Monoclonal antibody that targets the unique sialoglycosylated cancer-related epitope of CD43

Abstract

The present invention relates to a monoclonal mouse antibody that targets a unique sialoglycosylated cancer-associated epitope of CD43, produced by hybridoma cells deposited under ICLC Accession Number ICLC PD no. 16001. The present invention further relates to an antibody comprising a heavy chain variable region comprising complementarity-determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences GFTFSSFGMH, YISSGSGNFYYVDTVKG, STYYHGSRGAMDY, SASSSVSSMYWY, DTSKMAS, and QQWSSYPPIT, respectively. Additionally, the present invention relates to antibodies that recognize the same epitope.

Description

[Technical field]

[0001] The present invention relates to the monoclonal mouse antibody produced by the hybridoma cells deposited under ICLC Accession Number ICLC PD n° 16001, related antibodies and binding molecules and uses thereof. [Background technology]

[0002] CD43 is a transmembrane protein and a specific leukocyte marker restricted to cells of the hematopoietic lineage. However, among these cells, CD43 is widely expressed in most peripheral and bone marrow-derived cell components. The precursor form of CD43 migrates with an apparent molecular weight of 54 kD. In the mature form, CD43 is heavily glycosylated and has a molecular weight of 115-200 kD. Overall, CD4 + Thymocytes and monocytes express the 115 kD form and are associated with activated CD4 + and CD8 + - T cells, B cells, neutrophils and platelets express the 130 kD form. CD43 is involved in multiple functions including cell adhesion, apoptosis and migration (Ostberg JR et al. Immunology today. 1998; 19:546-50).

[0003] Glycoproteins such as glycosylated CD43 play a major role in cell signaling, immune recognition and cell-cell interactions due to the glycan branches that confer structural variability and binding specificity to lectin ligands (Ohtsubo K. et al. Cell 2006; 126, 855-867). Mucin-type glycoproteins are characterized by a high content of O-linked carbohydrate chains (O-glycans) and are expressed on the membranes of secretory or hematopoietic and epithelial cells. O-glycan biosynthesis begins in the Golgi apparatus with the addition of N-acetylgalactosamine (GalNAc) to serine or threonine residues by polypeptide-N-acetylgalactosaminyltransferase (GalNAc transferase) generating UDP-N-acetyl-D-galactosamine:Tn antigen structure of O-glycan. Subsequent elongation of the O-linked glycan branches by the addition of other carbohydrates such as galactose, fucose and sialic acid catalyzed by tissue-specific glycosyltransferases results in the synthesis of a complex array of O-glycan structures differing in the nature and length of the O-linked carbohydrate chain (Wopereis S. et al. Clin. Chem 2006; 52, 574-600). In addition, oligosaccharides can be modified by sialylation, fucosylation, sulfatation, methylation or acetylation. Shortening of O-glycan structures and aberrant expression of certain O-glycans occur in cancer cells, suggesting that aberrant glycosylation may contribute to cancer progression by modifying cell signaling, adhesion and antigenicity (Hakomori S. et al. Proc. Natl. Acad. Sci. USA 2002; 99, 10231-10233; Brockhausen I. EMBO Rep. 7 2006; 599-604).

[0004] Oncofetal antigens (OA) are primarily glycoproteins and products of one or more genes that are normally highly expressed only during fetal development and gradually repressed during differentiation, and therefore not expressed in adult tissues. Their re-expression in adults is the result of aberrant activation of regulated genes, as occurs in cancer. Oncofetal epitopes (OEs) are the parts of OA that are recognized by antibodies. Identification of OAs or specific oncofetal epitopes (OEs) whose expression is restricted to cancer tissues may not only be useful for detecting early carcinogenic processes, but most importantly, may be essential for developing new immunotherapeutic approaches against human cancers. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 4,816,567 [Patent Document 2] U.S. Patent No. 4,816,397 [Patent Document 3] WO 1988 / 001649 A1 [Patent Document 4] WO 1993 / 011161 A1 [Patent Document 5] WO 1999 / 057150 A2 [Patent Document 6] European Patent No. 1293514 B1 [Patent Document 7] US Patent Application Publication No. 2007031436 [Patent Document 8] WO 1990 / 012592 A1 [Patent Document 9] WO 2007 / 030642 A2 [Patent Document 10] WO 2004 / 067038 A1 [Patent Document 11] WO 2004 / 003183 A1 [Patent Document 12] US Patent Application Publication No. 2005 / 0074426 A1

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Non-licensed literature

[0006] [Non-licensed document 1] Ostberg JR et al. Immunology today. 1998;19:546~50 pages [Non-licensed document 2] Ohtsubo K. et al. Cell 2006; pages 126, 855~867 [Non-licensed document 3] Wopereis S. et al. Clin. Chem 2006; pages 52, 574~600

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[0007] The aim of the present invention is to provide antibodies that allow the detection of OE and the development of novel immunotherapeutic approaches.

[0008] The problem is solved by the antibody according to the invention. The data presented in the examples show that the epitopes recognized by the antibodies produced by the hybridoma cells deposited according to the invention, due to their specific pattern of restricted expression in fetal tissues and re-expression in malignant lesions, can be considered as OE. The latter therefore represent potentially suitable targets for innovative immunotherapeutic strategies to treat human cancers.

[0009] Furthermore, a chimeric antigen receptor (CAR) has been developed that comprises an intracellular domain containing the CD3 ζ chain, a signal transduction domain of the T cell receptor, and an scFv of an antibody-based binding molecule according to the invention linked to two costimulatory domains, CD28 and 41BB. + The lymphocytes induce significant cytotoxicity against cells expressing the epitope recognized by the antibody produced by the hybridoma cells deposited according to the present invention.

[0010] Even though the effects of the murine antibodies of the invention have already been partly described (Tassone et al., Tissue Antigens, 1994; De Laurentiis et al, Mol Cel Proteomics 2011; Cecco et al, Tissue Antigens, 1998; De Laurentiis et al, Int J Biol Macromol, 2006; Tassone et al, Int J Oncol, 2002; Tassone et al, Anticancer Res, 2002), the antibodies themselves or their sequences and the epitopes to which they bind have not been publicly available until now. [Means for solving the problem]

[0011] There is provided a mouse antibody produced by the hybridoma cells deposited under ICLC PD n° 16001. In addition, there is provided an antibody which recognizes the same epitope as the antibody produced by the hybridoma cells deposited under ICLC PD n° 16001.

[0012] The present invention further relates to an antibody comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the amino acid sequences GFTFSSFGMH, YISSGSGNFYYVDTVKG, STYYHGSRGAMDY, SASSSVSSMYWY, DTSKMAS and QQWSSYPPIT, respectively.

[0013] Preferably, the antibody is a monoclonal antibody.

[0014] Further provided is an antibody produced by the hybridoma cells deposited under ICLC PD no. 16001 or a binding molecule derived from the above-mentioned antibody of the invention.

[0015] Furthermore, a chimeric antigen receptor is provided, which comprises an intracellular domain comprising the CD3 zeta chain, a signalling domain of a T cell receptor and an scFv binding molecule according to the invention linked to two co-stimulatory domains CD28 and 41BB.

[0016] Further provided is an expression vector comprising a nucleic acid sequence encoding a chimeric antigen receptor according to the invention, an antibody according to the invention or a binding molecule according to the invention.

[0017] The present invention relates to a CD3 chimeric antigen receptor comprising the chimeric antigen receptor of the present invention. + Further provided is a lymphocyte, a NK lymphocyte, a cytokine-induced killer (CIK) cell, a γδ lymphocyte or a NKT cell, or an expression vector according to the invention.

[0018] The antibody according to the invention or the binding molecule according to the invention or the CD3 according to the invention + A pharmaceutical composition comprising lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, gamma delta lymphocytes or NKT cells is provided.

[0019] Nucleic acid encoding an antibody according to the invention or a binding molecule according to the invention is provided.

[0020] A hybridoma cell producing an antibody according to the invention is provided.

[0021] A method for producing an antibody according to the invention is provided, comprising isolating said antibody from hybridoma cells deposited under ICLC PD n°16001.

[0022] There is provided a method for identifying or isolating T-cell acute lymphoblastic leukemia cells, T lymphoma cells, Waldenström's macroglobulinemia cells or tumor associated macrophages, comprising the step of contacting a cell sample comprising said cells with an antibody according to the invention or a binding molecule according to the invention.

[0023] CD3 expressing the chimeric antigen receptor according to the present invention +A method for producing lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, gamma delta lymphocytes or NKT cells, comprising: + Methods are provided which comprise the introduction of an expression vector according to the invention into lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, γδ lymphocytes or NKT cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] In a first aspect, the present invention relates to a monoclonal mouse antibody produced by the hybridoma cells deposited under ICLC PD n°16001.

[0025] The hybridoma cells were deposited at the Centro Biotecnologie Avanzate (CBA), Interlay Cell Line Collection (ICLC), Largo Rosanna, 10, 16132, Genoa, Italy under the accession number ICLC PD n° 16001 on August 4, 2016. The antibodies were tested in the examples below. As shown in the examples, the antibodies bind to specific sialoglycosylated epitopes on CD43.

[0026] In this first aspect, the present invention further relates to an antibody comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the amino acid sequences GFTFSSFGMH, YISSGSGNFYYVDTVKG, STYYHGSRGAMDY, SASSSVSSMYWY, DTSKMAS and QQWSSYPPIT, respectively.

[0027] These sequences are also given in SEQ ID NOs: 1-6.

[0028] The above CDR sequences, as determined by sequencing, are those derived from the monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC PD no. 16001.

[0029] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), and Chothia et al., J. Mol. Biol. 196:901-917 (1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues encompassing the CDRs defined in each of the above cited references are set forth for comparison. Preferably, the term "CDR" refers to the CDRs defined by Kabat based on sequence comparisons. CDRH1, CDRH2 and CDRH3 refer to the heavy chain CDRs, and CDRL1, CDRL2 and CDRL3 refer to the light chain CDRs.

[0030] The monoclonal antibody may have framework sequences from any species, preferably it may have a murine or human framework.

[0031] As used herein, the term "framework (FR) amino acid residues" refers to 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 CDRs (e.g., using the Kabat definition of CDRs).

[0032] Methods for producing monoclonal antibodies with the above-mentioned CDR sequences are known in the art and include introducing nucleic acid sequences encoding the CDRs into an appropriate expression vector encoding the desired framework sequences. Further methods are described below.

[0033] In a second aspect, the present invention relates to an antibody which recognises the same epitope as the antibody according to the first aspect.

[0034] Generally, and as generally known in the art, an antibody is a protein that belongs to the immunoglobulin protein family and is composed of a variable region that consists of framework regions and complementarity determining regions as defined above. In nature, antibodies are produced by plasma cells in response to a specific antigen. Generally, each antibody has two identical heavy chain immunoglobulins and two identical light chain immunoglobulins. Each heavy chain and each light chain may have variable and constant regions. The constant region of the heavy chain may be one of the five types of mammalian Ig heavy chains: α, δ, ε, γ and μ. The type of heavy chain present usually defines the class (isotype) of the antibody: IgA, IgD, IgE, IgG and IgM antibodies, respectively. Similarly, the constant region of the light chain may be one of the two types of mammalian Ig light chains: κ and λ. The variable regions of the heavy and light chains are usually made of a unique combination of multiple protein sequences that allow binding to a specific antigen.

[0035] According to the present invention, the term "antibody" also encompasses isolated antibodies.

[0036] Generally, each heavy chain is connected to one of the light chains, whereby the variable regions of the heavy and light chains combine to form one of two identical antigen-binding sites, and their constant regions combine to form the constant region of the antibody. Furthermore, both constructs of one heavy chain and one light chain may be connected via the constant region of the heavy chain to form a "Y" shaped molecule, where the two arms represent the antigen-binding variable regions and the base represents the constant region.

[0037] The antibody according to the second aspect may be a complete antibody, which means that it usually comprises three or four constant domains of the heavy chain and one constant domain of the light chain and the respective variable domains, wherein each domain may contain further modifications such as mutations, deletions or insertions, which modifications do not change the overall domain structure.

[0038] Furthermore, the antibodies according to the second aspect of the invention may form homo- or heterodimers or homo- or heteromultimers, where "dimer" and "multimer" mean that two and at least three antibodies, respectively, may combine to form a complex. The prefix "homo" means that the complex may be in the form of the same antibody molecule, whereas the prefix "hetero" means that the complex may be in the form of different antibody molecules.

[0039] In general, the term "antibody" is intended to include all of the immunoglobulin isotypes mentioned above, i.e., the antibody may be an IgA, IgD, IgE, IgG or IgM antibody, including any subclass of isotype. Preferably, the antibody is an IgG antibody, more preferably, the antibody is an IgG1 antibody. Since the antibody may be recombinantly expressed and produced, the antibody may comprise constant regions of two different heavy chains, e.g. one IgG1 and one IgG2 heavy chain, or heavy chains from different species. However, the heavy chains are preferably from the same species. Furthermore, the antibody may comprise a lambda or kappa light chain.

[0040] An antibody recognizing the same epitope as one of the antibodies of the first aspect of the invention may further be an antibody comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 have the amino acid sequences GFTFSSFGMH, YISSGSGNFYYVDTVKG, STYYHGSRGAMDY, SASSSVSSMYWY, DTSKMAS and QQWSSYPPIT, respectively.

[0041] Furthermore, an antibody that recognizes the same epitope as one of the antibodies of the first aspect of the invention may be an antibody in which the CDRs have at least one conservative amino acid exchange compared to the above sequences, e.g. a similar amino acid that has a similar chemical structure and properties and / or function to the original amino acid.

[0042] An antibody which recognises the same epitope as one of the antibodies of the first aspect of the invention may be an antibody with increased or decreased affinity or specificity compared to one of the antibodies of the first aspect of the invention. Such antibodies are readily obtainable by methods known in the art and further described herein below.

[0043] Generally, the antibody according to the second aspect of the invention may have a sequence, particularly in its variable region, which is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g. at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the sequence of the monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC PD n° 16001.

[0044] Typically, the antibody according to the invention may be a monoclonal, bispecific or multispecific antibody. Such antibodies are known in the art. In the context of the present invention, the term "monoclonal" may be understood in the broadest sense to describe an antibody produced by a single clone of B lymphocytes or an antibody having the same or similar amino acid sequence. Herein, the term "bispecific" may be understood in the broadest sense to describe an antibody that interacts with two different epitopes. A bispecific antibody may be derived from two monoclonal antibodies. Optionally, these two different epitopes may be located on the same antigen, but may also be located on two different antigens. Herein, the term "multispecific" may be understood in the broadest sense to describe an antibody that interacts with three or more different types of epitopes. Optionally, these epitopes may be located on the same antigen or on two or more antigens.

[0045] Preferably, the antibody according to aspect 2 of the invention is a monoclonal antibody. Furthermore, the antibody according to aspect 2 of the invention is preferably a bispecific or multispecific antibody.

[0046] Methods for producing antibodies are well known to those skilled in the art. Preferably, the antibodies are produced by generating hybridoma cells. Methods for producing hybridoma cells and for producing antibodies using hybridoma cells are well known to those skilled in the art. Generally, mice are injected with a desired antigen, killed a few days later, and spleen cells secreting antibodies against the desired antigen are isolated. Generally, fusion of these antibody-secreting spleen cells with immortalized non-secreting myeloma cells results in hybridoma cells. These hybridoma cells are then usually screened to select hybridomas producing the desired antibodies. The selected hybridomas may then be cultured in vivo or in vitro, and the desired antibodies may be isolated.

[0047] Bifunctional or bispecific antibodies may have antigen-binding sites of different specificity. Various forms of bispecific antibodies and their production are known to those skilled in the art. For example, these include BSIgG, which is an IgG molecule containing two unique heavy chains and two unique light chains secreted by so-called "hybrid hybridomas", and heteroantibody conjugates produced by chemical conjugation of antibodies or antibody fragments of different specificities (Segal DM et al. Current Opin. Immunol. 1999, 11:558-562; Van Spriel AB et al. Immunology Today 2000, 21:391-397).

[0048] Bispecific antibodies may be generated to deliver cells, cytotoxins or drugs to specific sites. An important use may be to deliver host cytotoxic cells such as NK or cytotoxic T cells to specific cell targets (PJ Lachmann, Clin. Exp. Immunol. 1990, 79: 315). Another important use may be to deliver cytotoxic proteins to specific cell targets (V. Raso, T. Griffin, Cancer Res. 1981, 41: 2073; S. Honda et al., Cytotechnology, 1990, 4: 59). A further important use may be to deliver anticancer non-protein drugs to specific cell targets (J. Corvalan et al., Intl. J. Cancer Suppl. 1988, 2: 22; M. Pimm et al., British J. of Cancer 1990, 61: 508). Such bispecific antibodies may be prepared by chemical cross-linking (M. Brennan et al., 1985, Science 229:81), disulfide exchange or production of hybrid hybridomas (quadromas), which may be constructed by fusing hybridomas secreting two different types of antibodies against two different antigens (Milstein and Cuello, Nature, 1983, 305:537-539).

[0049] In the context of the present invention, the term "epitope" may be understood in its broadest sense as one or more of the antigen-binding regions of an antibody, a portion of the CD43 molecule that can be recognized and bound by the antibody produced by the hybridoma cell deposited under ICLC PD no. 16001. The portion of the antibody that binds to the epitope is called the paratope. In many cases, the epitope has conformational properties that specifically generate a binding site for the paratope.

[0050] Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and typically have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0051] Furthermore, it is understood and appreciated by those skilled in the art that the interaction between an epitope and an antibody may be based generally on the primary structure of the antigen, i.e., on the contiguous sequence of amino acids. Typically, the interaction may be based on the secondary, tertiary or quaternary structure of the epitope and on post-translational modifications such as glycosylation. The interaction between an epitope and an antibody may further be based on the three-dimensional structure and resulting surface features of the antigen, and may include non-contiguous sections of amino acid sequence that include amino acids at positions distant from the interaction with the antibody.

[0052] If two antibodies recognize the same or sterically overlapping epitopes, the antibodies recognize the "same epitope" as the antibody according to the first embodiment. In general, the most widely used and rapid method to determine whether two epitopes recognize the same or sterically overlapping epitopes is the competitive assay, which may be configured in a whole number of different formats, usually using either labeled antigen or labeled antibody. For example, antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzymatic labels.

[0053] An antibody that recognizes the "same epitope" as an antibody according to the first aspect typically refers to an antibody that blocks 50% or more of the binding of a reference antibody to an antigen in a competitive assay; conversely, the reference antibody typically blocks 50% or more of the binding of the antibody to the antigen in a competitive assay.

[0054] In general, the epitope recognised and bound by the antibody produced by the hybridoma cell deposited in ICLC PD n°16001 may be identified by any suitable epitope mapping method known in the art in combination with the antibody produced by the hybridoma cell deposited in ICLC PD n°16001.

[0055] Examples of such methods include screening peptides of various lengths derived from CD43 for binding to antibodies produced by hybridoma cells deposited under ICLC PD n°16001, wherein the smallest fragment capable of specifically binding to the antibody usually contains the sequence of the epitope recognized by the antibody. Generally, the CD43 peptides may be produced by synthesis or by proteolytic digestion of CD43. Methods for identifying peptides bound to antibodies, such as mass spectrometry, are well known to those skilled in the art. In another example, NMR spectroscopy can be used to identify residues that interact with the antibodies of the present invention. For example, uniformly 15N- and 2H-labeled CD43 peptides can be mixed with unlabeled antibodies, and the amino acids of the labeled peptides that interact with the unlabeled antibodies can be detected as positions within the NMR spectral changes. Generally, the difference between two spectra enables the identification of amino acids in CD43 that are involved in the interaction with the antibody. Preferably, mass spectrometry is used to identify the peptides bound to the antibody.

[0056] As an example, the epitope recognized and bound by the antibody produced by the hybridoma cells deposited under ICLC PD n°16001 may be identified by a method comprising amplifying various DNA fragments of CD43 DNA by polymerase chain reaction (PCR), incorporating these fragments into an expression vector containing a connection with a histidine fusion protein, and detecting the epitope, for example, by Western blot after protein expression.

[0057] In a preferred embodiment, the antibody according to aspect 1 or 2 recognizes an epitope comprising GalNAc O-linked to human CD43.

[0058] In a further example, to determine the site on CD43 recognized and bound by the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001, deletion mutations may be introduced by PCR methods into an expression vector cloned with CD43 to prepare a mutant series, such as an Escherichia coli (E. coli) mutant series expressing proteins with various deletion sites in CD43. These E. coli mutants may be cultured and induced for expression. Western blot analysis may be performed using cell lysates as antigens.

[0059] Further methods for identifying the epitope recognized and bound by the antibody produced by the hybridoma cells deposited under ICLC PD no. 16001 may include detection by immunoassay, such as enzyme-linked immunosorbent assay (ELISA).

[0060] In the context of the present invention, the term "affinity" can be understood in the broadest sense as the strength of interaction between an epitope and an epitope-binding site of an antibody. Methods for determining the absolute value of antibody affinity, i.e., affinity constant, are well known to those skilled in the art. However, the relative value of antibody affinity can also be generally determined, i.e., the affinity of two antibodies is compared without determining their absolute value. Methods for comparing antibody affinity are well known to those skilled in the art. For example, flow cytometry can be used, in which cells bearing the desired epitope can be contacted independently with different antibodies, which are then marked with immunofluorescent secondary antibodies. Usually, the intensity of the antibody signals can be compared after detection by flow cytometry.

[0061] Methods for identifying an antibody according to the second aspect that recognizes the same epitope as an antibody according to the first aspect are well known to those skilled in the art. For example, an antibody according to the second aspect may be identified by phage display based on an antibody library.

[0062] As a result, the antibodies of the present invention which recognize the same epitope may be human antibodies.

[0063] In another preferred embodiment, the antibody according to the second aspect is a chimeric antibody. In a more preferred embodiment, the antibody according to the second aspect is a chimeric antibody according to the first aspect.

[0064] Chimeric antibodies are antibodies in which at least one region of an immunoglobulin from one species has been fused by genetic engineering to another region of an immunoglobulin from another species, thereby reducing their immunogenicity (see, e.g., U.S. Pat. Nos. 4,816,567 and 4,816,397).

[0065] In another preferred embodiment, the antibody according to the second aspect is a humanized antibody. In a more preferred embodiment, the antibody according to the second aspect is a chimeric or humanized antibody according to the antibody of the first aspect.

[0066] Generally, a humanized antibody is a specific type of chimeric antibody. For example, a humanized antibody may be produced by grafting the DNA of a human antibody into a mouse antibody framework-encoding DNA or by grafting the DNA of a mouse antibody into a human antibody framework-encoding DNA. Preferably, the DNA of a human antibody is grafted into the mouse antibody framework-encoding DNA. Generally, DNA grafting involves grafting one or more DNA sequences into the target antibody framework-encoding DNA. Optionally, the variable and constant regions as well as the heavy and light chains may be partially or fully humanized. Preferably, the heavy and light chain variable regions of the mouse antibody are humanized. More preferably, the heavy and light chain variable regions of the mouse antibody are humanized by changing the DNA sequences encoding 1 to 50, preferably 1 to 30, more preferably 1 to 20 amino acids. The grafted DNA may generally include the DNA regions of the six hypervariable loops that determine the antigen specificity, also called complementarity determining regions (CDRs), or the DNA regions that do not contain the CDRs, or both. Preferably, humanization involves the transplantation of DNA that does not contain the CDRs.

[0067] Generally, the resulting DNA construct may then be used to express and produce antibodies that are typically less or not immunogenic compared to the parent non-human antibody. This includes the production of modified antibodies, such as deglycosylated or defucosylated antibodies. Such methods are well known in the art.

[0068] As a result, an antibody of the present invention that recognizes the same epitope may be a deglycosylated antibody or a defucosylated antibody.

[0069] In another preferred embodiment, the monoclonal antibody according to the antibody of aspect 2 is capable of inducing antibody-dependent cellular cytotoxicity (ADCC) against the EGIL T3 subgroup of T-cell acute lymphoblastic leukemia (T-ALL), T-cell lymphoblastic lymphoma cells, and Waldenstrom's macroglobulinemia (WM) cells.

[0070] Lymphocytes are a group of white blood cells that are mediators of humoral and cell-mediated immunity. There are two groups of lymphocytes: B cells and T cells.

[0071] Like many other cell types, B and T cells can develop abnormally into B and T cell tumors. There are many different tumors because of the many developmental steps that develop B and T cells. Both B and T cells arise from lymphoid progenitor cells.

[0072] In the case of B cells, these lymphoid progenitor cells progress through many stages of B cell development, each involving a definable specific cell type, until plasma cells are formed. One of these stages is the so-called "IgM-secreting B cells", which eventually develop into antibody-producing plasma cells. Tumors arising from "IgM-secreting B cells" are called "Waldenström's macroglobulinemia" (WM). WM is a rare, indolent, and incurable disease. WM is characterized by the bone marrow accumulation of clonal IgM-secreting lymphoplasmocytic cells.

[0073] T cells develop from lymphoid progenitor cells into mature T cells in a few developmental stages. Tumors can specifically evolve from mature T cells or lymphoid progenitor cells, the latter leading to B- or T-cell acute lymphoblastic leukemia, (B-ALL) and (T-ALL), respectively. T-cell phenotype T-ALL accounts for approximately 20% of all acute lymphoblastic leukemia cases, occurring more frequently in adults than in children. T-ALL is closely related to T-cell lymphoblastic lymphoma (T-LBL), and the differential diagnosis between the two diseases is based on localization to specific sites, often bone marrow in T-ALL or secondary lymphoid organs in T-LBL. The European Group for the Immunological Characterization of Leukemias (EGIL) has classified T-ALL into four subgroups based on immunophenotype (Bene MC, Leukemia 1995;9:1783): 1) EGIL T1 (pro-), characterized by cytoplasmic positivity for CD3 (cCD3) and surface expression of CD7; 2) EGIL T2 (pre-), characterized by cCD3, CD7 positivity and CD2 or CD5 positivity; 3) EGFR T3 (cortical), characterized by positivity for cCD3, CD1a, and the presence or absence of surface CD3 (sCD3) 4) EGIL T4 (mature leukemia), characterized by cCD3 and sCD3 positivity and CD1a negativity.

[0074] As used herein, the term "antibody-dependent cellular cytotoxicity (ADCC)" refers to the killing of cells that are bound and marked by an antibody by cytotoxic effector cells, such as natural killer (NK) cells.

[0075] To test whether an antibody is capable of inducing ADCC, the following assay can be used: A degranulation assay is performed by co-culturing peripheral blood mononuclear cells (PBMCs) from healthy donors, which contain the effector cells, with target cells expressing the epitope in the presence of different concentrations of antibody. 4Target cells were seeded in 96-well round-bottom plates and incubated at 37°C, 5% CO in the presence of different concentrations of antibody (0, 10, 50, 100 and 200ug / mL) or control IgG1. 2 The cells were then incubated with 0.4 × 10 6 PBMCs (fixed effector cells (E):target cells (T) = 10:1) were added to each well along with 20 μL / mL phycoerythrin (PE)-conjugated anti-CD107a monoclonal antibody (mAb) (BD), and the cells were incubated at 37 °C, 5% CO 2 The cells are then incubated at 4°C for 3 h. After 1 h, 6 μg / mL monensin is added to each well (GolgiStop, BD). At the end of the incubation period, the cells are stained with allophycocyanin (APC)-conjugated anti-CD56 and peridinin chlorophyll protein complex (PerCp)-conjugated anti-CD3 and analyzed on an ATTUNE NxT flow cytometer (THERMO Scientific). CD3 - / CD56 + / CD107a + NK cells (CD3 - / CD56 + )(CD107a + ) is measured. - / CD56 + / CD107a + The expansion of cells thus confirms the potential of the antibody to induce ADCC.The data obtained allows designing an immune targeting approach, which is an urgent and unmet clinical need, for example, in T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma.Additional methods for testing whether an antibody has the ability to induce ADCC can also be used and are well known to those skilled in the art.

[0076] In a third aspect, the present invention relates to a binding molecule derived from an antibody according to aspect 1 or aspect 2.

[0077] According to the invention, the binding molecule is a molecule derived from the monoclonal mouse antibody produced by the hybridoma cells deposited under ICLC PD no 16001. Preferably, the binding molecule is an immunoglobulin-containing molecule, i.e. it comprises at least one immunoglobulin (Ig) domain.

[0078] In a preferred embodiment, the binding molecule of the invention is selected from the group consisting of single chain antibodies. In a more preferred embodiment, the binding molecule is selected from the group consisting of single chain variable fragments (scFv), multimers of scFv such as diabodies, triabodies or tetrabodies, antibody fragments, preferably Fabs, tandabs and flexibodies.

[0079] The structure of antibodies, in particular the function of their CDRs, is generally known in the art (Carter PJ. Potent antibody therapeutics by design. Nature Rev. Immunol. 6:343-357, 2006). Single-chain Fvs (scFvs) and multimers thereof, tandabs, diabodies and flexibodies are generally standard antibody formats known in the art, e.g. from WO 1988 / 001649 A1, WO 1993 / 011161 A1, WO 1999 / 057150 A2 and EP 1293514 B1.

[0080] In scFv, the two antigen-binding variable regions (V H Fv and V LFv) are generally artificially connected by a linker peptide and are called single-chain variable fragments or single-chain antibodies (Bird, et al. (1988) Science 242:423-426; Orlandi, et al (1989) Proc Natl Acad Sci USA 86:3833-3837; Clarkson et al., Nature 352:624-628(1991)). The antigen-binding site may be composed of the variable domains of the light and heavy chains of a monoclonal antibody. Some studies have shown that scFv fragments may actually have the full essential antigen-binding affinity of one binding site of a whole antibody.

[0081] In the context of this invention, diabodies are scFvs with two binding specificities and can be either monospecific, bivalent, or bispecific, bivalent.

[0082] Tandabs and flexibodies are further antibody formats defined, for example, in US Patent Application Publication No. 2007031436 and EP Patent No. 1293514 B1, respectively.

[0083] Antibody fragments containing the idiotype of the protein can be generated by techniques known in the art. For example, these fragments include, but are not limited to, F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule, Fab' fragments which can be produced by reducing the disulfide bridges of F(ab')2 fragments, Fab fragments which can be produced by treating the antibody molecule with papain and a reducing agent, and Fv fragments.

[0084] The antibody or binding molecule of the present invention may be further linked to an active agent, preferably a toxin, a nanoparticle, a cytokine or a radionucleotide. Such antibody conjugates are known in the art (Wu AM, Senter PD. Nature Biotechnol. 23:1137-1146, 2005; Pastan et al. Annu. Rev. Med. 58:221-237, 2007; WO 1990 / 012592 A1; WO 2007 / 030642 A2; WO 2004 / 067038 A1; WO 2004 / 003183 A1; US ​​Patent Publication No. 2005 / 0074426 A1; WO 1994 / 004189 A1).

[0085] In a further aspect thereof, the present invention further relates to a chimeric antigen receptor (CAR) comprising the binding molecule of aspect 3 linked to an intracellular domain, preferably comprising one or more signaling domains.

[0086] Preferably, the invention relates to a chimeric antigen receptor (CAR) comprising an intracellular region comprising the CD3 zeta chain, a signalling region of a T cell receptor and an scFv of a preferred embodiment of a binding molecule of aspect 3 linked to two co-stimulatory domains CD28 and 4-1BB.

[0087] The CAR according to the invention, when expressed in T cells or NK cells, is a valid tool for targeting malignant cells carrying epitopes recognized and bound by the monoclonal antibody of embodiment 1 or embodiment 2. As used herein, the term "chimeric antigen receptor" (CAR) refers to a synthetic receptor that comprises a targeting moiety associated with one or more signaling domains in a single fusion molecule. Generally, the binding moiety of the CAR comprises an scFv, but may also comprise other binding entities. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domain of the CAR may be derived from the cytoplasmic region of the CD3 zeta or Fc receptor gamma chain, but may also be derived from other cytoplasmic regions. First generation CARs have been shown to successfully redirect the cytotoxicity of T cells. Adding signaling domains from costimulatory molecules, as well as transmembrane and hinge domains, has resulted in second and third generation CARs that can redirect T cells against malignant cells expressing CD19, resulting in some successful therapeutic studies in humans (Porter DL et al., N Eng J Med, 2011).

[0088] In a fifth aspect, the present invention relates to an expression vector comprising a nucleic acid sequence encoding a chimeric antigen receptor according to aspect 4, an antibody according to aspects 1 and 2 or a binding molecule according to aspect 3.

[0089] 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 the protein encoded by the nucleic acid sequence, i.e., chimeric antigen receptor, antibody, or binding molecule. Thus, an expression vector generally contains regulatory sequences, such as promoter and enhancer regions, and a polyadenylation site, to direct the efficient transcription of the nucleic acid sequence carried on the expression vector. An expression vector may further contain additional necessary or useful regions, such as a selectable marker for selection in eukaryotic or prokaryotic cells, a purification tag for purifying the resulting protein, a multiple cloning site, or an origin of replication.

[0090] Generally, the expression vector may be a virus or a non-virus vector. Generally, various virus vectors, such as retrovirus vectors, for example lentivirus or adenovirus vectors, or plasmids can be used. In a preferred embodiment, the expression vector according to aspect 5 is a virus vector. In a more preferred embodiment, the expression vector is a lentivirus vector.

[0091] In a sixth aspect, the present invention relates to a chimeric antigen receptor agonist (CAGR) comprising a chimeric antigen receptor agonist (CAGR) according to aspect 4 or an expression vector according to aspect 5. + The present invention relates to a lymphocyte, a NK lymphocyte, a cytokine-induced killer (CIK) cell, a gamma delta lymphocyte, a NKT cell or another immune effector cell.

[0092] In general, CD3 is a complex of four signaling chains that associate with the α:β heterodimer of the T cell receptor in a functional T cell receptor complex. The CD3 complex is normally required for T cell receptor signaling. In general, CD3 + The lymphocyte population contains only thymocytes and T cells. CD3 + Detection of the cells can be accomplished, for example, by flow cytometry.

[0093] In a seventh aspect, the present invention relates to a monoclonal antibody according to aspect 1 or 2 or a binding molecule according to aspect 3 or a CD3 + The present invention relates to pharmaceutical compositions comprising lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, gamma delta lymphocytes, NKT cells or other immune effector cells.

[0094] As used herein, the term "pharmaceutical composition" may be used interchangeably with the term "drug."

[0095] Antibodies, binding molecules or CD3 in pharmaceutical compositions + The content of lymphocytes is not limited as long as it is useful for treatment or prevention, but preferably contains 0.0000001 to 10% by mass per total composition. Furthermore, the present invention also includes an antibody, a binding molecule or a CD3 +Lymphocytes are preferably utilized in a carrier. The choice of carrier may depend on the route of administration and the 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 a pharma- ceutically acceptable salt is used in the carrier to make the composition isotonic. Examples of carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. Preferably, acceptable excipients, carriers, or stabilizers are non-toxic at the dosages and concentrations utilized, and include buffers such as citrate, phosphate, and other organic acids; salt-forming counterions, such as sodium and potassium; low molecular weight (>10 amino acid residues) polypeptides; proteins, such as serum albumin or gelatin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates, including glucose, mannose, or dextrin; monosaccharides; disaccharides; other sugars, such as sucrose, mannose, glycerol ... Examples of suitable carriers include ethanol, trehalose or sorbitol; chelating agents such as EDTA; non-ionic surfactants such as Tween, Pluronic or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and / or preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. Suitable carriers and their formulations are described in detail in Remington's Pharmaceutical Sciences, 17th Edition, 1985, Mack Publishing Co. The composition may contain at least one additional active compound, such as a chemotherapeutic agent.

[0096] Preferably, the antibody, binding molecule, CD3 + The lymphocytes and / or active compound are 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.

[0097] In an eighth aspect, the present invention relates to a nucleic acid or polynucleotide encoding an antibody according to aspect 1 or 2 or a binding molecule according to aspect 3.

[0098] Also provided herein are polynucleotides encoding antibodies that are optimized, for example, by codon / RNA optimization, replacement with a heterologous signal sequence, and deletion of mRNA destabilizing elements. Thus, methods of generating optimized nucleic acids encoding antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and / or deleting inhibitory regions in the mRNA can be performed, for example, by adapting the optimization methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. For example, potential splice sites and destabilizing elements (e.g., A / T or A / U rich elements) in the RNA can be mutated without modifying the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. Modifications can take advantage of, for example, the degeneracy of the genetic code, which uses alternative codons for the same amino acid. In some embodiments, it may be desirable to make conservative mutations, e.g., modify one or more codons to encode a similar amino acid with a similar chemical structure and properties and / or function as the original amino acid. Such methods may increase expression of the antibody or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more, compared to expression of an antibody encoded by a non-optimized polynucleotide.

[0099] In certain embodiments, an optimized polynucleotide sequence encoding an antibody or fragment thereof (e.g., VL and / or VH domain) described herein can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an antibody or fragment thereof (e.g., VL and / or VH domain) described herein. In certain embodiments, an optimized nucleotide sequence encoding an antibody or fragment thereof described herein hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an antibody or fragment thereof described herein. In certain embodiments, an optimized nucleotide sequence encoding an antibody or fragment thereof described herein hybridizes under high stringency, medium stringency or lower stringency hybridization conditions to an antisense polynucleotide of a non-optimized nucleotide sequence encoding an antibody or fragment thereof described herein. Information related to hybridization conditions is described, see, e.g., U.S. Patent Application Publication No. 2005 / 0048549 (e.g., paragraphs 72-73).

[0100] The polynucleotides of the present invention can be obtained and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. The nucleotide sequences encoding the antibodies described herein and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled to generate nucleic acids encoding the antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6), which briefly involves the synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligation of the oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

[0101] Alternatively, polynucleotides encoding the antibodies described herein can be generated from nucleic acid of a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, genomic DNA obtained from hybridoma cells can be used to perform a PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the known sequence to produce the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acid comprising sequences encoding the light and / or heavy chains of the antibody. Such PCR amplification methods can be used to obtain nucleic acid comprising sequences encoding the light and / or heavy chain variable regions of the antibody. The amplified nucleic acid can be cloned into a vector for expression in a host cell and further cloning to generate, for example, chimeric and humanized antibodies.

[0102] If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acid encoding the immunoglobulin can be obtained from an appropriate source (e.g., an antibody cDNA library or a cDNA library generated from any tissue or cell that expresses the antibody, such as hybridoma cells selected to express the antibody described herein, or nucleic acid isolated therefrom, preferably polyA+ RNA) by chemical synthesis or PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence to identify cDNA clones, for example, from a cDNA library encoding the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0103] DNA encoding the antibodies of the invention described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector that is then transfected into host cells, such as E. coli cells that do not otherwise produce immunoglobulin proteins, monkey COS cells, Chinese Hamster Ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells, to obtain the synthesis of the antibody in the recombinant host cells.

[0104] To generate whole antibodies, the VH or VL sequences within the scFv clone or other clones can be amplified using PCR primers that contain the VH or VL nucleotide sequence, a restriction site, and flanking sequences to protect the restriction site. Using cloning techniques known to those of skill in the art, the PCR amplified VH domain can be cloned into a vector expressing a heavy chain constant region, e.g., the human γ4 constant region, and the PCR amplified VL domain can be cloned into a vector expressing a light chain constant region, e.g., the human κ or λ constant region. In certain embodiments, the vector for expressing the VH or VL domain contains a promoter, a secretion signal, a cloning site for the variable region, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. Using techniques known to those of skill in the art, the heavy chain conversion vector and the light chain conversion vector are then co-transfected into cell lines to generate stable or transient cell lines expressing full-length antibodies, e.g., IgG.

[0105] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the murine sequences, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0106] Site-directed or high-density mutagenesis of the variable regions or other mutagenesis methods can be used to optimize the specificity, affinity, etc. of monoclonal antibodies. In particular, affinity maturation and chain shuffling strategies (Marks et al., 1992, Bio / Technology 10:779-783) are known in the art and can be used to generate high affinity human antibodies.

[0107] In a ninth aspect, the present invention relates to a hybridoma cell producing a monoclonal antibody according to the antibody of aspect 1 or 2.

[0108] In a tenth aspect, the present invention relates to the hybridoma deposited under ICLC PD n°16001.

[0109] In an eleventh aspect, the present invention relates to a method for producing a monoclonal antibody according to aspect 1 or 2, said method comprising a step of isolating said antibody from a hybridoma cell deposited under ICLC PD n° 16001.

[0110] In a twelfth aspect, the present invention relates to a method for identifying or isolating T-cell acute lymphoblastic leukaemia cells, T lymphoma cells, Waldenström's macroglobulinemia cells or tumour associated macrophages, comprising the step of contacting a cell sample comprising said cells with a monoclonal antibody according to aspect 1 or 2 or with a binding molecule according to aspect 3.

[0111] In general, macrophages are the most representative non-malignant cells in the tumor microenvironment. These tumor-associated macrophages (TAMs) are believed to acquire pro-tumor inflammatory and immunosuppressive phenotypes, favoring chemotherapy resistance, angiogenesis, cell motility, and intravasation / extravasation. Therefore, targeting TAMs may represent a novel therapeutic and yet unexplored clinical option to improve the efficacy of current anti-cancer treatments.

[0112] Antibody or binding molecule based methods for identifying or isolating specific cells such as T-cell acute lymphoblastic leukemia cells, T-lymphoma cells, Waldenström's macroglobulinemia cells or tumor associated macrophages are generally known to the person skilled in the art, such as methods based on fluorescent cell sorting by flow cytometry, magnetic cell isolation or single cell sorting, e.g. by a cell sorter.

[0113] In a thirteenth aspect, the present invention provides a method for the treatment of CD3 +A method for producing a CD3+ lymphocyte expressing a chimeric antigen receptor according to the chimeric antigen receptor of embodiment 4, comprising the step of introducing an expression vector according to embodiment 5 into lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, γδ lymphocytes, NKT cells or other immune effector cells. + The present invention relates to a method for producing lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, gamma delta lymphocytes, NKT cells or other immune effector cells.

[0114] This also includes the possibility of producing single cells or introducing expression vectors into single cells.

[0115] In a fourteenth aspect, the present invention relates to a method for the treatment of CD45 + , CD3 + , CD8 - , CD127 + , CCR7 + The present invention relates to a method for identifying or isolating T lymphocytes, the method comprising the step of contacting a cell sample comprising said T lymphocytes with an antibody according to aspect 1 or 2 or a binding molecule according to aspect 3.

[0116] A preferred embodiment comprises an antibody according to aspect 1 or 2, a binding molecule according to aspect 3, an expression vector according to aspect 5, a CD3+ antibody according to aspect 6 for use in a method for the treatment of T-cell acute lymphoblastic leukemia or Waldenström's macroglobulinemia. + lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, γδ lymphocytes, NKT cells or other immune effector cells, or a pharmaceutical composition according to aspect 7.

[0117] In addition, other malignancies may also be treated, the cells expressing the epitope recognised by the antibodies of aspects 1 and 2 of the present invention.

[0118] Additional preferred embodiments include CD45 + , CD3 + , CD8 - , CD127 + , CCR7 +An antibody according to embodiment 1 or 2 or a binding molecule according to embodiment 3 for use in a method for identifying or isolating T lymphocytes.

[0119] Another preferred embodiment is an antibody according to aspect 1 or 2 or a binding molecule according to aspect 3 for use in a method for isolating or identifying T-cell acute lymphoblastic leukemia cells, T lymphoma cells, Waldenström's macroglobulinemia cells or tumor-associated macrophages.

[0120] A further preferred embodiment is an antibody according to aspect 1 or 2 or a binding molecule according to aspect 3 for use in a method for the diagnosis of T-cell acute lymphoblastic leukemia or Waldenström's macroglobulinemia.

[0121] In addition, other malignancies may be diagnosed, the cells expressing the epitopes recognised by the antibodies of aspects 1 and 2 of the invention.

[0122] In the following the invention is further described by means of figures and examples, which are intended to be illustrative and not limiting of the invention. [Brief description of the drawings]

[0123] [Figure 1] Figure 1 shows the expression of epitopes of the antibodies produced by the hybridoma cells deposited according to the invention in peripheral blood mononuclear cells of a panel of healthy donors and a comparison with state of the art CD43 detection antibodies. The upper scatter plot shows data obtained by flow cytometry. The x-axis shows forward scatter detection (FSC) and the y-axis illustrates side scatter (SSC). Each point corresponds to one cell. The lower histogram illustrates the phycoerythrin signal intensity on the x-axis. The y-axis relates the signal intensity to a maximum signal intensity of 100% of the unstained sample. The unfilled curve represents the unstained control, the horizontally striped curve represents the scrambled IgG1 stained cells (i.e. negative control), the checkered curve represents the mAb UN1 stained cells and the diagonal striped curve represents the CD43 stained cells. [Diagram 2] Figure 2 shows cell populations recognized by antibodies produced by hybridoma cells deposited according to the present invention. Figure 2 shows four scatter plots. The two left scatter plots belong to lymphocytes. The two right scatter plots are from lymphocytes detected by antibodies produced by hybridoma cells deposited according to the present invention. In the top two scatter plots, the x-axis represents CD4 signal intensity and the y-axis illustrates CD8 signal intensity. In the bottom two scatter plots, the x-axis represents CD45ro signal intensity and the y-axis represents CCR7 signal intensity. [Diagram 3] 1 shows two histograms: the upper histogram shows the expression of epitope UN1 detected by the antibody (mAb UN1) produced by the hybridoma cells deposited according to the invention in the BCWM.1 cell line; the lower histogram shows UN1 expression in the MWCL.1 cell line; the unfilled curve represents the unstained control, the horizontally striped curve represents the secondary mAb stained cells, the vertically striped curve represents the scrambled IgG + secondary stained cells, and the diagonal striped curve represents the cells stained with mAb UN1. [Figure 4] 1 shows tumor associated macrophages (TAMs) recognized by antibodies produced by hybridoma cells deposited according to the present invention, the white arrows indicating TAMs infiltrating a colorectal cancer specimen. [Diagram 5] Figure 1 shows THP1-derived macrophages stained with control IgG1 in the absence of tumor cells (first column), UMG1-CH (chimeric antibody according to embodiment 2 of the invention, in which the original mouse Fc region is replaced with a fully human IgG1 Fc region) in the absence of tumor cells (second column) and UMG1-CH in the presence of PANC1 pancreatic cancer cell line (third column and special left). First row represents DAPI staining, second row represents antibody + alexa-fluor 488 labeled secondary antibody, and third row represents overlay images. [Figure 6]Figure 1 shows the results of a degranulation assay evaluating ADCC in HPB- ALL (left graph) and H9 cell line (right graph). The numbers on the x-axis represent the different samples, meaning: no target (1), E+T (2), negative control (NC) 200 μg / mL (3), UMG1-CH 10 μg / mL (4), UMG1-CH 50 μg / mL (5), UMG1-CH 100 μg / mL (6), UMG1-CH 200 μg / mL (7), positive control (PC) 200 μg / mL (8). The y-axis represents the percentage of CD107a+ NK cells affected by ADCC relative to the total number of CD107a+ NK cells tested per sample. [Figure 7] FIG. 1 shows the results of a degranulation assay assessing ADCC in the BCWM.1 cell line. The numbers on the x-axis represent the different samples. The numbering is according to FIG. 6. The y-axis represents the percentage of CD107a+ NK cells affected by ADCC relative to the total number of CD107a+ NK cells tested per sample. [Figure 8] FIG. 1 shows that CD3+ expressing lymphocytes (CAR-T) were able to release significantly higher amounts of interferon gamma (IFNγ) in the presence of H9 cells. The y-axis records the concentration of IFNγ expressed in ng / mL. The x-axis records: non-transduced T cells (1), T cells transduced with a control CAR (2) and T cells transduced with UMG-1 CAR (3). [Figure 9] FIG. 1 shows that CAR-T was able to release significantly higher amounts of interleukin 2 (IL-2) in the presence of H9 cells. The y-axis represents the concentration of IL2 expressed in ng / mL. The x-axis records: untransduced T cells (1), T cells transduced with a control CAR (2) and T cells transduced with UMG-1 CAR (3). [Figure 10] FIG. 1 shows that CAR-T was able to induce selective killing of H9 cells. The y-axis records the dead / live cell ratio. The x-axis records: H9 alone (1), H9 in the presence of non-transduced T cells (2), H9 in the presence of T cells transduced with a control CAR (3) and H9 in the presence of T cells transduced with UMG-1 CAR (4). [Figure 11] FIG. 1 shows tumor volume curves from an in vivo experiment comparing control IgG1 versus a humanized version of UN1-mAb (h-UN1) and a defucosylated version of UN1-mAb (ah-UN1).

[0124] [ka] EXAMPLES

[0125] The following examples are provided for the purpose of illustrating the present invention, but should not be construed as limiting the present invention. The examples include technical features, and it goes without saying that the present invention relates to any combination of the technical features shown in this illustrative section.

[0126] Example 1 Expression of epitopes of antibodies produced by the hybridoma cells deposited according to the present invention in peripheral blood mononuclear cells of a panel of healthy donors and comparison with state-of-the-art CD43 detection antibodies First, peripheral blood mononuclear cells (PBMCs) from different healthy donors were obtained by Ficoll gradient separation. Then, cells were seeded in 5 mL tubes and stained with 1 μg / mL of mAb (mAb UN1) produced by hybridoma cells deposited under ICLC accession number ICLC PD n°16001 or 1 μg / mL of scrambled mouse IgG1 antibody in 100 μL of binding solution (phosphate buffered saline (PBS) + 0.5% fetal bovine serum (FBS)) and incubated at 4° C. for 30 minutes. The cells were then washed twice in binding solution and stained with fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 30 minutes at 4° C. in the dark. Afterwards, the cells were washed twice in binding solution and acquired on an ATTUNE NxT flow cytometer (THERMO Scientific). One tube for each donor was left unstained and one tube for each donor was stained only with FITC-conjugated secondary antibody. Overall, the antibodies produced by the hybridoma cells deposited according to the present invention were able to recognize variable lymphocyte subpopulations (range: 0-15%) of different donors. Moreover, the antibodies did not show any reactivity with all other cell populations within PBMCs, including bone marrow-derived cells, which were therefore negative for the expression of the respective antigens (see FIG. 1, above).

[0127] In contrast, when the same PBMCs were assayed for CD43 expression by using a commercially available clone (S7 from Beckton Dickinson), all lymphoid and myeloid cells were found to be positive (see Figure 1, bottom).

[0128] As a result, the antibodies produced by the hybridoma cells deposited according to the present invention exhibit a specific and restricted pattern of reactivity and properties not possessed by preexisting antibodies against CD43.

[0129] Example 2 Cell population recognized by the antibody produced by the hybridoma cell deposited according to the present invention To characterize the lymphocyte subpopulations detected by the antibodies produced by the hybridoma cells deposited according to the invention, immunomagnetic sorting of the respective lymphocytes was performed (Easysep do-it-yourself, Stemcell technologies). Briefly, 15 μg of the antibody was mixed with the components provided by the manufacturer to obtain a solution ready for immunomagnetic separation. This solution was added to PBMCs of three different donors with at least 10% lymphocytes detected by the antibody, and after FcR blocking, the cells were incubated for 15 min at room temperature (rt). Then, EasySep® Magnetic Nanoparticles were added to the solution and the cells were incubated for another 10 min at rt. The solution was then placed in a magnet and unbound cells were removed. The cells detected by the antibodies according to the invention were almost all CD45 + CD3 + CD4 + CD8 - CD127 + CCR7 + T lymphocytes, the majority of which are CD45 - (See Figure 2 and Table 1).

[0130] [Table 1]

[0131] Example 3 The antibody (mAb UN1) produced by the hybridoma cells deposited according to the present invention recognizes T-ALL and Waldenstrom's macroglobulinemia cell lines. Various cancer cell lines (Table 2) were evaluated for expression of UN1 (MM: multiple myeloma). Briefly, cells were seeded in 5 mL tubes and stained with mAb UN1 1 μg / mL or scrambled mouse IgG1 antibody 1 μg / mL in 100 μL of binding solution (phosphate buffered saline (PBS) + 0.5% fetal bovine serum (FBS)) and incubated at 4° C. for 30 min. Cells were then washed twice in binding solution and stained with fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 30 min at 4° C. in the dark. Afterwards, cells were washed twice in binding solution and acquired on an ATTUNE NxT flow cytometer (THERMO Scientific). One tube for each cell line was left unstained and one tube for each cell line was stained with FITC-conjugated secondary antibody only. It was observed that T-ALL cell lines belonging to the EGIL T3 classification and Waldenstrom macroglobulinemia (Figure 3) were all positive for UN1 expression.

[0132] [Table 2]

[0133] Example 4 The antibody (mAb UN1) produced by the hybridoma cells deposited according to the present invention recognizes tumor-associated macrophages. As previously described, CD43 is a specific leukocyte marker that is largely restricted to different cells of the hematopoietic lineage. Specific epitopes on CD43 isoforms bound by mAb UN1 were found to be highly expressed by tumor-associated macrophages (TAMs).

[0134] By evaluating specimens from different types of cancer by immunohistochemistry (Table 3, Figure 4), UN1 + Macrophages are a highly infiltrating component of most tumors and have been observed to have a specific and particular highly invasive malignant potential in pancreatic and ovarian cancer.

[0135] Furthermore, we assessed whether UN1 expression was altered in the presence or absence of co-cultured cancer cells in a model of macrophage differentiation.

[0136] For this purpose, THP1 monocytic leukemia cells were used, which, as previously shown, do not express UN1. To obtain differentiated unpolarized human M0 macrophages (THP1-M), these cells were cultured for 48 hours in the appropriate complete medium in the presence of 50 ng / mL phorbol 12-myristate 13-acetate (PMA). The medium was then replaced with fresh medium without PMA. At this stage, selected wells were added with the PANC1 pancreatic cancer cell line at a 1:1 ratio for 48 hours. All cells were then prepared for immunofluorescence analysis. Briefly, after fixation, THP1-M were stained with UMG1-CH or human IgG1 control and incubated overnight at 4°C. FITC anti-human secondary mAb was then added to the cells for 2 hours. After washing, anti-fade mounting medium (Vectashield, Vectorlabs) containing DAPI was added to the cells and coverslipped for reading.

[0137] As shown in Figure 5, right, THP1-derived macrophages stained with control IgG1 were completely negative, whereas those stained with UMG1-CH were weakly positive. Interestingly, in the presence of PANC1, THP1-derived macrophages became strongly positive for UN1 expression. A detailed view of the interaction between THP1-derived macrophages (white arrows) and PANC1 (red arrows) is shown (Figure 5, left). These findings demonstrate that UN1-specific epitopes are significantly upregulated when macrophages are co-cultured and interact with cancer cells within a reconstituted tumor microenvironment. This upregulation is per se plausible as a representative target that is convenient for therapeutic approaches focused on tumor-associated macrophage purification. Beyond this plausible potential as a therapeutic tool, mAb UN1 may also offer utility for detection, analysis of prognostic role, and predictive studies.

[0138] [Table 3]

[0139] Example 5 The chimeric mAb UMG1-CH (according to embodiment 2 of this invention) is an active immunotherapeutic tool for T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma. To determine the potential activity of mAb UMG1-CH as an immunotherapeutic tool, its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) was first evaluated. For this purpose, degranulation assays by co-culturing PBMCs from healthy donors (effector cells) with HPB-ALL or H9 T-ALL cell lines (target cells) in the presence of different concentrations of mAb UMG1-CH were performed as follows:

[0140] 4×10 4 Target cells were seeded in 96-well round-bottom plates and incubated at 37 °C, 5% CO in the presence of different concentrations of mAb UMG1-CH (0, 10, 50, 100, 200 μg / mL) or chimeric negative or positive control IgG1 (NC and PC, 200 μg / mL, respectively) at the highest dose (200 μg / mL). 2 The cells were then incubated with 0.4 × 10 6 PBMCs (fixed E:T = 10:1) were added to each well along with 20 μL / mL PE-conjugated anti-CD107a mAb (BD), and the cells were incubated at 37 °C, 5% CO 2 The cells were then incubated for 3 hours at 4°C for 1 hour. After 1 hour, 6 μg / mL monensin was added to each well (GolgiStop, BD). At the end of the incubation period, the cells were stained with APC-conjugated anti-CD56 and PerCp-conjugated anti-CD3 and analyzed on an ATTUNE NxT flow cytometer (THERMO Scientific). CD3 - / CD56 + / CD107a + The cells were found to expand significantly, thus confirming the potential of mAb UMG1-CH as an ADCC inducer (FIG. 6).

[0141] These data make it possible to design immune targeting approaches, which represent an urgent unmet clinical need in T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma.

[0142] Example 6 The chimeric mAb UMG1-CH (embodiment 2 of this invention) is a potent immunotherapeutic tool for Waldenström's macroglobulinemia. To investigate the immunotherapeutic potential of mAb UMG1-CH, its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated. For this purpose, degranulation assays were performed by co-culturing purified NK cells (effector cells) from healthy donors and the BCWM.1 cell line (target cells) in the presence of different concentrations of mAb UMG1-CH or negative / positive controls. mAb cetuximab was chosen as a negative control and mAb rituximab as a positive control. In particular, 10 5 Target cells were seeded in 96-well round-bottom plates and incubated at 37°C, 5% CO in the presence of different concentrations of mAb UMG1-CH (0, 10, 50, 100, 200 μg / mL), 200 μg / mL cetuximab, or 200 μg / mL rituximab. 2 After incubation at 10 °C for 30 min, 10 5 NK cells (fixed E:T = 1:1) were added to each well along with 20 μL / mL PE-conjugated anti-CD107a mAb (BD), and the cells were incubated at 37 °C, 5% CO 2 The cells were then incubated for 2 hours at 4°C for 1 hour. After 1 hour, 6 μg / mL monensin was added to each well (GolgiStop, BD). At the end of the incubation period, the cells were stained with APC-conjugated anti-CD56 and PerCp-conjugated anti-CD3 and analyzed on an ATTUNE NxT flow cytometer (THERMO Scientific). CD3 - / CD56 + / CD107a +It was found that the cells expanded significantly, reaching exactly the same effect as obtained with rituximab, thus confirming the potential of the mAb as an ADCC inducer (FIG. 7).

[0143] Example 7 The chimeric antigen receptor (CAR)-UMG1 induces significant cytotoxicity against cells expressing the epitope of the antibody produced by the hybridoma cells deposited according to the present invention. To further improve the immunotherapeutic potential of the antibodies produced by the hybridoma cells deposited according to the present invention as immunotherapeutic tools, a third generation CAR was developed. In particular, CAR-T was designed by coupling an extracellular domain consisting of an scFv derived from the sequence of an antibody with an intracellular region consisting of the CD3ζ chain, the signaling region of the TCR, and two costimulatory domains CD28 and 4-1BB, thus mimicking physiological T cell activation. For this purpose, an antibody scFv was cloned into a CAR cassette together with three selected costimulatory domains to generate a lentiviral vector. The viral particles were then used to infect CD3 cells from healthy donors at a multiplicity of infection (MOI) of 5. + Lymphocytes were transduced and transduction efficiency was assessed by flow cytometry (approximately 38%). CAR-Ts against the epitopes of the antibodies were finally assayed for their ability to release IFNγ and IL-2 in the presence of target cells, as well as for selective cytotoxicity. As shown in Figures 8 and 9, CAR-UMG1 was able to release significantly higher amounts of interferon gamma (IFNγ) and interleukin 2 (IL-2) only in the presence of H9 cells. In addition, only CAR-UMG1 was able to induce selective killing of H9 cells (see Figure 10), thus demonstrating the ability of the resulting CAR to recognize H9 cells and induce T cell activation.

[0144] Example 8 In this example, the tumor volume curves of in vivo experiments comparing the humanized version of control IgG1 against UN1-mAb (h-UN1) and the defucosylated version of UN1-mAb (a-h-UN1) are recorded. In this experiment, 15 NOD-SCID-γ-chain-null (NSG) mice were subcutaneously implanted with 5×10 6 cells of HPB-ALL. The mice were then randomized and given 15 mg / kg of control IgG1, h-UN1 or a-h-UN1 intraperitoneally once a week starting on day 1 until death, tumor volume > 2000 mm 3 or toxicity became unacceptable. Tumor volume was determined every other day, and the average volume of each group at each time point was recorded in Figure 11. As of day 29, both h-UN1 and a-h-UN1 showed a significant reduction in disease burden, confirming the strong in vivo activity of both antibodies. [Table 4]

Claims

1. A monoclonal mouse antibody produced by hybridoma cells deposited under ICLC accession number ICLC PD16001.

2. An antibody against CD43, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the amino acid sequences GFTFSSFGMH, YISSGSGNFYYVDTVKG, STYYHGSRGAMDY, SASSSVSSMYWY, DTSKMAS and QQWSSYPPIT, respectively.

3. 3. The antibody of claim 2, which is a monoclonal or bispecific antibody, or a chimeric or humanized antibody.

4. The antibody of claim 3, which is a chimeric or humanized antibody of the antibody of claim 1.

5. The antibody of claim 3 or 4, which is capable of inducing antibody-dependent cellular cytotoxicity (ADCC) against the EGIL T3 subgroup of T-cell acute lymphoblastic leukemia (T-ALL) cells, T-cell lymphoblastic lymphoma cells, and Waldenstrom's macroglobulinemia (WM) cells.

6. 6. The antibody of claim 1, which recognizes an epitope comprising O-linked GalNAc on human CD43.

7. 7. A CD43 binding molecule comprising at least one immunoglobulin (Ig) domain of an antibody according to any one of claims 1 to 6, wherein the at least one immunoglobulin (Ig) domain binds to CD43 and comprises a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the amino acid sequences GFTFSSFGMH, YISSGSGNFYYVDTVKG, STYYHGSRGAMDY, SASSSVSSMYWY, DTSKMAS and QQWSSYPPIT, respectively.

8. The CD43 binding molecule of claim 7, selected from the group consisting of single chain antibodies.

9. The CD43 binding molecule of claim 7, which is selected from the group consisting of scFv, multimers of scFv, and antibody fragments selected from Fab, tandab, flexibody and F(ab')2.

10. The CD43 binding molecule of claim 9, which is a multimer of scFvs selected from diabodies, triabodies and tetrabodies.

11. 11. A chimeric antigen receptor comprising the CD43 binding molecule of any one of claims 7 to 10 linked to an intracellular domain.

12. The chimeric antigen receptor of claim 11, wherein the intracellular domain comprises one or more signaling domains.

13. 13. An expression vector comprising a nucleic acid sequence encoding the chimeric antigen receptor of claim 11 or 12, the antibody of any one of claims 1 to 6 or the CD43 binding molecule of any one of claims 7 to 10.

14. 14. The expression vector of claim 13, which is a viral or non-viral vector.

15. The expression vector of claim 14 which is a lentiviral vector.

16. A CD3 comprising the chimeric antigen receptor of claim 11 or 12 or the expression vector of any one of claims 13 to 15. + Lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, gamma delta lymphocytes or NKT cells.

17. The antibody according to any one of claims 1 to 6, the CD43 binding molecule according to any one of claims 7 to 10, or the CD3 + A pharmaceutical composition comprising lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, gamma delta lymphocytes or NKT cells.

18. 11. A nucleic acid encoding an antibody according to any one of claims 1 to 6 or a CD43 binding molecule according to any one of claims 7 to 10.

19. Hybridoma deposited under ICLC accession number ICLC PD16001.

20. 3. A method for producing the antibody of claim 1 or 2, comprising isolating said antibody from hybridoma cells deposited under ICLC Accession No. ICLC PD16001.

21. CD45 + , CD3 + , CD8 - , CD127 + and / or CCR7 + A method for identifying or isolating T lymphocytes, the method comprising the step of contacting a cell sample containing said T lymphocytes with an antibody described in any one of claims 1 to 6 or a CD43 binding molecule described in any one of claims 7 to 10.

22. 11. A method for identifying or isolating T-cell acute lymphoblastic leukemia cells, T lymphoma cells, Waldenstrom's macroglobulinemia cells or tumor associated macrophages, comprising the step of contacting a cell sample comprising said cells with a monoclonal antibody according to any one of claims 1 to 6 or a CD43 binding molecule according to any one of claims 7 to 10.

23. A CD3 expressing the chimeric antigen receptor of claim 11 or 12. + A method for producing lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, gamma delta lymphocytes or NKT cells, comprising: + 16. A method comprising introducing an expression vector according to any one of claims 13 to 15 into a lymphocyte, a NK lymphocyte, a cytokine-induced killer (CIK) cell, a gamma delta lymphocyte or a NKT cell.

24. The antibody according to any one of claims 1 to 6, the CD43 binding molecule according to any one of claims 7 to 10, the expression vector according to any one of claims 13 to 15, or the CD3 binding molecule according to claim 16 for use in a method for the treatment of T-cell acute lymphoblastic leukemia or Waldenström's macroglobulinemia. + 18. A lymphocyte, a NK lymphocyte, a cytokine-induced killer (CIK) cell, a gamma delta lymphocyte or a NKT cell, or the pharmaceutical composition of claim 17.

25. CD45 + , CD3 + , CD8 - , CD127 + and / or CCR7 + 11. An antibody according to any one of claims 1 to 6 or a CD43 binding molecule according to any one of claims 7 to 10 for use in a method for identifying or isolating T lymphocytes.

26. 11. An antibody according to any one of claims 1 to 6 or a CD43 binding molecule according to any one of claims 7 to 10 for use in a method for isolating or identifying T-cell acute lymphoblastic leukemia cells, T lymphoma cells, Waldenström's macroglobulinemia cells or tumor-associated macrophages.

27. 11. An antibody according to any one of claims 1 to 6 or a CD43 binding molecule according to any one of claims 7 to 10 for use in a method for diagnosing T-cell acute lymphoblastic leukemia or Waldenström's macroglobulinemia.

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