The invention relates to a monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC accession number ICLC PD n° 16001, related antibodies and binding molecules and uses thereof.

Background of the invention

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 on most of peripheral and bone marrow- derived cell components. The precursor form of CD43 migrates with an apparent molecular weight of 54 kD. In its mature form, CD43 is heavily glycosylated having a molecular weight between 115 and 200 kD. Overall, CD4 ⁺ thymocytes and monocytes express the 115 kD form, while activated CD4 ⁺ and CD8 ⁺ -T cells, B cells, neutrophils and platelets express a 130 kD form. CD43 is involved in multiple functions, such as cell adhesion, apoptosis and migration (Ostberg JR et al. Immunology today. 1998;19:546-50). Glycoproteins, such as glycosylated CD43, play a major role in cell signaling, immune recognition, and cell-cell interaction because of their glycan branches conferring structure 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 secreted or expressed on the membrane of hematopoietic and epithelial cells. O-glycan biosynthesis initiates in the Golgi apparatus with the attachment of N-acetyl-galactosamine (GalNAc) to serine or threonine residues by an UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) generating the Tn antigen structure of O-glycans. Subsequent elongation of the O-linked glycan branch is catalyzed by tissue-specific glycosyltransferases through the addition of other carbohydrates, such as galactose, fucose, and sialic acid, which results in the synthesis of a complex array of O-glycan structures that differ for the nature and length of O-linked carbohydrate chains (Wopereis S. et al. Clin. Chem 2006; 52, 574-600). In addition, oligosaccharides can be modified by sialylation, fucosylation, sulfatation, methylation, or acetylation. Truncation of O-glycan structures as well as abnormal expression of specific 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. U.S.A. 2002; 99, 10231-10233, Brockhausen I. EMBO Rep. 7 2006; 599-604). Oncofetal antigens (OAs) are primarily glycoproteins and products of one or more genes that normally are highly expressed only during fetal development, progressively repressed during differentiation and therefore not expressed in adult tissues. Their re-expression in adults is the result of aberrant activation of controlled genes as it occurs in cancer. Oncofetal epitopes (OEs) are the part of the OA that is recognized by the antibody. Identification of OAs or specific oncofetal epitopes (OEs), whose expression is confined to cancer tissue, may not only serve in detecting early oncogenic processes, but most importantly, may be crucial in developing new immunotherapeutic approaches to human cancer.

The object of the present invention is to provide an antibody allowing the detection of an OE and the development of novel immunotherapeutic approaches.

The problem is solved by an antibody according to the invention. The data shown in the examples indicate that due to the specific pattern of restricted expression in fetal tissues and re-expression in malignancies, the epitope recognized by the antibody produced by the hybridoma cell deposited according to the invention can be considered to be an OE. The latter represents therefore a potentially suitable target for innovative immunotherapeutic strategies for treatment of human cancer.

Further, a chimeric antigen receptor (CAR) comprising the scFv of a binding molecule based on the antibody according to the invention linked to an intracellular region comprising the CD3ζ chain, the signaling region of the T cell receptor, and the two co-stimulatory domains CD28 and 4 IBB was developed. CD3 ⁺ lymphocytes expressing the CAR according to the invention induce significant cytotoxicity against cells expressing the epitope recognized by the antibody produced by the hybridoma cell deposited according to the invention. Even though the effect of the murine antibody of the invention has in part already been 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 antibody itself or its sequence as well as the epitope, which it binds to, have never been made available to the public.

Summary of the invention

A mouse antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 is provided. In addition, an antibody, which recognises the same epitope as the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 is provided. The 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, CDRL 1 , CDRL2, and CDRL3 comprise the amino acid sequences GFTFSSFGM H, YISSGSGNFYYVDTV G, STYYHGSRGAM DY, SASSSVSS YWY, DTSKMAS, and QQWSSYPPIT , respectively.

Preferably, said antibody is a monoclonal antibody. Further, a binding molecule derived from the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 or from the above antibody of the invention is provided.

Moreover, a chimeric antigen receptor is provided, which comprises an scFv binding molecule according to the invention linked to an intracellular region comprising the CD3ζ chain, the signaling region of the T cell receptor, and to the two co-stimulatory domains CD28 and 41BB.

Furthermore, an expression vector is provided that comprises a nucleic acid sequence, which encodes the chimeric antigen receptor according to the invention, the antibody according to the invention or the binding molecule according to the invention.

The invention further provides a CD3 ⁺ lymphocyte, an NK lymphocyte, a Cytokine induced killer (CIK) cell, a gamma-delta lymphocyte, or an NKT cell comprising the chimeric antigen receptor according to the invention or the expression vector according to the invention. A pharmaceutical composition is provided comprising the antibody according to the invention or the binding molecule according to the invention or the CD3 ⁺ lymphocyte an NK lymphocyte, a Cytokine induced killer (CIK) cell, a gamma-delta lymphocyte, or an NKT cell according to the invention.

A nucleic acid is provided, encoding the antibody according to the invention or the binding molecule according to the invention.

A hybridoma cell is provided that produces the antibody according to the invention.

A method for producing the antibody according to the invention is provided, which comprises isolating said antibody from the hybridoma cell deposited under ICLC PD n° 16001. A method for the identification or isolation of T-cell acute lymbhoblastic leukemia cells, T lymphoma cells, Waldenstrom's Macroglobulinemia cells or tumor-associated macrophages is provided, which comprises contacting a cell sample comprising said cells with the antibody according to the invention or with the binding molecule according to the invention.

A method for producing CD3 ⁺ lymphocytes, NK lymphocytes, Cytokine induced killer (CIK) cells, gamma-delta lymphocytes, or NKT cells expressing a chimeric antigen receptor according to the invention is provided comprising the introduction of the expression vector according to the invention into said CD3 ⁺ lymphocytes, NK lymphocytes, Cytokine induced killer (CIK) cells, gamma-delta lymphocytes, or an NKT cells.

Detailed description of the invention In a first aspect, the invention relates to a monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC PD n° 16001.

The hybridoma cell was deposited at Centro Biotecnologie Avanzate (CBA), Interlay Cell Line Collection (ICLC), Largo Rosanna, 10, 16132 Genova, Italy under accession number ICLC PD n° 16001 on August 4, 2016. The antibody was tested in the examples given below. As shown in the examples, the antibody binds to a specific sialoglycosilated epitope on CD43.

In this first aspect, the invention further relates to an antibody, comprising a heavy chain variable region comprising complementarity determining regions CDRHl, CDRH2 and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRHl, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences GFTFSSFGM H, Y I S SG SG FY Y V DT V G , STY Y HGSRG AM DY, SASSSVSSMYWY, DTSKMAS, and QQWSSYPPIT , respectively. These sequences are also given in SEQ IDs NO. 1-6.

The CDR sequences mentioned above are the CDR sequences from the monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC PD n° 16001, as determined by sequencing. As used herein, the term "CDR" or "complementarity determining region" means the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et ah, Sequences of protein of immunological interest. (1991), and by Chothia et ah, J. Mol. Biol. 196:901-917 (1987) and by MacCallum et al, J. Mol. Biol. 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth for comparison. Preferably, the term "CDR" is a CDR as defined by Kabat, based on sequence comparisons. CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs, and CDRLl, CDRL2 and CDRL3 denote the light chain CDRs.

This monoclonal antibody may have framework sequences 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. The term "framework region" or "FR region" as used herein, includes the 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).

Methods for producing a monoclonal antibody with the CDR sequences as mentioned above are known in the art and include the introduction of the nucleic acid sequences encoding the CDRs into suitable expression vectors encoding the desired framework sequences. Further methods are described below.

In a second aspect, the invention relates to an antibody which recognizes the same epitope as the antibody according to the first aspect. Typically, and as generally known in the art, an antibody is a protein belonging to the protein family of immunoglobulins and is composed in its variable regions of framework regions and complementarity determining regions as defined above. Naturally, antibodies are produced by plasma cells in response to a certain antigen. In general, each antibody has two identical heavy chain immunoglobulins and two identical light chain immunoglobulins. Each heavy and each light chain may have a variable and a constant region. The constant region of a heavy chain may be one of five types of mammalian Ig heavy chains: α, δ, ε, γ and μ. The type of the heavy chain present usually defines the class (isotype) of the antibody: IgA, IgD, IgE, IgG and IgM antibodies, respectively. Similarly, the constant region of a light chain may be one of two types of mammalian Ig light chains: κ and λ. The variable regions of heavy and light chains are usually made of a unique combination of numerous protein sequences allowing the binding to a particular antigen.

According to the invention, the term "antibody" also covers isolated an isolated antibody. In general, each heavy chain is connected to one of the light chains, whereby the variable regions of a heavy and a light chain combine to form one of the two identical antigen-binding sites and their constant regions combine to form the constant region of the antibody. Further, both constructs of one heavy and one light chain may be connected via the constant regions of their heavy chains, forming a "Y"-shaped molecule, whereby the two arms depict the antigen-binding variable region and the stem depicts the constant region.

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

Further, the antibody according to the second aspect of the present invention may form a homo- or heterodimer or a homo- or heteromultimer, whereby "dimer" and "multimer" means that two and at least three antibodies, respectively, may combine to form a complex. The prefix "homo" means that a complex may be formed of identical antibody molecules, whereby the prefix "hetero" means that a complex may be formed of different antibody molecules. In general, the term "antibody" is intended to comprise all above-mentioned immunoglobulin isotypes, i.e. the antibody may be an IgA, IgD, IgE, IgG or IgM antibody, including any subclass of these isotypes. Preferably, the antibody is an IgG antibody, more preferred the antibody is an IgGl antibody. Since the antibody may be expressed and produced recombinantly, the antibody may also comprise two different constant regions of heavy chains, e.g. one IgGl and one IgG2 heavy chain, or heavy chains from different species. However, the heavy chains preferably are from the same species. Furthermore, the antibody may comprise either a lambda or a kappa light chain.

The antibody which recognizes the same epitope as one of the antibodies of the first aspect of the invention my further be 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, CDRL 1 , CDRL2, and CDRL3 have the amino acid sequences GFTFSSFGM H, YISSGSGN FY Y VDTV G, STYYHGSRG AM DY, SASSSVSSMYWY, DTSKMAS, and QQWSSYPPIT , respectively.

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

The antibody which recognizes the same epitope as one of the antibodies of the first aspect of the invention may also be an antibody which has an increased or lowered affinity or specificity in comparison to one of the antibodies of the first aspect of the invention. Such antibodies are readily obtained by methods known in the art and further described herein below.

Generally, the antibody according to the second aspect of the invention may have a sequence, especially in its variable regions, that 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 that of monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC PD n° 16001.

Usually, the antibody according to the invention may be a monoclonal, a bispecific or a multispecific antibody. Such antibodies are known in the art. As used in the context of the present invention, the term "monoclonal" may be understood in the broadest sense describing antibodies produced by a single clone of B lymphocytes or antibodies having the same or a similar aminoacid sequence. The term "bispecific", as used herein, may be understood in the broadest sense describing antibodies interacting with two different epitopes. The bispecific antibody may be derived from two monoclonal antibodies. Optionally, these two different epitopes may be localized on the same antigen, but they may also be localized on two different antigens. The term "multispecific", as used herein, may be understood in the broadest sense describing antibodies interacting with three or more different types of epitopes. Optionally, these epitopes may be localized on the same antigen or on two or more antigens. Preferably, the antibody according to aspect two of the present invention is a monoclonal antibody. Further, the antibody according to aspect two of the present invention preferably is a bispecific or a multispecific antibody.

Methods for the production of antibodies are well known to the person skilled in the art. Preferably, antibodies are produced by making hybridoma cells. Methods for the production of hybridoma cells as well as methods for the production of antibodies with the help of hybridoma cells are well-known to the person skilled in the art. Generally, mice are injected with the desired antigen and killed after a few days in order to isolate the spleen cells secreting the antibody against the desired antigen. In general, fusion of these antibody-secreting spleen cells with immortal non-secreting myeloma cells results to hybridoma cells. These hybridoma cells are then usually screened and the hybridoma producing the desired antibody is selected. The selected hybridoma may then be cultured in vivo or in vitro and the desired antibody can be isolated. Bifunctional, or bispecific, antibodies may have antigen binding sites of different specificities. Various forms of bispecific antibodies and their production are known to the person skilled in the art. For example, these include BSIgG, which are IgG molecules comprising two distinct heavy chains and two distinct light chains that are secreted by so-called "hybrid hybridomas", and heteroantibody conjugates produced by the 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). 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 cellular targets. (P. J. Lachmann, Clin. Exp. Immunol. 1990, 79: 315). Another important use may be the delivery of cytotoxic proteins to specific cellular 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 anti-cancer non-protein drugs to specific cellular 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 the production of hybrid-hybridomas (quadromas). Quadromas may be constructed by fusing hybridomas that secrete two different types of antibodies against two different antigens (Milstein and Cuello, Nature, 1983, 305: 537-539).

The term "epitope", as used in the context of the present invention, may be understood in the broadest sense as a portion of a CD43 molecule capable of being recognized by and bound by the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 at one or more of the antibody's antigen binding regions. The part of an antibody that binds to the epitope is called a paratope. In many cases, epitopes have conformational properties that specifically generate binding sites for the paratope.

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

Further, it is understood and appreciated by one skilled in the art that the interaction between the epitope and the antibody may generally be based on the primary structure of the antigen, i.e. a continuous sequence of amino acids. Usually, the interaction may also be based on the secondary structure, the tertiary structure or the quaternary structure of the epitope as well as post-translational modifications, such as glycosylation. The interaction between the epitope and the antibody may further be based on the three-dimensional structure and resulting surface features of the antigen, which may involve a discontinuous section of the amino acid sequence comprising amino acids at distant locations into the interaction with the antibody.

An antibody recognises "the same epitope" as the antibody according the first aspect, when the two antibodies recognise identical or sterically overlapping epitopes. In general, the most widely used and rapid methods for determining whether two epitopes recognise identical or sterically overlapping epitopes are competition assays, which usually may be configured in all number of different formats, using either labeled antigen or labeled antibody. For example, the 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 enzyme labels.

An antibody that recognises "the same epitope" as the antibody according to the first aspect usually refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody usually blocks binding of the antibody to its antigen in a competition assay by 50% or more.

In general, the epitope recognized by and bound by the antibody produced by the hybridoma cell deposited under 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 under ICLC PD n° 16001.

Examples of such a method include screening peptides of varying lengths derived from CD43 for binding to the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001, whereby the smallest fragment that can specifically bind to the antibody usually contains the sequence of the epitope recognised by the antibody. In general, CD43 peptides may be produced synthetically or by proteolytic digestion of CD43. Methods for the identification of peptides binding to the antibody are well-known to the person skilled in the art, such as mass spectrometric analysis. In another example, NMR spectroscopy, can be used to identify residues which interact with an antibody of the present invention. For example, a CD43 peptide that has been uniformly 15N and 2H labelled can be mixed with an unlabelled antibody and those amino acids in the labelled peptide that interact with the unlabelled antibody can be detected as their position within the NMR spectra change. Typically, the difference between the two spectra enables the identification of the amino acids in CD43 that are involved in the interaction with the antibody. Preferably, mass spectrometric analysis is used for the identification of peptides binding to the antibody.

Exemplarily, the epitope recognized by and bound by the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 may also be identified by a method comprising amplification of various DNA fragments of CD43 DNA by polymerase chain reaction (PCR), integration of these fragments into an expression vector comprising their connection to a histidine fusion protein and, following protein expression, detection of the epitope, for example by Western Blot. In a preferred embodiment, the antibody according to aspects one or two recognises an epitope comprising a GalNAc which is O-linked to human CD43.

In a further example, in order to determine the site on CD43 recognized by and bound by the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 , an expression vector cloned with CD43 may be introduced with deletion mutation by PCR method to prepare mutant series, such as Escherichia coli (E. coli) mutant series, that express proteins having various deleted sites in CD43. These E. coli mutants may be cultured and induced for expression. Western blot analysis may be carried out using the cell lysate as an antigen. Further methods for the identification of the epitope recognized by and bound by the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 may comprise detection via immunoassays, such as enzyme-linked immunosorbent assay (ELISA).

The term "affinity", as used in the context of the present invention, may be understood in the broadest sense as the strength of the interaction between an epitope and an epitope-binding site of an antibody. Methods for determining an absolute value for antibody affinity, i.e. the affinity constant, are well known to the person skilled in the art. However, also relative values of antibody affinities may generally be determined, i.e. the affinity of two antibodies is compared without determining their absolute values. Methods for comparing the affinities of antibodies are well-known to the person skilled in the art. For example, flow cytometry may be used, whereby cells having the desired epitope may independently be brought into contact with different antibodies, which are subsequently marked with an immunofluorescent secondary antibody. Usually, after detection with flow cytometry, the intensity of the signals of the antibodies can be compared. Methods for the identification of antibodies according to the second aspect, which recognise the same epitope as the antibody according to the antibody of the first aspect, are well-known to the person skilled in the art. For example, antibodies according to the second aspect may be identified by phage display based on antibody libraries. Consequently, the antibody of the invention recognizing the same epitope may also be a human antibody. 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. A chimeric antibody is an antibody, in which at least one region of an immunoglobulin of a species is fused to another region of an immunoglobulin of another species by genetic engineering in order to reduce its immunogenicity (see, e.g., U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397).

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.

In general, humanized antibodies are a particular type of chimeric antibodies. For example, humanized antibodies may be produced by grafting DNA of a human antibody into the mouse antibody framework coding DNA or by grafting DNA of a mouse antibody into human antibody framework coding DNA. Preferably, DNA of a human antibody is grafted into the mouse antibody framework coding DNA. In general, grafting of DNA comprises grafting of one or more DNA sequences into the target antibody framework coding DNA. Optionally, the variable and constant regions as well as heavy and light chains may be partially or fully humanized. Preferably, the heavy chain variable region and the light chain variable region of a mouse antibody are humanized. More preferably, the heavy chain variable region and the light chain variable region of a mouse antibody are humanized by changing a DNA sequence encoding 1 to 50, preferably, 1 to 30, more preferably 1 to 20 amino acids. In The DNA grafted may generally comprise DNA regions of the six hypervariable loops determining antigen specificity, also called complementarity-determining regions (CDR), or DNA regions not comprising a CDR, or both. Preferably, the humanization comprises grafting of DNA not comprising CDRs.

In general, the resulting DNA construct may then be used to express and produce antibodies that are usually less or not immunogenic in comparison to the non-human parental antibody. This includes the production of modified antibodies such as aglycosylated antibodies or afucosylated antibodies. Such methods are well-known in the art.

Consequently, the antibody of the invention recognizing the same epitope may also be an aglycosylated antibody or a afucosylated antibody.

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

Lymphocytes belong to the group of white blood cells and are mediators of humoral and cell-mediated immunity. There are two groups of lymphocytes, B-cells and T-cells.

Just like many other cell types, B- and T-cells, can abnormally develop to B- and T-cell tumors. Due to the numerous developmental stages of developing B- and T-cells, there are various kinds of tumors. Both, B-cells and T-cells, originate from lymphoid progenitor cells.

In the case of B-cells, this lymphoid progenitor cell develops via many B cell developmental stages each comprising a certain definable cell type until a plasma cell is formed. One of these stages includes the so-called "IgM-secreting B cell", which finally develops into an antibody-producing plasma cell. A tumor originating from an "IgM-secreting B-cell" is called "Waldenstrom's macroglobulinemia" (WM). WM is a rare, indolent and incurable disease. It is characterized by bone marrow accumulation of clonal IgM secreting lymphoplasmacytic cells.

T-cells develop from lymphoid progenitor cells to mature T-cells in only a few developmental stages. Tumors may especially 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. The T-cell phenotype T-ALL accounts for about 20% of all acute lymphoblastic leukemia cases and occurs more often in adults than in children. T-ALL is closely related to T-cell lymphoblastic lymphoma (T-LBL) and differential diagnosis between the two diseases is based on prevalent localization in specific sites, such as bone marrow in T-ALL or secondary lymphoid organ in T-LBL. The European Group for the Immunological Characterization of Leukemias (EGIL) classified T-ALL in four subgroups according to their immunophenotype (Bene MC, Leukemia 1995;9:1783):

1) EGIL Tl (pro-), characterized by cytoplasmic positivity for CD3 (cCD3) and surface expression of CD7;

2) EGIL T2 (pre-) characterized by positivity for cCD3, CD7 and positivity of CD2 or CD5;

3) EGIL T3 (cortical) characterized by positivity for cCD3, CDla and the presence or the absence of surface CD3 (sCD3) and

4) EGIL T4 (mature leukemia), characterized by the positivity for cCD3 and sCD3 and negative for CDla. The term "Antibody-dependent cellular cytotoxicity (ADCC)", as used herein, is the killing of a cell bound and marked by antibodies by a cytotoxic effector cell, such as natural killer (NK) cells. In order to examine, whether an antibody is capable of inducing ADCC, the following assay can be used. A degranulation assay by co-culturing peripheral blood mononuclear cells (PBMCs) from healthy donors, which include the effector cells, with target cells expressing the epitope in the presence of different antibody concentrations is performed. 4 xlO ⁴ target cells are seeded in 96 wells round-bottom plate and cultured for 30 minutes at 37°C 5% CO ₂ in the presence of different concentrations of antibody (0, 10, 50, 100 and 200 ug/ml) or control IgGl. Subsequently, 0.4 xlO ⁶ PBMCs (fixed effector cells (E): target cells (T) =10:1) from the same donor are added to each well together with 20 μΐ/ml of Phycoerythrin (PE)-conjugated anti-CD 107a monoclonal antibody (mAb) (BD) and cells are then incubated at 37°C 5% CO ₂ for 3h. After lh, 6 μg/ml monensin is added to each well (GolgiStop, BD). At the end of the incubation period, 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). By detecting CD37CD56 ⁺ /CD107a ⁺ cells, NK cells (CD37CD56 ⁺ ) inducing target cells lysis (CD107a ⁺ ) are measured. An increase of CD37CD56 /CD107a ⁺ cells according to increasing antibody concentrations therefore confirms the potential of an antibody to induce ADCC. The resulting data allow to design immune targeting approaches, which e.g. are an urgent and unmet clinical need in T cell acute lymphoblastic leukemias/lymphoblastic lymphomas. Further methods to examine, whether an antibody is capable of inducing ADCC, can also be used and are well-known to the person skilled in the art.

In a third aspect, the invention relates to a binding molecule derived from an antibody according to aspect one or aspect two.

According to the invention, a binding molecule is a molecule derived from the monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC PD n° 16001. Preferably, the binding molecule is an immunoglobulin comprising molecule, i.e. it comprises at least one Immunoglobulin (Ig) domain.

In a preferred embodiment the binding molecule of the invention is being selected from the group consisting of single chain antibodies. In a more preferred embodiment, the binding molecule is being selected from the group consisting of a single chain variable fragment (scFv), a multimer of a scFv, such as a diabody, a triabody or a tetrabody, antibody fragments, preferably a Fab, a tandab, and a flexibody. The structure of an antibody and especially the function of its CDRs are generally known in the art (Carter PJ. Potent antibody therapeutics by design. Nature Rev. Immunol. 6:343-357, 2006). Single chain Fv (scFv) and multimers thereof, tandabs, diabodies and flexibodies are in general standard antibody formats known in the art, e.g. from WO 1988/001649 Al, WO 1993/011161 Al, WO 1999/057150 A2 and EP1293514B1.

In a scFv, the two antigen binding variable regions of the light and heavy chain (V _H Fv and V _L Fv) of an antibody are in general artificially connected by a linker peptide, designated as single chain variable fragment or single chain antibody (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 can be made up of the variable domains of light and heavy chains of a monoclonal antibody. Several investigations have shown that the scFv fragment may have indeed the full intrinsic antigen binding affinity of one binding site of the whole antibody.

In the context of this invention, diabodies are scFv with two binding specificities and can either be monospecific and bivalent or bispecific and bivalent. Tandabs and flexibodies are further antibody formats which are e.g. defined in US2007031436 and EP1293514B1, respectively.

Antibody fragments that contain the idiotypes of the protein can be generated by techniques known in the art. For example, such fragments include, but are not limited to, the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragment that can be generated by reducing the disulfide bridges of the F(ab')2 fragment; the Fab fragment that can be generated by treating the antibody molecular with papain and a reducing agent; and Fv fragments.

The antibody or binding molecule of the invention can further be linked to an active substance, 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 Al, WO 2007/030642 A2, WO 2004/067038 Al, WO 2004/003183 Al, US 2005/0074426 Al, WO 1994/004189 Al). The invention further relates in its further aspect to a chimeric antigen receptor (CAR) comprising a binding molecule of aspect three linked to an intracellular domain preferably comprising one or more signaling domains.

Preferably, the invention relates to a chimeric antigen receptor (CAR) comprising the scFv of the preferred embodiment of the binding molecule of aspect three linked to an intracellular region comprising the CD3ζ chain, the signaling region of the T cell receptor, and to the two co-stimulatory domains CD28 and 4-1BB. The CAR according to the invention is a relevant tool for targeting malignant cells bearing the epito e recognized and bound by the monoclonal antibody of aspect one or aspect two, when expressed in T- cells or NK cells. The term "Chimeric antigen receptors" (CAR), as used herein, refers to synthetic receptors comprising a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR comprises scFv, but it may also comprise other binding entities. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for CARs can be derived from the cytoplasmic region of the CD3ζ or the Fc receptor gamma chains, but may also be derived from other cytoplasmic regions. First generation CARs have been shown to successfully redirect T-cell cytotoxicity. Signaling domains from co-stimulatory molecules, as well as transmembrane and hinge domains have been added to form CARs of second and third generations, leading to some successful therapeutic trials in humans, where T-cells could be redirected against malignant cells expressing CD19 (Porter DL et al., N Eng J Med, 2011).

In a fifth aspect, the invention relates to an expression vector comprising a nucleic acid sequence which encodes the chimeric antigen receptor according to aspect four, the antibody according to the aspects one and two or the binding molecule according to the binding molecule according to aspect three.

Generally, expression vectors are plasmids which are 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. the chimeric antigen receptor, the antibody or the binding molecule. Therefore, the expression vector in general comprises regulatory sequences, such as promoter and enhancer regions, as well as a polyadenylation site in order to direct efficient transcription of the nucleic acid sequence on the expression vector. The expression vector may further comprise additional necessary or useful regions, such as a selectable marker for selection in eukaryotic or prokaryotic cells, a purification tag for the purification of the resulting protein, a multiple cloning site or an origin of replication.

Usually, the expression vector may be a viral or a non- viral vector. In general, various kinds of viral vectors, such as retroviral vectors, e.g. lentiviral or adenoviral vectors, or plasmids may be used. In a preferred embodiment, the expression vector according to aspect five is a viral vector. In a more preferred embodiment, the expression vector is a lentiviral vector. In a sixth aspect, the invention relates to a CD3 lymphocyte, an NK lymphocyte, a Cytokine induced killer (CIK) cell, a gamma-delta lymphocyte, an NKT cell or another immune effector cell comprising the chimeric antigen according to aspect four or the expression vector according to aspect five. Generally, CD3 is a complex of four signaling chains associated to the α:β heterodimer of the T-cell receptor in a functional T-cell receptor complex. The CD3 complex is usually required for T-cell receptor signaling. In general, the group of CD3 ⁺ lymphocytes exclusively contains thymocytes and T- cells. Detection of CD3 ⁺ cells can be achieved by e.g. flow-cytometry. In a seventh aspect, the invention relates to a pharmaceutical composition comprising the monoclonal antibody according to aspects 1 or 2 or the binding molecule according to aspect three or the CD3 ⁺ lymphocyte, the NK lymphocyte, the Cytokine induced killer (CIK) cell, the gamma-delta lymphocyte, the NKT cell or the other immune effector cellaccording to aspect six. The term "pharmaceutical composition", as used herein, may be interchangeably used with the term "drug".

The content of the antibody, the binding molecule or the CD3 ⁺ lymphocyte in the pharmaceutical composition is not limited as far as it is useful for treatment or prevention, but preferably contains 0.0000001-10% by weight per total composition. Further, the antibody, the binding molecule or the CD3 ⁺ lymphocyte described herein are preferably employed in a carrier. The choice of carrier may depend upon route of administration and concentration of the active agent(s) and the carrier may be in the form of a lyophilised composition or an aqueous solution. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to render the composition isotonic. Examples of the carrier include but are not limited to saline, Ringer's solution and dextrose solution. Preferably, acceptable excipients, carriers, or stabilisers are non-toxic at the dosages and concentrations employed, including buffers such as citrate, phosphate, and other organic acids; salt-forming counter- ions, e.g. sodium and potassium; low molecular weight (> 10 amino acid residues) polypeptides; proteins, e.g. serum albumin, or gelatine; hydrophilic polymers, e.g. polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrins; monosaccharides; disaccharides; other sugars, e.g. sucrose, mannitol, trehalose or sorbitol; chelating agents, e.g. EDTA; non-ionic surfactants, e.g. Tween, Pluronics or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and/or preservatives, e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g. methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Suitable carriers and their formulations are described in greater 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.

Preferably, the antibody, the binding molecule, the CD3 ⁺ lymphocyte and/or the 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 to be administered.

In an eighth aspect, the invention relates to a nucleic acid or polynucleotide, encoding the antibody according to aspects one or two or the binding molecule according to aspect three.

Also provided herein are polynucleotides encoding an antibody that are optimized, e.g., by codon/RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods to generate optimized nucleic acids encoding an antibody or a fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and/or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly. For example, potential splice sites and instability elements (e.g., A/T or A/U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression. The alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid. In some embodiments, it can be desirable to alter one or more codons to encode a conservative mutation, e.g., a similar amino acid with similar chemical structure and properties and/or function as the original amino acid. Such methods can increase expression of an 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 relative to the expression of an antibody encoded by polynucleotides that have not been optimized.

In certain embodiments, an optimized polynucleotide sequence encoding an antibody described herein or a fragment thereof (e.g., VL domain and/or VH domain) can hybridize to an antisense (e.g., complementary) polynucleotide of an unoptimized polynucleotide sequence encoding an antibody described herein or a fragment thereof (e.g., VL domain and/or VH domain). In specific embodiments, an optimized nucleotide sequence encoding an antibody described herein or a fragment hybridizes under high stringency conditions to antisense polynucleotide of an unoptimized polynucleotide sequence encoding an antibody described herein or a fragment thereof. In a specific embodiment, an optimized nucleotide sequence encoding an antibody described herein or a fragment thereof hybridizes under high stringency, intermediate or lower stringency hybridization conditions to an antisense polynucleotide of an unoptimized nucleotide sequence encoding an antibody described herein or a fragment thereof. Information regarding hybridization conditions has been described, see, e.g., U.S. Patent Application Publication No. US 2005/0048549 (e.g., paragraphs 72-73). The polynucleotides of the invention can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Nucleotide sequences encoding 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 particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody. Such a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides {e.g., as described in Kutmeier G et ah, (1994), BioTechniques 17: 242-6), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described herein can be generated from nucleic acid from a suitable source {e.g., a hybridoma) using methods well known in the art {e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the light chain and/or heavy chain of an antibody. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the variable light chain region and/or the variable heavy chain region of an antibody. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source {e.g., an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody described herein) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

DNA encoding the antibodies of the invention described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the antibodies in the recombinant host cells.

To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones or other clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a heavy chain constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a light chain constant region, e.g., human kappa or lambda constant regions. In certain embodiments, the vectors for expressing the VH or VL domains comprise a promoter, a secretion signal, a cloning site for the variable region, constant domains, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co- transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.

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

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

In a ninth aspect, the invention relates to a hybridoma cell that produces the monoclonal antibody according to the antibody of aspects one or two. In a tenth aspect, the invention relates to the hybridoma deposited under ICLC PD n° 16001. In an eleventh aspect, the invention relates to a method for producing the monoclonal antibody according to aspects one or two, said method comprising isolating said antibody from the hybridoma cell deposited under ICLC PD n° 16001. In a twelfth aspect, the invention relates to a method for the identification or isolation of T-cell acute lymbhoblastic leukemia cells, T lymphoma cells, Waldenstrom's Macroglobulinemia cells or tumor- associated macrophages, comprising contacting a cell sample comprising said cells with the monoclonal antibody according to aspects one or two or with the binding molecule according to aspect three.

In general, macrophages are the most represented non-malignant cells in the tumor microenvironment. These tumor associated macrophages (TAM) are considered to acquire a pro-tumoral inflammatory and immune-suppressive phenotype and to favor chemo-resistance, angiogenesis, cell motility and intra/extravasation. Therefore, targeting TAM may represent a novel therapeutic and still unexplored clinical option to improve the efficacy of current anticancer treatments.

Methods for the identification or isolation of specific cells, such as T-cell acute lymbhoblastic leukemia cells, T lymphoma cells, Waldenstrom's Macroglobulinemia cells or tumor-associated macrophages, based on antibodies or binding molecules in general are well-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 cell sorters.

In a thirteenth aspect, the invention relates to a method for producing CD3 ⁺ lymphocytes, NK lymphocyte, the Cytokine induced killer (CIK) cells, gamma-delta lymphocytes, NKT cells or the other immune effector cells expressing a chimeric antigen receptor according to the chimeric antigen receptor of aspect four comprising the introduction of the expression vector according to the expression vector of aspect five into said CD3 ⁺ lymphocytes, NK lymphocyte, the Cytokine induced killer (CIK) cells, gamma-delta lymphocytes, NKT cells or the other immune effector cells. This includes also the possibility to produce a single cell or introducing the expression vector into a single cell.

In a fourteenth aspect, the invention relates to a method for the identification or isolation of CD45 ⁺ , CD3 ⁺ , CD8 ^" , CD127 ⁺ , CCR7 ⁺ T lymphocytes, comprising contacting a cell sample comprising said T lymphocytes with the antibody according to aspects 1 or 2 or with the binding molecule according to aspect three. A preferred embodiment is the antibody according to the antibody of aspects one or two, the binding molecule according to aspect three, the expression vector according to aspect five, the CD3 ⁺ lymphocyte, the NK lymphocyte, the Cytokine induced killer (CIK) cell, the gamma-delta lymphocyte, the NKT cell or the other immune effector cell according to aspect six or the pharmaceutical composition according to aspect seven for use in a method for the treatment of T-cell acute lymphoblastic leukemia or Waldenstrom's Macroglobulinemia.

In addition, also other malignancies could be treated wherein the cells express the epitope recognized by the antibody of the aspect 1 and 2 of the invention.

An additional preferred embodiment is the antibody according to aspects one or two or the binding molecule according to aspect three for use in a method for the identification or isolation of CD45 ⁺ , CD3 ⁺ , CD8 ^" , CD127 ⁺ , CCR7 ⁺ T lymphocytes. Another preferred embodiment is the antibody according to the antibody of aspects one or two or the binding molecule according to aspect three for use in a method for the isolation or identification of T- cell acute lymbhoblastic leukemia cells, T lymphoma cells, Waldenstrom's macroglobulinemia cells or tumor-associated macrophages. An additional preferred embodiment is the antibody according to the antibody of aspects one or two or the binding molecule according to aspect three for use in a method for the diagnosis of T-cell acute lymphoblastic leukemia or Waldenstrom's Macroglobulinemia.

In addition, also other malignancies could be diagnosed wherein the cells express the epitope recognized by the antibody of the aspect 1 and 2 of the invention.

In the following, the invention is further described by the figures and examples, which are intended to explain, but not to limit the invention. Description of the figures

Figure 1 : Expression of the epitope of the antibody produced by the hybridoma cell deposited according to the invention on peripheral blood mononuclear cells of a panel of healthy donors and comparison to a state-of-the-art CD43 -detecting antibody.

The top scatterplot presents data obtained by flow cytometry. The x-axis presents the forward scatter detected (FSC), the y-axis depicts the side scatter (SSC). Each dot corresponds to one cell. The histogram below depicts on the x-axis the phycoerythrin signal intensity. The y-axis relates the signal intensities to the maximum signal intensity of the unstained sample being 100%. The unfilled curve represents the unstained control, the curve filled with horizontal stripes represents the scramble IgGl stained cells (i.e. the negative control), the curve filled with crossed stripes represents the mAb UNI stained cells and the curve filled with diagonal stripes represents the CD43 stained cells)

Figure 2: Cell population recognized by the antibody produced by the hybridoma cell deposited according to the invention.

Figure 2 shows four scatterplots. The two scatterplots on the left belong to lymphocytes. The two scatterplots on the right are from lymphocytes detected by the antibody produced by the hybridoma cell deposited according to the invention. In the two scatterplots on the top, the x-axis represents CD4 signal intensity, while the y-axis depicts CD8 signal intensity. In the two scatterplots on the bottom, the x-axis represents CD45ro signal intensity and the y-axis represents CCR7 signal intensity.

Figure 3 shows two histograms. The histogram on the top presents the expression of the epitope UNI detected by the antibody produced by the hybridoma cell deposited according to the invention (mAb UNI) on BCWM.l cell line. The histogram on the bottom presents UNI expression on MWCL. l cell line. The unfilled curve represents the unstained control, the curve filled with horizontal stripes represents the secondary mAb stained cells, the curve filled with vertical stripes represents the scramble IgG plus secondary stained cells and the curve filled with diagonal stripes represents cells stained by mAb UNI .

Figure 4: tumor associated macrophages (TAM) recognized by the antibody produced by the hybridoma cell deposited according to the invention. White arrows indicate TAM infiltrating a specimen of colorectal carcinoma.

Figure 5: THP1 -derived macrophages stained with: control IgGl in absence of tumor cells (first row), UMG1-CH (chimeric antibody according to aspect 2 of this invention, where the original murine Fc region was replaced with a fully human IgGl Fc region) in absence of tumor cells (second row) and UMG1-CH in presence of PANC1 pancreatic cancer cell line (third row and particular on the left). The first column represents the DAPI staining, the second column the antibody plus alexa-fluor 488 labeled secondary antibody and the third column represents the superimposed image. Figure 6: Results of the degranulation assay to evaluate ADCC in HPB-ALL (left diagram) and H9 cell lines (right diagram). The numbers on the x-axis represent the different samples, meaning: no target (1), E+T (2), Negative control (NC) 200 μ^ιηΐ (3), UMG1-CH 10 μ^ιηΐ (4), UMG1-CH 50 μg/ml (5), UMG1-CH 100 μ^ιηΐ (6), UMG1-CH 200 μ^ιηΐ (7), Positive control (PC) 200 μ^ιηΐ (8). The y-axis represents the percentage of CD107a ⁺ NK cells affected by ADCC related to the whole number of CD107a ⁺ NK cells tested per sample.

Figure 7: Results of the degranulation assay to evaluate ADCC in BCWM. l cell line. The numbers on x-axis represent the different samples. The numbering is according to that of Figure 6. The y-axis represents the percentage of CD107a ⁺ NK cells affected by ADCC related to the whole number of CD107a ⁺ NK cells tested per sample.

Figure 8: CD3 ⁺ expressing lymphocytes (CAR-T) were able to release significantly higher amount of Interferon gamma (IFNy) in the presence of H9 cells. The y-axis report the concentration of IFNy expressed in ng/ml. The x-axis reports: non-transduced T cells (1), T cells transduced with a control CAR (2) and T cells transduced with UMG-1 CAR (3).

Figure 9: CAR-T were able to release significantly higher amount 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 reports: non-transduced T cells (1), T cells transduced with a control CAR (2) and T cells transduced with UMG-1 CAR (3).

Figure 10: CAR-T were able to induce selective killing of H9 cells. The y-axis reports the dead/live cells ratio. The x-axis reports: 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 : This figure shows tumor volume curves of an in vivo experiment comparing a control IgGl versus the humanized version of UNl-mAb (h-UNl) and the afucosylated version of UNl-mAb (a-h- UNI).

-·- Control IgGl

→- h-UNl

a-h-UNl Examples

The following examples are provided for the purpose of illustrating the invention, but should not be construed as limiting the invention. The examples comprise technical features, and it will be appreciated that the invention also relates to any combinations of the technical features presented in this exemplifying section.

Example 1 Expression of the epitope of the antibody produced by the hybridoma cell deposited according to the invention on peripheral blood mononuclear cells of a panel of healthy donors and comparison to a state-of-the-art CD43 -detecting antibody

First, peripheral blood mononuclear cells (PBMCs) from different healthy donors were obtained by Ficoll gradient separation. Subsequently, cells were seeded in 5 ml tubes and stained with 1 μg/ml of mAb produced by the hybridoma cell deposited under ICLC accession number ICLC PD n° 16001(mAb UNI) or 1 μg/ml of a scramble murine IgGl antibody in 100 μΐ of binding solution (phosphate buffered saline (PBS) + 0.5% fetal bovine serum (FBS)) and incubated at 4°C for 30 minutes. Cells were then washed 2 times in binding solution and stained with a Fluorescein isothiocyanate (FITC)-conjugated secondary antibody at 4°C in the dark for 30 minutes. Subsequently, cells were washed 2 times 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 with the FITC-conjugated secondary antibody only. Overall, the antibody produced by the hybridoma cell deposited according to the invention was able to recognize a variable lymphocyte subpopulation (range: 0-15%) of different donors. Moreover, it did not show any reactivity with all other cell populations within the PBMCs, including myeloid derived cells, which therefore were negative for expression of the respective antigen (see Figure 1, top).

In contrast, when we assayed the same PBMCs for CD43 expression by using a commercial clone (S7 from Beckton Dickinson), all lymphocytes and myeloid cells were found to be positive (see Figure 1, bottom).

Consequently, the antibody produced by the hybridoma cell deposited according to the invention exhibits a specific restricted pattern of reactivity and properties that already existing antibodies against CD43 do not have. Example 2

Cell population recognized by the antibody produced by the hybridoma cell deposited according to the invention

To characterize the lymphocyte sub-population detected by the antibody produced by the hybridoma cell deposited according to the invention, we performed an immune-magnetic sorting of the respective lymphocytes (Easysep do-it-yourself, Stemcell technologies). Briefly, 15 μg of the antibody were mixed with components provided by the manufacturer to obtain a solution ready for immunomagnetic separation. This solution was added to PBMCs from 3 different donors having at least 10% of lymphocytes detected by the antibody, after FcR blocking, and cells were incubated at room temperature (r.t.) for 15 minutes. Subsequently, EasySep® Magnetic Nanoparticles were added to the solution and cells were incubated for further 10 minutes at r.t. The solution was then placed in a magnet and unbound cells were removed. Cells detected by the antibody according to the invention were almost all CD45 ⁺ CD3 ⁺ CD4 ⁺ CD8 ^~ CD127 ⁺ CCR7 ⁺ T lymphocytes with the majority of them CD45ro ^" (see Figure 2 and Table 1).

Table 1 : Marker of UNI antigen-positive cells

Example 3

The antibody produced by the hybridoma cell deposited according to the invention (mAb UNI) recognizes T-ALL and Waldenstrom's Macroglobulinemia cell lines.

Various cancer cell lines (Table 2) were evaluated for the expression of UNI (MM: multiple myeloma). Briefly, cells were seeded in 5 ml tubes and stained with 1 μg/ml of mAb UNI or 1 μg/ml of a scramble murine IgGl antibody in 100 μΐ of binding solution (phosphate buffered saline (PBS) + 0.5% fetal bovine serum (FBS)) and incubated at 4°C for 30 minutes. Cells were then washed 2 times in binding solution and stained with a Fluorescein isothiocyanate (FITC)-conjugated secondary antibody at 4°C in the dark for 30 minutes. Subsequently, cells were washed 2 times 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 the FITC-conjugated secondary antibody only. It was observed that T-ALL cell lines belonging to EGIL T3 classification and Waldenstrom's macroglobulinemia (Figure 3) were all positive for UNI expression.

Table 2: UNI expression of cancer cell lines.

Cell lines Cancer type UI

H929 MM -

AMO MM -

U266 MM -

KMS11 MM -

8226 MM -

JURKAT T-ALL -

CEM T-ALL +/ ^■

HSB2 T-ALL -

Thpl Monocytic leukemia -

MOJ Glioblastome -

SKMEL Melanome -

HCC Breast -

MCF-7 Breast -

5637 Bladder -

CAP AN 1 Pancreatic -

BXPC 3 Pancreatic Example 4

The antibody produced by the hybridoma cell deposited according to the invention (mAb UNI) recognizes tumor-associated macrophages.

As already described, CD43 is a specific leukocyte marker generally restricted to different cells of the hematopoietic lineage. It as found that the specific epitope bound by mAb UNI and on CD43 isoform is highly expressed by tumor-associated macrophages (TAM).

By evaluating specimen from different kinds of cancer through immunohistochemistry, (Table 3, Figure 4), it was observed that UN1+ macrophages are a high infiltrating component of most of tumors, with a specific and particular high grade of infiltration in pancreatic and ovarian cancer.

Moreover, it was evaluated in a model of macrophages differentiation whether UNI expression changes in the presence or absence of co-cultured cancer cells.

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

As shown in Figure 5 on the right, THP1 -derived macrophages stained with control IgGl were completely negative, while those stained with UMG1-CH were weakly positive. Interestingly, in presence of PANCl, THP1 -derived macrophages became bright for UNI expression. A particular of the interaction between THP1 -derived macrophages (white arrow) and PANCl (red arrow) is shown (Figure 5, on the left). These findings demonstrate that the UNI specific epitope is significantly upregulated when macrophages are co-cultured and interact with cancer cells within a reconstituted tumor microenvironment. This upregulation is relevant by itself as well represented target for therapeutic approaches focalized to tumor-associated macrophage purging. Beyond this relevant potential as therapeutic tool, mAb UNI, might also provide usefulness for detection, analysis of prognostic role and predictive studies. Table 3: UN1 ⁺ TAM infiltration in various tumors

Cancer Type UN1 ⁺ TAM infiltration

(+: low; ++: medium; +++: high)

Hodgkin Lymphoma

Non-Hodgkin

Lymphoma

Colorectal cancer

Lung cancer

Breast cancer

Ovarian cancer ++- Bladder cancer

Example 5

Chimeric mAb UMG1-CH (according to aspect 2 of this invention) is an active immunotherapeutic tool for T cell acute lymphoblastic leukemias/lymphoblastic lymphomas.

To determine the potential activity of mAb UMG1 -CH as immunotherapeutic tool, its ability to induce antibody-dependent cell mediated cytotoxicity (ADCC) was evaluated first. To this end, a degranulation assay by co-culturing PBMCs from healthy donors (effector cells) with HPB-ALL or H9 T-ALL cell lines (target cells) was performed in the presence of different concentration of mAb UMG1-CH as follows: 4 x10 target cells were seeded in 96 wells round-bottom plate and cultured for 30 minutes at 37°C 5% C0 ₂ in the presence of different concentrations of mAb UMG1-CH (0, 10, 50, 100, 200 μ^ητΐ) or chimeric negative or positive control (NC and PC respectively, 200 μg/ml each) IgGl at the highest dose (200 μg/ml). Subsequently, 0.4 xlO ⁶ PBMCs (fixed E:T=10: 1) from the same donor were added to each well together with 20 μΐ/ml of PE-conjugated anti-CD 107a mAb (BD) and cells were then incubated at 37°C 5% CO ₂ for 3h. After lh, 6 μg/ml monensin was added to each well (GolgiStop, BD). At the end of the incubation period, 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+ cells were found to significantly increase according to mAb UMGl-CH concentration thus confirming the potential of mAb UMGl-CH as ADCC inducer (Figure 6).

These data allow to design immune targeting approaches, which are an urgent and unmet clinical need in T cell acute lymphoblastic leukemias/lymphoblastic lymphomas.

Example 6

Chimeric mAb UMGl-CH (aspect two of this invention) is an active immunotherapeutic tool for Waldenstrom's Macroglobulinemia.

To investigate the immunotherapeutic potential of mAb UMGl-CH, we evaluated its ability to induce antibody-dependent cell mediated cytotoxicity (ADCC). To this end, we performed a degranulation assay by co-culturing purified NK cells from healthy donors (effector cells) and BCWM.l cell line (target cells) in the presence of different concentrations of mAb UMGl-CH or negative/positive controls. We selected the mAb cetuximab as negative control and the mAb rituximab as positive control. Specifically, 10 ⁵ target cells were seeded in 96 wells round-bottom plate and cultured for 30 minutes at 37°C 5% C0 ₂ in the presence of different concentration of mAb UMGl-CH (0, 10, 50, 100, 200 μg/ml), 200 ug/ml cetuximab or 200 μg/ml rituximab. Subsequently, 10 ⁵ NK cells (fixed E:T=1 :1) from the same donor were added to each well together with 20 μΐ/ml of PE-conjugated anti-CD 107a mAb (BD) and cells were then incubated at 37°C 5% CO ₂ for 2h. After lh, 6 μg/ml monensin was added to each well (GolgiStop, BD). At the end of the incubation period 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+ cells were found to significantly increase according to mAb UMGl-CH concentration, reaching exactly the same effect obtained with rituximab, thus confirming the potential of the mAb as ADCC inducer (Figure 7).

Example 7 Chimeric antigen receptor (CAR)-UMG1 induces significant cytoxicity against cells expressing the epitope of the antibody produced by the hybridoma cell deposited according to the invention.

To further improve the immunotherapeutic potential of the antibody produced by the hybridoma cell deposited according to the invention as immunotherapeutic tool, a third generation CAR was developed. Specifically, a CAR-T was designed by coupling an extracellular domain, consisting of a scFv derived from the sequence of the antibody, with an intracellular region consisting of the CD3ζ chain, the signaling region of the TCR, and 2 co-stimulatory domains, CD28 and 4- IBB, thus mimicking physiological T-cell activation. To this end, the antibody scFv was cloned with the 3 selected co-stimulatory domains into a CAR cassette to generate a lentivirus vector. Subsequently, viral particles were used to transduce CD3 ⁺ lymphocytes from healthy donors at a multiplicity of infection (MO I) of 5 and transduction efficiency was evaluated by flow cytometry (about 38%). CAR- T against the epitope of the antibody were finally assayed for their ability to release IFNy and IL-2 in the presence of target cells and for their selective cytotoxicity capability. As shown in Figure 8 and Figure 9, CAR-UMG1 was able to release significantly higher amounts of Interferon gamma (IFNy) and Interleukin 2 (IL-2) only in the presence of H9 cells. Additionally, 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 inducing T-cell activation.

Example 8

This example reports tumor volume curves of an in vivo experiment comparing a control IgGl versus the humanized version of UNl-mAb (h-UNl) and the afucosylated version of UNl-mAb (a-h-UNl). In this experiment 15 NOD-SCID-g-chain-null (NSG) mice have been subcutaneously engrafted with 5x10 ^Λ 6 HPB-ALL cells. Mice were then randomized to receive weekly intra-peritoneal administration of 15mg/kg of control IgGl, h-UNl or a-h-UNl starting from dayl until death, tumor volume >2000mm ^A 3 or unacceptable toxicity. Tumor volume was assessed every other days and average volume of each group at each time point is reported in the Figure 11. Starting from day 29 both h-UNl and a-h-UNl presented a significantly reduced disease burden, confirming in vivo the strong activity of both antibodies.

1/1

Print Out (Original in Electronic Form)

(This sheet is not part of and does not count as a sheet of the international application)

Indications are Made All designations

FOR RECEIVING OFFICE USE ONLY -4 This form was received with the

international application:

YES

(yes or no)

-4-1 Authorized officer

Benzler, Annemarie

FOR INTERNATIONAL BUREAU USE ONLY -5 This form was received by the

international Bureau on:

-5-1 Authorized officer