NEW MONOCLONAL ANTIBODY OF IgM ISOTYPE ANTI-HLA-DR POTENTIATING THE ACTIVITY OF IgG1 OR IgM ISOTYPE ANTIBODIES ANTI-CD20

Abstract

The present invention relates to a novel anti-HLA-DR IgM monoclonal antibody and its use alone or in combination with anti-CD20 IgG1 or IgM antibodies in the treatment of lymphoma, and in particular chronic lymphocytic leukemia (CLL). Figure 4B

Description

Title of the invention: NOVEL MONOCLONAL ANTIBODY OF THE IgM ISOTYPE ANTL HLA-DR ENHANCING THE ACTIVITY OF IgGl OR IgM ISOTYPE ANTIBODIES ANTI-CD20 Technical field of the invention

[0001] The present invention relates to a new monoclonal antibody of IgM isotype anti-HLA-DR and its use alone or in addition to IgGl or IgM anti-CD20 isotype antibodies, in the treatment of malignant hematological diseases of B phenotype, such as chronic lymphocytic leukemia (CLL) and B non-Hodgkin lymphomas.

[0002] The present invention has an application in human health, for example in immuno-hematology, hemato-oncology and oncology, and malignant lymphoid B-cell hemopathies.

[0003] In the description below, references in brackets ([ ]) refer to the list of references at the end of the text. State of the art

[0004] Over the past few decades, immunotherapy has become one of the most remarkable advances in the field of cancer treatment, particularly of lymphoid malignancies, essentially of B phenotype. Unlike traditional approaches such as chemotherapy and radiotherapy, which are mainly aimed at directly eliminating tumor cells, immunotherapy harnesses the power of the immune system to fight the disease.

[0005] The remarkable successes of immunotherapy in the treatment of certain types of cancer have aroused great interest and opened up new perspectives in the field of drug research and development.

[0006] For anti-tumor immunotherapy, humanized or human divalent IgGl monoclonal antibodies (mAbs) represent the majority (80%) of antibodies approved for various oncological and hematological indications.

[0007] An example of current immunotherapy is that which targets the CD20 protein, or cluster of differentiation 20, which is a transmembrane glycoprotein expressed primarily on the surface of mature B lymphocytes. Its main role is to act as a crucial regulator of B lymphocyte proliferation and differentiation, as well as in their activation and immune function. It has become an important therapeutic target in the treatment of various diseases, in particular B-type lymphoproliferative disorders such as B-cell non-Hodgkin lymphomas and chronic lymphocytic leukemia.

[0008] Thus, the treatment of B-cell lymphoid malignancies involves the use of anti-CD20 monoclonal antibodies, which allow for the elimination of pathological and clonal B lymphocytes while preserving the functions of other immune cells, particularly T and NK lymphocytes. For example, rituximab (RTX) (type I anti-CD20 monoclonal antibody) has demonstrated significant clinical activity against the majority of B-cell neoplasms, such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). RTX can be used alone in indolent B-cell non-Hodgkin lymphomas, such as FL, as immunotherapy, or in combination with chemotherapy in aggressive B-cell non-Hodgkin lymphomas as immunochemotherapy.

[0009] In chronic lymphocytic leukemia (clonal disease of CD20+ CD19+ CD5+ CD23+ B lymphocytes), the reference anti-CD20 monoclonal antibody is Obinituzumab (OBZ) (type II anti-CD20 monoclonal antibody), used, according to current clinical recommendations, in combination with a Bruton's tyrosine kinase inhibitor (BTKi) or with an inhibitor of the B-cell lymphoma 2 oncogene, Bcl-2 (venetoclax).

[0010] Today, chemo-free approaches are becoming the norm in clinical practice and clinical research protocols, as well as the preferred choice for patients, compared to immunochemotherapy. When an anti-CD20 monoclonal antibody (mAb) is used as monotherapy, for example, rituximab (RTX), measurable residual disease persists after treatment (incidence of 67%) [1-3]. Continuing the chemo-free therapeutic approach requires enhancing the bioactivity of anti-CD20 mAbs or other anti-B-cell mAbs (anti-CD19, anti-CD38 (daratumumab and isatuximab), anti-CD47, etc.) without causing toxicity.Beyond their use as a single therapeutic agent or as reference antibodies in clinical recommendations, the current step is to consider their combination with a tumor-targeting therapy such as an iBTK, venetoclax or with a chromatin modulator or another agent (alisertib, TGF-[3, FOXO1 or famesyltransferase inhibitor) [4-10].

[0011] Despite its very promising advantages, immunotherapy presents challenges, including variation in patient response, the identification of predictive biomarker(s) of clinical response to an immunotherapeutic agent and / or targeted therapy, and the possible emergence of biological resistance (e.g., downregulation of the epitope). Achieving measurable residual disease (MRD) or minimal residual disease (MRD) is a major challenge for ensuring a favorable clinical outcome for the patient. favorable on disease-free survival or "progression-free survival" or PFS, and overall survival or "overall survival" or OS.

[0012] Mechanisms of resistance to IgGl isotype mAbs can exist naturally (primary resistance) or arise after treatment (acquired resistance in residual tumor cells). The level of expression of surface markers such as CD20 density and high expression of CD55 and CD59 proteins induces resistance to RTX. Resistance to RTX also involves the overexpression of Bcl-2 or Bcl-xL molecules, as anti-apoptotic factors.

[0013] In CLL, OBZ is the standard antibody therapy used, according to clinical guidelines, in combination with anti-Bcl2 venetoclax (treatment limited to 2 years, achievable non-measurable minimal residual disease (incidence of 15-25%), very expensive) or with BTK inhibitors (continuous lifelong treatment, persistent MDR (incidence of 70%), very expensive) [1-3]. OBZ alone or in combination with BTK inhibitors is effective but leads to measurable and always detectable minimal residual disease, requiring long-term treatment and resulting in significant medical and economic costs.

[0014] The current challenge in immunotherapy research is to define the best combination of immunotherapy and / or targeted therapy(ies), without the addition of chemotherapy and without introducing additional toxicity. Furthermore, in the context of using therapeutic monoclonal antibodies (mAbs), the question arises as to the best isotype (IgG, IgA, IgM) of monoclonal antibody to use at the patient's bedside. To date, IgG1, with its divalent structure, represents the majority (80%) of approved and authorized antibodies for various hematological and oncological indications.

[0015] IgM monoclonal antibodies have entered the therapeutic arena. Due to their natural format, which can be either pentameric or hexameric, their bioactivity and anti-tumor potency can be considered potentially superior to those of IgG, due to their superior complement-dependent cytotoxicity (CDC) and their multivalence, as they have the advantage of possessing numerous binding sites for their target (10 or 12 sites, respectively). This multivalence of IgM is capable of inducing strong multimerization of epitope targets on the cell surface, cell agglutination capabilities, and thus inducing intracellular signal transduction pathways and / or cytokine responses (interferon-gamma, IL-12, etc.), leading to a significant reduction in the viability of tumor cells and their death.

[0016] The IgM isotype was thus introduced into the generation of monospecific and bispecific antibodies against solid and hematological malignancies (lgM-8444, DR5 receptor crosslinking agonist) and against refractory and resistant non-Hodgkin lymphomas and CLL (anti-CD20 x CD3 IgM) (IgM BioSciences). PAT-SM6 was also evaluated in a phase 1 trial in relapsed or refractory multiple myeloma [11-15].

[0017] Special cases of refractory patients: In CLL, allogeneic bone marrow transplantation may be considered: - in patients refractory to immunological therapies and / or targeted therapies and / or immunochemotherapy and / or in patients with a TP53 or del(17p) mutation. - in patients with CLL evolving into Richter's syndrome after an isotopic response following immunochemotherapy treatment for Richter's syndrome.

[0018] In both cases, bispecific antibodies (BsAbs) and CAR-T cells (chimeric antigen receptor T cells) may constitute an alternative to allogeneic bone marrow transplantation. The curative nature of these approaches is uncertain in the medium and long term. These immunological approaches may be considered in patients with comorbidities that preclude allogeneic bone marrow transplantation. However, they are not without toxicity: cytokine release syndrome (CRS), encephalopathy related to the use of CAR-T cells

[16] .

[0019] BsAbs are antibodies with two binding sites directed against two different antigens or two different epitopes of the same antigen. With treatment using epcoritamab (bispecific anti-CD3xanti-CD20 IgGl), CRS is observed in 90% of patients (40% grade 1 and 50% grade 2). A first BsAb based on anti-CD3 / anti-CD19 scFv (BiTE, blinatumomab) has received marketing authorization, but this molecule has a very short half-life (must be used by continuous infusion) and can present significant side effects. Neurological events, including tremors, seizures, and changes in mental status, can be observed in 52% of patients, mainly grade 1 or 2, with 11% grade 3 and 4 (2%) grade 4 [17-19]. Doses below 60 pg / m2 / day resulted in poor response rates and a dose of 90 pg / m2 / day was limited by neurotoxicity [17-19].In CLL, the efficacy of blinatumomab was analyzed in a phase II trial in combination with nivolumab (anti-PD-1 antibody) and ibrutinib (BTK inhibitor) and showed disappointing results (response rate of 22% at two months of treatment).

[0020] In CLL and Richter's syndrome, another BsAb monoclonal antibody is in phase Ib / II trials in the EPCORE-CLL trial: ebcoritamab (Tepkinly*), a bivalent CD20 / CD3 BiTe antibody

[20] . Its future clinical indication is geared towards ebcoritamab-venetoclax synergy. The BsAb monoclonal antibody, glofitamab, a bivalent CD20 / CD3 BiTe antibody (Columvi*), could also find a future place in the treatment of refractory CLL [21-22],

[0021] CAR-T cells represent another promising area of ​​investigation in adoptive cell therapies, combining the strengths of T cells and antibodies to stimulate the anti-tumor activity of T cells. CD19 CAR-T cells have been widely used in B-cell lymphoid malignancies, and there is little experience with their use in CLL. In CLL, their efficacy is uncertain and controversial due to the exhausted phenotype of T lymphocytes and the possible loss of CD19 via a shaving phenomenon, creating resistance to CAR-T cell therapy.

[0022] A combination of anti-CD5 IgGl and anti-HLA-DR IgGl (HLA-DR is a class II MHC cell surface receptor encoded by the human leukocyte antigen complex on the 6p21.31 region of chromosome 6, whose main function is to present peptide antigens, potentially of foreign origin, to the immune system in order to induce or suppress T (helper) lymphocyte responses that eventually lead to the production of antibodies against the same peptide antigen) was tested against B-cell malignancies (International Application WO2010145895)

[23] . Experiments were performed in vivo, with the injection of JOK-1 cells expressing human CD5 (transfected cells) (cell line, not patient cells).The results obtained were very promising insofar as mice treated with the anti-CD5 anti-HLA-DR combination did not develop disease, unlike mice treated with other combinations: anti-HLA-DR x anti-CD71 or anti-CD20, mAb RTX alone. Description of the invention.

[0023] Anti-CD20 antibodies are very effective, but minimal residual disease (MRD) is still detectable and measurable. The inventors therefore sought to improve immunotherapies based on the CD20 molecule by avoiding the formation of disease resistance mechanisms against this type of agent (e.g., resistance to the use of anti-CD20 IgGl isotype antibodies such as RTX and OBZ) while reducing the risk of disease recurrence, the risk of which can be assessed by measuring minimal residual disease using RT-qPCR on blood and / or bone marrow.

[0024] To this end, the Inventors sought an immunotherapeutic partner to enhance anti-CD20 bioactivity and induce non-measurable residual disease, thereby enabling a non-toxic, short-duration treatment. The primary objective is to increase the efficacy of type I (e.g., RTX) anti-CD20 IgGl mAbs for non-CLL B-cell malignancies and type II (e.g., OBZ, antibodies) for non-CLL B-cell malignancies. reference) for B-cell CLL and / or anti-CD20 IgM antibodies; and to overcome resistance to anti-CD20 antibodies.

[0025] The Inventors have demonstrated in a completely unexpected manner that the use of an IgM isotype anti-HLA-DR monoclonal antibody (heavy chain VHantiHLADR-Cmu, SEQ ID NO: 1; light chain VLantiHLADR-Ck, SEQ ID NO: 2; VHanti-HLADR, SEQ ID NO: 3; VLanti-HLADR, SEQ ID NO: 4) in combination with an IgGl isotype anti-CD20 monoclonal antibody of type I (rituximab, RTX) or II, (Obinituzumab, OBZ), or with an IgM isotype anti-CD20 monoclonal antibody, improves the efficacy of anti-CD20 monoclonal antibodies. Anti-HLA-DR IgM, even at low doses or isodoses relative to anti-HLA-DR IgG, when combined with anti-CD20, is capable of complementing the anti-tumor B-cell response of anti-CD20 and achieving residual disease that is not measurable in vivo. Anti-HLA-DR IgM showed no in vitro toxicity on HLA-DR+ HUVEC cells, unlike other anti-HLA-DR IgG isotype antibodies such as the IMMU-114 (L243) antibody.No toxicity was observed during in vivo experiments in the CLL-PDX NGS mouse model or the chicken embryo.

[0026] The Inventors therefore propose a new treatment for CLL and B-cell malignant lymphoid hemopathies (non-Hodgkin B-cell lymphomas) based on a combination of two antibodies, anti-CD20 (e.g., OBZ or RTX) and anti-HLA-DR IgM, which leads to residual disease that is no longer measurable, unlike the reference treatment with anti-CD20 ITGGl (OBZ or RTX), and better management of B-cell lymphoid hemopathies and patients, particularly when the disease progresses and the patient becomes resistant to OBZ / RTX ITGGl.

[0027] The present invention therefore relates to an anti-HLA-DR IgM monoclonal antibody in pentameric or hexameric form. For example, the anti-HLA-DR IgM monoclonal antibody recognizes the same epitope as the murine IgGl monoclonal antibody IMMU357 (Beckman Coulter), but with a higher affinity for the HLA-DR molecule. The IgGl IMMU357 isotype is a monoclonal antibody used in diagnostics.

[0028] According to a particular embodiment of the present invention, the anti-HLA-DR IgM isotype monoclonal antibody is in hexameric form, and exhibits greater efficacy than the anti-HLA-DR IgGl isotype T.

[0029] According to a particular embodiment of the present invention, the anti-HLA-DR IgM isotype monoclonal antibody according to the present invention is capable of potentiating the activity of anti-CD20 IgGl isotype monoclonal antibodies and / or anti-CD20 IgM isotype monoclonal antibodies.

[0030] The present invention further relates to a pharmaceutical composition comprising the anti-HLA-DR IgM isotype monoclonal antibody according to the present invention, at least one anti-CD20 IgGl isotype monoclonal antibody and / or at least one anti-CD20 IgM isotype monoclonal antibody, and a pharmaceutically acceptable excipient.

[0031] According to a particular embodiment of the present invention, said at least one monoclonal antibody of IgGl anti-CD20 is an anti-CD20 of type I.

[0032] According to a particular embodiment of the present invention, said at least one monoclonal antibody of IgGl isotype anti-CD20 is an anti-CD20 type II.

[0033] The present invention further relates to an anti-HLA-DR IgM isotype monoclonal antibody according to the present invention or a pharmaceutical composition according to the present invention, for use as a medicinal product.

[0034] The present invention further relates to an anti-HLA-DR IgM isotype monoclonal antibody according to the present invention or a pharmaceutical composition according to the present invention, for use in the treatment of lymphoma, for example, B-type non-Hodgkin lymphoma or chronic lymphocytic leukemia (CLL). BRIEF DESCRIPTION OF THE FIGURES

[0035] [Fig. 1] represents the epitope map of the anti-HLA-DR IgM isotype monoclonal antibody. Residues predicted to belong to the epitope are indicated on the sequence and on the structure of the target, the alpha (HLA-DRA) and beta (HLA-DRB) subunits of the HLA-DR molecule (SEQ ID NO: 5). They are divided into four categories according to their crude probability of belonging to the epitope, in shades of gray from lightest to darkest, corresponding to low to high probability. Regions of weak (0, light gray) to strong (3, dark gray) interactions are represented by lines below the amino acid sequences.

[0036] [Fig. 2] shows a comparison of the antigenic recognition specificity of the anti-HLA-DR IgM monoclonal antibody with four commercial anti-HLA-DR IgG isotype antibodies used for diagnosis. The histograms represent the overlay of MFIs obtained by cytometric analysis for Daudi cell line B cells (HLA-DR positive) labeled with commercial anti-HLA-DR IgG isotype antibodies (LN3 and L243 from Biolegend; and IM1638U and IMMU-357 from Beckman) coupled to FITC. For each clone tested, cells were either pre-incubated or not with the anti-HLA-DR mAbs in Fab'2, IgG, and IgM formats. The vertical dashed line represents the median fluorescence of the commercial clone alone.

[0037] [Fig. 3] shows the comparative membrane distribution of the HLA-DR epitope, according to the isotype of the anti-HLA-DR agonist monoclonal antibody, IgM or IgG isotype. CLL cells are incubated with FITC-conjugated anti-HLA-DR IgGl and IgM monoclonal antibodies at 2 pg / ml for 4 hours on glass coverslips in serum-free RPMI medium. After fixation, the cells are covered with a mounting medium. containing DAPI (nuclear labeling) and analyzed by fluorescence microscopy. The figure shows representative images obtained with anti-HLA-DR IgG or IgM mAbs, the nuclei (DAPI), and the fusion of the two fluorescences (MERGE). Scale bar 10 µm.

[0038] [Fig. 4A] represents the comparative cell viability of clonal B lymphocytes observed after 6 hours (H6) of treatment with anti-HLA-DR IgG and IgM isotypes. CLL cells were incubated for 6 hours with anti-HLA-DR IgG and IgM monoclonal antibodies at a concentration of 2 pg / ml. B cell cytotoxicity was then analyzed by flow cytometry using an anti-CD19-AF700 antibody for specific B cell detection and annexin V coupled to FITC for dead cell detection. Normalized B cell mortality is shown for each condition. Bars represent mean ± SEM. Statistical significance of differences between groups was determined using the unpaired t-test (*p < 0.05).

[0039] [Fig. 4B] represents the comparative cell viability of clonal B lymphocytes observed after 6 hours (H6) of treatment with anti-CD20 IgGl alone or in combination with the anti-HLA-DR IgM isotype. CLL cells were incubated for 6 hours with anti-CD20 IgGl mAbs, anti-HLA-DR IgM, or the combination of both, all at an isodose concentration of 2 pg / ml. B cell cytotoxicity was then analyzed by flow cytometry with dead cell labeling using an anti-CD19-AF700 antibody for specific B cell detection and annexin V coupled to FITC for dead cell detection. Normalized B cell mortality is shown for each condition. Bars represent mean ± SEM. The statistical significance of the differences between the groups was determined using the unpaired t-test (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0040] [Fig. 5A] represents the impact of the tumor microenvironment on the bioactivity of IgGl or IgM anti-CD20 and IgM anti-HLA-DR monoclonal antibodies. Autologous serum (AS) reflects the immunosuppressive activity of the tumor microenvironment (TM). AS contains molecules with immunosuppressive activity (soluble HLA-DR molecules that induce immunological tolerance, TGF-beta, soluble CD48, etc.) and survival factors (nurse-like cells, etc.). B lymphocyte cell viability after treatment with anti-CD20 rituximab (RTX) monoclonal antibodies of the IgGl and IgM anti-CD20 isotypes alone or in combination with RTX IgGl and IgM with the IgM anti-HLA-DR isotype in fetal bovine serum (FBS) and in anaphylactic acid (AGA) from patients was determined after incubation of CLL cells for 6 hours with the anti-CD20 RTX IgGl and IgM monoclonal antibodies, anti-HLA-DR IgM, or a combination of both, all at an isodose concentration of 2 pg / ml. B cell cytotoxicity was then assessed. Analyzed by flow cytometry (FC) using an anti-CD19-AF700 antibody for specific B-cell detection and annexin V coupled to FITC for dead-cell detection. Normalized B-cell mortality is shown for each condition. Bars represent mean ± SEM.

[0041] [Fig. 5B] illustrates the impact of the tumor microenvironment on the bioactivity of anti-CD20 IgGl or IgM monoclonal antibodies. CLL PBMCs were pre-incubated for 16 hours, with or without CpG ODN (1.5 pg / ml) and CD40 ligand (CD40L) (1 pg / ml), to induce a microenvironment conducive to B cell clonal survival, mirroring the in vivo survival effect of the tumor microenvironment. Following this, the cells were treated with the mAbs for 5 hours at a dose of 2 pg / ml. B cell death was assessed by annexin V staining.

[0042] [Fig. 6A] represents the study of the impact of the chronobiology of treatment with anti-CD20 IgGl and anti-HLA-DR IgM on the synergistic activity of the IgG plus IgM combination. Cell death induced by the anti-CD20 RTX and OBZ IgGl isotype monoclonal antibodies in combination with anti-HLA-DR IgM was determined after incubating CLL cells for 6 hours with the monoclonal antibodies at 2 pg / ml, with IgG applied 30 minutes before IgM or vice versa. Comparative cytotoxicity on tumor B cells was then analyzed by flow cytometry using an anti-CD19-AF700 antibody for the specific detection of B lymphocytes and annexin V coupled to FITC for the detection of dead cells. The cytotoxicity index (%) is shown for each condition.

[0043] [Fig. 6B] represents the effects of anti-CD20 and anti-HLA-DR IgGl chronobiology on BB homotype cell aggregation. The modification of cultured cells induced by the anti-CD20 RTX and OBZ IgGl monoclonal antibodies in association with anti-HLA-DR IgM was observed after 1 day and 3 days of incubation of CLL PBMCs with the monoclonal antibodies at 2 pg / ml by depositing IgGl 30 minutes before IgM or vice versa.

[0044] [Fig. 7A] shows the impact of in vivo treatment with rituximab (RTX) on minimal residual disease (MRD), compared with its combination with anti-HLA-DR IgM. Detection of human tumor cells (CLL PBMCs) in the mouse xenograft model was evaluated in CLL-PDX NGS mice (n = 5 per group). Group 1: control (0.9% NaCl). Group 2: Rituximab RTX. Group 3: anti-HLA-DR IgM. Group 4: Rituximab RTX / anti-HLA-DR IgM. Animals were sacrificed 5 days after treatment with the mAbs (Day 5). The presence of CLL clonal B cells in the spleen was determined by real-time RT-qPCR using primers specific for human Alu (hAlu) sequences normalized to mouse [3-Actin] (m[3-Actin] expression on total DNA extracts (a). Using a standard curve generated with a number of cells The known minimal residual disease (MRD) (b) and a normalized detectable cell ratio on the control were calculated (c). Data are expressed as mean ± SD, with one point representing one mouse. The statistical significance of differences between groups was determined by a one-way ANOVA test (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0045] [Fig. 7B] shows the impact of in vivo treatment with the antibody obinutuzumab (OBZ) on MRD, compared with its combination with anti-HLA-DR IgM. Detection of human tumor cells (CLL PBMCs) in the mouse xenograft model was evaluated in CLL-PDX NGS mice (n = 5 per group). Group 1: control (0.9% NaCl). Group 2: Obinutuzumab OBZ. Group 3: anti-HLA-DR IgM. Group 4: Obinutuzumab OBZ / anti-HLA-DR IgM. Animals were sacrificed 5 days after treatment with the mAbs (Day 5). The presence of CLL clonal B cells in the spleen was determined by real-time RT-qPCR using primers specific for human Alu (hAlu) sequences normalized to mouse [3-Actin] (m[3-Actin] expression on total DNA extracts (a). Using a standard curve generated with a known number of cells, the MRD (b) and a ratio of detected cells normalized to the control were calculated (c).Data are expressed as mean ± SD, with one point representing one mouse. The statistical significance of differences between groups was determined by a one-way ANOVA test (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). EXAMPLES EXAMPLE 1: MATERIALS AND METHODS

[0046] - Production of recombinant anti-HLADR IgM [24-25].

[0047] The original anti-HLA-DR monoclonal antibody was generated in mice using human cells expressing HLADR as the antigen. Complementary DNA (cDNA) encoding the variable domains VH and VL of this antibody was isolated from murine hybridoma (International Application WO2010 / 145895)

[23] . This cDNA enabled the production of this antibody in recombinant form under the IgG1 and IgM isotypes.

[0048] An anti-HLA-DR IgM monoclonal antibody in its hexameric form (12 Fab domains instead of 2 with an IgG1) was produced using the baculovirus / insect cell expression system. For this purpose, the cDNAs encoding the variable regions VH and VL of the anti-HLADR antibody were cloned into two specific transfer vectors (pVT light chain and pVT heavy chain) already containing the complementary DNAs encoding the constant kappa or mu regions as described in Juliant et al. 2013

[24] . Recombinant double viruses were generated after co-transfection of Sf9 cells (ATCC CRL1711) with the two loaded transfer vectors and viral DNA according to the technology described in International Application WO2019 / 081858

[25] . The recombinant viruses were cloned by plaque lysis. The genome of the recombinant viruses was checked by Southern blot, and the sequences integrated into the viral DNA were verified by sequencing after PCR amplification. After 3 days of incubation at 28°C, the recombinant antibodies secreted during the infection of serum-free cultured Sf9 cells were purified from the culture supernatant on a protein A column. The purity of the antibodies was verified by polyacrylamide gel electrophoresis and silver staining. - The cells.

[0049] All experiments were conducted with primary tumor cells from patients (with their informed consent), in vitro and in vivo in mice, and not with established tumor cell lines (B cell lines). The clinical presentation of each patient with CLL is precisely known; knowledge of the phenotypic, cytogenetic, and molecular parameters allows for correlation studies between the treatment of interest and the response to it.

[0050] - Identification of residues participating in the epitope recognized by the antibody monoclonal of IgM isotype anti-HLA-DR used.

[0051] The determination of the epitope recognized by the anti-HLADR antibody was carried out by the company Mabsilico (https: / / www.mabsilico.com) using a bioinformatics approach.

[0052] While there is only one α chain (HLADRA UNIPROT P01903.DRA_HUMAN), there are numerous variants of the [3] chain. The work carried out to identify the epitope was performed using as a model a heterodimer composed of the α chain and the [3] chain HLA-DRB1 (HLADRB UNIPROT P0191 l.DRBl_HUMAN), which is the most represented (over 80%). From 3D models of the target and the antibody, docking positions identified by bioinformatics are classified according to their crude probability of belonging to the epitope, from dark gray for the highest probability to light gray for the lowest (but still significant) probability.

[0053] - Comparison of the specificity and individuality of antigenic recognition of the anti-HLA-DR IgM isotype monoclonal antibody used with 4 commercial antibodies.

[0054] Four commercial anti-HLA-DR IgGl isotype antibodies (LN3 and L243 from Biolegend; and IM1638U and IMMU-357 from Beckman) coupled to FITC (fluorescein isothiocyanate) were incubated at a saturating dose with Daudi B cell line (400,000 Daudi cells per test, in PBS1X 4°C) previously incubated for 15 minutes at 4°C with a high dose (4 pg) of anti-HLA-DR IgM, anti-HLA-DR IgG, and a Fab'2 anti-HLA-DR (derived from IgM or IgG), or nothing. After incubation with commercial antibodies for 15 minutes, the cells are washed and analyzed by flow cytometry (Navios, Beckman; FL1 laser for FITC detection).

[0055] The results show that the three antibody formats interfere with the binding of commercial antibodies in the following decreasing order of significance: IMMU-357»IM1638U>LN3>L243 ([Fig.2]). Therefore, the anti-HLA-DR IgM has an epitope identical to or overlapping with IMMU-357.

[0056] It should be noted that the L243 antibody (IMMU114) has been used for clinical trials [27-34], - Immunofluorescence on slide

[0057] CLL cells are suspended at 2 million per milliliter in RPMI culture medium without fetal bovine serum (FBS) on glass coverslips (16 mm in diameter) placed in 12-well culture plates containing anti-HLA-DR IgG and IgM monoclonal antibodies coupled to FITC at 2 pg / ml. After 4 to 6 hours of incubation, the well contents are rinsed with phosphate-buffered (PBS Ix) and the cells are fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After two washes with PBS Ix, the preparations are covered with mounting medium containing DAPI for nuclear staining. The slides were examined with the Axio Imager 2 (Zeiss). Images were acquired with a 63x objective and analyzed using ImageJ software.

[0058] - Bioactivity: effect of mAbs on the viability of tumor B lymphocytes and determination of the cytotoxic activity of antibodies

[0059] CLL cells are suspended at 1 million per millilitre in RPMI culture medium, 10% FBS. Anti-HLA-DR antibodies of IgG and IgM isotype and anti-CD20 RTX and OBZ are incubated with the cells at a concentration of 2 pg / ml.

[0060] According to the experiments, B lymphocyte viability was compared following 3 regimens of administration of mAbs allowing the chronobiological analysis of the treatments: - Synchronous IgGl and IgM treatment - Treatment with ITgl for 30 minutes followed by IgM - Treatment with IgM for 30 minutes followed by IgGl.

[0061] In order to evaluate the impact of the microenvironment, in some experiments CLL PBMCs were pre-incubated for 16 hours, with or without CpG ODN (CpG oligodeoxynucleotides) (1.5 pg / ml) and CD40 ligand (CD40L) (1 pg / ml), to provide a microenvironment conducive to the survival of clonal B cells, similar to the tumor microenvironment in vivo. Following this, the cells were treated with the AcMs were administered for 6 hours at a dose of 2 pg / ml. B cell death was assessed by annexin V staining.

[0062] Cell death analysis is performed after 6 hours of incubation (at H6), by flow cytometry using an anti-CD19-AF700 antibody for the specific detection of B lymphocytes and annexin V coupled to FITC for the detection of dead cells (Excitation: 488 nm; Emission: 350 nm). Data are collected and analyzed using Kaluza software. Normalized cell death is calculated using this formula (I): (I) % normalized cell death = 100 - ((%T*100) / %C) where “%T” is the % of live cells in the test tube and “%C” is the % of viability in the control condition.

[0063] The cytotoxicity index combining 3 parameters representative of cytotoxicity is calculated according to formula (II): (II) Cytotoxicity Index (CI) = (% MTT - % MTC) + (% BC - % BT) + (%NCC-%NCT) with % MTT and MTC, the % of total mortality in the test and control tubes (without antibodies); % BC and BT, the % of LB in the control and test tubes; % NCC and NCT, the % of cells of normal size / structure in the test and control tubes.

[0064] - Detection of MRD in in vivo chronobiology: the CLL-PDX mouse model NGS

[0065] On day 0, CLL-PDX NGS mice were intravenously (IV) injected with fresh CLL PBMCs (3 x 10⁷ cells / mouse). On the first day, the mice were randomly divided into four groups (5 mice per group) and immunotherapy was administered with an IV dose of each monoclonal antibody. Two experiments were conducted combining the anti-CD20 IgG1 and anti-HLA-DR IgM monoclonal antibodies, first with rituximab (RTX) and then with obozoolitin (OBZ). CLL-PDX NGS mice received IV 0.9% NaCl (group 1), 7.5 mg / kg of RTX or 7.5 mg / kg of OBZ (group 2), 7.5 mg / kg of anti-HLA-DR IgM (group 3), and IV RTX (7.5 mg / kg) or OBZ (7.5 mg / kg) followed by anti-HLA-DR IgM (7.5 mg / kg) < 2 hours after IgG1 (group 4). CLL-PDX NGS mice were sacrificed on day 5 after the single-dose treatment administered on day 0.

[0066] The presence of CLL tumor B cells was assessed by RT-qPCR in the liver, spleen, lungs and bone marrow, using primers specific for human Alu sequences.

[0067] - Quantitative detection of human cells by q-PCR

[0068] The detection of primary human B tumor cells in the spleen, bone marrow, lungs, and liver of mice was based on the quantitative detection of human Alu sequences in DNA extracts from mouse organs using a protocol developed by Funakoshi et al., 2017

[35] . For DNA extraction, one hundred microliters of homogenized organ in PBS were used. The QIAGEN BioSprint 15 DNA Blood & Tissue kit (Ref: 940017) was used. in accordance with the manufacturer's instructions. To detect human cells in mouse tissues, primers specific to human Alu sequences (Forward: 5'-GGTGAAACCCCGTCTACT-3' (SEQ ID NO: 6); Reverse: 5'-GGTTCAAGCGATTCTCGC-3' (SEQ ID NO: 7)) were used to amplify human Alu sequences present in genomic DNA extracted from the samples. Mouse-specific [3-actin] primers (m[3-actin] were used as housekeeping genes (forward: 5'-AAGGCCAACCGTGAAAAGAT-3' (SEQ ID NO: 8); reverse: 5'-GTGGTACGACCAGGGATAC-3' (SEQ ID NO: 9)

[36] . Real-time RT-qPCR to amplify and detect huAlu sequences was performed on 40 ng of genomic DNA with 0.1 pM of each primer and 5 µl of SYBR green Master Mix (Thermofisher) in a final volume of 10 µl.A QuantStudio 6 Flex Real Time System (Applied Biosystems) was used to monitor acquisitions under the following conditions: 50°C for 2 min and 95°C for 10 min, followed by 50 cycles at 95°C for 30 sec, 60°C for 1 min, a melting curve step at 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. A quantitative measurement of amplifiable mouse DNA was obtained by amplifying the mouse genomic DNA sequence of [3-actin] with m[3-actin primers (forward and reverse), using the same PCR conditions. The cycle threshold (Ct) of each sample was recorded as a quantitative measure of the amount of PCR product in the sample. Where indicated, the Alu signal has been normalized with respect to the relative amount of m[3-actin] and expressed as ACt = (Ctm[3-actin- Ctalu).Changes in the Alu signal relative to the total amount of genomic DNA (i.e., changes in the amount of human DNA in mouse tissue) were expressed as AACt = ACtcontrol - ACttreatment. Relative changes in detection were then calculated as 2-AACt. Each assay included a negative control, a positive control, a control without a DNA template, and duplicate experimental samples. Data processing and statistical analysis were performed using GraphPad Prism. EXAMPLE 2: RESULTS

[0069] - Identification of residues participating in the epitope recognized by the antibody anti-HLA-DR IgM isotype monoclonal used

[0070] The HLA-DR molecule is composed of two alpha and beta subunits. From the best-ranked docking positions, the target residues were noted according to their probability of belonging to the epitope. The residues expected to belong to the epitope are indicated on the sequence and on the target structure ([Fig. 1]).

[0071] They are divided into 4 categories according to their raw probability of belonging to the epitope, from dark gray for the highest probability to light gray for the lowest (but still significant) probability. For technical reasons, the The target is renumbered as a single chain. Furthermore, not all amino acids in the target chains are visible in the 3D structure. Residues predicted to belong to the epitope and validation peptides are indicated on the target sequence.

[0072] From these residues, four main interaction regions were defined on the target. The interaction regions are indicated on the target structure. It is interesting to note that the anti-HLA-DR mAb preferentially recognizes an epitope of the alpha chain of HLA-DR, but also certain amino acids of the beta chain.

[0073] - Membrane clustering effect (oligomerization) of the HLA- epitope DR induced by the anti-HLA-DR monoclonal antibody of the IgM isotype and biological effects related to epitope clustering

[0074] The anti-HLADR antibody under an IgM isotype exhibits very different properties compared to IgGl of the same specificity, in particular the oligomerization of the target not observed by the anti-HLA-DR IgGl agonist.

[0075] Observable: The membrane biodistribution of the HLA-DR epitope after treatment with anti-HLA-DR monoclonal antibodies of the IgG or IgM isotype was analyzed by immunofluorescence. It highlights the capacity and specificity of inductive oligomerization of the epitope induced by the agonist isotype of the anti-HLA-DR IgM ([Fig. 3]).

[0076] The results show that the anti-HLA-DR IgGl isotype induces only a random and disorganized distribution of the HLA-DR epitope within the membrane of CLL B cells. In contrast, the anti-HLA-DR IgM isotype rapidly induces (< 3 hours) the grouping of HLA-DR molecules into epitope-membrane clusters specifically in areas of intercellular contact.

[0077] This oligomerization leads to the recruitment of HLA-DR within lipid rafts and to the induction of numerous other new biological activities linked to this epitope clustering: - Interaction with the cytoskeleton, reorganization of F-actin and the cytoskeleton; - Strong colocalization of cortactin and F-actin in the membrane structures of podosomes involved in strong homotypic BB lymphocyte adhesion; - Strong homotypic aggregation (HA) and lymphocyte clustering of B cells clearly visible in optical microscopy and real-time imaging with the Incucyte™. - Interaction with novel signal transduction pathways inducing B lymphocyte cell death (activation of N0X2, production of oxygen free radicals and creation of lethal B cell oxidative stress, phosphorylation of JNK1 / 2, ERK1-2 and Bcl-2 proteins, activation of Beclin 1, significant intrinsic B cell production of interferon-gamma); - All these biological events leading to the death of the tumor target cell, the B lymphocyte.

[0078] We have shown the correlation that existed between homotypic aggregation and B lymphocyte cell death as well as the correlation between the percentage of HA and reduced cell viability (induction of large clusters with 100% B cell death within these clusters).

[0079] - Comparative bioactivity of the anti-HLA-DR isotype IgM monoclonal antibody versus IgG anti-HLADR

[0080] Observable: treatment with the anti-HLA-DR IgM isotype induces greater anti-tumor efficacy compared to the anti-HLA-DR IgGl isotype ([Fig.4A]).

[0081] When the anti-HLA-DR monoclonal antibody was used in the hexameric IgM isotype, a substantial and significant reduction in the viability of tumor B cells was observed. Efficacy was evaluated 6 hours (H6) after exposure to a single dose of monoclonal antibody. The anti-HLA-DR IgGl isotype is less effective than ITgM in inducing the death of CLL B cells: the anti-HLA-DR IgGl antibody induced the death of 14.7 ± 2.8% of CLL B cells at H6 (n = 24), and a significant increase to 26.6 ± 3% was observed with the anti-HLA-DR IgM antibody (*p < 0.05; n = 36).

[0082] - Combinations of type I and type II anti-CD20 antibodies plus anti-HLA mAb- DR: The anti-HLA-DR IgM monoclonal antibody potentiates the anti-tumor activity of anti-CD20 IgGl monoclonal antibodies on CLL B cells

[0083] Observable: Treatment with a low dose (< 2 pg / ml) of anti-HLA-DR IgM is synergistic with anti-CD20 monoclonal antibodies ([Fig. 4B]) and amplifies the bioactivity of an anti-CD20 monoclonal antibody, whether type I or type IL. Such amplification of the activity of type I and type II anti-CD20 monoclonal antibodies by anti-HLA-DR IgM is observed even at low doses of IgM as low as 0.5 pg / ml, demonstrating synergistic activity at low doses of IgM in vitro and in vivo (ITgM dose < 2 pg / ml). This observed "low-dose IgM" synergistic activity suggests a potential pharmacokinetic and pharmacodynamic benefit in vivo.

[0084] Efficacy was evaluated at 6 hours (H6) after exposure to a single dose of AcMs.

[0085] Compared to H6 controls, 2 pg / ml of anti-CD20 mAbs induced a mean normalized CLL B cell death of 10 ± 1.2% for RTX (n = 36) to 28.6 ± 5.1% for OBZ (n = 17). The combination of anti-HLA-DR IgGl and anti-CD20 IgGl did not increase cellular cytotoxicity, with approximately 20% cell death (data not shown). Conversely, treatment with the combined anti-HLA-DR IgM isotype significantly increased cell death detected at H6, compared with anti-CD20 IgGl alone (39.14 ± 4.14% for RTX + IgM; 50.06 ± 5.18% for OBZ + IgM).

[0086] The combination of anti-CD20 IgGl plus anti-HLA-DR IgM creates an amplifying antitumor activity against the B lymphocyte clone, an activity that is synergistic and not additive. The convergence of the respective bioactivities of these two mAbs is based on the amplification of the NADPH oxidase pathway, a B lymphocyte membrane protein, NOX2, whose synergistic activation by IgGl and IgM generates reactive oxygen species (ROS). ROS lead to the induction of death in neoplastic clonal B lymphocytes, a B cell death independent of caspase activation. This is a regulated, caspase-independent necrotic cell death induced by ROS, which are responsible for lethal oxidative stress associated with intramitochondrial calcium transfer, which we measured using the Rhod-2 probe. The importance of intramitochondrial calcium transfer is correlated with IgM treatment and its intensity is correlated with the observed B lymphocyte death.Oxidative stress and intramitochondrial calcium transfer lead to B lymphocyte mitochondrial dysfunction and lethal mitochondrial morphological damage.

[0087] We have regularly observed lethal mitochondrial lesions in electron microscopy studies, at H6 of treatment, by the combination of IgGl mAbs then IgM: edematous ballooning of all mitochondria, severe involution of mitochondrial cristae, mitochondrial rupture).

[0088] These major and lethal mitochondrial morphological and functional alterations constitute the property of the synergy of IgGl anti-CD20 and IgM anti-HLA-DR, generate B lymphocyte death by induced and regulated necrosis, preceded by edema of the mitochondria and the peri-nuclear membrane.

[0089] - Anti-HLA-DR x anti-CD20 combinations: Anti-HLA- IgM isotype mAb DR potentiating the anti-tumor activity of an anti-CD20 IgGl or IgM monoclonal antibody (derived from RTX). Impact of the tumor microenvironment and autologous serum.

[0090] SA reproduces the tumor microenvironment (soluble immunosuppressive factors and survival factors) with greater fidelity, compared with SVF.

[0091] Observable: the use of autologous serum (AS) from patients with CLL decreases the effectiveness of mAbs alone or in combination IgGl plus IgM anti-HLA-DR, compared with the use of FBS 10% (in vitro reference serum).

[0092] Importantly, the use of an anti-CD20 IgM with the anti-HLA-DR IgM not only renders the combination insensitive to the inhibitory effect of SA but increases its bio-activity in this condition of immune resistance.

[0093] The bio-activity of the mAbs is evaluated at 6 hours (H6) after exposure to a single dose of antibody.

[0094] In SA, compared to the use of SVF 10%, the levels of activity loss are as follows: AcM RTX anti-CD20: average loss of -2.5% for IgGl and average loss of -6.2% for IgM anti-CD20. IgM anti-HLA-DR: a decrease of approximately -2.7%. For the combination of RTX IgG + anti-HLA-DR IgM: a decrease of approximately -9.5%.

[0095] A low dose (< 2 pg / ml) of anti-HLA-DR IgM induces synergistic efficacy with anti-CD20 IgGl type I and type II mAbs, both in 10% FBS and in autologous patient serum (n = 3) ([Fig.5A]).

[0096] In 10% FBS, compared to H6 controls, 2 pg / ml of RTX anti-CD20 mAb induces a low cytotoxicity index against CLL tumor B cells, representing an increase of approximately 5.4% for RTX IgGl and 13.8% for RTX IgM. In FBS, the addition of anti-HLA-DR IgM to anti-CD20 mAbs significantly increases B cell death, compared to IgGl and anti-CD20 IgM alone: ​​mean gain of +32.8% for RTX IgG + anti-HLA-DR IgM; mean gain of +17.6% for RTX IgM + anti-HLA-DR IgM.

[0097] In SA, the anti-B lymphocyte cytotoxicity index with the anti-CD20 RTX is approximately 3.1% for IgG1 and 7.6% for the anti-CD20 IgM isotype. Similar to the 10% FBS regimen, in SA, the combination of anti-CD20 monoclonal antibodies with anti-HLA-DR IgM potentiates their effect: mean gain of +25.6% for RTX IgG + IgM anti-HLA-DR; mean gain of +32.5% for RTX IgM + IgM anti-HLA-DR. The combination of RTX IgM with the anti-HLA-DR IgM monoclonal antibody increases the efficacy of the synergy: mean gain of 8.6%.

[0098] B cells are pretreated for 16 hours with the combination of CpG ODN (CpG oligodeoxynucleotides) and CD40 ligand (CD40L) to provide an environment conducive to cell survival by inducing resistance in clonal B cells. These cells are then treated with the monoclonal antibodies IgG1 anti-CD20 and IgM anti-HLA-DR, alone or in combination. Mortality is then assessed by flow cytometry and normalized to the control condition without CpG / CD40L.

[0099] Comparing the normalized cell death of B cells without pretreatment with CpG ODN / CD40L, a decrease in basal cell death is observed in the presence of CpG ODN / CD40L ([Fig. 5B]). Pretreatment with CpG ODN and CD40L effectively leads to improved survival of tumor B lymphocytes and a significant reduction in their spontaneous mortality (experimental positive control). A significant spontaneous mortality of CLL B lymphocytes > 30% is observed after a cell freeze / thaw cycle and confirms the resistance effect on B cell death induced by pretreatment with CpG ODN and CD40L (and even the positive control of untreated cells in the CpG / CD40L condition).

[0100] The efficacy of mAbs with or without pretreatment is evaluated in parallel. Anti-CD20 mAb treatments are insufficient to overcome the resistance induced by the pretreated culture condition. Conversely, the anti-HLA-DR IgM mAb alone or in combination with anti-CD20 IgGl allows for the significant induction of substantial cell death in CpG / CD40L compared to the effect of anti-CD20 IgGl, without any benefit from the combination with an IgGl (average of the conditions without CpG / CD40L with anti-CD20: 1.93%; with IgM: 6.3%; with CpG / CD40L and anti-CD20: an average decrease of -0.6%, with IgM: an increase of approximately 15.9%).

[0101] The bioactivity of the IgM monoclonal antibody remains unchanged and induces the same B-cell mortality threshold, including under tumor resistance conditions, unlike type I and II anti-CD20 antibodies. This highlights the fact that IgM is relatively insensitive to the tumor microenvironment, which, conversely, induces resistance to IgG treatment.

[0102] - Chronobiology of in vitro and in vivo anti-HLA-DR IgM x IgGl treatment anti-CD20 (OBZ) in vitro chronobiology

[0103] B lymphocyte viability was analyzed chronobiologically to optimize the anti-tumor response. B lymphocyte viability was compared according to the following three mAb administration regimens: - Synchronous IgGl and IgM treatment - Treatment with IgGl for 30 minutes followed by IgM - Treatment with IgM for 30 minutes followed by IgGl

[0104] Observable: we show that treatment with IgGl followed by treatment with anti-HLA-DR IgM (after 30 minutes of exposure to TIgGl) induces strong homotypic BB aggregation and significantly higher cell death, compared to other chronobiological forms, synchronous IgGl and IgM treatment and IgM then IgGl ([Fig.6A]) ([Fig.6B]).

[0105] Efficacy was evaluated at 6 hours (T6) by cytometry after exposure to a single dose of AcMs.

[0106] A low dose (< 2 pg / ml up to 0.5 pg / ml) of anti-HLA-DR IgM led to synergistic efficacy with anti-CD20 IgGl mAbs (RTX and OBZ) (FBS 10%), with IgG chronobiology before IgM (RTX, n = 3; OBZ, n = 4) ([Fig.6A]).

[0107] For the RTX + IgM anti-HLA-DR combination, the anti-B lymphocyte cytotoxicity index is increased by +21.5%, by depositing ITgG before IgM (cytotoxicity index at H6 for IgG then IgM = 47.5% and IgM then IgG = 26%).

[0108] For the OBZ + IgM anti-HLA-DR combination, the anti-B lymphocyte cytotoxicity index is increased by +11.2%, by depositing IgG before IgM (cytotoxicity index at H6 for IgG then IgM = 78% and IgM then IgG = 66.8%).

[0109] Real-time imaging assessments with image capture at H and J3 (IncuCyte technique) show the clear difference between the two chronobiologies with a clear increase in homotypic BB cell aggregation, with the IgG chronobiology 30 minutes before IgM ([Fig.6B]). Evaluation of MRD: In vivo chronobiology

[0110] The objective of achieving unmeasurable residual disease is very important in the medical management of hematological malignancies. Indeed, tumor cells that survive treatment are capable of new genetic modifications, leading to relapse and the transient efficacy of mAbs due to the development of acquired resistance.

[0111] Observable: In vivo treatment of CLL-PDX NGS mice with the combination of RTX mAbs (or OBZ mAbs) and anti-HLA-DR IgM induces a strong anti-tumor response against CLL clonal B lymphocytes. MRD analysis shows a significantly greater anti-tumor response induced by the combination of RTX (or OBZ) IgGl plus IgM than by RTX (or OBZ) monotherapy.

[0112] Indeed, using the PDX-CLL NGS murine model, we show that residual tumor disease remains detectable after treatment with anti-CD20 IgGl isotype mAbs used as monotherapy (RTX and OBZ), whereas the association with OBZ x IgM HLA-DR renders residual disease undetectable and non-measurable in the spleen ([Fig.7A] and 7B).

[0113] The anti-HLA-DR IgM isotype combined with OBZ induced in vivo non-measurable residual disease of CLL. This result demonstrates the efficacy of reducing MRD in a B-cell lymphoid malignancy by combining type I or type II anti-CD20 IgGl with a low dose of anti-HLA-DR IgM monoclonal antibody.

[0114] The IgGl RTX isotype combined with the anti-HLA-DR IgM isotype reduces, in vivo, the level of MRD in the spleen by 17-fold. The IgGl OBZ isotype combined with the anti-HLA-DR IgM isotype reduces, in vivo, the level of MRD in the spleen by 2-fold ([Fig. 7A] bc and 7B bc).

[0115] The reduction in MRD was confirmed in vivo in two different models: chicken embryo tumor xenograft and mouse xenograft with PBMCs from CLL patients (PDX-CLL NGS mouse model). References list

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Claims

Demands

1.

2. IgM isotype anti-HLA-DR monoclonal antibody. IgM isotype anti-HLA-DR monoclonal antibody according to claim 1, wherein said antibody is in hexameric form.

3. Monoclonal antibody of anti-HLA-DR isotype IgM according to claim 1 or 2, wherein said antibody potentiates the activity of anti-CD20 isotype IgGl monoclonal antibodies and / or anti-CD20 isotype IgM monoclonal antibodies.

4. Pharmaceutical composition comprising the anti-HLA-DR IgM isotype monoclonal antibody according to any one of claims 1 to 3, at least one anti-CD20 IgGl isotype monoclonal antibody and / or at least one anti-CD20 IgM isotype monoclonal antibody, and a pharmaceutically acceptable excipient.

5. Pharmaceutical composition according to claim 4, wherein said at least one monoclonal antibody of IgGl is an anti-CD20 type I.

6. Pharmaceutical composition according to claim 4, wherein said at least one monoclonal antibody of IgGl isotype is an anti-CD20 type II.

7. Anti-HLA-DR IgM isotype monoclonal antibody according to any one of claims 1 to 3 or pharmaceutical composition according to any one of claims 4 to 6, for use as a medicinal product.

8. Anti-HLA-DR IgM isotype monoclonal antibody according to any one of claims 1 to 3 or pharmaceutical composition according to any one of claims 4 to 6, for use in the treatment of lymphoma.

9. Anti-HLA-DR IgM isotype monoclonal antibody according to any one of claims 1 to 3 or pharmaceutical composition according to any one of claims 4 to 6, for use according to claim 8, wherein the lymphoma is selected from non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL).

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