Melanoma and its treatment methods targeting human antibodies

Abstract

The present invention relates in particular to antibodies or antigen-binding fragments thereof that target melanoma cancer, as well as pharmaceutical compositions containing such antibodies and their use.

Description

Technical Field

[0001] The present invention relates to an antibody or an antigen-binding fragment thereof that targets melanoma cancer, as well as a pharmaceutical composition containing said antibody and methods of using them.

Background Art

[0002] Malignant melanoma is the most lethal form of skin cancer and arises from malignant melanocytes in the basal layer of the epidermis. Cutaneous melanoma accounts for 4% of all skin cancer diagnoses, yet accounts for 75% of skin cancer deaths. The incidence of melanoma has been increasing at an accelerating rate among Caucasians over the past few decades. Every year, 132,000 cases of melanoma are diagnosed, and the advanced forms of the disease are highly resistant to treatment.

[0003] Therefore, there is an urgent need for new therapies and early diagnosis.

[0004] To treat melanoma, several antibody immunotherapies that act via different mechanisms against different targets have been developed. Using monoclonal antibodies that recognize melanoma-associated antigens such as CSPG4 / MCSP and target factors of the tumor-associated vasculature (VEGF, bevacizumab) exemplifies a translational approach aimed at reducing melanoma growth and metastasis. However, clinical trials using bevacizumab have shown only 3% complete response and 14% partial response, and bevacizumab treatment induced early hypertension in responsive patients.

[0005] Recent insights into the mechanisms of immune regulation and the interaction between tumors and immune cells have helped identify checkpoint molecules on immune (CTLA4, PD-1) and tumor (PD-L1) cells as promising therapeutic targets to re-engage the immune system and enhance existing anti-tumor immune responses.

[0006] This effect is applicable to both T and B cells that promote TCR-directed tumor recognition (TCR-directed tumor recognition), humoral, and ADCC, via antibodies secreted from the patient's immune repertoire. Antibodies such as pembrolizumab, nivolumab (PD-1 inhibitor), or atezolizumab (anti-PD-L1) have improved treatment outcomes for various cancers, including metastatic melanoma. Patients with metastatic melanoma treated with nivolumab showed a 40% response rate, a 43% survival rate in the first two years, and a mean survival time of 16.8 months. Nevertheless, 60% of patients do not respond to this treatment. Furthermore, there are numerous serious and unpredictable side effects of immune checkpoint inhibitor treatment (diabetes, hepatitis, colitis, vitiligo), which hinder broad use in the absence of predictive markers. Another immunotherapy strategy used in cancer, particularly melanoma, is vaccination with autologous dendritic cells (DCs) that elicit therapeutic effects in vivo through immune system stimulation.

[0007] Cancer vaccines aim to induce and / or enhance the adaptive immune response against tumors. Both T and B cell clones are activated and proliferate in vivo in response to vaccination, and specific effector T cells can reduce tumor mass and induce tumor-specific memory T cells to control tumor progression and recurrence.

[0008] Immune checkpoint inhibitor (ICI) therapy can elicit a robust immunological response to tumor and B-cell infiltration in tertiary lymphoid tissue in melanoma patients, which correlates with a better prognosis.

[0009] While immunotherapy itself has already significantly improved outcomes for melanoma patients, a considerable proportion of patients still do not respond to these treatments and would benefit from certain adoptive cell therapies or other immunotherapies.

[0010] Effective immunotherapy for melanoma remains in need, particularly immunotherapies that are low-toxicity and compatible with the human immune response and physiological functions. [Overview of the project]

[0011] NGS-based B cell receptor (BCR) repertoire analysis revealed that novel / enriched B cell clones appeared in the peripheral blood of responders after nivolumab treatment, specifically in the memory B cell component.

[0012] A melanoma-targeting antibody was discovered in melanoma patients who were in a complete response state after nivolumab treatment, resulting in the development of a novel antibody.

[0013] These antibodies can be advantageously used in immunotherapy-based strategies targeting melanoma, such as nude antibodies for ADCs, CAR-carrying cells, and ADCCs, and bispecific T-cell engagers (BiTEs), which have the advantage of being human-derived and thus conforming to the human immune response, contributing to ICI-induced clinical responses in melanoma patients.

[0014] The antibodies of the present invention were derived from the analysis of the B cell receptor repertoire and subsequent relative statistical analysis of NGS sequencing data of memory B cell components present in the peripheral blood of subjects responding to or partially responding to nivolumab treatment.

[0015] This invention is: a. Heavy chain variable domains (VH) including the following: i. SEQ ID NO: CDR1 sequence of an amino acid sequence selected from the group consisting of 8, 1, and 4; ii. CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 2, and 5; and iii. CDR3 sequences of amino acid sequences selected from the group consisting of SEQ ID NO: 10, 3, 6, and 7; and / or b. Light chain variable domains (VLs) including the following: i. CDR1 sequence of amino acid sequence SEQ ID NO: 11; ii. SEQ ID NO: CDR2 sequence of amino acid sequence 12; and iii. CDR3 sequence of amino acid sequence SEQ ID NO: 13 The present invention provides an isolated antibody or its antigen-binding fragment containing [the specified substance].

[0016] Preferably, the isolated antibody or its antigen-binding fragment is used as a CDR: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their respective Kabat, IMGT, Chothia, AbM, or Contact CDR or SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their respective Kabat, IMGT, Chothia, AbM, or Contact CDR or SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their respective Kabat, IMGT, Chothia, AbM, or Contact CDR or SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their respective Kabat, IMGT, Chothia, AbM, or Contact CDR. Includes.

[0017] In a preferred embodiment, the antibody or its antigen-binding fragment includes a heavy chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.1, CDR2 containing the amino acid sequence of SEQ ID N.2, and CDR3 containing the amino acid sequence of SEQ ID N.3, and a light chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.11, CDR2 containing the amino acid sequence of SEQ ID N.12, and CDR3 containing the amino acid sequence of SEQ ID N.13. Each of the CDRs may also vary by one or two amino acids, as long as antibody activity is maintained.

[0018] In a preferred embodiment, the antibody or its antigen-binding fragment includes a heavy chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.4, CDR2 containing the amino acid sequence of SEQ ID N.5, and CDR3 containing the amino acid sequence of SEQ ID N.6, and a light chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.11, CDR2 containing the amino acid sequence of SEQ ID N.12, and CDR3 containing the amino acid sequence of SEQ ID N.13. Each of the CDRs may also have one or two amino acid mutations, as long as antibody activity is maintained.

[0019] In a preferred embodiment, the antibody or its antigen-binding fragment includes a heavy chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.1, CDR2 containing the amino acid sequence of SEQ ID N.2, and CDR3 containing the amino acid sequence of SEQ ID N.7, and a light chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.11, CDR2 containing the amino acid sequence of SEQ ID N.12, and CDR3 containing the amino acid sequence of SEQ ID N.13. Each of the CDRs may also have one or two amino acid mutations, as long as antibody activity is maintained.

[0020] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity determining region CDR1 comprising the amino acid sequence of SEQ ID N.8, a CDR2 comprising the amino acid sequence of SEQ ID N.9, and a CDR3 comprising the amino acid sequence of SEQ ID N.10, and a light chain variable region comprising a complementarity determining region CDR1 comprising the amino acid sequence of SEQ ID N.11, a CDR2 comprising the amino acid sequence of SEQ ID N.12, and a CDR3 comprising the amino acid sequence of SEQ ID N.13. Each said CDR may also have a mutation of 1 or 2 amino acids as long as the antibody activity is retained.

[0021] Preferably, the isolated antibody or antigen-binding fragment thereof comprises a CDR shown in any of Tables 1 to 12, more preferably, a CDR shown in Tables 1 and 2.

[0022] Preferably, the antibody or antigen-binding fragment thereof specifically binds to an antigen present on melanoma cancer cells.

[0023] Preferably, the isolated antibody or antigen-binding fragment thereof a. a heavy chain variable domain sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 127, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126; b. a light chain variable domain sequence of the amino acid sequence of SEQ ID NO: 128; or c. the heavy chain variable domain sequence of (a) and the light chain variable domain sequence of (b) comprises.

[0024] Preferably, the isolated antibody comprises a heavy chain comprising or consisting of the sequence of SEQ ID NO: 124 and a light chain comprising or consisting of the sequence of SEQ ID NO: 128; E; a heavy chain comprising or consisting of the sequence of SEQ ID NO: 125 and a light chain comprising or consisting of the sequence of SEQ ID NO: 128; G; a heavy chain comprising or consisting of the sequence of SEQ ID NO: 126 and a light chain comprising or consisting of the sequence of SEQ ID NO: 128; H; or a heavy chain comprising or consisting of the sequence of SEQ ID NO: 127 and a light chain comprising or consisting of the sequence of SEQ ID NO: 128; I.

[0025] Preferably, the isolated antibody comprises a heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, and a light chain encoded by the nucleotide sequence of SEQ ID NO: 133.

[0026] Preferably, the isolated antibody is E and comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 129 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 133; or is G and comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 130 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 133; or is H and comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 131 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 133; or is I and comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 132 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 133.

[0027] Preferably, the isolated antibody is herein named I and comprises the sequences shown as antibody I in Tables 1 to 14.

[0028] Preferably, the isolated antibody or its antigen-binding fragment of the present invention is a human antibody.

[0029] More preferably, the isolated antibody or its antigen-binding fragment is an IgG1, IgG2, IgG3, or IgG4 antibody, preferably an IgG1 kappa antibody, an IgG1 lambda antibody, an IgG4 kappa antibody, or an IgG4 lambda antibody, and preferably the IgG1, IgG2, IgG3, or IgG4 is human IgG1, human IgG2, human IgG3, or human IgG4.

[0030] The present invention provides isolated polynucleotides comprising at least one antibody sequence encoding an antibody or an antigen-binding fragment thereof as defined above, preferably the polynucleotide being cDNA. Preferably, such polynucleotides comprise sequences SEQ ID N.129 to SEQ ID NO: 133, particularly those disclosed in Tables 15 to 16.

[0031] In some embodiments, the polynucleotide is: - The sequence of SEQ ID NO. 129 encoding the heavy chain and the sequence of SEQ ID NO. 133 encoding the light chain; or - The sequence of SEQ ID NO. 130 encoding the heavy chain and the sequence of SEQ ID NO. 133 encoding the light chain; or - The sequence of SEQ ID NO. 131 encoding the heavy chain and the sequence of SEQ ID NO. 133 encoding the light chain; or - The sequence of SEQ ID NO. 132 encoding the heavy chain and the sequence of SEQ ID NO. 133 encoding the light chain. Includes.

[0032] The present invention relates to a vector comprising the polynucleotide defined above, preferably the vector being selected from the group consisting of plasmids, viral busters, non-episomal mammalian vectors, expression vectors, and recombinant expression vectors.

[0033] The present invention further provides isolated cells comprising the polynucleotides defined above or the vectors defined above, preferably the isolated cells being hybridomas, Chinese hamster ovary cells (CHO), or human fetal kidney cells (HEK293).

[0034] The present invention further provides antibodies or antigen-binding fragments thereof or isolated polynucleotides or vectors or isolated cells, as defined above, for use as pharmaceuticals, preferably for the treatment of cancer, preferably for the treatment of melanoma.

[0035] The present invention also provides a pharmaceutical composition comprising, preferably for use in the treatment of melanoma, an isolated antibody or its antigen-binding fragment or isolated polynucleotide or vector or isolated cell as defined above, and at least one pharmaceutically acceptable carrier.

[0036] The present invention provides a method for treating melanoma, comprising administering to a subject who requires a pharmaceutical composition comprising the isolated antibody or its antigen-binding fragment or isolated polynucleotide or vector or isolated cells and a pharmaceutically acceptable carrier, or administering to a subject who requires the isolated antibody or its antigen-binding fragment or isolated polynucleotide or vector or isolated cells, as defined above.

[0037] The present invention also provides a method for producing an antibody or an antigen-binding fragment thereof, which includes obtaining the cells defined above and producing an antibody or an antigen-binding fragment thereof.

[0038] Methods for producing antibody molecules, expression vectors, nucleic acid molecules encoding host cells, and antibody molecules are also provided. Immunoconjugates, multiple or bispecific antibody molecules, and pharmaceutical compositions comprising antibody molecules are also provided.

[0039] The antibodies disclosed herein can be used alone or in combination with other drugs or therapeutic agents to treat, prevent and / or diagnose melanoma.

[0040] In addition, disclosed herein are methods and compositions comprising a combination of the antibody of the present invention with a drug and / or immunotherapy for treating melanoma.

[0041] Additional therapies may be selected from among those used to treat melanoma, including ICIs, chemotherapy drugs, mutant B-RAF targeting agents, anti-angiogenic drugs, and adoptive cell therapy.

[0042] Furthermore, an object of the present invention is a chimeric antigen receptor (CAR) comprising an antigen-binding fragment of the antibody of the present invention, preferably linked to an intracellular domain of a T cell that includes one or more signaling domains. Preferably, the present invention relates to a chimeric antigen receptor (CAR) comprising a CD3ξ chain, an intracellular domain including a signaling region of a T cell receptor, and an scFV of the antibody of the present invention bound to two costimulatory domains, CD28 and 4-1BB. When expressed on T cells or NK cells, for example, the CAR according to the present invention is a suitable means for targeting malignant cells that are recognized and bound by the antibody of the present invention.

[0043] A further object of the present invention is a bispecific T cell engager (BiTE) comprising at least an antigen-binding fragment of the antibody of the present invention. The BiTE can be manufactured by general knowledge of the art.

[0044] A further object of the present invention is an antibody-drug conjugate or ADC comprising the antibody of the present invention or its antigen-binding fragment conjugated with a cytotoxic agent, thereby advantageously combining the ability of the antibody of the present invention to target melanoma cells with the cancer-killing ability of the cytotoxic agent.

[0045] A further object of the present invention is the antibody of the present invention for use in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cell-mediated cytotoxicity (CDC) in patients with cancer, preferably melanoma. In particular, in patients with cancer, cancer cells are recognized and bound by the antibody of the present invention.

[0046] A further object of the present invention is an antibody or antigen-binding fragment thereof for use in adoptive cell therapy, particularly in tumor-infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR) therapy, chimeric antigen receptor (CAR) T cell therapy, or natural killer (NK) cell therapy. [Brief explanation of the drawing]

[0047] [Figure 1] Workflow for an experimental approach to antibody identification from peripheral blood of melanoma patients. [Figure 2] A Venn diagram that helps identify clone types that are common between two replicas of a POST sample and are absent (or very rare) in the replica of the PRE sample. [Figure 3] Screening of binding properties of nine candidate antibodies against melanoma cell lines and PDXs. The bar represents the doubling of MFI compared to the value obtained with the isotype control antibody. [Figure 4] Binding results of selected candidate antibodies on living cells. Data are expressed as the percentage of cells positive for the isotype antibody used as a control. [Figure 5] Binding results of selected candidate antibodies on living cells. Data are expressed as the multiplication rate of MFI compared to isotype control. [Modes for carrying out the invention]

[0048] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including” or “includes”; or “containing” or “contains,” and are comprehensive or open-ended, not excluding additional, unmentioned elements, components, or stages. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0049] In the present invention, “at least 80% identity” means that the identity may be at least 80%, or 85%, 90%, 95%, or 100% sequence identity with respect to the reference sequence. This applies to all percentages of identity mentioned. In the present invention, “at least 95% identity” means that the identity may be at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the reference sequence. This applies to all percentages of identity mentioned. “at least 98% identity” means that the identity may be at least 98%, 99%, or 100% sequence identity with respect to the reference sequence. This applies to all percentages of identity mentioned. Preferably, the percentage of identity relates to the entire length of the reference sequence.

[0050] As used herein, the term “antibody” is used in the most broadly understood sense in the art, encompassing all polypeptides described as antibodies. For example, as used herein, the term “antibody” includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as the fragment exhibits the desired antigen-binding activity (antigen-binding fragment). This term has the broadest meaning in the art and includes all known meanings, including, but is not limited to, divalent monospecific monoclonal antibodies, divalent bispecific antibodies, trivalent trispecific antibodies, F(ab) fragments, F(ab)'2 fragments, scFv fragments, diabodies, single-domain antibodies, camelid VHH single-domain antibodies, tandabs, and flexibodies.

[0051] The terms used herein, such as “antigen-binding fragment” of an antibody, or equivalent “antigen-binding moiety” of an antibody, include naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptides or glycoproteins that comprise a portion of an antibody and specifically bind to an antigen to form a complex. Antigen-binding fragments of antibodies may originate from full-length antibody molecules using any appropriate standard technique, such as protease digestion or genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercially available sources, DNA libraries (e.g., including phage-antibody libraries), or can be synthesized. The DNA may be manipulated by using chemical or molecular biological techniques, such as sequencing, arranging one or more variable and / or constant domains in a suitable configuration, or introducing codons, creating cysteine ​​residues, or modifying, adding, or deleting amino acids. According to the present invention, the antigen-binding fragment of an antibody is, in particular, a fragment that preserves the binding activity of a full-length antibody.

[0052] Similar to full-length antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two distinct variable domains, each capable of specifically binding to a different antigen or a different epitope of the same antigen.

[0053] In certain embodiments, the antigen-binding fragment of an antibody comprises at least one variable domain covalently bound to at least one constant domain. A non-limiting, typical configuration of variable and constant domains that may be found in an antigen-binding fragment of an antibody is: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and steady domains, including any of the typical configurations described above, the variable and steady domains may be directly coupled to each other or coupled by a complete or partial hinge or linker region. In various embodiments, the hinge region consists of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in flexible or semi-flexible linkages between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, in various embodiments, the antigen-binding fragment of the antibody may contain homodimers or heterodimers (or other polymers) of either of the above variable and constant domain configurations (e.g., by disulfide bonds) non-covalently bonded to each other and / or in one or more monomeric VH or VL domains.

[0054] The term "antigen-binding fragment" in relation to antibodies further includes single-domain antibodies.

[0055] A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. In some embodiments, the single-domain antibody is derived from a variable domain (also called a nanobody or VHH fragment) of an antibody heavy chain from a camelid. In some embodiments, the single-domain antibody is an autonomous human heavy chain variable domain (aVH) or VNAR fragment derived from a shark.

[0056] Non-specific examples of antigen-binding fragments are: (i) Fab fragment; (ii) F(ab)'2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv(scFv) molecule; (vi) dAb fragment; and (vii) minimal recognition unit or constrained FR3-CDR3-FR4 peptide consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as the CDR3 peptide). This includes domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR graft antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent and divalent nanobodies), small modular immunotherapy products (SMIPs), and other modified molecules such as shark variable IgNAR domains, which are also included in the expression “antigen-binding fragment” as used herein.

[0057] The antigen-binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or within a framework sequence. In an antigen-binding fragment having a VH domain bound to a VL domain, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and may include VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0058] The antibody or binding molecule of the present invention can be further bound to an active substance, preferably nanoparticles or radioactive nucleotides.

[0059] As used herein, the term “antigen-binding molecule” refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules include antibodies, including antigen-binding antibody fragments, and scaffold antigen-binding proteins.

[0060] The term "antigen-binding moiety" refers to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. Antigen-binding molecules include antibodies and their antigen-binding fragments, such as scFv, which can specifically bind to antigens on target cells. In certain embodiments, antigen-binding proteins can guide the target to which they are bound, such as cells, to a target site.

[0061] In addition, antigen-binding moieties capable of specifically binding to target cell antigens include, as defined below, engineered repeat proteins or binding domains based on engineered repeat domains, such as engineered ankyrin repeat proteins (DARPin) (see, e.g., WO 2002 / 020565) or lipocalin (anticalin). Engineered ankyrin repeat proteins (DARPin) are derived from ankyrin, a family of proteins that mediate the attachment of endogenous membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and beta-turns. They can be manipulated to bind to different target antigens by randomizing the residues in the first alpha-helices and beta-turns of each repeat.

[0062] These bonding interfaces can be increased by increasing the number of modules (affinity maturation method). For details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028.

[0063] In some embodiments, the antibodies and antigen-binding molecules provided herein are modified to increase or decrease the degree to which the antigen-binding portion is glycosylated. Glycosylated variants of the molecules may be conveniently obtained by altering the amino acid sequence such that one or more glycosylation sites are created or removed. At sites containing an Fc region of the antigen-binding molecule, the carbohydrate attached thereto may be altered. In one embodiment, a variant of the antigen-binding molecule is provided having a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. Such fucosylated variants may have improved ADCC function, see, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.) or US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Further variants of the antigen-binding molecule of the present invention include those having bisected oligosaccharides, for example, those in which a bisected oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, for example, WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may improve CDC function and are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0064] In certain embodiments, it may be desirable to produce cysteine-modified variants of the antibody or antigen-binding molecule of the present invention, for example, “thioMAbs” in which one or more residues of the molecule are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur in the accessible sites of the molecule. By substituting these residues with cysteine, the reactive thiol group is positioned in the accessible site of the antibody and may be used to conjugate the antibody to other parts, such as the drug moiety or linker-drug moiety, in order to produce an immune complex. In certain embodiments, one or more of the following residues may be substituted with cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the heavy chain Fc region. Cysteine-modified antigen-binding molecules may be produced, for example, as described in US Patent No. 7,521,541.

[0065] In certain embodiments, the antibody or antigen-binding molecules provided herein may be further modified to include additional non-proteinoid moieties that are known and readily available in the art. Suitable moieties for derivatization of antibody or antigen-binding molecules include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxolane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may offer advantages in production due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if multiple polymers are attached, they may be the same or different molecules. In general, the number and / or types of polymers used in derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used in treatment under defined conditions.

[0066] In another embodiment, a complex of an antibody and a non-proteinaceous moiety is provided, which may be selectively heated by radiation exposure. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-proteinaceous moiety to a temperature in which cells proximal to the antibody-non-proteinaceous moiety are killed.

[0067] In another embodiment, an immune complex of an antibody or antigen-binding molecule provided herein may be obtained. The “immune complex” is an antibody conjugated with one or more heterologous molecules and includes, but is not limited to, a cytotoxic agent. Preferably, the immune complex is an antibody-drug conjugate (ADC) and includes an antibody conjugated with a cytotoxic agent or its antigen-binding fragment. The cytotoxic agent is preferably an agent effective against melanoma cancer, or its active portion, for example, an agent approved for the treatment of melanoma, such as vemurafenib, aldesleukin, or dacarbazine.

[0068] The constant region of an antibody is crucial for its ability to immobilize complement and mediate cell-dependent cytotoxicity. Therefore, the antibody isotype may be selected based on whether it is desirable for the antibody to mediate cytotoxicity. In one embodiment, the constant regions are IgG1, IgG2, IgG3, and IgG4.

[0069] The present invention includes antibodies having one or more mutations in the hinge, CH2, or CH3 region that may be desirable, for example, to improve the yield of a desired antibody morphology in production, in various embodiments. In some embodiments, for example, the antibodies described herein include a human IgG4 constant region. In certain embodiments, the IgG4 constant region has a single amino acid substitution in the hinge region of the human IgG4 hinge that reduces the Fab arm exchange to a level typically observed with the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105).

[0070] In one embodiment, the antibody comprises one or more mutations that increase the serum half-life in a constant region, and includes methods described in US Patent Nos. 7,083,784, 8,323,962 and Dall'Aqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Hinton et al., J. Immunology 176:346-356 (2006); Yeung et al., J. Immunology 182:7663-7671 (2009); and Petkova et al., I. Immunology, 18: 1759-1769 (2006), the entirety of which is incorporated herein by reference.

[0071] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies characterized by the present invention still include, in various embodiments, for example, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo), for example, in CDRs and in some embodiments in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies derived from germline cells of other mammalian species, such as mice, in which the CDR sequence has been grafted onto a human framework sequence.

[0072] As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, including, for example, antibodies transfected and expressed in host cells using a recombinant expression vector (described below), antibodies isolated from a recombinant combined human antibody library (described below), antibodies isolated from animals (e.g., mice) transgenic with human immunoglobulin genes (Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295, the entire collection of which is incorporated herein by reference), or antibodies prepared, expressed, produced, or isolated by other means, including splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if animal transgenicity for human Ig sequences is used, in vivo somatic mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to the VH and VL sequences of the human germline, and may not naturally exist in the human antibody germline repertoire in vivo.

[0073] As used herein, the term “isolated antibody” means an antibody identified, isolated, and / or removed from at least one component of its natural environment. For example, an antibody isolated or removed from at least one component of an organism, or from a tissue or cell in which antibodies naturally exist or are naturally produced, is an “isolated antibody.” In various embodiments, isolated antibodies also include in situ antibodies in recombinant cells. In other embodiments, isolated antibodies are antibodies subjected to at least one purification or isolation step. In various embodiments, isolated antibodies may substantially contain no other intracellular material and / or chemical substances.

[0074] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, also known as a paratope. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes may be conformational or linear. Conformational epitopes are generated from spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated from adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl groups on the antigen.

[0075] The present invention comprises, in various embodiments, antibodies and methods relating to the use of antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more frameworks and / or CDR regions are mutated to a residue corresponding to the germline sequence from which the antibody was induced, or to a residue corresponding to another human germline sequence, or to a conserved amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as “germline mutations”).

[0076] Multiple antibodies and antigen-binding fragments may be constructed containing one or more individual germline mutations or combinations thereof. In one embodiment, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to residues found in the original germline from which the antibody was induced. In another embodiment, only specific residues, for example, only mutant residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutant residues found within CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In yet another embodiment, one or more of the framework and / or CDR residues are mutated to a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally induced). Furthermore, the antibody may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, certain individual residues are mutated to corresponding residues in a particular germline, while other specific residues different from the original germline are maintained or mutated to corresponding residues in a different germline sequence. After acquisition, antibodies or antigen-binding fragments containing one or more germline mutations can be easily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, (in some cases) improved or enhanced antagonistic or agonic biological properties, or decreased immunogenicity. The use of antibodies and antigen-binding fragments obtained by conventional methods is included in the present invention.

[0077] The present invention includes antibodies comprising any variant of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, and having one or more conserved substitutions. For example, the present invention includes antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, and 4 or fewer conserved amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0078] As used herein, the term "bioequivalent" refers to a molecule that, after administration at the same molar dose and under similar conditions (e.g., the same route of administration), exhibits similar bioavailability (rate and degree of availability) to a comparator molecule, such that its effects can be expected to be essentially the same as those of a comparator molecule in terms of both efficacy and safety. Two pharmaceuticals containing antibodies are bioequivalent if they are pharmaceutically identical, that is, they contain the same amount of active ingredient, are in the same dosage form, are administered via the same route of administration, and meet the same or equivalent criteria. Bioequivalence can be determined, for example, by in vivo studies comparing the pharmacokinetic parameters of two compositions. Parameters commonly used in bioequivalence studies include the maximum plasma concentration (Cmax) and the area under the drug plasma concentration-time curve (AUC).

[0079] In one embodiment, the present invention relates to an antibody comprising a heavy chain variable region containing a sequence selected from the group consisting of SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127, and a light chain variable region containing the sequence of SEQ ID NO: 128.

[0080] In one embodiment, one or more CDRs (or all CDRs in general) have one, two, three, four, five, six or more changes, such as amino acid substitutions or deletions, compared to the amino acid sequences shown in Tables 1 to 12, particularly Tables 1 and 2.

[0081] In one embodiment, the antibody includes substitutions by light chain CDRs, for example, one or more substitutions in CDR1, CDR2 and / or CDR3 of the light chain.

[0082] In one embodiment, the antibody includes heavy chain CDR substitutions, for example, one or more substitutions in the heavy chain CDR1, CDR2 and / or CDR3.

[0083] Preferably, the term "CDR" is a CDR as defined by Kabat, where CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0084] In another embodiment, the antibody molecule comprises at least one, two, or three CDRs by Kabat et al. (e.g., at least one, two, or three CDRs by Kabat definition as shown in Tables 3 to 6) derived from the heavy chain variable region of the antibodies described herein; or a sequence substantially identical to any of the aforementioned sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more identical); or at least one amino acid modification compared to one, two, or three CDRs by Kabat et al. shown in Tables 1, 3 to 6, but not exceeding two, three, or four modifications (e.g., substitutions, deletions, insertions, e.g., conservative substitutions).

[0085] In another embodiment, the antibody molecule comprises at least one, two, or three CDRs by Kabat et al. derived from the light chain variable region of the antibodies described herein (e.g., at least one, two, or three CDRs by Kabat definition as shown in Table 11); or at least one amino acid modification, but not exceeding two, three, or four, compared to one, two, or three CDRs by Kabat et al. shown in Table 2, 11 (e.g., substitutions, deletions, insertions, e.g., conservative substitutions).

[0086] In yet another embodiment, the antibody molecule comprises all six CDRs by Kabat et al. regarding heavy and light chain variable regions as described herein (e.g., all six CDRs by Kabat definition as described in Tables 1 to 6, 11); or, compared to all six CDRs by Kabat, at least one amino acid modification, but not exceeding two, three, or four (e.g., substitution, deletion, insertion, e.g., conservative substitution).

[0087] In another embodiment, the antibody comprises CDRs of at least one, two, or three heavy chain variable regions as defined by Chothia, AbM, Contact, or IMGT, as disclosed in Tables 7 to 10; and at least one amino acid modification, but not exceeding two, three, or four (e.g., substitutions, deletions, insertions, e.g., conservative substitutions).

[0088] In another embodiment, the antibody comprises CDRs of at least one, two, or three light chain variable regions as defined by Chothia, AbM, Contact, or IMGT, as disclosed in Table 12; and at least one amino acid modification, but not exceeding two, three, or four modifications (e.g., substitutions, deletions, insertions, e.g., conservative substitutions).

[0089] In one embodiment, the antibody molecule comprises a combination of Kabat, Chothia, AbM, Contact, or CDR as defined by the IMGT definition.

[0090] Preferred antibodies are antibodies E, G, H, and I as defined in Tables 1 to 14, or encoded by the nucleotide sequences in Tables 15 and 16.

[0091] In one embodiment, the antibody is (i) A heavy chain variable region (VH) comprising: a VHCDR1 amino acid sequence selected from SEQ ID NO: 1, 4, and 8; a VHCDR2 amino acid sequence selected from SEQ ID NO: 2, 5, and 9; and a VHCDR3 amino acid sequence selected from SEQ ID NO: 3, 6, 7, and 10; and (ii) The light chain variable region (VL) containing the VLCDR1 amino acid sequence of SEQ ID NO: 11; the VLCDR2 amino acid sequence of SEQ ID NO: 12; and the VLCDR3 amino acid sequence of SEQ ID NO: 13. Includes.

[0092] In one embodiment, the light chain or heavy chain variable framework of the antibody (e.g., a region including at least FR1, FR2, FR3, and optionally FR4) (a) A light chain or heavy chain variable framework comprising at least 80%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% of amino acid residues derived from a human light chain or heavy chain variable framework, for example, from a human mature antibody, a human germline sequence, or a human consensus sequence; (b) A light chain or heavy chain variable framework comprising at least 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of amino acid residues derived from a human light chain or heavy chain variable framework, for example, from a human mature antibody, a human germline sequence, or a human consensus sequence; (c) Non-human frameworks (e.g., rodent frameworks); or (d) A modified non-human framework, for example, by removing antigenic or cytotoxic determinants, such as by immunodepletion or partial humanization; You can choose from the following. In one embodiment, the light chain or heavy chain variable framework region (in particular FR1, FR2, and / or FR3) includes at least 70, 75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, 99% identical or identical light chain or heavy chain variable framework sequence to the framework of the VL or VH segment of the human germline gene. Preferably, the light chain or heavy chain variable framework is as shown in Table 3-6 for the heavy chain and in Table 11 for the light chain.

[0093] In one embodiment, the antibody molecule includes a heavy chain variable domain having at least 1, 2, 3, 4, 5, 6, 7, 10, 15, 20 or more variations, for example, amino acid substitutions or deletions relating to the sequence of SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO: 127.

[0094] In one embodiment, the antibody molecule includes a light chain variable domain having at least 1, 2, 3, 4, 5, 6, 7, 10, 15, 20 or more variations, for example, amino acid substitutions or deletions relating to the sequence of SEQ ID N.128. In a particular embodiment, the heavy chain or light chain variable region of the antibody molecule, or both, includes a nucleic acid sequence described herein or a nucleic acid that hybridizes to a nucleic acid sequence described herein, in particular an amino acid sequence encoded by a nucleic acid sequence or its complement, such as those shown in Tables 15 and 16, for example, under low, medium, or high strictness, or under other hybridization conditions described herein.

[0095] In another embodiment, the antibody molecule includes a variable region having at least one, two, three, or four antigen-binding regions, for example, amino acid sequences as shown in Tables 1 to 12, or substantially identical sequences (e.g., sequences that are at least about 85%, 90%, 95%, 99% or more identical, or sequences that differ from the sequences listed in Tables 1 to 12 by only 1, 2, 5, 10, or 15 amino acid residues or less).

[0096] In another embodiment, the antibody molecule comprises VH and / or VL domains encoded by nucleic acids having nucleotide sequences as shown in Tables 15 and 16, or substantially identical sequences, for example, sequences identical by at least about 85%, 90%, 95%, 99%, or more, or sequences differing by only 3, 6, 15, 30, or 45 nucleotides from the sequences shown in Tables 15 and 16.

[0097] In yet another embodiment, the antibody molecule has a heavy chain constant region (Fc) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, preferably selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, more preferably selected from the heavy chain constant region of IgG1 or IgG4 (e.g., human IgG1, IgG2, or IgG4). In one embodiment, the heavy chain constant region is human IgG1. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, the light chain constant regions of kappa or lambda. In one embodiment, the constant region is modified and mutated, for example, to modify the properties of the antibody molecule (e.g., to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, complement function, half-life, aggregation, and stability).

[0098] In one embodiment, the antibody is either isolated or recombinant.

[0099] In one embodiment, the antibody is a humanized or human antibody molecule.

[0100] The antibody of the present invention may have a framework sequence derived from any species. Preferably, it may have a mouse or human framework. As used herein, the term “framework (FR) amino acid residues” refers to those amino acids in the framework region of an immunoglobulin chain. As used herein, the term “framework region” or “FR region” includes amino acid residues that are part of the variable region but are not part of the CDR (for example, using the Kabat definition of CDR).

[0101] Monoclonal antibodies containing any of the CDR sequences of the antibodies used herein are also within the scope of the present invention.

[0102] Methods for producing monoclonal antibodies having the CDR sequence described above are known in the art and include introducing a nucleic acid sequence encoding the CDR into a suitable expression vector encoding a desired framework sequence.

[0103] The antibodies according to the present invention may form homodimers or heterodimers or homopolymers, where “dimer” and “polymer” mean that two and at least three antibodies may bind to form a complex, respectively. The prefix “homo” means that the complex may be formed from the same antibody molecule, and the prefix “hetero” means that the complex may be formed from different antibody molecules. Generally, the term “antibody” is intended to include all of the above immunoglobulin isotypes; that is, the antibody may be an IgA, IgD, IgE, IgG, or IgM antibody containing any of the subclasses of these isotypes. Preferably, the antibody is an IgG antibody, and more preferably an IgG1 antibody. The antibody may be expressed and produced by recombination, and the antibody may include two different heavy chains, for example, one IgG1 and one IgG2 heavy chain, or constant regions of heavy chains from different species, however, the heavy chains are preferably derived from the same species.

[0104] To generate the entire antibody, PCR primers containing a VH or VL nucleotide sequence, a restriction site, and an adjacent sequence to protect the restriction site can be used to amplify the VH or VL sequence in an scFv clone or other clone. Using cloning techniques well known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, e.g., a human gamma-4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, e.g., a human kappa or lambda constant region. In one embodiment, the vector for expressing the VH or VL domain includes a promoter, a secretion signal, a cloning site for a variable region, a constant region, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a single vector expressing the required constant region. The heavy chain and light chain conversion vectors are then co-transfected into a cell line to generate a stable or transient cell line expressing a full-length antibody, e.g., IgG, using techniques well known to those skilled in the art.

[0105] The present invention also features nucleic acid molecules comprising nucleotide sequences encoding one or both of the heavy chain and light chain variable regions, CDRs, hypervariable loops, and framework regions of antibody molecules as described herein.

[0106] In one embodiment, the nucleotide sequence encoding the antibody molecule is an optimized codon.

[0107] For example, the present invention features first and second nucleic acids that encode the heavy chain and light chain variable regions of an antibody molecule selected from one or more of antibodies E, G, H, and I, respectively, which are encoded by, for example, the nucleotide sequences defined in Tables 1 to 14 or Tables 15 and 16, or substantially identical sequences. For example, the nucleic acid comprises a nucleotide sequence as described in Tables 15 and 16, or a substantially identical sequence (for example, at least about 85%, 90%, 95%, 99%, or more identical, or a sequence that differs from the sequences shown in Tables 15 and 16 by 3, 6, 15, 30, or 45 or fewer nucleotides).

[0108] In other embodiments, the nucleic acid molecule includes a nucleotide sequence encoding a heavy chain variable domain and / or a heavy chain constant region, which includes an amino acid sequence of any of antibodies E, G, H, and I, as defined in Table 13 or encoded by the nucleotide sequences in Table 15, or a sequence substantially identical to any of the aforementioned sequences (e.g., a sequence identical by at least about 85%, 90%, 95%, 99%, or more).

[0109] In other embodiments, the nucleic acid molecule includes a nucleotide sequence encoding a light chain variable domain and / or a light chain constant region, which includes an amino acid sequence of any of antibodies E, G, H, and I, as defined in Table 14 or encoded by the nucleotide sequences in Table 16, or a sequence substantially identical to any of the aforementioned sequences (e.g., a sequence identical by at least about 85%, 90%, 95%, 99%, or more).

[0110] The aforementioned nucleotide sequences encoding the variable domains and constant regions of the heavy and light chains may be present in a separate nucleic acid molecule or in the same nucleic acid molecule. In one embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a leader sequence.

[0111] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding at least one, two, or three CDRs derived from a heavy chain variable region having an amino acid sequence as described in Tables 1 to 10, or a sequence substantially homologous thereto (e.g., at least about 85%, 90%, 95%, 99%, or more identical thereto, and / or having one, two, three, or more substitutions, insertions, or deletions, e.g., sequences having conserved substitutions).

[0112] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding at least one, two, or three CDRs derived from a light chain variable region having an amino acid sequence as described in Tables 11 to 12, or a sequence substantially homologous thereto (e.g., at least about 85%, 90%, 95%, 99%, or more identical thereto, and / or having one, two, three, or more substitutions, insertions, or deletions, e.g., sequences having conserved substitutions).

[0113] In yet another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding at least one, two, three, four, five, or six CDRs or hypervariable loops derived from heavy chain and light chain variable regions having the amino acid sequences as described in Tables 1 to 12 or substantially homologous thereto (e.g., at least about 85%, 90%, 95%, 99%, or more identical thereto, and / or having one, two, three, or more substitutions, insertions, or deletions, e.g., sequences having conserved substitutions).

[0114] In another embodiment, the nucleic acid molecule includes one or more heavy chain framework regions (e.g., any of VHFW1 (type a), VHFW1 (type b), VHFW1 (type c), VHFW1 (type d), VHFW2 (type a), VHFW2 (type a'), VHFW2 (type b), VHFW2 (type c), VHFW2 (type d), VHFW2 (type e), VHFW3 (type a), VHFW3 (type b), VHFW3 (type c), VHFW3 (type d), VHFW3 (type e), or VHFW4, or any combination thereof, for example, a combination of frameworks described herein for any of antibodies E, G, H, and I as defined in Tables 3 to 6, or a sequence substantially identical thereto).

[0115] In another embodiment, the nucleic acid molecule includes one or more light chain framework regions of antibodies E, G, H, and I as defined in Table 11 (e.g., any of VLFW1 (type a), VLFW1 (type b), VLFW1 (type c), VLFW1 (type d), VLFW1 (type e), VLFW1 (type f), VLFW2 (type a), VLFW2 (type c), VLFW3 (type a), VLFW3 (type b), VLFW3 (type c), VLFW3 (type d), VLFW3 (type e), VLFW3 (type f), VLFW3 (type g), or VHFW4, or any combination thereof, e.g., the framework combinations described herein), or a substantially identical sequence. In another embodiment, the nucleic acid molecule includes one or more heavy chain framework regions and one or more light chain framework regions as described herein. The heavy chain and light chain framework regions may be located in the same vector or in different vectors.

[0116] In other embodiments, the application features host cells and vectors comprising nucleic acids described herein or modified for codon optimization by known methods. The nucleic acids may be present in a single vector or in a separate vector, in the same host cell or in a different host cell. The host cells may be eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells, or prokaryotic cells, e.g., Escherichia coli. Mammalian cells, for example, may be cultured cells or cell lines. Typical mammalian cells include lymphocyte cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, oocytes, and cells from transgenic animals, e.g., mammary epithelial cells.

[0117] Generally, an expression vector is a plasmid used to introduce a desired nucleic acid sequence, such as a gene, into a target cell, resulting in the transcription and translation of a protein encoded by the nucleic acid sequence, i.e., a chimeric antigen receptor, antibody, or binding molecule. Therefore, expression vectors generally contain not only regulatory sequences such as promoter and enhancer regions, but also polyadenylation sites to direct the efficient transcription of the nucleic acid sequence on the expression vector. An expression vector may further include additional necessary or useful regions, such as selectable markers for selection in eukaryotic or prokaryotic cells, purification tags for purifying the resulting protein, multiple cloning sites, or origins of replication.

[0118] Typically, the expression vector may be a virus or a non-viral vector. Generally, various types of viral vectors may be used, such as retroviral vectors, e.g., lentivirus or adenovirus vectors, or plasmids. In a preferred embodiment, the expression vector in aspect 5 is a viral vector. In a more preferred embodiment, the expression vector is a lentiviral vector.

[0119] In another embodiment, the present invention provides a pharmaceutical composition comprising, for example, a pharmaceutically acceptable carrier, excipient or stabilizer and at least one of the antibodies described herein. In one embodiment, the composition, for example, the pharmaceutical composition comprises a combination of an antibody molecule and one or more agents described herein, for example, a therapeutic agent or a combination with other antibody molecules. In one embodiment, the antibody molecule is labeled or conjugated with a therapeutic agent.

[0120] This disclosure provides pharmaceutical compositions containing these antibodies, and methods for using these compositions.

[0121] Antibodies are administered to subjects in various embodiments of formulations containing appropriate carriers, excipients, or other agents that improve transfer, delivery, resistance, etc., and are suitable for intravenous or subcutaneous injection.

[0122] The injection preparation may be prepared by publicly known methods. For example, the injection preparation may be prepared by dissolving, suspending, or emulsifying the antibody or salt thereof described above in a sterile aqueous medium or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, which may be used in combination with appropriate solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 20 or 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection preparation thus prepared can be filled into a suitable ampoule.

[0123] The content of the antibody or its antigen-binding fragment in the pharmaceutical composition is not limited as long as it is useful for treatment or prevention, but preferably contains 0.0000001-10% by mass per total composition. Furthermore, the antibodies or their antigen-binding fragments described herein are preferably used within a carrier. The selection of the individual depends on the route of administration and concentration of the active agent, and the carrier may be in the form of a lyophilized composition or an aqueous solution. Generally, an appropriate amount of pharmaceutically acceptable salt is used as the carrier to make the composition isotonic. Examples of carriers include, but are not limited to, physiological saline, Ringer's solution, and dextrose solution. Preferably, the accepted excipients, carriers, or stabilizers are nontoxic at the dose and concentration used and include buffers such as citric acid, phosphoric acid, and other organic acids; counterions that form salts, e.g., sodium and potassium; low molecular weight (>10 amino acid residues) polypeptides; proteins, e.g., serum albumin or gelatin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrin; monosaccharides; disaccharides; other sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; chelating agents, e.g., EDTA; nonionic surfactants, e.g., Tween, Pluronics, or polyethylene glycol; antioxidants including methionine, ascorbic acid, and tocopherol; and / or preservatives, e.g., octadecyldimethylbenzylammonium chloride (chloride); The composition includes hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. Suitable carriers and their formulations are described in more detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co. The composition may also contain at least one further active compound, such as a chemotherapeutic agent.

[0124] Preferably, the antibody or its antigen-binding fragment is included in an effective amount. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is administered.

[0125] The antibodies according to the present invention can be administered to a subject using any acceptable device and mechanism. For example, administration can be achieved using a syringe and needle, or using a reusable pen and / or auto-injector delivery device. According to the present invention, “subject” means a human subject or human patient.

[0126] In embodiments, the antibody is intended for use in the treatment of cancer. The cancer can be selected from skin cancer, preferably melanoma, colon cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, brain tumor, gastric cancer, and liver cancer. In particular, it is a cancer in which cancer cells are bound to the antibody of the present invention. Preferably, it is a cancer in which cancer cells express antigens that are also present in melanoma cancer cells.

[0127] In a preferred embodiment, the antibody is intended for use in the treatment of melanoma.

[0128] Accordingly, in another embodiment, a method for treating melanoma in a subject is provided. The method comprises administering an antibody molecule disclosed herein (e.g., a therapeutically effective amount of antibody molecule) to a subject, either alone or in combination with one or more agents or procedures, thereby treating the melanoma in the subject.

[0129] In one embodiment, the antibody molecule inhibits, reduces, neutralizes, or blocks melanoma in a subject. The subject may be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having one of the disorders disclosed herein). In one embodiment, the subject requires inhibition, reduction, neutralization, or blockage of melanoma. In one embodiment, the subject has or is at risk of having melanoma cancer.

[0130] A further object of the present invention is an in vitro method for screening subjects suffering from cancer, particularly melanoma, to predict the responsiveness of the subject to a treatment involving the administration of an antibody or derivative thereof disclosed herein, the method comprising at least the step of contacting and establishing at least one antibody disclosed herein with a biological sample isolated from the subject. Preferably, if binding is detected between the antibody and the sample, the subject is likely to respond to treatment with the antibody or derivative thereof. The isolated biological sample may be a fluid sample such as blood, plasma, or saliva, or a tissue sample such as a tumor tissue sample. The derivative of the antibody may be, for example, a chimeric antigen receptor (CAR), an antibody-drug conjugate, or a bispecific T cell engager (BiTE).

[0131] "Melanoma" or "melanomatous cancer" refers to skin cancer that arises from pigment-producing cells known as melanocytes. "Malignant melanoma" is used as a synonym herein. Melanoma can be of any type.

[0132] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to the death of cells that have been bound to and labeled with an antibody by cytotoxic effector cells such as natural killer (NK) cells.

[0133] As used herein, the term "complement-dependent cell damage (CDC)" refers to the induction of the classical complement pathway by the binding of antibodies to antigens on the surface of target cells, leading to the death of the target cells.

[0134] ADCC and CDC are well-known therapies involving the use of antibodies. The antibodies according to the present invention can be advantageously used in the treatment of ADCC and CDC in subjects with cancer. Such therapies can be carried out with general knowledge in the field.Individuals with: Lo Nigro C, Macagno M, Sangiolo D, Bertolaccini L, Aglietta M, Merlano MC. NK-mediated antibody-dependent cell-mediated cytotoxicity in solid tumors: biological evidence and clinical perspectives. Ann Transl Med. 2019 Mar;7(5):1 doi: 10.21037 / atm. PMID: 31019955; PMCID: PMC6462666; Wang W, Erbe AK, Hank JA, Morris ZS, Sondel PM. NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy. Front Immunol. 2015 Jul 27;6:368. doi: 10.3389 / fimmu.2015.00368. PMID: 26284063; PMCID: PMC4515552; Hernandez , A. , Parmentier , J. , Wang , Y. , Cheng , J. , Bornstein , GG (2012). Monoclonal Antibody Lead Characterization: In Vitro and In Vivo Methods. In: Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 907. Humana Press, Totowa, NJ; Taylor RP, Lindorfer MA The role of complement in mAb-based therapies of cancer. Methods. 2014 Jan 1 ;65(1):18-27. doi: 10.1016 / j.ymeth.2013.07.027. Epub 2013 Jul 22. PMID:23886909.

[0135] As used herein, the term “chimeric antigen receptor (CAR)” refers to a synthetic receptor containing a target moiety that binds to one or more single domains in a single fusion molecule. Generally, the binding moiety of a CAR contains scFv, but may also contain other conjugates. Binding moieties based on receptor or ligand domains have also been successfully utilized. The signaling domain of a CAR may be derived from the cytoplasmic region of the CD3ξ or Fc receptor gamma chain, but may also be derived from other cytoplasmic regions. First-generation CARs have been shown to successfully retarget T cell cytotoxicity. The addition of signaling domains derived from costimulatory molecules, as well as transmembrane and hinge domains, has formed second and third-generation CARs that can retarget T cells against malignant cells expressing CD19, resulting in several successful therapeutic trials in humans (Porter DL et al., N Eng J Med, 2011).

[0136] As used herein, the term “adoptive cell therapy” refers to treatments that use cells of the human immune system to eliminate cancer. In particular, cells of the immune system, such as T cells or natural killer cells, may be genetically engineered to enhance their ability to fight cancer. Examples of adoptive cell therapies include tumor-infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR) therapy, chimeric antigen receptor (CAR) T cell therapy, or natural killer (NK) cell therapy. The antibodies or antigen-binding fragments of the present invention can be advantageously used in these types of adoptive cell therapies, particularly by engineering cells of the immune system, such as T cells and NK cells, with the antibodies or antigen-binding fragments of the present invention to provide them with the ability to recognize melanoma cells.

[0137] The antibodies disclosed herein can be used alone or in combination in the methods of the present invention.

[0138] array The CDR sequences of the four antibodies of the present invention are reported in Table 1-2 below. [Table 1] [Table 2]

[0139] The definition of CDR is also provided using the annotation tool from http: / / www.abysis.org / , based on the full-length VH and VL amino acid sequences, as defined in Tables 13 and 14.

[0140] For example, the VH amino acid sequence of any of the antibodies disclosed herein is inserted into an annotation tool, providing a CDR sequence defined by Kabat.

[0141] Table 3-6 below reports the CDR and framework regions of the heavy chain variable region (VH) as defined by Kabat for each antibody.

[0142] In the table, HFR refers to the heavy chain framework region, and LFR refers to the light chain framework region.

[0143] Antibody E [Table 3]

[0144] Antibody G [Table 4]

[0145] Antibody H [Table 5]

[0146] Antibody I [Table 6]

[0147] Furthermore, the VH amino acid sequence of any antibody disclosed herein may be inserted into an annotation tool to provide a CDR sequence defined by Chothia, AbM, Kabat, Contact, and IMGT definitions.

[0148] Table 7 below shows the CDR sequences in Chothia, AbM, Kabat, Contact, and IMGT relative to VH of E. [Table 7-1] [Table 7-2]

[0149] Table 8 below shows the CDR sequences in Chothia, AbM, Kabat, Contact, and IMGT relative to VH of G. [Table 8-1] [Table 8-2]

[0150] Table 9 below shows the CDR sequences for H in Chothia, AbM, Kabat, Contact, and IMGT relative to VH. [Table 9-1] [Table 9-2]

[0151] Table 10 below shows the CDR sequences in Chothia, AbM, Kabat, Contact, and IMGT relative to VH of I. [Table 10-1] [Table 10-2]

[0152] Table 11 shows the CDR and framework regions of the light chain variable region (VL) as defined by Kabat, which are common to antibodies E, G, H, and I. [Table 11]

[0153] The definition of CDR, also provided using the annotation tool from http: / / www.abysis.org / based on the full-length VL amino acid sequences of antibodies E, G, H, and I, is also reported. Table 12 below shows the CDR sequences in Chothia, AbM, Kabat, Contact, and IMGT for the VL of E, G, H, and I. [Table 12-1] [Table 12-2]

[0154] Table 13 below reports the VH amino acid sequences of the example antibodies. [Table 13]

[0155] Table 14 below reports the VL amino acid sequences of the example antibodies. [Table 14]

[0156] Table 15 below reports the VH nucleotide sequences of the example antibodies. [Table 15] Table 16 below reports the VL nucleotide sequences of the example antibodies. [Table 16] [Examples]

[0157] method Sample collection and processing The experimental workflow is shown in Figure 1.

[0158] Peripheral blood mononuclear cells (PMBCs) collected from melanoma patients and stored in the IRST Biobank (CRB) are used with ethical approval from the local ethics committee (CEROM) (Prot.n.3534 / 51 / 2018, 10 / 05 / 2018). Clinical information, as well as the sample collection date and the number of PMBCs and purified mB cells collected in the CRB, are reported in Table 17.

[0159] [Table 17] Table 17 shows the patient cohort used in the study. PRE and POST represent blood samples taken before and after nivolumab treatment, respectively. PD, PR, and CR are clinical outcomes defining progressive disease, partial response, and complete response, respectively.

[0160] Samples were collected before (PRE) and after (POST) first-line nivolumab treatment in melanoma patients (Figure 1). Diagnosis and clinical reassessment are shown in Table 1. Specifically, we used PBMCs collected during PRE and POST from partial responders (PR, Patient IDs n. 7 and 9), complete responders (CR, Patient IDs n. 3 and 8), and non-responding melanoma patients (PD, Patient IDs n. 1 and 4). Memory B cells (mB cells) were isolated using Miltenyi's memory B cell isolation kit (130-093-546). mB cells were resuspended in RLT buffer to ensure RNA stability, and RNA samples were sent to MiLaboratories (https: / / milaboratories.com) for B cell receptor repertoire analysis via previously reported NGS (Turchaninova, et al. High-quality full-length immunoglobulin profiling with unique molecular barcoding. Nat Protoc 11, 1599-1616 (2016)).

[0161] NGS data analysis performed by MiLaboratories was integrated with our in-house analysis using VDJviz software (https: / / vdjviz.cdr3.net / ) to search for de novo / enriched mB cell clones occurring only in patients who have received a partial or complete clinical response after nivolumab treatment.

[0162] NGS sequencing was performed in two runs: the first run aimed to identify VH CDR3 sequences derived only from mB cells expressing any isotype (IgD, IgE, IgA, IgG), and the second run aimed to identify the entire VH sequence derived only from IgG with higher sensitivity. For the first analysis, bioinformatics and statistical analyses were performed considering clones enriched / newly formed after nivolumab treatment (see Table 18).

[0163] To design a variable heavy chain (VH) with a specific CDR3, we employed specific FR1, CDR1, FR2, CDR2, FR3, and FR4 sequences as reported in Table 19 (clones H and I).

[0164] [Table 18] Table 18 shows significant changes observed from the initial heavy chain CDR3 sequencing run. Two CDR3 sequences, namely the patient 7-derived sequence (CDR3 sequence: CARDASSGSYAGRAHFDYW (SEQ ID NO.134)) and the patient 8-derived sequence (CDR3 sequence: CARHRRAGAHFFDYW (SEQ ID NO.135)), both derived from IgM isotype mB clones, were newly formed after nivolumab treatment.

[0165] A second analysis of IgG-derived sequences based on low / high clonal frequency and enrichment / derivation criteria after ICI treatment identified seven additional candidates (AG; Table 19). [Table 19] Table 19: Details of the nine candidates identified by sequencing.

[0166] Tests of antibody production and binding To produce full-length human IgG in the human IgG1 / k format, the producing cell lines were co-transfected with HEK293 or CHO-producing cells using random (non-binder) kappa variable light chains (VLs) (the same for all candidates, sequences reported in the right column of Table 4) and the VH chains of all candidate antibodies (see Table 19).

[0167] The antibodies released into the supernatant were purified with AmMag™ Protein A Magnetic Beads and resuspended in PBS at pH 7.2. A total of nine antibodies were produced, and their binding affinity to the following melanoma cell lines, M14 (UCLA-SO-M14) and SK-MEL-28 (HTB-72), or patient-derived xenograft (PDX) cells (RiMi, LuDi, SaBr) kindly provided by Professor Lanfrancone Luisa of the European Institute of Oncology (IEO, Milan) was tested. The M14 cell line was grown in DMEM low glucose:HAM-F12 1:1 + 10% fetal bovine serum (FBS, Euroclone, L-glutamine, insulin, and penicillin / streptomycin (p / s) antibiotic at a final concentration of 10 mM). SK-MEL-28 cell lines were maintained in EMEM (Gibco) and 10% FBS and p / s. PDX cell lines were grown in IMDM glutamax (Thermofisher) and 10% FBS and p / s. To evaluate the specificity of candidate antibodies against tumor cells, the antibodies were also tested for their ability to bind to melanocytes (Normal Human Epidermal Melanocytes 2 (NHEM 2), Promocell). In the initial series of experiments, target cells were fixed with 1% formaldehyde (FA) in PBS for 10 minutes. After blocking with 1% BSA / PBS solution for 30 minutes, candidate antibodies were incubated with target cells at a final concentration of 25 ng / μL in 1% BSA / PBS solution, and with human IgG1 / k isotype antibody used as a control at the same final concentration. The secondary antibody used was a goat anti-human IgGFc fragment conjugated with Alexa Fluor 488 (109-546-008, Jackson Immunoresearch). Selected candidate antibodies (E, G, H, and I, see Table 4) were also prepared in mouse / human chimeric IgG2a / k format and retested in live (unfixed) cells to confirm their binding properties.In this case, a mouse isotype antibody (IgG2a / k) was used as a control, and goat anti-mouse (H+L) (Invitrogen) conjugated with Alexa Fluor 488 was used as a secondary detection antibody. Binding measurements were evaluated using an AttuneNxt flow cytometer autosampler (Thermofisher) capable of processing multiple 96-well plates.

[0168] Immunohistochemical analysis of melanoma and normal tissue specimens The selected antibodies H and I were also tested for binding to normal tissue in the FDA Standard Tissue Array (T823470, AMSBIO), as well as to a melanoma tissue microarray (ME481c, AMSBIO) containing 40 cases of malignant melanoma and 8 cases of normal skin tissue.

[0169] Candidate antibodies H and I were tested at different concentrations and protocols, optimizing IHC staining using Roche Ventana and Benchmark Ultra. Antibody I was used at a concentration of 20 ng / μL using the CC2 protocol (ket fast RED), which involves citrate buffer (pH=6), casein for 8 minutes, and antigen unmasking for 24 minutes.

[0170] Instead, the "H" antibody was used at a concentration of 10 ng / μL using the CC1 protocol (ket fast RED), which involves EDTA (pH=8), casein for 8 minutes, and antigen unmasking for 36 minutes.

[0171] The day after the Ventana instrument session, the slides were removed and placed in a special tray that had been thoroughly cleaned with running water and mild detergent. This step is important to completely remove any residual liquid coverslip oil (LCS(oily)) that would otherwise bias the analysis of the bonds on the slides.

[0172] Next, the slides were washed with desalted water for 1 minute and dried on a 60°C stove for 5 minutes before incubation at room temperature for 2 hours. After drying, the slides were immersed in xylene for a few seconds and then covered with a cover slip and mounted with Histo Mount before drying overnight at room temperature.

[0173] result Example 1 Identification of candidate antibodies From the initial sequencing run analysis, we identified two VH-CDR3 sequences in patient numbers 7 and 8 (partial and full responders, respectively) that were newly formed after nivolumab treatment, significantly bound, and both were originally produced as IgM isotypes in mB cells (see Table 18). After reconstruction using the FR1-CDR1-FR2-CDR2-FR3 and FR4 sequences shown in Tables 5 and 6, these candidate antibodies were named "H" and "I".

[0174] From a second IgG-specific sequencing run, following in-house analysis based on the criteria described in Materials and Methods, we selected seven IgG-VH sequences from particularly abundant newly formed mB cell clones after nivolumab treatment, which we named AF (Table 20 and Figure 2).

[0175] [Table 20-1] [Table 20-2] [Table 20-3] [Table 20-4] Table 20 shows the clonal types selected from the second IgG-specific sequencing run. The frequency and number of each sample are reported. Clonal types with high frequency / number and absence / low frequency / number in POST samples are selected from responder patients 3 and 8.

[0176] Example 2 Binding and specificity of candidate antibodies The binding and specificity of candidate antibodies were tested using several melanoma cell lines and control human melanocytes.

[0177] Initial screening was performed on 1% formalin-fixed melanoma cell lines and cells derived from melanoma patients (PDX). Specifically, a total of 10 antibodies, 9 candidate antibodies (AIs), and isotype controls were tested for binding to melanoma cell lines M14, SK-Mel-28, and two PDXs, namely RiMi and SaBr. Complement antibodies E, G, H, and I were selected for further analysis due to cross-reactivity and binding to multiple melanoma cell lines and PDXs (Figure 3).

[0178] To confirm these results, we retested the selected candidate antibodies E, G, H, and I in unfixed live cells. Another PDX cell line (LuDi) and healthy corresponding control melanocytes were also included in this second test experiment. As shown in Figures 4 and 5, antibodies E, H, and I confirmed previous evidence of binding in terms of positive rate and mean fluorescence intensity (MFI), while antibody G did not. The same clones also showed binding to the reference tissue, melanocytes.

[0179] To test the binding of candidate antibodies in a larger cohort of melanoma cases and to evaluate potential off-targeting, we performed immunohistochemical analysis based on FFPE tissue microarrays (TMA) of the aforementioned malignant melanoma and normal tissues. Binding was assessed by experienced pathologists who attributed positive score results to each case series. For melanoma binding, antibody I signaling was consistently and almost without exception detected in 37 out of 40 cases (92%), with weak signals in 82% and intermediate signals in 10%, in the cytoplasm of malignant cells. Antibody H signaling was detected in 38 out of 40 cases (95%), with weak signals in 10%, intermediate signals in 50%, and strong signals in 20% of the cases tested. Neither antibody bound to the negative control sample (pheochromocytoma) on the slides, while weak (I) to intermediate (H) signals were observed against normal skin tissue of the control.

[0180] Evaluation of antibody I binding in normal tissues showed no signal in bone marrow, esophagus, ovaries, peripheral nerves, placenta, skin, salivary glands, stomach, and testicular tissue; a very weak signal in colon, kidney, small intestine, and prostate tissue; and an intermediate signal in brain, skeletal muscle, salivary glands, and cardiac tissue. Evaluation of antibody H binding in normal tissues is ongoing.

[0181] conclusion While immunotherapy itself has already improved outcomes for many melanoma patients, a significant proportion of patients remain unresponsive to these therapies and would likely benefit from certain adoptive cell therapies or other immunotherapies. In this study, we utilized the BCR repertoire of memory B cells recovered from the peripheral blood of melanoma patients treated with ICI as a screening approach to identify candidate human antibodies for tumor targeting. Specifically, we identified four novel subsets of melanoma-targeting antibodies generated after nivolumab treatment in patients with a fully progressing response. These antibodies have potential applications in melanoma-targeted strategy-based immunotherapies (i.e., nude antibodies for ADCs, CAR-carrying cells, ADCCs, etc.). The added value of these biomolecules lies in their in vivo production, their form already compatible with human immune responses, and their high potential to contribute to ICI-induced clinical responses in melanoma patients.

Claims

1. a. Heavy chain variable domains (VH) including the following: i. SEQ ID NO: CDR1 sequence of an amino acid sequence selected from the group consisting of 8, 1, and 4; ii. CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 2, and 5; and iii. CDR3 sequences of amino acid sequences selected from the group consisting of SEQ ID NO: 10, 3, 6, and 7; and / or b. Light chain variable domains (VLs) including the following: i. CDR1 sequence of amino acid sequence SEQ ID NO: 11; ii. SEQ ID NO: CDR2 sequence of amino acid sequence 12; and iii. CDR3 sequence of amino acid sequence SEQ ID NO: 13 An isolated antibody or its antigen-binding fragment containing [the specified substance].

2. SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their respective Kabat, IMGT, Chothia, AbM, or Contact CDR or SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their respective Kabat, IMGT, Chothia, AbM, or Contact CDR or SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their respective Kabat, IMGT, Chothia, AbM, or Contact CDR or SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their respective Kabat, IMGT, Chothia, AbM, or Contact CDR. The antibody or antigen-binding fragment thereof according to claim 1, comprising as a CDR.

3. An antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a complementarity-determining region CDR1 comprising the amino acid sequence of SEQ ID N.8, CDR2 comprising the amino acid sequence of SEQ ID N.9, and CDR3 comprising the amino acid sequence of SEQ ID N.10, and a light chain variable region comprising a complementarity-determining region CDR1 comprising the amino acid sequence of SEQ ID N.11, CDR2 comprising the amino acid sequence of SEQ ID N.12, and CDR3 comprising the amino acid sequence of SEQ ID N.13; or The antibody or its antigen-binding fragment comprises a heavy chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.1, CDR2 containing the amino acid sequence of SEQ ID N.2, and CDR3 containing the amino acid sequence of SEQ ID N.3, and a light chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.11, CDR2 containing the amino acid sequence of SEQ ID N.12, and CDR3 containing the amino acid sequence of SEQ ID N.13; or The antibody or its antigen-binding fragment includes a heavy chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.4, CDR2 containing the amino acid sequence of SEQ ID N.5, and CDR3 containing the amino acid sequence of SEQ ID N.6, and a light chain variable region comprising a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.11, CDR2 containing the amino acid sequence of SEQ ID N.12, and CDR3 containing the amino acid sequence of SEQ ID N.13; The antibody or antigen-binding fragment comprises a heavy chain variable region including a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.1, CDR2 containing the amino acid sequence of SEQ ID N.2, and CDR3 containing the amino acid sequence of SEQ ID N.7, and a light chain variable region including a complementarity-determining region CDR1 containing the amino acid sequence of SEQ ID N.11, CDR2 containing the amino acid sequence of SEQ ID N.12, and CDR3 containing the amino acid sequence of SEQ ID N.13, Each of the CDRs may also undergo one or two amino acid mutations, as long as antibody activity is maintained, and is the antibody or its antigen-binding fragment.

4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a CDR shown in any one of Tables 1 to 12.

5. a. Heavy chain variable domain sequences of amino acid sequences selected from the group consisting of SEQ ID NO: 127, SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126; b. SEQ ID NO: 128: Light chain variable domain sequence of the amino acid sequence; or c. Heavy chain variable domain sequence of (a) and light chain variable domain sequence of (b) An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising:

6. The isolated antibodies include: I, containing a heavy chain containing or consisting of the sequence of SEQ ID NO: 127 and a light chain containing or consisting of the sequence of SEQ ID NO: 128; E, containing a heavy chain containing or consisting of the sequence of SEQ ID NO: 124 and a light chain containing or consisting of the sequence of SEQ ID NO: 128; G, containing a heavy chain containing or consisting of the sequence of SEQ ID NO: 125 and a light chain containing or consisting of the sequence of SEQ ID NO: 128; or H, containing a heavy chain containing or consisting of the sequence of SEQ ID NO: 126 and a light chain containing or consisting of the sequence of SEQ ID NO:

128. The antibody or antigen-binding fragment thereof according to claim 5.

7. An antibody or antigen-binding fragment according to any one of claims 1 to 6, comprising a heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 132, SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 131, and a light chain encoded by the nucleotide sequence of SEQ ID N.

133.

8. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the isolated antibody or antigen-binding fragment thereof is an IgG1, IgG2, IgG3, or IgG4 antibody, preferably an IgG1 kappa antibody, an IgG1 lambda antibody, an IgG4 kappa antibody, or an IgG4 lambda antibody, and preferably the IgG1, IgG2, IgG3, or IgG4 is human IgG1, human IgG2, human IgG3, or human IgG4.

9. An isolated polynucleotide comprising at least one antibody sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the polynucleotide is preferably cDNA.

10. A vector comprising a polynucleotide as described in claim 9, wherein the vector is preferably selected from the group consisting of plasmids, viral busters, non-episomal mammalian vectors, expression vectors, and recombinant expression vectors.

11. Isolated cells comprising the polynucleotide described in claim 9 or the vector described in claim 10, preferably hybridomas, Chinese hamster ovary cells (CHO), or human fetal kidney cells (HEK293).

12. A chimeric antigen receptor (CAR) comprising an antigen-binding fragment of an antibody according to any one of claims 1 to 8, ligated to the intracellular domain of a T cell, wherein the intracellular domain preferably comprises one or more signaling domains.

13. An antibody-drug conjugate (ADC) comprising an antibody according to any one of claims 1 to 8 or an antigen-binding fragment thereof, conjugated with a cytotoxic agent.

14. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a polynucleotide according to claim 9, or a vector according to claim 10, or a cell according to claim 11, or a CAR according to claim 12, or an ADC according to claim 13, for use as a pharmaceutical.

15. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a polynucleotide according to claim 9, or a vector according to claim 10, or a cell according to claim 11, or a CAR according to claim 12, or an ADC according to claim 13, for use in the treatment of melanoma.

16. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a polynucleotide according to claim 9, or a vector according to claim 10, or a cell according to claim 11, or a CAR according to claim 12, or an ADC according to claim 13, and at least one pharmaceutically acceptable carrier, preferably for use in the treatment of melanoma.

17. An antibody according to any one of claims 1 to 8, for use in a subject with cancer to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC), wherein the cancer is preferably melanoma, or for use in adoptive cell therapy for the treatment of melanoma.

18. An in vitro method for screening subjects suffering from cancer, preferably melanoma, in order to predict the responsiveness of the subject to a treatment comprising the administration of an antibody or a derivative thereof, as disclosed in any one of claims 1 to 8, The method comprising at least the step of contacting and installing an antibody according to any one of claims 1 to 8 with a biological sample isolated from the subject.

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