(OAcGD2)

The present patent application claims the priority of the European patent application EP16001564.0 filed on July 15 ^th , 2016, which is herein incorporated by reference.

FIELD OF THE INVENTION:

The present invention provides novel antibodies and their uses in cancer therapies and diagnosis.

BACKGROUND OF THE INVENTION:

GD2, a disialoganglioside, is an oncofetal antigen that is expressed in the fetus, which is also found in neural stem cells, and mesenchymal stem cells.

Ganglioside GD2 is also a tumor-associated surface antigen found in a broad spectrum of human cancers and stem cells including neuroblastoma, glioma, retinoblastoma, Ewing's family of tumors, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, small cell lung cancer and melanoma. Nevertheless, it is also found on stem cells, neurons, some nerve fibers and basal layer of the skin.

On this basis, the antibody dinutuximab (UNITED THERAPEUTICS) directed against GD2 has been authorized by the FDA and EMA, and recently the antibody dinutuximab beta (APEIRON) has been recently authorized by the EMA, which are both for the treatment of neuroblastoma. However, dinutuximab given in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 2 (IL-2) and isotretinoin (RA) induce severe side effects especially intense neuropathic pain in 85% of patients (despite pretreatment with analgesics including morphine sulfate infusion), serious sensory and motor neuropathies related to the expression of GD2 on nerve and brain cells. Severe (Grade 3) pain occurred in 52% vs. 5% of patients when compared to standard treatment with isotretinoin. Toxicity to central nervous system has also been reported and is worrisome to clinicians. Now, because of its neurotoxicity, its use has been restricted to high-risk neuroblastoma in pediatric patients. This on-target toxicity is highly detrimental to patient quality of life, limits the efficacy of anti-GD2 therapy, and impairs the development of next generation immunotherapies targeting GD2 positive cancer. Despite significant advances in neuroblastoma treatment, high-risk NB is associated with a poor prognosis, and there is a strong need for more effective and less-toxic drugs and strategies to treat NB and other cancer.

It has been previously demonstrated that a modified form of GD2 - i.e. O- acetylated GD2 ganglioside (OAcGD2) - has, in contrast to GD2, a safer expression pattern, with no expression in the peripheral nerves, pituitary gland or human brain cells but, as GD2, OAcGD2 is expressed on tumors. As a result, it has been shown in the international patent application PCT WO 2008/043777 that the administration of a mouse therapeutic antibody (8B6) targeting this OAcGD2 is not associated with any neurotoxicity, especially due to the absence of expression of this cancer antigen on healthy cells, notably on peripheral nerves. A human-mouse chimeric antibody, named c8B6, have been first generated. This specific antibody shows no cross-reaction, neither with GD2, nor with others gangliosides and shows the absence of OAcGD2 antigen expression in the normal brain tissue. In animal models, anti-OAcGD2 chimeric antibodies display similar anti-tumor activity than anti-GD2 monoclonal antibodies (mAbs), while avoiding their toxicity, indicating that OAcGD2 is a better tumor- associated antigen than GD2 and that anti-OAcGD2 mAbs are best-in-class antibodies capable to reduce the uncomfortable side effects commonly associated with anti-GD2 mAb therapies and improve quality of life of patients. However, chimeric antibodies may cause immunogenicity and reduce anti-OAcGD2 efficiency. This problem may be overcome by generating "human", "humanized' or "humaneered" antibodies. Now, humanized anti-OAcGD2 antibodies are needed.

Humanized antibodies generally have at least three potential advantages over mouse or in some cases chimeric antibodies for use in human therapy:

(1) Because the effector portion is human, it may interact better with the other parts of the human immune system (e.g., destroy the target cells more efficiently by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC)); (2) The human immune system should not recognize the framework or constant region of the humanized antibody as foreign, and therefore the antibody response against such an injected antibody should be less than against a totally foreign mouse antibody or a partially foreign chimeric antibody; and (3) Injected mouse antibodies have been reported to have a half- life in the human circulation much shorter than the half-life of human antibodies. Injected humanized antibodies will presumably have a half-life more similar to naturally occurring human antibodies, allowing smaller and less frequent doses to be given.

Nevertheless, the obtaining of humanized antibody is not so simple since humanization process results most of the time in loss of antigen binding affinity and/or loss of specificity. As CDR regions confer both affinity and specificity for antigen, the modification of these regions to reduce potential immunogenicity might be problematic.

Thus, the antibody humanization has to be designed so as to retain specificity and affinity of the antibody for the antigen, while producing molecule with minimal immunogenicity in humans.

Now, this process is so complex that the obtaining of such humanized antibody is never evident.

SUMMARY OF THE INVENTION:

Now, the inventors have surprisingly shown that only specific substitutions maintain the binding affinity, whether the immunogenicity of the antibody is improved or potentially improved. Thus, the inventors provide with new humanized anti- OAcGD2 antibodies (OGD201) with such substitutions.

Consequently, the present invention relates to an antibody, functional fragment, and derivative thereof, which binds specifically to the OAcGD2 ganglioside, said antibody comprising: a) a humanized light chain variable region (VL) polypeptide having the amino acid sequence SEQ id n°l 12; and b) a humanized heavy chain variable region (VH) having the amino acid sequence SEQ id n°76.

The present invention also relates to a pharmaceutical composition comprising at least one of such antibody, and a pharmaceutically acceptable carrier. Additionally, the present invention relates to a method for treating and/or preventing a cancer expressing the OAcGD2 comprising providing to a patient in need thereof such a pharmaceutical composition which comprises at least one said antibody, or at least one functional fragment or derivative thereof.

The present invention relates to a pharmaceutical composition comprising at least one of such antibody, or at least one such functional fragment or derivative thereof for use in a method for treating and/or preventing cancer expressing the OAcGD2 ganglioside.

The present invention relates to a method for diagnosing a cancer expressing the OAcGD2 ganglioside in a subject comprising the step of contacting a biological sample of said subject with at least one antibody as described herein, functional fragment, or derivative thereof, for determining the OAcGD2 ganglioside expression level in said biological sample, wherein a detectable OAcGD2 expression level is indicative of such a cancer.

Finally, the present invention relates to a kit for diagnosing a cancer expressing the OAcGD2 ganglioside in a subject, which comprises at least one of such antibody, or at least one such functional fragment or derivative thereof.

BRIEF DESCRIPTION OF THE FIGURES:

Figure 1 presents the binding titration assays on IMR5 cells of VH humanized anti-OAcGD2 (8B6 VL) mAb as compared to the mouse antibody. Figure 2 presents the binding titration assays on IMR5 cells of VL humanized anti-OAcGD2 (8B6 VH) mAb as compared to the mouse antibody. Figure 3 presents the binding titration assays on IMR5 cells of VH-VL humanized anti-OAcGD2 n Ab as compared to the mouse antibody.

DETAILED DESCRIPTION:

In a first aspect, the present invention concerns an antibody, functional fragment, and derivative thereof, which binds specifically to the OAcGD2 ganglioside, said antibody comprising: a) a humanized light chain variable region (VL) polypeptide having the amino acid sequence SEQ id n°l 12; and b) a humanized heavy chain variable region (VH) having the amino acid sequence SEQ id n°76.

In a first preferred embodiment, the humanized light chain variable region (VL) polypeptide is selected in the group comprising VL30BH (SEQ id n°134) and VL28Bs01/A2 (SEQ id n°135).

Preferably, the humanized heavy chain variable region (VH) polypeptide is selected in the group comprising VH72Bmax (SEQ id n°131), VH49B (SEQ id n°132), and VH49Bmax (SEQ id n°133).

In fact, the inventors surprisingly established that VH72Bmax, VH49B, VH49Bmax, VL30BH, and VL28Bs01/A2 have both a binding affinity comparable to their mouse counterpart, and a weaken immunogenicity. In a second preferred embodiment, the humanized light chain variable region (VL) polypeptide is selected in the group comprising VLl (SEQ id n°8), VL2 (SEQ id n°9), VL3 (SEQ id n°10), VL4 (SEQ id n°l 1 ), VL28BH (SEQ id n°140), VL30BH (SEQ id n°134) and VL28Bs01/A2 (SEQ id n° 135).

Preferably, the humanized heavy chain variable region (VH) polypeptide is selected in the group comprising VH 1 (SEQ id n°3), VH2 (SEQ id n°4), and VH3 (SEQ id n°5), VH72BCDR (SEQ id n°136), VH72BH (SEQ id n°137), VH49BCDR (SEQ id n°138), VH49BH (SEQ id n°139), VH72Bmax (SEQ id n°131 ), VH49B (SEQ id n° 132), and VH49Bmax (SEQ id n° 133).

In fact, the inventors surprisingly established that all these sequences show both a binding affinity comparable to their mouse counterpart, and a weaken immunogenicity. An antibody is an immunoglobulin molecule corresponding to a tetramer comprising four polypeptide chains, two identical heavy (H) chains (about 50-70 kDa when full length) and two identical light (L) chains (about 25 kDa when full length) inter-connected by disulfide bonds. Light chains are classified as kappa and lambda.

The term "antibody", as used herein, refers to a monoclonal antibody per se. Each heavy chain is comprised of a N-term heavy chain variable region

(abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains (CHI, CH2, and CH3) for IgG, with a hinge domain between CHI and CH2 domains.

Each light chain is comprised of a N-term light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino- terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain is in accordance with well- known numbering systems including IMGT, Kabat and Chothia systems (IMGT, The International Immunogenetics Information System®, LEFRANC et al, Nucleic Acids Research, vol. 27, p : 209-212, 1999; KABAT, sequences of Proteins of Immunological Interest, 5 ^th edition. U.S Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH publication, No91-3242, 991; CLOTHIA & LESK, JMol Biol, vol.196(4), p:901 -917, 1987). The functional ability of the antibody to bind a particular antigen depends on the variable regions of each light/heavy chain pair, and is largely determined by the CDRs. According to another preferred embodiment, the humanized light chain variable region (VL) polypeptide is the amino acid sequence SEQ id n°29.

According to another preferred embodiment, the humanized light chain variable region (VL) polypeptide is the amino acid sequence SEQ id n°7. According to another preferred embodiment, the humanized heavy chain variable region (VH) polypeptide is the amino acid sequences SEQ id n°12.

According to still another preferred embodiment, the humanized heavy chain variable region (VH) polypeptide is the amino acid sequences SEQ id n°2.

The term "functional fragments" as used herein refers to antibody fragments, which bind specifically to the OAcGD2 ganglioside. Such fragments can be simply identified by the skilled person and comprise, as an example, ScFv fragment, F _a b fragment (e.g., by papain digestion), F _a b' fragment (e.g., by pepsin digestion and partial reduction), F( _ab ') ₂ fragment (e.g., by pepsin digestion), F _ac b (e.g., by plasmin digestion), and also F _v and F _< j (e.g., by pepsin digestion, partial reduction and re-aggregation) fragments are encompassed by the invention.

Such fragments can be produced by enzymatic cleavage, synthetic or recombinant technique using well known method in the art, such as described in STANWORTH et al (Handbook of Experimental Immunology, vol.1, chapter 8, Blackwell Scientific Publications, 1978). Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a F( _a b') ₂ heavy chain portion can be designed to include DNA sequences encoding the CHi domain and/or hinge region of the heavy chain. The various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.

The expression "binding specifically to the O-acetylated GD2 ganglioside" refers to a binding affinity (¾) of less than 5 x 10 ^"6 M at 25°C for the O-acetylated GD2 ganglioside, preferably a !Q of equal to or less than 5 x 10 ^"7 M, more preferably a Kj of equal to or less than 1 x 10 ^" 7 M or even 5 x 10 ^" 8 M. Such affinity can be simply measured by techniques available in the art, e.g. Scatchard assay, competition ELISA, BIACORE assay or ΚΓΝΕΧΑ assay.

The expression "binding specifically to the O-acetylated GD2 ganglioside" refers to a binding affinity (¾) of more than 5 x 10 ^"5 M at 25 °C for GD2 ganglisoside, preferably a ¾ of more than 10 ^*5 M for GD2 ganglioside.

Now, these fragments comprise at least the variable regions of the heavy and light chains described previously.

These fragments can be soluble, but also anchored within the cytoplasmic membrane, as a single-chain variable part of a chimeric antigen receptor (CAR). The term "chimeric antigen receptors (CARs)," as used herein, refers to an artificial hybrid polypeptide comprising at least one antigen binding domain of an antibody and at least one effector cell signaling domain. Such CARs encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell (e.g. T cells, NK cells and NKT cells). CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell in a non-MHC-restricted manner, thus exploiting the antigen-binding properties of monoclonal antibodies. When expressed in T-cells, CARs recognize unprocessed antigens independently of their expression of major histocompatibility antigen which is unlike the physiologic T-Cell Receptors (TCR), thus bypassing two major mechanisms of tumor escape, the downregulation of HLA expression or proteosomal antigen processing. The binding of CARs to a specific antigen elicits an immune response.

In particular aspects, CARs comprise an ectodomain, a transmembrane domain and an endodomain. Now, the arrangement could be multimeric, such as a diabody or also multimers (e.g., the multi-chain chimeric antigen receptor described in International Patent application PCT WO 2016/016343).

The ectodomain corresponds to the antigen binding domain and to the spacer domain (stalk region). The antigen binding domain is preferably a single-chain variable fragment (scFv). Such scFv is a genetically engineered antibody fragment that usually consists of the heavy chain and light chain of an immunoglobulin, or parts thereof such as VH and VL, joined together by a flexible peptide linker as disclosed as an example in PLUCKTHU (The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer- Verlag, New York, p:269-315, 1994). The flexible peptide linker can be a peptide of between 6 to 40 amino acid residues. The use of small amino acids such as alanine and glycine are of use in creating flexible linker. Exemplary flexible linkers include glycine polymers (G)n, glycine- serine polymers such as for example (GS)n, GSGGS _n (SEQ id n°1 13)n, GGGS _n (SEQ id n°1 14)n and GGGGS _{n (} SEQ id n°1 15)n, where n is an integer of at least one, glycine-alanine polymers or glycine- serine polymers, or other flexible linkers known in the art. As example of useful polymers, one can cite GGGGSGGGGSGGGGS ((G4S)3 ; SEQ id n°1 16), GSTSGSGKPGSGEGSTKG (CD19 linker; SEQ id n°1 17), GGSSRSSSSGGGGSGGGG (18mer; SEQ id n°1 18),

GGGGSGGGGSGGGGSGGGGS ((G4S)4 ; SEQ id n°1 19),

KESGSVSSEQLAQFRSLD (SEQ id n° 120), EGKSSGSGSESKST (SEQ id n°121), GSAGSAAGSGEF (SEQ id n°122), GGGGGGGG (SEQ id n°123) or GGGGGG (SEQ id n°124). Finally, these scFv fragments can be obtained by methods well known to those skilled in the art, such as described by GILLILAND et al. {Tissue Antigens, vol.47, p: l-20, 1996). The term "stalk region" also called as "spacer or hinge domain" as used herein refers to any oligo- or polypeptide that functions to link the transmembrane domain to the ectodomain. In particular, stalk region are used to provide more flexibility and accessibility for the ectodomain. The spacer elements play a predominantly structural role in the CAR. The spacer physically separates the targeting moiety from the T-cell membrane. The optimum distance required is likely to be different for each antigen. To enable efficient target access, a spacer appears to be required if a CAR binds an epitope that lies close to the target cell membrane, or when an antigen is complex in size and glycosylation status. Human IgG-derived spacers (Hinge-CH2-CH3) are commonly used due to their stabilizing action on CAR expression but interactions between the Fc domain of the spacer and Fc gamma receptors (FcgRs) on myeloid cells can lead to activation-induced cell death of T-cells and limited persistence in- vivo. This can be overcome by deleting or modifying regions of the constant heavy (CH)2 domain that are essential for FcgR binding thereby improving CAR T-cell persistence and anti-tumour activity in-vivo in pre-clinical models. Other Hinge domains commonly used include those derivated from CD28 or CD8 or other truncated fragments from Human IgG-derived spacers. In a preferred embodiment, the CAR comprises a stalk region between the ectodomain and the transmembrane domain. A stalk region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. This stalk region may be derived from all or part of naturally occurring molecules, such as part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, this stalk region may be a synthetic sequence.

The transmembrane domain is a membrane anchor domain and also a linker between the ectodomain and the endomain. This transmembrane domain may be a human IgG4Fc hinge region, a Fc region, a CD4 transmembrane domain, a T cell receptor transmembrane, or other transmembrane domains from other human transmembrane signaling proteins, such as CD16, TCR Zeta chain (CD3Q, CD28 and CD8 and erythropoietin receptor, and mutants thereof. Preferably, this transmembrane domain is a T cell receptor transmembrane domain. Preferably, the T cell receptor transmembrane domain is issued from a transmembrane protein able to form a complex with the T cell receptor for antigen (TCR). Preferably, the T cell receptor transmembrane domain comprises part or all of one or more of TCR Zeta chain (CD3Q, CD28, OX40/CD134, 4-1 BB/CD 137/TNFRSF9, FcsRIy, ICOS/CD278, ILRB/CD122, IL-2RG/CD 132, CD27, DAP 10 and CD40.

The endodomain is an intracellular signaling domain, which is responsible for intracellular signaling following the binding of the ectodomain to the target antigen resulting in the activation of the immune cell. In other word, the intracellular signaling domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the chimeric receptor is expressed. The term "effector function" refers to a specialized function of a T cell, which can be a cytolytic activity or helper activity including the secretion of cytokines. Thus the term "intracellular signaling domain" refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain will be employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. Preferred examples of signal transducing domain for use in multi -chain CAR can be the cytoplasmic sequences of the Fc receptor or T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivate or variant of these sequences and any synthetic sequence that as the same functional capability. Signal transduction domain comprises two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases. Examples of ITAM used in the invention can include as non limiting examples those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, the signaling transducing domain of the multi -chain CAR can comprise the CD3zeta signaling domain, or the intracytoplasmic domain of the FcsRI beta or gamma chains.

In particular embodiment the signal transduction domain of the multi-chain CAR of the present invention comprises a co-stimulatory signal molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient immune response. "Co-stimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory molecule on a T-cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation activation, differentiation and the like. A co-stimulatory ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4), an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, DAP- 10, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.

A "co-stimulatory molecule" refers to the cognate binding partner on a T-cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as, but not limited to proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and Toll ligand receptor. Examples of costimulatory molecules include CD27, CD28, CD8, 4- IBB (CD 137), OX40, CD30, CD40, PD-1, ICOS, DAP- 10, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like.

As used herein, the term "derivatives" refers to an amino acid sequence having a percentage of identity of at least 90%, preferably at least 95 %, most preferably at least 98 % (i.e. corresponding to about 10, 5 and 2 amino acids substitutions respectively) with an amino acid sequence selected in the group consisting of SEQ id n°: 2, 3, 4, 5, 7, 8, 9, 10, 1 1, 12, 29, 40, 41, 42, 43, 44, 45, 46, 76 and 112, preferably of at least 99 % (i.e. corresponding to about 1 amino acid substitution). Such derivatives can be simply identified by the skilled person in view of its personal knowledge and of the teaching of the present patent application. It will also be understood that natural amino acids may be replaced by chemically modified amino acids. Typically, such chemically modified amino acids increase the polypeptide half-life.

As used herein, "percentage of identity" between two amino acids sequences, means the percentage of identical amino-acids, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the amino acids sequences. As used herein, "best alignment" or "optimal alignment", means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two amino acids sequences are usually realized by comparing these sequences that have been previously aligned according to the best alignment; this comparison is realized on segments of comparison in order to identify and compare the local regions of similarity. The best sequences alignment to perform comparison can be realized, beside by a manual way, by using the local homology algorithm developed by Smith and Waterman (Ad. App. Math., vol.2, p:482, 1981), by using the global homology algorithm developed by Neddleman and Wunsch (J. Mol. Biol., vol.48, p:443, 1970), by using the method of similarities developed by Pearson and Lipmolan (Proc. Natl. Acad. Sci. USA, vol.85, p:2444, 1988), by using computer softwares using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, WI USA), by using the MUSCLE multiple alignment algorithms (Edgar, Robert C, Nucleic Acids Research, vol. 32, p: 1792, 2004). To get the best local alignment, one can preferably use the BLAST software with the BLOSUM 62 matrix. The identity percentage between two sequences of amino acids is determined by comparing these two sequences optimally aligned, the amino acids sequences being able to encompass additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.

The antibody of the invention is produced recombinantly. The antibody may or may not be glycosylated, though glycosylated antibodies are preferred. In a preferred embodiment, the antibody of the invention may be low fucose.

The antibody of the invention encompasses immunoconjugates.

As used herein, the term "immunoconjugate" refers to a conjugate molecule comprising at least one antibody, a functional fragment or derivative thereof, bound to a second molecule, preferably an immunomodulating agent, a cytotoxic agent or a radioisotope. Such immunoconjugate may be Antibody Drug Conjugates (ADCs), Immunocytokines (ICK) or Antibody Radio Conjugates (ARC). Now, this second molecule may be an antibody having a binding specificity for another antigen, the formed immunoconjugate being a bispecific antibody like a BiTEs (Bi-specific T-cell engagers). Said antibody or fragment thereof is complexed or covalently bound (e.g. fusion protein) to said second molecule. Preferably, said antibody or fragment thereof is bound to said second molecule by covalent linkage.

A second aspect of the invention is related to a pharmaceutical composition comprising at least one antibody as described herein, at least one functional fragment, or at least one derivative thereof, and a pharmaceutically acceptable carrier for use in therapy.

The expression "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar undesirable reactions, such as gastric upset, dizziness and the like when administered to a human. Preferably, as used herein, the expression "pharmaceutically acceptable" means approvable by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "carrier" refers to a solvent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

Said composition may be in any pharmaceutical form suitable for administration to a patient, including but not limited to solutions, suspensions, lyophilized powders, capsule and tablets. Now, the route of administration of the composition of the invention is preferably parenteral; as used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, intraperitoneal, rectal, vaginal, mucosal, intrathecal, intracranial, or intratumoral administration. Thus, the pharmaceutical composition contains vehicles which are pharmaceutically acceptable for a formulation intended to be injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

Most preferably, the composition is in any pharmaceutical form suitable for intravenous administration to a patient.

The antibody, functional fragment or derivative of the invention may be solubilized in a buffer or water or incorporated in emulsions, microemulsions, hydrogels (e.g. PLGA-PEG-PLGA triblock copolymers-based hydrogels), in microspheres, in nanospheres, in microparticles, in nanoparticles (e.g. poly(lactic-co-glycolic acid) microparticles (e.g. poly lactic acid (PLA) ; poly (lactide-co-glycolic acid) (PLGA) ; polyglutamate microspheres, nanospheres, microparticles or nanoparticles), in liposomes, or other galenic formulations. In all cases, the formulation must be sterile and fluid to the extent of acceptable syringability. 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.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The antibody, functional fragment or derivative of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The antibodies of the invention may also be modified, by pegylation as an example, so as to increase its biodisponibility.

The proper fluidity can be maintained, for example, 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. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate, gelatin, polyols, and half-life enhancing covalent and non-covalent formulations.

There are numerous causes of peptide instability or degradation, including hydrolysis and denaturation. Hydrophobic interaction may cause clumping of molecules together (i.e. aggregation). Stabilizers may be added to reduce or prevent such problems. Stabilizers include cyclodextrine and derivatives thereof (see U.S. Pat.

No.5, 730,969). Suitable preservatives such as sucrose, mannitol, sorbitol, trehalose, dextran and glycerin can also be added to stabilize the final formulation. A stabilizer selected from ionic and non-ionic surfactants, D-glucose, D-galactose, D-xylose, D- galacturonic acid, trehalose, dextrans, hydroxyethyl starches, and mixtures thereof may be added to the formulation. Addition of alkali metal salt or magnesium chloride may stabilize a peptide. The peptide may also be stabilized by contacting it with a saccharide selected from the group consisting of dextran, chondroitin sulphuric acid, starch, glycogen, dextrin, and alginic acid salt. Other sugars that can be added include monosaccharides, disaccharides, sugar alcohols, and mixtures thereof (E.g., glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol, xylitol). Polyols may stabilize a peptide, and are water-miscible or water-soluble. Suitable polyols may be polyhydroxy alcohols, monosaccharides and disaccharides including mannitol, glycerol, ethylene glycol, propylene glycol, trimethyl glycol, vinyl pyrrolidone, glucose, fructose, arabinose, mannose, maltose, sucrose, and polymers thereof. Various excipients may also stabilize peptides, including serum albumin, amino acids, heparin, fatty acids and phospholipids, surfactants, metals, polyols, reducing agents, metal chelating agents, polyvinyl pyrrolidone, hydrolysed gelatin, and ammonium sulfate.

In another object, the composition as defined previously is for use in a method for preventing and/or treating cancer expressing the OAcGD2 ganglioside in a subject. As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine or a primate, and most preferably a human.

In the context of the invention, the term "treating cancer expressing OAcGD2 ganglioside ", as used herein, means the inhibition of the growth of such cancer cells. Preferably such treatment also leads to the regression of tumor growth or metastasis spread, i.e., the decrease in size of a measurable tumor. Most preferably, such treatment leads to the complete regression of the tumor.

Said cancer expressing the OAcGD2 ganglioside are selected in the group comprising neuroblastoma, glioma, retinoblastoma, Ewing's family of tumors, sarcoma (i.e. rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, and fibrosarcoma), small cell lung cancer, breast cancer, melanoma, metastatic renal carcinoma, head and neck cancer and hematological cancers (i.e. leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma and myeloma).

The antibody of the invention is contained in said pharmaceutical composition in an amount effective to achieve the intended purpose, and in dosages suitable for the chosen route of administration.

An "effective amount" of the conjugate is an amount which is sufficient to induce the regression of tumor growth or metastasis spread. The doses used for the administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. Naturally, the form of the pharmaceutical composition, the route of administration, the dosage and the regimen naturally depend on the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc. The ranges of effective doses provided below are not intended to limit the invention and represent preferred dose ranges. However, the preferred dose can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.

According to a preferred embodiment, the antibody, functional fragment or derivative thereof can be by administrated by injection at a dose comprised between 2 and 2,000 mg/m ² of subject, preferably at a dose comprised between 5 and 1,000 mg/m , and most preferably at a dose comprised between 10 and 500 mg/m .

According to another preferred embodiment, the antibody, functional fragment or derivative thereof of the invention is in the form a chimeric antigen receptor (CAR). Thus, the amount of transduced cells (such as T cells, NKT cells or NK cells) administered should take into account the route of administration and should be such that a sufficient number of the transduced cells will be introduced so as to achieve the desired therapeutic response. Furthermore, the amounts of each active agent included in the compositions described herein (e.g., the amount per each cell to be contacted or the amount per certain body weight) can vary in different applications. In general, the concentration of transduced T cells desirably should be sufficient to provide in the subject being treated at least from about 1 x 10 ⁴ to about 1 x 10 ⁹ transduced cells per m ² , even more desirably, from about 1 x 10 ⁶ or 1 x 10 ⁷ to about 5 x 10 ⁸ transduced cells,

g although any suitable amount can be utilized either above, e.g., greater than 5 x 10 cells, or below, e.g., less than 1 x 10 ⁷ cells. The dosing schedule can be based on well- established cell-based therapies (see, e.g., TOPALIAN and ROSENBERG, 1987; U.S. Pat. No. 4,690,915), or an alternate continuous infusion strategy can be employed.

These values provide general guidance of the range of transduced T cells to be utilized by the practitioner upon optimizing the method of the present invention for practice of the invention. The recitation herein of such ranges by no means precludes the use of a higher or lower amount of a component, as might be warranted in a particular application. For example, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on inter-individual differences in pharmacokinetics, drug disposition, and metabolism. One skilled in the art readily can make any necessary adjustments in accordance with the exigencies of the particular situation. A third aspect of the present invention concerns a method for treating and/or preventing a cancer expressing the OAcGD2 ganglioside in a subject comprising the step of administrating to a subject in need thereof an effective amount of at least one antibody, functional fragment, or derivative thereof.

Preferably, a subject refers to a mammal and most preferably a human. Said cancer expressing the OAcGD2 ganglioside are selected in the group comprising neuroblastoma, glioma, retinoblastoma, Ewing's family of tumors, sarcoma (i.e. rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, and fibrosarcoma), small cell lung cancer, breast cancer, melanoma, metastatic renal carcinoma, head and neck cancer and hematological cancers (i.e. leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma and myeloma).

A forth aspect of the present invention concerns a method, preferably an in vitro method for diagnosing a cancer expressing the OAcGD2 ganglioside in a subject comprising the step of contacting a biological sample of said subject with at least one antibody as described herein, functional fragment, or derivative thereof, for determining the OAcGD2 ganglioside expression level in said biological sample, wherein a detectable OAcGD2 ganglioside expression level is indicative of such a cancer.

Preferably, said subject refers to a mammal and most preferably a human.

As used herein, a biological sample refers to a sample potentially comprising cancer cells such as a blood sample or a cancer biopsy. Said cancer expressing the OAcGD2 ganglioside is selected in the group comprising neuroblastoma, glioma, retinoblastoma, Ewing's family of tumors, sarcoma (i.e. rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, and fibrosarcoma), small cell lung cancer, breast cancer, melanoma, metastatic renal carcinoma, head and neck cancer and hematological cancers (i.e. leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma and myeloma).

In a preferred embodiment, the method of the invention further comprises the step of comparing said OAcGD2 ganglioside expression with a control. As used herein said "control" refers to the OAcGD2 ganglioside expression level in a control sample corresponding to cells of a biological sample from a healthy subject.

In a fifth aspect, the present invention relates to a kit for diagnosing a cancer expressing the OAcGD2 ganglioside in a subject, which comprises at least one antibody, functional fragment or derivative as described previously and eventually means useful to the administration of said antibody, functional fragment or derivative thereof or said formulation to said subject.

As described previously, said antibody, functional fragment or derivative thereof of said kit may refer to an immunoconjugate suitable to be directly detected by means of imaging techniques for use in a in vivo method for diagnosing a cancer expressing the OAcGD2 ganglioside in a subject, for example an immunoconjugate comprising a fluorophore or a radioisotope, notably for a diagnostic by imaging and monitoring the responsiveness of a subject.

As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term "fragmented kit" refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. The term "fragmented kit" is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term "kit" includes both fragmented and combined kits.

The present kits can also include one or more reagents, buffers, hybridization media, solid supports, databases, computer programs for calculating dispensation orders and/or disposable lab equipment, such as multi-well plates, in order to readily facilitate implementation of the present methods. Enzymes that can be included in the present kits include nucleotide polymerases and the like. Solid supports can include beads and the like whereas molecular weight markers can include conjugatable markers, for example biotin and streptavidin or the like. In one embodiment, the kit is made up of instructions for carrying out the method described herein. The instructions can be provided in any intelligible form through a tangible medium, such as printed on paper, computer readable media, or the like.

In the following, the invention is described in more detail with reference to amino acid sequences, nucleic acid sequences and examples. However, no limitation of the invention is intended by the details of the examples. Rather, the invention pertains to any embodiment which comprises details which are not explicitly mentioned in the examples herein, but which the skilled person finds without undue effort.

EXAMPLES 1) Humanization 1.1 1 ^st round:

The 8B6 sequence has been humanized using CDR-grafting method by using the most closed identified human germline. Then, the binding of the obtained humanized antibodies (OGD201) was tested by their incubation at different concentrations -i.e. 0.01 to 10 μg/ml- on IMR5 cells -i.e. expressing OAcGD2 at their surface- in PBS, 1%BSA for 45 minutes on ice. After incubation and washes, antibody binding was detected by incubation with a goat anti-mouse IgG coupled with FITC (Southern Biotech) for 30 min on ice. Finally, cell fluorescence was analyzed by flow cytometer.

The results in relation with the only one functional germline are presented in table 1 and in figures 1 and 2.

Table 1

OGD201 VL4 78 l|CRSSQSLL

(SEQ id n°ll) LLIYKVSNRL

IK

CDRs are represented in bold in the reference and consensus sequences, and mutated amino-acid mutated are highlighted in grey.

The figure 1 presents the binding titration assays on IMR5 cells of VH humanized anti- OAcGD2 mAb (OGD201 VH3 (Seq id n°5), OGD201 VH2 (Seq id n°4), and OGD201 VH1 (Seq id n°3)) as compared to the mouse antibody (8B6).

The figure 2 presents the binding titration assays on IMR5 cells of VL humanized anti- OAcGD2 mAb (OGD201 VL1 (Seq id n°8), OGD201 VL2 (Seq id n°9), OGD201 VL3 (Seq id n°10), and OGD201 VL4 (Seq id n°l l )) as compared to the mouse antibody (8B6).

The figure 3 presents the binding titration assays on IMR5 cells of VH-VL humanized anti-OAcGD2 mAbs as compared to the mouse antibody (8B6). These VH-VL humanized anti-OAcGD2 mAb combine the VH humanized anti-OAcGD2 mAb (OGD201 VH3 (Seq id n°5), OGD201 VH2 (Seq id n°4), or OGD201 VH1 (Seq id n°3)) with the VL humanized anti-OAcGD2 mAb (OGD201 VL1 (Seq id n°8), OGD201 VL2 (Seq id n°9), OGD201 VL3 (Seq id n° 10), and OGD201 VL4 (Seq id n° l l )).

The results show that on the numerous tested human germline (data not shown), the inventors identified a first VH and VL consensus sequence having at least 83% and 84% of identity with a human germline. The obtained consensus sequence are nearly 10%) and 7% more humanized than the original antibody respectively and have simultaneously a very good binding to OAcGD2 (See Figures 1 , 2 and 3).

1.2 Optimization:

In the aim to increase the antibody humanization degree, the inventors initiate the testing of point mutations on the 8B6 sequences to select those not affecting the binding to OAcGD2. Then, the OAcGD2 binding of the obtained humanized antibodies (OGD201 ) was tested as described previously. The results in relation with the tested point mutations are presented in table 2.

Name OAcGD2 Amino-acid sequence

binding

OGD201 VH4 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°13) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH5 + EVKLVESGGG LVLPGDSLRL SCATSEFT FT DYYMTWVRQP (SEQ id n°14) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TI SRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH6 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°15) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 H7 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°16) PRKALEWLgF IRNRANGYTT EYNPSVKGRF TISRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH8 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°17) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH9 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°18) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH10 PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

(SEQ id n°19)

OGD201 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH11 PRKALEWLGF IRNRANGYTT EYNPSVKGRF T I SRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

(SEQ id n°20)

OGD201 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH12 PRKALEWLGF IRNRANGYTT EYNPSVKGRF TI SRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

(SEQ id n°21)

OGD201 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH13 PRKALEWLGF IRNRANGYTT EYNPSVKGRF T I SRDNSQSg

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

(SEQ id n°22)

OGD201 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH14 PRKALEWLGF IRNRANGYTT EYNPSVKGRF T I SRDNSQS I jgYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS (SEQ id n°23)

OGD201 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH15 PRKALEWLGF IRNRANGYTT EYNPSVKGRF T ISRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

(SEQ id n°24)

OGD201 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH16 PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQS I

LYLQMNTLRT EDSAg-YYCAR VSNWAFDYWG QGTTLTVSS (SEQ id n°25)

OGD201 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH17 PRKALEWLGF IRNRANGYTT EYNPSVKGRF T ISRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

(SEQ id n°26)

OGD201 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH18 PRKALEWLGF I RNRANGYTT EYNPSVKGRF T ISRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTBLTVSS

(SEQ id n°27)

OGD201 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP VH19 PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQS I

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

CDRs are represented in bold in the consensus sequences, and mutated amino-acid mutated are highlighted in grey.

The inventors identified new positions which can be mutated/humanized without affecting the OAcGD2 binding. On the basis of these results, the inventors established a new consensus sequences enabling the obtaining of more humanized antibodies degree (consensus sequences 2).

1.3 Humanization 2 ^nd round: On the basis of the optimization step done previously and of the corresponding consensus sequences, the inventors designed new humanized VH and VL sequences that were tested for their binding to OAcGD2.

CDRs are represented n o an mutate am no-ac mutate are g g te in grey.

Then, the binding of the obtained humanized antibodies is tested by their incubation at different concentrations -i.e. 0.01 to 10 μg/ml- on IMR5 cells -i.e. expressing OAcGD2 at their surface- in PBS, 1%BSA for 45 minutes on ice. After incubation and washes, antibody binding is detected by incubation with a goat anti-mouse IgG coupled with FITC (Southern Biotech) for 30 min on ice. Finally, cell fluorescence is analyzed by flow cytometer.

1.4 Optimization:

So as to increase again the antibody humanization degree, the inventors initiate a new round of point mutations in the CDR this time so as to select those not affecting the binding to OAcGD2. Then, the OAcGD2 binding of the obtained antibodies is tested as described previously.

The tested point mutations are presented in table 4.

Table 4 Name 0AcGD2 Amino-acid sequence

binding

OGD201 VH22 + EVKLVESGGG LVLPGDSLRL SCATSgFTFT DYYMTWVRQP (SEQ id n°47) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VS WAFDYWG QGTTLTVSS

OGD201 VH23 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°48) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH24 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°49) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH25 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DjHYMTWVRQP (SEQ id n°50) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH26 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°51) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH27 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMDWVRQP (SEQ id n°52) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH28 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMSWVRQP (SEQ id n°53) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH29 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMHWVRQP (SEQ id n°54) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH30 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMNWVRQP (SEQ id n°55) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH51 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°126) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH31 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°56) PRKALEWLGS IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH32 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°57) PRKALEWLGg IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH33 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°58) PRKALEWLGF gRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH34 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°59) PRKALEWLGF IR _j SRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH35 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°60) PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH36 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°61) PRKALEWLGF IRNSANGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH37 + EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°62) PRKALEWLGF IRNR§NGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH38 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP (SEQ id n°63) PRKALEWLGF IRNRAYGYTT EYNPSVKGRF TISRDNSQSI

LYLQMNTLRT EDSATYYCAR VSNWAFDYWG QGTTLTVSS

OGD201 VH52 EVKLVESGGG LVLPGDSLRL SCATSEFTFT DYYMTWVRQP

PRKALEWLGF IRNRANGYTT EYNPSVKGRF TISRDNSQSI (SEQ id n°78) SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL21 + DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KSNGNTFLHW (SEQ id n°79) YLQKSGQSPK LLIYKVS RL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 L22 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNgGNTFLHW (SEQ id n°80) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL23 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNjSNTFLHW (SEQ id n°8lj YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL24 + DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGYTFLHW (SEQ id n°82) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL25 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGYTFLHW (SEQ id n°83) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL26 + DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNjgFLHW (SEQ id n°84) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL27 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNjSFLHW (SEQ id n°85) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL28 + DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLjgW (SEQ id n°86) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL29 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLSW (SEQ id°87) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL30 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLSW (SEQ id°88) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL31 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLDW (SEQ id°89) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL32 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLjYW (SEQ id°90) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL33 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLjgW (SEQ id°91) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL34 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLHW (SEQ id°92) YLQKSGQjAPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL35 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLHW (SEQ id°93) YLQKSGQSPK LLIYKVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 L36 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLHW (SEQ id n°94) YLQKSGQSPK LLIYEVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL37 + DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLHW (SEQ id n°95) YLQKSGQSPK LLIYEVSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

OGD201 VL38 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLHW (SEQ id n°96) YLQKSGQSPK LLIYKSSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK ·

OGD201 VL39 + DVVMTQTPLS LPVSLGDQAS ISCRSSQSLL KNNGNTFLHW (SEQ id n°97) YLQKSGQSPK LLIYKjGSNRL SGVPDRFSGS GSGTYFTLKI

SRVEAEDLGV YFCSQSTHIP YTFGGGTKLE IK

CDRs are represented in bold and mutated amino-acid mutated are highlight in grey.

1.5 Humanization 3" round: On the basis of the optimization step done previously and of the corresponding consensus sequences, the inventors designed new humanized VH and VL sequences that were tested for their binding to OAcGD2.

n° 35) I I I _{; ;} I

CDRs are represented in bold and mutated amino -acid mutated are highlighted in grey.

Then, the binding of the obtained humanized antibodies is tested by their incubation at different concentrations -i.e. 0.01 to 10 μg/ml- on IMR5 cells -i.e. expressing OAcGD2 at their surface- in PBS, 1%BSA for 45 minutes on ice. After incubation and washes, antibody binding is detected by incubation with a goat anti-mouse IgG coupled with FITC (SOUTHERN BIOTECH) for 30 min on ice. Finally, cell fluorescence is analyzed by flow cytometer.

Surprisingly, the results show that, whereas VH72Bmax, VH49B, VH49Bmax, VL30BH, and VL28Bs01/A2 have been far modified as compared to their mouse counterpart, they have got a binding affinity comparable to this mouse counterpart, VH49Bmax and VL28Bs01/A2 having the better one.

2) scFv constructs

The selected humanized VH and VL sequences are fused together in VH-Linker-VL or VL-Linker-VH orientation using the linkers disclosed in table 5. For simplifying the step of purification of these polypeptides various tags could be added in C-terminal extremity, preferably a His Tag (HHHHHH; SEQ id n°125).

Table 5: Linker

"Name" Linker sequence

(G4S)3 GGGGSGGGGSGGGGS

(SEQ id n°116)

CD19 linker GSTSGSGKPGSGEGSTKG

(SEQ id n°117)

18mer GGSS SSSSGGGGSGGGG

(SEQ id n°118)

(G4S)4 GGGGSGGGGSGGGGSGGGGS

(SEQ id n°119)

(SEQ id n°120) KESGSVSSEQLAQFRSLD

(SEQ id n°121) EGKSSGSGSESKST

(SEQ id n°122) GSAGSAAGSGEF

(SEQ id n°123) GGGGGGGG

(SEQ id n°124) GGGGGG Then, the designed scFv fragments are produced in E. coli system or mammalian including CHO, Sp2/0, HEK 293 and further purified by affinity chromatography depending of the used tags (e.g. nickel chelated with nickel ions for His tag).

3) KA determination

The affinity of humanized antibodies and fragments thereof (scFv) is assessed using a BIACORE T200 BIOSENSOR (GE HEALTHCARE). For these experiments, gangliosides (OAcGD2, GD2, GM2) are directly immobilized onto the CM5 biosensor chip via hydrophobic interaction. For this, ganglioside diluted mixtures (5(^g/ml) are injected (75μ1) at a flow rate of 5μ1/ηιίη during 15min.

Then, increasing concentration (6.25 to 200nM) of purified antibody or fragment thereof (scFv) diluted in HBS-E buffer containing 250 mM NaCl are prepared. The samples (60μ1) to be tested are injected over the sensor surface at a flow rate of 30μ1/ηιπι over 2min.

Finally, the binding data are analyzed by a bivalent analysis model and default parameter settings for the rate constants using the BIACORE T-200 evaluation software.

OGD201 VH9 18 1.80E-07

OGD201 VH10 19 2.87E-07

OGD201 VH11 20 2.72E-07

OGD201 VH 12 21 ND

OGD201 VH14 23 2.58E-07

OGD201 VH17 26 ND

4) Cytotoxic activity determination

Direct cytotoxicity of purified humanized antibodies or fragments thereof is analyzed by propidium iodide incorporation (PI). In IP assays, 1 x 10 ⁵ IMR5 cells are incubated 24h at 37°C in a 48-well plate. 40 μg/ml of antibodies or fragments thereof are added and incubated 16h at 37°C. After incubation, PI at l (^g/ml in PBS is added and the fluorescence is immediately analyzed by flow cytometry. Percentage of death cells were represented as mean ± SD in triplicate. ADCC activity was determined as follow. Tumor cells were labeled with membrane dye PKH-26 (Sigma Aldrich) according to the manufacturer's instructions. Labeled cells (10 ⁴ cells in 100 μΐ.) were incubated with 50 μL· of antibodies in 96-well microtiter plates. The human cells line NK-92-RFcgIII+, or total PBMC, were used as effector cells. Effector cells (50 μΙ_,) at the indicated effector-to-target ratio were added to the tumor cells and incubated for 24 hours at 37°C. Cell death within the PKH-26+ target cell population was then assessed by the addition of TOPRO-3 iodide (TP3) (Life Technologies). The double-positive TP3+, PKH26+ dead target cell population was detected by flow cytometry. The percentage of specific lysis was calculated as: 100 x (non viable double-positive target cells)/(non viable double-positive target cells + viable PKH26+ target cells).

5) Immunogenicity determination

To study the immunogenicity potential of the antibodies, we used the PROPRESENT® Antigen Presentation Assays (PROIMMUNE) or EPISCREEN (ANTITOPE) or EPIBASE T CELL assays (LONZA) or IMMUNO'LINE (PLATINE) as well. Briefly, a panel of HLA-typed, healthy donor peripheral blood mononuclear cell (PBMC) samples are prepared from tissue bank (selected to reflect HLA distribution of choice). Then, monocytes from donor PBMC are cultured in defined media and differentiated to produce dendritic cells (DC). For analysis for T cell activation, we used the DC loaded with the test antigen (i.e. antibodies to be tested) in presence to CD4+ T cells. Finally, T cell activation is measured by T cell proliferation assays (i.e. CFSE, H ) and cytokine secretion (i.e. IL-2, IL-6, IL-8, INFy). Significant T cell responses are determined by parametric and non- parametric statistical analysis. Results are benchmarked to internal control. To identifying sequence of interest, we used harvested DC, and purified corresponding HLA molecules. Associated peptides are eluted. The peptide samples are analyzed by sequencing mass spectrometry and the obtained data are then compared against a protein database library consisting of the sequence of interest and the international protein index (IPI) of the organism of choice. The peptides are ranked by significance according to a probability based algorithm and the data are verified by searching against a scrambled decoy database to reduce false positives.

Finally, the obtained data enable to determine the potential immunogenicity in human of the tested antibodies.

6) Cytokine release Assay

Total PBMC are incubated with humanized antibodies and fragments thereof (scFv) or with the control mouse antibody (8B6) and the cytokines secretion (IFN gamma, TNF alpha, IL-6, IL-2, IL-10, IL-12, IL-13, IL-17,, IL-1 beta, ...) is analyzed by the Bio-Plex Precision Pro™ human cytokine immunoassay (BIORAD) according to the manufacturer's instructions. For more details, see FI CO et al. (Cytokine, vol.66, p:143-145, 2014).

Finally, the cytokine expression profiles enable to determine the potential immunogenicity in human of the tested antibodies. Surprisingly, the immunogenicity results show that, whereas VH72Bmax, VH49B, VH49Bmax, VL30BH, and VL28Bs01/A2 have a binding affinity for OAcGD2 and GD2 gangliosides comparable to their mouse counterpart, they have a weaken immunogenicity, VH72max and VH49Bmax having the weakest one. Now and still surprisingly, the immunogenicity results also show that, whereas VHl, VH2, VH3, VH72BCDR, VH72BH, VH49BCDR, VH49BH, VLl, VL2, VL3, VL4, and VL28BH have a binding affinity as good as their mouse counterpart, they have also a smallest immunogenicity.