are expressed with signal sequences and are thus released to the culture media. However, if the anti-GD2 antibody chains are not naturally secreted by host cells, the anti- GD2 antibody chains can be released by treatment with mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification technique, column chromatography, ion exchange purification technique, gel electrophoresis and the like (See generally Scopes, Protein Purification (Springer-Verlag, N.Y., 1982).

[00114] Polynucleotides encoding anti-GD2 antibodies, e.g., the anti-GD2 antibody coding sequences, can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. See, e.g., U.S. Pat. Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences for light and/or heavy chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or P-lactoglobulin. For production of transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

[00115] Single-Chain Antibodies. In one embodiment, the anti-GD2 antibody of the present technology is a single-chain anti-GD2 antibody. According to the present technology, techniques can be adapted for the production of single-chain antibodies specific to a GD2 antigen (See, e.g., U.S. Pat. No. 4,946,778). Examples of techniques which can be used to produce single-chain Fvs and antibodies of the present technology include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al, Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[00116] Chimeric and Humanized Antibodies. In one embodiment, the anti-GD2 antibody of the present technology is a chimeric anti-GD2 antibody. In one embodiment, the anti-GD2 antibody of the present technology is a humanized anti-GD2 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody.

[00117] Recombinant anti-GD2 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques, and are within the scope of the present technology. For some uses, including in vivo use of the anti-GD2 antibody of the present technology in humans as well as use of these agents in in vitro detection assays, it is possible to use chimeric or humanized anti-GD2 antibodies. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, e.g., but are not limited to, methods described in International Application No. PCT/US86/02269; U.S. Pat. No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO 86/01533; U.S. Pat. Nos. 4,816,567; 5,225,539; European Patent No. 125023; Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214- 218; Nishimura, et al., 1987. Cancer Res. 47: 999-1005; Wood, et al., 1985. Nature 314: 446- 449; Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202-1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321 : 552-525; Verhoeyan, et al., 1988. Science 239: 1534; Morrison, Science 229: 1202, 1985; Oi et al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. Pat. No. 5,807,715; and Beidler, et al., 1988. J. Immunol. 141 : 4053-4060. For example, antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. No. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., Molecular Immunology, 28: 489-498, 1991; Studnicka et aL, Protein Engineering 7: 805-814, 1994; Roguska et al. , PNAS 91 : 969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332). In one embodiment, a cDNA encoding a murine anti-GD2 monoclonal antibody is digested with a restriction enzyme selected specifically to remove the sequence encoding the Fc constant region, and the equivalent portion of a cDNA encoding a human Fc constant region is substituted (See Robinson et al., PCT/US86/02269; Akira et aL, European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et aL, WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et aL, European Patent Application 125,023; Better et aL (1988) Science 240: 1041-1043; Liu et al. (1987) roc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et aL (1987) Cancer Res 47: 999-1005; Wood et aL (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; U.S. Pat. No. 6,180,370; U.S.

Pat. Nos. 6,300,064; 6,696,248; 6,706,484; 6,828,422.

[00118] In one embodiment, the present technology provides the construction of humanized anti-GD2 antibodies that are unlikely to induce a human anti-mouse antibody (hereinafter referred to as “HAMA”) response, while still having an effective antibody effector function. As used herein, the terms “human” and “humanized”, in relation to antibodies, relate to any antibody which is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject. In one embodiment, the present technology provides for a humanized anti-GD2 antibodies, heavy and light chain immunoglobulins.

[00119] CDR Antibodies. In some embodiments, the anti-GD2 antibody of the present technology is an anti-GD2 CDR antibody. Generally the donor and acceptor antibodies used to generate the anti-GD2 CDR antibody are monoclonal antibodies from different species; typically the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody. The graft may be of a single CDR (or even a portion of a single CDR) within a single VH or VL of the acceptor antibody, or can be of multiple CDRs (or portions thereof) within one or both of the VH and VL. Frequently, all three CDRs in all variable domains of the acceptor antibody will be replaced with the corresponding donor CDRs, though one needs to replace only as many as necessary to permit adequate binding of the resulting CDR-grafted antibody to GD2 antigen. Methods for generating CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; and Winter U.S. 5,225,539; and EP 0682040. Methods useful to prepare VH and VL polypeptides are taught by Winter et al., U.S. Pat. Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161;

6,545,142; EP 0368684; EP0451216; and EP0120694.

[00120] After selecting suitable framework region candidates from the same family and/or the same family member, either or both the heavy and light chain variable regions are produced by grafting the CDRs from the originating species into the hybrid framework regions. Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with regard to either of the above aspects can be accomplished using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (z.e., frameworks based on the target species and CDRs from the originating species) can be produced by oligonucleotide synthesis and/or PCR. The nucleic acid encoding CDR regions can also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with suitable ligation enzymes. Alternatively, the framework regions of the variable chains of the originating species antibody can be changed by site- directed mutagenesis.

[00121] Since the hybrids are constructed from choices among multiple candidates corresponding to each framework region, there exist many combinations of sequences which are amenable to construction in accordance with the principles described herein. Accordingly, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[00122] This process typically does not alter the acceptor antibody’s FRs flanking the grafted CDRs. However, one skilled in the art can sometimes improve antigen binding affinity of the resulting anti-GD2 CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (See, e.g., US 5,585,089, especially columns 12- 16). Or one skilled in the art can start with the donor FR and modify it to be more similar to the acceptor FR or a human consensus FR. Techniques for making these modifications are known in the art. Particularly if the resulting FR fits a human consensus FR for that position, or is at least 90% or more identical to such a consensus FR, doing so may not increase the antigenicity of the resulting modified anti-GD2 CDR-grafted antibody significantly compared to the same antibody with a fully human FR.

[00123] Fc Modifications. In some embodiments, the anti-GD2 antibodies of the present technology comprise a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region (or the parental Fc region), such that said molecule has an altered affinity for an Fc receptor (e.g., an FcyR), provided that said variant Fc region does not have a substitution at positions that make a direct contact with Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that make a direct contact with an Fc receptor such as an FcyR, include amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F/G) loop.

[00124] In some embodiments, an anti-GD2 antibody of the present technology has an altered affinity for activating and/or inhibitory receptors, having a variant Fc region with one or more amino acid modifications, wherein said one or more amino acid modification is a N297 substitution with alanine, or a K322 substitution with alanine.

[00125] Glycosylation Modifications. In some embodiments, anti-GD2 antibodies of the present technology have an Fc region with variant glycosylation as compared to a parent Fc region. In some embodiments, variant glycosylation includes the absence of fucose; in some embodiments, variant glycosylation results from expression in GnTl -deficient CHO cells.

[00126] In some embodiments, the antibodies of the present technology, may have a modified glycosylation site relative to an appropriate reference antibody that binds to an antigen of interest (e.g., GD2), without altering the functionality of the antibody, e.g., binding activity to the antigen. As used herein, "glycosylation sites" include any specific amino acid sequence in an antibody to which an oligosaccharide (z.e., carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach.

[00127] Oligosaccharide side chains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine. For example, an Fc-gly coform (hGD2-IgGln) that lacks certain oligosaccharides including fucose and terminal N- acetylglucosamine may be produced in special CHO cells and exhibit enhanced ADCC effector function.

[00128] In some embodiments, the carbohydrate content of an anti-GD2 antibody disclosed herein is modified by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, e.g., U.S. Patent No. 6,218,149; EP 0359096B1; U.S.

Patent Publication No. US 2002/0028486; International Patent Application Publication WO 03/035835; U.S. Patent Publication No. 2003/0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511 ; all of which are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In some certain embodiments, the present technology includes deleting the glycosylation site of the Fc region of an antibody, by modifying position 297 from asparagine to alanine.

[00129] Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example N- acetylglucosaminyltransferase III (GnTIII), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740;

Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application Serial No. 10/277,370; U.S. Patent Application Serial No. 10/113,929;

International Patent Application Publications WO 00/61739A1 ; WO 01/292246A1; WO 02/311140A1; WO 02/30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton, N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., International Patent Application Publication WO 00/061739; U.S. Patent Application Publication No. 2003/0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[00130] Formulations of Pharmaceutical Compositions. According to the methods of the present technology, the anti-GD2 antibodies of the present technology can be incorporated into pharmaceutical compositions suitable for administration. The pharmaceutical compositions may comprise an anti-GD2 antibody of the present technology and one or more excipients of pharmaceutical grade, such as diluents, fillers, stabilizers, salts, pH regulating agents, tonicity regulating agents etc. [00131] The pharmaceutical compositions generally comprise recombinant or substantially purified antibody and a pharmaceutically-acceptable carrier in a form suitable for administration to a subject. Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the antibody compositions (See, e.g., Remington’ s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18 ^th ed., 1990). The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[00132] The terms “pharmaceutically-acceptable,” “physiologically-tolerable,” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition. For example, “pharmaceutically- acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. “Pharmaceutically-acceptable salts and esters” means salts and esters that are pharmaceutically-acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the composition are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically-acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the anti-GD2 antibody, e.g., Ci-6 alkyl esters. When there are two acidic groups present, a pharmaceutically-acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified. An anti-GD2 antibody named in this technology can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such anti-GD2 antibody is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically-acceptable salts and esters. Also, certain embodiments of the present technology can be present in more than one stereoisomeric form, and the naming of such anti- GD2 antibody is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers. A person of ordinary skill in the art, would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present technology.

[00133] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the anti-GD2 antibody, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[00134] A pharmaceutical composition of the present technology is formulated to be compatible with its intended route of administration. The anti-GD2 antibody compositions of the present technology can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; or intramuscular routes, or as inhalants. The anti-GD2 antibody can optionally be administered in combination with other agents that are at least partly effective in treating various GD2-associated cancers.

[00135] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[00136] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic compounds, e.g., sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g., aluminum monostearate and gelatin.

[00137] Sterile injectable solutions can be prepared by incorporating an anti-GD2 antibody of the present technology in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the anti-GD2 antibody into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The antibodies of the present technology can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[00138] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the anti-GD2 antibody can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding compounds, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring.

[00139] For administration by inhalation, the anti-GD2 antibody is delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[00140] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the anti-GD2 antibody is formulated into ointments, salves, gels, or creams as generally known in the art.

[00141] The anti-GD2 antibody can also be prepared as pharmaceutical compositions in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. [00142] In one embodiment, the anti-GD2 antibody is prepared with carriers that will protect the anti-GD2 antibody against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically-acceptable carriers. These can be prepared according to methods known to those skilled in the art, e.g., as described in U.S. Pat. No. 4,522,811.

Prognostic Methods of the Present Technology

[00143] In one aspect, the present disclosure provides a method for identifying a cancer patient that will show positive prognosis after receiving a GD2 vaccine comprising: detecting levels of anti-GD2-IgA and/or anti-GD2-IgG3 antibodies in a biological sample obtained from the cancer patient that are elevated compared to a predetermined threshold or a reference sample. In some embodiments, the method further comprises administering the GD2 vaccine to the cancer patient one or more times after the anti-GD2-IgA and/or anti- GD2-IgG3 antibodies are detected in the biological sample. Additionally or alternatively, in some embodiments, the method further comprises detecting levels of anti-GD2-IgGl antibodies in a biological sample obtained from the cancer patient that are elevated compared to a predetermined threshold or a reference sample. The biological sample may comprise peripheral blood mononuclear cells, whole blood, serum or plasma.

[00144] The levels of anti-GD2 antibodies can be assessed by any suitable method. Binding assay methods include, e.g., ELISA, radioimmunoassays, scintillation proximity assays, fluorescence energy transfer assays, liquid chromatography, membrane filtration assays, and the like. Biophysical assays for the direct measurement of GD2 binding to anti- GD2 antibody are, e.g., nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chips) and the like. Specific binding is determined by standard assays known in the art, e.g., radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy and the like. [00145] Additionally or alternatively, in some embodiments, the GD2 vaccine comprises GD2 conjugated to a carrier, such as keyhole limpet hemocyanin (KLH). In certain embodiments, the GD2 vaccine further comprises GD3 conjugated to a carrier, e.g., KLH. In some embodiments, the GD2 vaccine has been formulated for parenteral administration. In any of the preceding embodiments of the methods described herein, the GD2 vaccine has been separately, sequentially or simultaneously co-administered with an adjuvant, such as QS21. Vaccine compositions comprising GD2 conjugated to KLH and optionally GD3 conjugated to KLH and further comprising QS21 as adjuvant are known in the art, e.g. from US 6,916,746 (incorporated by reference) and such compositions may be used according to the present technology.

[00146] In one embodiment, the vaccine comprises GD2 conjugated to a carrier (e.g., KLH), an adjuvant (e.g., QS21), and optionally GD3 conjugated to a carrier (e.g., KLH), wherein the vaccine is administered in one or more administrations in a first phase, each administration separated from the next administration by a period in the range of one to two weeks; followed by one or more administration in a second phase, each administration separated from the next administration by a period in the range of one week to 6 months.

[00147] In some embodiments, each dose of the vaccine composition comprises: GD2 conjugated to KLH, in an amount of 10-50 pg, preferably in the range of 20-40 pg, and most preferred around 30 pg; GD3 conjugated to KLH, in an amount of 10-50 pg, preferably in the range of 20-40 pg, and most preferred around 30 pg; and QS21 in an amount of 50-300 pg/m ² , preferably in the range of 100-200 pg/m ² , and most preferred around 150 pg/m ² . In certain embodiments, the dose of the vaccine composition comprises: GD2 conjugated to KLH, in an amount of 30 pg; GD3 conjugated to KLH, in an amount of 30 pg; and QS21 in an amount of 150 pg/m ² .

[00148] The first phase, the prime phase, is intended to elicit a first immune response against GD2 and/or GD3, and it consists of at least one administration of the vaccine composition to the individual being treated according to the method. Typical, the first phase comprises two or more administrations of vaccine compositions, e.g. 2, 3, 4 or even more administrations; given with short intervals such as with 4-10 days between the administrations. A preferred first phase comprises 2 or three administrations of the vaccine composition, where the 2 or three administrations are given with one weeks intervals. [00149] The presence the anti-GD2 and optionally anti-GD3 antibodies in patients at the end of the first phase may be detected using methods known in the art. However, it is also possible to proceed to the second phase without analyzing for the presence of an immune response. The second phase, the boost phase, it intended to “mature” the immune response by increasing the titer of GD2 and GD3 antibodies in plasma, and further by inducing subclass and isotype switches, as known in the art.

[00150] The second phase comprises a number of administrations of the vaccine composition, such as 2-10 administrations, and the administrations may be given with longer intervals, such as in the range of 2 weeks to 20 weeks. The first administration of the second phase it typically given 2-4 weeks after the last administration of the first phase. In one embodiment the second phase comprises 3-6 administrations given with continuous increasing intervals, so the first administration of the second phase may be given e.g. 2-4 weeks after the previous administration, i.e. the last administration in the first phase, where the last administration in the second phase may be given 20 weeks after the previous administration. The presence the anti-GD2 and optionally anti-GD3 antibodies in patients at the end of the second phase may be detected using methods known in the art.

[00151] P-glucans are polymers containing P-l,3-linked and P-l,4-D-glucose molecules with 1,6-linked side-chains. ^31,32 Different P-glucan preparations have vastly different properties. ³⁶ GMP grade 1,3-1, 4 (NCT00492167) and 1,3-1, 6 (NCT00037011) purified P- glucans, administered orally, have proven safe in patients. ⁴¹ A special gel formulation of yeast derived P-glucan was tested in mice and was able to show a strong adjuvant effect in enhancing anti-ganglioside antibody response. ⁴² The safety and adjuvant effect when given orally was demonstrated in a phase I trial (ClinicalTrials.gov NCT00492167). ⁴³

[00152] Accordingly, in any of the above embodiments of the methods disclosed herein, the GD2 vaccine has been separately, sequentially or simultaneously co-administered with an effective amount of a yeast beta-glucan. The beta-glucan is selected among glucans containing P-1, 3 -linked and P-l,4-D-glucose molecules with 1,6-linked side-chains. Methods for recovering such glucans are available in the art. The present technology is not limited to any particular method of recovering the glucans, but any such method capable of providing the glucan in a satisfactory purity for use in a pharmaceutical composition may be used according to the present technology. In some embodiments, the yeast beta-glucan comprises a plurality of P-(l,3) side chains linked to a P-(l,3) backbone via P~(l,6) linkages, and has a range of average molecular weights from about 6 kDa to about 30 kDa. In certain embodiments, the yeast beta-glucan is formulated for oral administration. The beta-glucan may be administered orally in amounts in the range of 20-200 mg/kg/day, preferably in the range of 30-100 mg/kg/day, and most preferred around 40 mg/kg/day.

[00153] In some embodiments of the methods disclosed herein, the yeast beta-glucan is administered one, two, three, four, or five times per day. In some embodiments, the yeast beta-glucan is administered more than five times per day. Additionally or alternatively, in some embodiments, the yeast beta-glucan is administered every day, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the yeast beta-glucan is administered weekly, bi-weekly, tri-weekly, or monthly. In some embodiments, the yeast beta-glucan is administered for a period of one, two, three, four, or five weeks. In some embodiments, the yeast beta-glucan is administered for six weeks or more. In some embodiments, the yeast beta-glucan is administered for twelve weeks or more. In some embodiments, the yeast beta-glucan is administered for a period of less than one year. In some embodiments, the yeast beta-glucan is administered for a period of more than one year. In some embodiments, the yeast beta-glucan is administered throughout the subject’s life.

[00154] In some embodiments of the methods of the present technology, the yeast betaglucan is administered daily for 1 week or more. In some embodiments of the methods of the present technology, the yeast beta-glucan is administered daily for 2 weeks or more. In some embodiments of the methods of the present technology, the yeast beta-glucan is administered daily for 3 weeks or more. In some embodiments of the methods of the present technology, the yeast beta-glucan is administered daily for 4 weeks or more. In some embodiments of the methods of the present technology, the yeast beta-glucan is administered daily for 6 weeks or more. In some embodiments of the methods of the present technology, the yeast beta-glucan is administered daily for 12 weeks or more. In some embodiments, the yeast beta-glucan is administered daily throughout the subject’s life. In certain embodiments, the yeast betaglucan is administered daily for one or more days (1-14 days), followed by one or more days (1-14 days) of no yeast beta-glucan treatment for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more cycles. [00155] The beta-glucan may be administered in two or more cycles, each cycles comprising daily administration of the glucan for a first period, typical 2 weeks, followed by a second period without administration, typical 2 weeks. The beta glucan may be administered in this regimen for a number of cycles, e.g. 5-15 cycles.

[00156] In certain embodiments, the administration regime comprises administering to a subject a vaccine composition comprising 30 pg GD2 conjugated to KLH; GD3 pg conjugated to KLH; and 150 pg/m ² QS21, wherein the vaccine is administered in three administrations in a first phase, each administration separated from the next administration by one two weeks; followed by four administrations in a second phase, at week 8, 20, 32 and 52, calculated from the first administration; and starting from week 6, administration of yeast beta-glucan for 13 cycles, each cycle consisting of daily administration of 40 mg/kg/day of beta-glucan for two weeks, followed by 2 weeks without administration of beta-glucan. See Fig. I-

[00157] In any of the above embodiments of the methods disclosed herein, the cancer expresses GD2 and/or GD3. In some embodiments, the cancer is selected from the group consisting of breast cancer, melanoma, small cell lung cancer, neuroblastoma, osteosarcoma, retinoblastoma, brain tumor, soft tissue sarcoma, Ewing’s sarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, and spindle cell sarcoma. In certain embodiments, the cancer is neuroblastoma. Additionally or alternatively, in some embodiments, the cancer patient shows increased progression free survival relative to a control cancer patient that has anti-GD2-IgA and/or anti-GD2-IgG3 antibody levels that are at or below the predetermined threshold.

[00158] The GD2 vaccine may be administered in a number of administrations, e.g. between 3 and 10 administrations; distributed over a time period in the range of 6-18 months, where the titer of anti-GD2 antibodies in the plasma of the patient at an early time, e.g. at a time between 6 and 12 weeks after initiation of the treatment is indicative of the outcome of the treatment. For example, a titer of anti-GD2 antibodies above approximately 150 ng/ml is indicative for a long progression-free survival of the patient, whereas for patients with a titer of anti-GD2 antibodies below approximately 150 ng/ml, the prognosis is poorer. [00159] In some embodiments, the method comprises 7 administrations of a vaccine composition comprising GD2 conjugated to KLH and optionally GD3 conjugated to KLH and QS21 at weeks 1, 2, 3, 8, 20, 32 and 52, and further starting from week 6 administration of yeast beta-glucan for 13 cycles, each cycle consisting of daily administration of 40 mg/kg/day of beta-glucan for two weeks, followed by 2 weeks without administration of beta-glucan , where the titer of anti-GD2 antibodies in the plasma of the patient at week 8 is indicative for the outcome of the outcome of the treatment.

[00160] In certain embodiments, the method comprises between 3 and 10 administrations of a vaccine composition comprising GD2 conjugated to KLH and optionally GD3 conjugated to KLH and QS21 distributed over a time period in the range of 6-18 months, where the presence of both anti-GD2-IgA antibodies and anti-GD2-IgG antibodies in the plasma of the patient at an early time, e.g. at a time between 6 and 12 weeks after initiation of the treatment is indicative for the outcome of the outcome of the treatment. As demonstrated herein, the presence of both anti-GD2-IgA antibodies and anti-GD2-IgG is indicative for a long progression-free survival of the patient, whereas for patients with only anti-GD2-IgA or anti-GD2-IgG, or neither, the prognosis is poorer.

[00161] In some embodiments, the method comprises 7 administrations of a vaccine composition comprising GD2 conjugated to KLH and optionally GD3 conjugated to KLH and QS21 at weeks 1, 2, 3, 8, 20, 32 and 52, and further starting from week 6 administration of yeast beta-glucan for 13 cycles, each cycle consisting of daily administration of 40 mg/kg/day of beta-glucan for two weeks, followed by 2 weeks without administration of beta-glucan, where the presence of both anti-GD2-IgA antibodies and anti-GD2-IgG antibodies in the plasma at week 8 is indicative for the outcome of the outcome of the treatment.

[00162] In certain embodiments, the method comprises between 3 and 10 administrations of a vaccine composition comprising GD2 conjugated to KLH and optionally GD3 conjugated to KLH and QS21 distributed over a time period in the range of 6-18 months, where the presence of both anti-GD2-IgGl and anti-GD2-IgG3 antibodies in the plasma of the patient at an early time, e.g. at a time between 6 and 12 weeks after initiation of the treatment is indicative for the outcome of the outcome of the treatment. As demonstrated herein, the presence of both IgGl and IgG3 is indicative for a long progression-free survival of the patient, whereas for patients with only anti-GD2-IgGl or anti-GD2-IgG3, the prognosis is poorer.

[00163] In some embodiments, the method comprises 7 administrations of a vaccine composition comprising GD2 conjugated to KLH and optionally GD3 conjugated to KLH and QS21 at weeks 1, 2, 3, 8, 20, 32 and 52, and further starting from week 6 administration of yeast beta-glucan for 13 cycles, each cycle consisting of daily administration of 40 mg/kg/day of beta-glucan for two weeks, followed by 2 weeks without administration of beta-glucan , where presence of both anti-GD2-IgGl and anti-GD2-IgG3 antibodies in the plasma of the patient at week 8 is indicative for the outcome of the outcome of the treatment.

[00164] The biomarkers described above allow an early confirmation of the efficacy of the treatment, that can be used by the responsible medical staff to improve the treatment even further. E.g., in case of a positive prognosis, such as an indication that a long progression free survival can be expected, the treating medical staff can continue the treatment as planned, whereas in case of a negative prognosis, the medical staff can use the information to adjust or change the treatment prescribed in order to initiate a treatment that may be better for the patient in question.

Therapeutic Methods of the Present Technology

[00165] The GD2-binding antibodies of the present technology may be used for treating, ameliorating and/or prevention a GD2-positive tumor in a patient in need thereof. The GD2 binding antibodies may be of the IgGl, IgG2, IgG3, IgG4, IgAl or IgA2 isotype or it may even be combination of any two or more of these isotype, such as a combination of an IgG and an IgA antibody. The methods of the present technology may comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-GD2 antibody described herein to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.

[00166] In one aspect, the present disclosure provides a method for treating, ameliorating and/or preventing cancer in a patient in need thereof comprising administering to the patient an effective amount of an anti-GD2 IgA antibody e.g., IgAl or IgA2). In some embodiments, the anti-GD2 IgA antibody is an anti-GD2 IgAl antibody or an anti-GD2 IgA2 antibody. Additionally or alternatively, in some embodiments, the anti-GD2 IgAl antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 8. Additionally or alternatively, in certain embodiments, the anti-GD2 IgA2 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 12. Additionally or alternatively, in some embodiments, the anti- GD2 IgA antibody is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.

[00167] In one aspect, the present disclosure provides a method for treating, ameliorating and/or preventing cancer in a subject in need thereof comprising administering to the subject an effective amount of an anti-GD2 IgG3 antibody. In some embodiments, the anti-GD2 IgG3 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 4. Additionally or alternatively, in some embodiments, the anti-GD2 IgG3 antibody is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.

[00168] In any and all embodiments of the methods disclosed herein, the methods further comprise sequentially, separately or simultaneously administering to the subject an effective amount of an anti-GD2 IgGl antibody. In some embodiments, the anti-GD2 IgGl antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 15 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 16. Additionally or alternatively, in some embodiments, the anti-GD2 IgGl antibody is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally or intranasally.

[00169] In yet another aspect, the present disclosure provides a method for treating or preventing cancer in a subject in need thereof comprising: administering an effective amount of a GD2 vaccine to a donor to induce production of anti-GD2 IgA antibodies in the donor; isolating the anti-GD2 IgA antibodies from the donor; and administering an effective amount of the anti-GD2 IgA antibodies to a recipient subject. In some embodiments, the method further comprises expanding the anti-GD2 IgA antibodies ex vivo prior to step (c). The donor and the recipient subject may be the same or distinct. Any and all embodiments of the vaccine regimens described herein (see section on Prognostic Methods, supra) may be implemented in the methods of the present technology.

[00170] In any of the above embodiments of the methods disclosed herein, the cancer expresses GD2 and/or GD3. The GD2-positive cancer may be any such cancer as known in the art. Examples of GD2-positive cancer include, but are not limited to, breast cancer, melanoma, small cell lung cancer, neuroblastoma, osteosarcoma, retinoblastoma, brain tumor, soft tissue sarcoma, Ewing’s sarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, and spindle cell sarcoma. In certain embodiments, the cancer is neuroblastoma.

[00171] The methods disclosed herein may further comprise separately, simultaneously or sequentially administering at least one additional agent. Examples of additional agents include, but are not limited to, at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an IL- 18 antibody or fragment, small molecule IL-18 antagonist or IL-18 receptor binding protein, an IL-1 antibody (including both IL-1 alpha and IL-1 beta) or fragment, a soluble IL-1 receptor antagonist, an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine, radiation therapy, an anti-angiogenic agent, a chemotherapeutic agent, Thalidomidea muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a fluoroquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid agent, a vitamin, a calcium related hormone, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g.,G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog dornase alpha (Pulmozyme), a cytokine or a cytokine antagonist. Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Lorna Linda, Calif. (2000), each of which is entirely incorporated herein by reference.

EXAMPLES

[00172] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. It will be clear to the person skilled in the art that aspects, embodiments, claims and any items of the present technology may be combined.

Example 1: Vaccination Regimen using GD2-GD3 Bivalent Vaccine

[00173] A vaccine formulation comprising GD2 conjugated to KLH, GD3 conjugated to KLH (30 pg of each) and 150 pg/m ² QS21 adjuvant was used. The vaccine formulation was administered subcutaneous to the patients, with 3 administrations separated by one week in the first part (priming phase) and 4 administrations in second part (boost phase) at week 8, 20, 32 and 52.

[00174] From week 6 further 40mg/kg/day of yeast beta-glucan was administered orally, daily in periods of 2 weeks followed by periods of 2 weeks without administration of betaglucan. The administration was continued for 13 cycles. The complete treatment regime is shown schematically on Fig. 1. A total of 102 patients were treated using this vaccine protocol, and the immune response was monitored.

Example 2: Analysis of Immune Responses

[00175] The immune response of the patients of the vaccine study described in Example 1 was analyzed and the anti-GD2-IgGl titer was measured by week 8.

[00176] It was found that a subgroup of patients had a high titer by week 8 (> 150 ng/ml anti-GD2-IgGl), and another subgroup had a lower titer (<150 ng/ml anti-GD2-IgGl). Patients were followed after the treatment, and disease progression was monitored. Progression-free survival (PFS) and Overall survival (OS) was recorded (Figs. 2A-2B). These results demonstrate that the anti-GD2 IgGl titer is an important biomarker for the prognosis for PFS and OS.

Example 3: Analysis of Immunoglobulin Subclasses and Isotypes

[00177] The subclasses and isotypes in the anti-GD2 antibody population in patients that had received the GD2/GD3 bivalent vaccine treatment were analyzed for by ELISA analysis.

[00178] The analysis revealed anti-GD2 IgA and IgG3 in patients, confirming the ability of the vaccine to induce an immune response in vivo. These results further confirmed that the treatment method was able to induce the subtype switch that is believed to be crucial in conferring a protective effect in the cancer patients. The frequency of IgG2 and IgG4 were low.

Example 4: Analysis of Immune Response Based on Immunoglobulin Subclasses and Isotypes

[00179] PFS for patients who received the vaccine treatment positively correlated with anti-GD2 IgA or IgG3 subclass (Figs. 6A-6B). These results demonstrate that the cooccurrence of anti-GD2-IgGl and anti-GD2-IgA antibodies (Fig. 6A) or anti-GD2-IgGl anti- GD2-IgG3 Figs. 6B) serves as a positive prognostic biomarker for PFS, compared with situations where only one of anti-GD2 IgGl, IgG3 or IgA is detected.

[00180] These results demonstrate that the combination of anti-GD2-IgA and anti-GD2- IgG, may be used to predict the outcome of the complete treatment procedure.

Example 5: Preparation of Anti-GD2 Antibodies in Different Subclasses and Isotypes [00181] Different isotypes of the humanized anti-GD2 antibody Hu3F8 were generated. Hu3F8 class switch was carried out by recombinant antibody strategies where the IgGl CH2 and CH3 domains were replaced by either the human IgG3 or the human IgA constant region domains. Ig subclasses IgGl, IgG2 and IgG4 were also made for comparison. Proteins were expressed in CHO cells and IgG3 purified by protein A affinity chromatography, while IgA antibodies purified using peptide M or protein L affinity column chromatography.

[00182] Hu3F8-IgG3, consisting of a light chain with the sequence disclosed in SEQ ID NO: 3, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 1; and a heavy chain disclosed in SEQ ID NO: 4, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 2. [00183] Hu3F8-IgAl, consisting of a light chain with the sequence disclosed in SEQ ID NO: 7, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 5; and a heavy chain disclosed in SEQ ID NO: 8, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 6.

[00184] Hu3F8-IgA2, consisting of a light chain with the sequence disclosed in SEQ ID NO: 11, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 9; and a heavy chain disclosed in SEQ ID NO: 12, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 10.

[00185] Hu3F8-IgGl, consisting of a light chain with the sequence disclosed in SEQ ID NO: 15, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 13; and a heavy chain disclosed in SEQ ID NO: 16, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 14.

[00186] Hu3F8-IgG4, consisting of a light chain with the sequence disclosed in SEQ ID NO: 19, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 17; and a heavy chain disclosed in SEQ ID NO: 20, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 18.

[00187] Hu3F8-IgG2, consisting of a light chain with the sequence disclosed in SEQ ID NO: 23, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 21; and a heavy chain disclosed in SEQ ID NO: 24, encoded by the nucleic acid sequence disclosed in SEQ ID NO: 22.

Example 6: In vitro PMN-ADCC and PBMC-ADCC using GD2 Binding IgGl and IgA Antibodies

[00188] Cell based lysis tests in IMR-32 neuroblastoma cells were conducted using hu3F8-IgGl and hu3F8-IgA as prepared in Example 5. Both hu3F8-IgGl and hu3F8-IgA showed high lysis frequency in both PMN-ADCC and PBMC-ADCC (Figs. 3A-3B). Figs. 4A-4B show PMN-ADCC and PBMC-ADCC mediated by hu3F8-IgG subclasses. These result demonstrate that anti-GD2 IgA is useful treating neuroblastoma and would be beneficial as a therapeutic agent because anti-GD2 IgA should have little-no side effects in terms of pain relative to anti-GD2 IgG. Example 7: Synergistic Cytotoxic Effects with GD2 Binding IgGl and IgA Antibodies

[00189] Cell based lysis tests in IMR-32 neuroblastoma cells were repeated using combinations of hu3F8-IgA and hu3F8-IgGl. The results shown in Figs. 5A-5B show that that there is a synergic effect of IgGl and IgA, and particular at low IgA concentration, the cell lysis can be significantly increased by IgGl.

EQUIVALENTS

[00190] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[00191] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00192] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[00193] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

REFERENCES

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