Anti-HLA-dr igm isotype monoclonal antibody potentiating the activity of Anti-CD20 igg1 or igm isotype antibodies

Abstract

The present invention relates to a novel anti-HLA-DR IgM isotype monoclonal antibody and to the use thereof alone or in addition to anti-CD20 IgG1 or IgM isotype antibodies, in the treatment of B-phenotype lymphoid hemopathies, and in particular of non-Hodgkin lymphoma B, chronic lymphocytic leukemia (CLL) and B-cell malignant lymphoid hemopathies in general.

Description

[0001]

[0002] Technical field of the invention

[0003] The present invention relates to a new anti-HLA-DR IgM isotype monoclonal antibody and its use alone or in addition to IgG 1 isotype or IgM anti-CD20 antibodies, in the treatment of malignant lymphoid hematological diseases of B phenotype, such as chronic lymphocytic leukemia (CLL) and B non-Hodgkin lymphomas.

[0004] The present invention has applications in human health, for example in immuno-hematology, hemato-oncology and oncology, and malignant B-cell lymphoid hemopathies.

[0005] In the description below, references in brackets ([ ]) refer to the list of references at the end of the text.

[0006] State of the art

[0007] Over the past few decades, immunotherapy has become one of the most remarkable advances in the field of cancer treatment, particularly for lymphoid malignancies, primarily of B-cell phenotype. Unlike traditional approaches such as chemotherapy and radiotherapy, which mainly aim to directly eliminate tumor cells, immunotherapy harnesses the power of the immune system to fight the disease.

[0008] The remarkable successes of immunotherapy in treating certain types of cancer have generated considerable interest and opened up new perspectives in the field of drug research and development.

[0009] For anti-tumor immunotherapy, humanized or human lgG1 divalent monoclonal antibodies (mAbs) represent the majority (80%) of antibodies approved for various oncological and hematological indications.

[0010] An example of current immunotherapy targets the CD20 protein, or cluster of differentiation 20, a transmembrane glycoprotein primarily expressed 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 their activation and immune function. CD20 acts as a calcium channel, sharing structural similarities with certain ion channels (CD20: a B-cell-specific tetraspan protein). The membrane protein CD20 regulates a calcium channel that acts synergistically with the B-cell receptor (BCR).

[0011] It has become an important therapeutic target in the treatment of various diseases, particularly B-cell lymphoproliferative disorders such as B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia. Thus, the treatment of B-cell lymphoid malignancies involves the use of anti-CD20 monoclonal antibodies that allow for the elimination of pathological and clonal B lymphocytes while preserving the functions of other immune cells, especially T and NK cells. For example, rituximab (RTX) (a 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 in immunotherapy or in combination with chemotherapy in aggressive B-cell non-Hodgkin lymphomas in the form of immunochemotherapy.

[0012] In chronic lymphocytic leukemia (a clonal disease of CD20+ CD19+ CD5+ CD23+ B lymphocytes), the reference anti-CD20 monoclonal antibody is obinutuzumab (OBZ) (a type II anti-CD20 monoclonal antibody), used, according to current clinical guidelines (FILO-LYSA 2025 recommendations), in combination with an inhibitor of the B-cell lymphoma type 2 oncogene, Bcl-2 (venetoclax). 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 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 strengthening the bioactivity of anti-CD20 monoclonal antibodies or any other anti-B lymphocyte monoclonal antibodies (anti-CD19, anti-CD38 (daratumumab and isatuximab), anti-CD47...), without causing toxicity. Beyond their use as a single therapeutic agent or as reference antibodies in clinical guidelines, the current step is to consider their combination with a therapy targeting a B lymphocyte tumor protein such as venetoclax (targeted therapy) or potentially in the future, with a chromatin modulator or another agent (alisertib, TGF-P inhibitor, FOXO1, or farnesyltransferase) [4-10]. Despite very promising advantages, immunotherapy presents challenges, including the 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., epitope downregulation).Achieving measurable residual disease (MRD) status is a major factor in ensuring a favorable clinical outcome for the patient, particularly regarding progression-free survival (PFS) and overall survival (OS). MRD status is a good predictor of PFS and the quality of remission of the hematological malignancy. This is especially true in cases of complete hematological remission, but with an MRD positivity > 10 minutes. 4 (i.e., intermediate MRD: 10') 4 -10' 2 ; or high MRD: > 10' 2 ), strengthening treatment is currently considered beneficial. Similarly, monitoring of MRD progression is advised when MRD is low or undetectable < 10' 4is obtained, which should prompt consideration of treatment if MRD becomes detectable again, prior to clinical relapse. Resistance mechanisms to lgG1 isotype mAbs can exist naturally (primary resistance) or appear 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. RTX resistance also involves the overexpression of Bcl-2 or Bcl-xL molecules, as anti-apoptotic factors.

[0013] In CLL, OBZ is the reference antibody treatment used, in accordance with the FILO-LYSA 2025 clinical recommendations, as part of the treatment of 1 ere line (in 1 ere (relapse, RTX is recommended), OBZ (or RTX in 1 errelapse) prescribed in combination with Venetoclax, which is a targeted anti-Bcl2 therapy: treatment is limited to 1-2 years, and measurable residual disease (MRD) is achievable (MDR negative: 62% for the RTX + Venetoclax combination in the MURANO clinical trial; 76% for the OBZ + Venetoclax combination in the CLL14 study). This combination of immunotherapy and targeted therapy is very expensive. The use of a BTK inhibitor (continuous lifelong treatment, with detectable residual disease persisting in 90% of cases for ibrutinib monotherapy) is also very expensive because BTK use is currently considered a continuous treatment [1-3]. A BTK inhibitor can also be combined with Venetoclax, but the risk of cardiac complications is real: hypertension, atrial fibrillation.Indeed, this combination is associated with competition for cytochrome P450, CYP3A4, which affects the hepatic metabolism of both molecules and may increase or alter their plasma concentrations. The combination of an iBTK and venetoclax significantly increases the cost of this combined targeted therapy. Treatment with an iBTK monotherapy is effective but often leads to measurable residual disease, requiring long-term treatment and resulting in a significant medical and economic cost. The combination of an iBTK (ibrutinib) and rituximab (RTX) leads to MRD negativity at 12 months in 8.3% of cases. The combination of an iBTK (ibrutinib) and obozoolitinib (OBZ) leads to MRD negativity in 30% of cases (ILLUMINATE study). The combination of an iBTK (Ibrutinib) and Venetoclax leads to MRD negativity in 75% of cases at 12 treatment cycles (CAPTIVATE study).

[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 bedside. To date, IgG1 antibodies, with their divalent structure, represent 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 greater complement-dependent cytotoxicity (CDC) and multivalence, as they offer the advantage of 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, cellular and molecular agglutination capabilities, and thus inducing intracellular signal transduction pathways (such as the ERK1 / 2 pathway) and / or cytokine responses (interferon-gamma, TNF-alpha, IL-12, etc.), leading to a significant reduction in the viability of tumor cells and their eventual death.

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

[0017] There are B lymphocyte pathologies resistant to anti-CD20 monoclonal antibodies (RTX or OBZ) or presenting an unmet medical need, such as: • P53 gene deletion CLL and doubly refractory (2R CLL) (high genetic risk and high risk of Richter transformation) or patients not tolerating reference treatments and / or having experienced major adverse effects, and elderly patients who cannot tolerate them and / or at risk of complications with these treatments (cardiac, infectious...), also referred to as "unfit" patients.

[0018] • Double or triple hit (DHL / THL) and dual expression (DEL) non-Hodgkin lymphomas, both of which lead to an amplification of the expression of the c-Myc oncogene (a proliferation factor with Ki-67 > 70%), as well as elderly patients with relapsed, refractory or resistant diffuse large B-cell lymphoma.

[0019] Special cases of refractory patients:

[0020] In CLL, allogeneic bone marrow transplantation may be considered:

[0021] - in patients refractory to immunotherapies and / or targeted therapies and / or immunochemotherapy and / or patients with a TP53 or del(17p) mutation. or

[0022] - in patients with CLL evolving into Richter's syndrome after an isotopic response following immunochemotherapy treatment for Richter's syndrome.

[0023] In both cases, bispecific antibodies (BsAbs) and CAR-T cells (chimeric antigen receptor T cells) may be an alternative to allogeneic bone marrow transplantation. The curative effect 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) and encephalopathy related to the use of CAR-T cells

[0016] .

[0024] 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 IgG 1 anti-CD3xanti-CD20), chronic retinopathy syndrome (CRS) is observed in 90% of patients (40% grade 1 and 50% grade 2). A first BsAb based on scFv anti-CD3 / anti-CD19 (BiTE, blinatumomab) has received marketing authorization, but this molecule has a very short half-life (must be administered by continuous infusion) and can have 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 2 / day resulted in poor response rates and a dose of 90 pg / m 2 / 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 (22% response rate at two months of treatment).

[0025] 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

[0020] . 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 role in the treatment of refractory CLL [21-22].

[0026] 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 limited experience with their use in chronic lymphocytic leukemia (CLL). In CLL, their efficacy is uncertain and controversial due to the exhausted phenotype of T lymphocytes and the possible loss of CD19 via shaving, creating resistance to CAR-T cell therapy.

[0027] We evaluated an immunological combination of an anti-CD5 IgG1 antibody in combination with an anti-HLA-DR IgG1 antibody. The HLA-DR protein is a class II MHC cell surface receptor encoded by the human leukocyte antigen complex on the 6p21.31 region of chromosome 6. Its main function is to present peptide antigens, potentially of foreign origin, to the immune system in order to induce CD4+ T lymphocyte (helper T lymphocyte) responses that potentially lead to the production of antibodies against the same peptide antigen. The anti-HLA-DR IgG1 antibody has been tested against B-cell malignancies (International Application WO2010145895)

[0023] . Experiments were carried out in vivo, with the injection of JOK-1 cells (cell line), expressing the human CD5 protein (stableally transfected cells).The results obtained were very promising insofar as mice treated with the combination of anti-CD5 + anti-HLA-DR monoclonal antibodies did not develop disease, unlike mice treated with other combinations: anti-HLA-DR x anti-CD71 or anti-CD20, RTX mAb alone.

[0028] Description of the invention

[0029] Anti-CD20 antibodies are very effective, but minimal residual disease (MRD) is still detectable and measurable. The inventors therefore sought to improve CD20-based immunotherapies by avoiding the formation of disease resistance mechanisms against this type of agent (e.g., resistance to the use of anti-CD20 lgG1 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 (ASO-PCR, or immunophenotyping of the tumor clone by flow cytometry, or more recently, next-generation sequencing, i.e., NGS studies) of blood and / or bone marrow cells.

[0030] To achieve this, the Inventors sought an immunotherapeutic partner to enhance anti-CD20 bioactivity, induce non-measurable residual disease, and implement a non-toxic, short-term treatment. The primary objective is to increase the efficacy of type I anti-CD20 IgG1 monoclonal antibodies (e.g., RTX) for non-CLL B-cell lymphoid malignancies and type II anti-CD20 IgG1 monoclonal antibodies (e.g., OBZ, reference antibody) for B-cell CLL, and / or anti-CD20 IgM monoclonal antibodies; and to overcome resistance to anti-CD20 antibodies.

[0031] Medical applications include: - B-cell lymphoid malignancies in general, but in particular pathologies resistant to anti-CD20 monoclonal antibodies (RTX or OBZ) or presenting an unmet medical need, such as:

[0032] • CLL with P53 gene deletion and double-refractory (CLL 2R) (high genetic risk and high risk of Richter transformation) or patients not tolerating reference treatments and / or having experienced major adverse effects, and elderly patients who cannot tolerate them and / or at risk of complications with these treatments (cardiac, infectious...), also referred to as "unfit" patients.

[0033] • Double or triple hit (DHL / THL) and dual expression (DEL) non-Hodgkin lymphomas, both of which lead to an amplification of the expression of the c-Myc oncogene (a proliferation factor with Ki-67 > 70%), as well as elderly patients with relapsed, refractory or resistant diffuse large B-cell lymphoma. The Inventors have demonstrated in a completely unexpected way that the use of an anti-HLA-DR IgM 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 anti-CD20 type I (rituximab, RTX) or II (Obinutuzumab, OBZ) IgM monoclonal antibody, or with an anti-CD20 IgM 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 in HLA-DR+ HUVEC cells, unlike other lgG1 anti-HLA-DR isotype antibodies such as IMMU-114 (L243). No toxicity was observed during in vivo experiments in the CLL-PDX NGS mouse model or the chicken embryo.

[0034] The Inventors therefore propose a new treatment for CLL and B-cell malignant lymphoid hemopathies (non-Hodgkin B-cell lymphomas), and in particular in clinical situations of B-cell malignant lymphoid hemopathies presenting an unmet medical need (see above), 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 IgG1 (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 IgG1.

[0035] The present invention 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 or epitope fragment as the murine IgG1 monoclonal antibody IMMU357 (Beckman Coulter), but with a higher affinity for the HLA-DR molecule. IgG1 IMMU357 is a diagnostic monoclonal antibody.

[0036] According to a particular embodiment of the present invention, the monoclonal antibody disotype IgM anti-HLA-DR is in pentameric or hexameric form, preferably pentameric, and exhibits greater efficacy than lisotype lgG1 anti-HLA-DR.

[0037] According to a particular embodiment of the present invention, the monoclonal antibody disotype IgM anti-HLA-DR according to the present invention has the function of potentiating the activity of monoclonal antibodies disotype lgG1 anti-CD20 and / or disotype IgM anti-CD20.

[0038] The present invention further relates to a pharmaceutical composition comprising the IgM anti-HLA-DR monoclonal antibody according to the present invention, at least one IgG1 anti-CD20 monoclonal antibody and / or at least one IgM anti-CD20 monoclonal antibody, and a pharmaceutically acceptable excipient.

[0039] According to a particular embodiment of the present invention, said at least one monoclonal antibody disotype lgG1 anti-CD20 is an anti-CD20 of type I.

[0040] According to a particular embodiment of the present invention, said at least one monoclonal antibody disotype lgG1 anti-CD20 is an anti-CD20 type II.

[0041] 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. 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-cell lymphoma, non-Hodgkin lymphoma (of B-cell phenotype), or B-cell chronic lymphocytic leukemia (CLL), and also in clinical situations of B-cell lymphomas presenting an unmet medical need (see above).

[0042] BRIEF DESCRIPTION OF THE FIGURES

[0043] Figure 1 represents the epitope map of the anti-HLA-DR 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, using a gray gradient 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.

[0044] Figure 2 compares the antigenic recognition specificity of the anti-HLA-DR monoclonal antibody IgM with four commercial anti-HLA-DR IgG antibodies used for diagnosis. The histograms represent the overlay of MFIs obtained by flow cytometry analysis for Daudi cell line B cells (HLA-DR positive) labeled with commercial anti-HLA-DR IgG I 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.

[0045] Figure 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. CLL cells are incubated with FITC-conjugated anti-HLA-DR IgG1 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 monoclonal antibodies, the nuclei (DAPI), and the fusion of the two fluorescences (MERGE). Scale bar: 10 µm.

[0046] Figure 4A represents the comparative B cell mortality of primary and patient-derived 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 an isodose 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).

[0047] Figure 4B represents the comparative B cell mortality of primary and patient-derived clonal B lymphocytes, observed after 6 hours (H6) of treatment with anti-CD20 lgG1 alone or in combination with the anti-HLA-DR IgM isotype. CLL cells were incubated for 6 hours with anti-CD20 lgG1 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).

[0048] Figure 5A illustrates the impact of the tumor microenvironment on the bioactivity of IgG1 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 derived from nurse-like cells. B lymphocyte cell viability after treatment with anti-CD20 rituximab (RTX) isotype lgG1 mAbs and anti-CD20 IgM alone or in combination with RTX IgGI and IgM with the anti-HLA-DR IgM isotype in fetal calf serum (FCS) and in SA from patients was determined after incubation of CLL cells for 6 hours with RTX anti-CD20 lgG1 and IgM 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 (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.

[0049] Figure 5B illustrates the impact of the tumor microenvironment on the bioactivity of anti-CD20 IgG1 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 survival, mirroring the in vivo survival effect of the tumor microenvironment. Following this, the cells were treated with the monoclonal antibodies for 5 hours at a dose of 2 pg / ml. B cell death was assessed by annexin V staining.

[0050] Figure 6A illustrates the study of the impact of the chronobiology of treatment with anti-CD20 IgG1 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 monoclonal antibodies of the lgG1 isotype 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. [Figure 6B] represents the effects of anti-CD20 and anti-HLA-DR IgM lgG1 chronobiology on BB homotype cell aggregation.The modification of cultured cells induced by anti-CD20 RTX and OBZ mAbs of IgG 1 isotype in association with anti-HLA-DR IgM was observed after 1 day and 3 days of incubation of CLL PBMCs with the mAbs at 2 pg / ml by depositing the IgG 30 minutes before the IgM or vice versa.

[0051] Figure 7A illustrates 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 monoclonal antibodies (D5). The presence of CLL clonal B cells in the spleen was determined by real-time RT-qPCR with primers specific for human Alu sequences (hAlu) normalized to mouse [3-Actin (m[3actin] expression on total DNA extracts (a).Using a standard curve generated with a known number of cells, the 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).

[0052] Figure 7B illustrates the impact of in vivo treatment with the antibody obinutuzumab (OBZ) on MRD, compared to 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 P-actin (m-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).

[0053] [Figure 8A] represents B cell viability post-treatment (at H6) with therapeutic anti-CD20, IgG, and IgM anti-HLA-DR monoclonal antibodies, used alone or in combination. Viability is identified by B lymphocyte depletion (% B cell) for combinations of type I or II anti-CD20 IgG (RX and OBZ antibodies) and anti-HLA-DR IgM, the same combinations with inverted isotypes, and "double IgM" combinations; compared to the antibodies used alone or to a negative control without antibodies.

[0054] [Figure 8B] represents cell death of "remaining" B cells (identified by a size / structure remaining normal), for anti-CD20 IgG type I or II combinations (RX and OBZ antibodies) and anti-HLA-DR IgM, the same combinations with reversed isotypes, and "double IgM" combinations, compared to antibodies used alone or a negative control without antibodies.

[0055] [Figure 8C] represents the importance of epitopes in the bio-activity of anti-CD20 IgG antibodies on primary tumor B cells from patients with CLL.

[0056] The role of IgG and IgM isotypes in anti-CD20 and anti-HLA-DR antibody combinations was evaluated using CLL PBMCs. CLL cells were suspended at 1 million per milliliter in RPMI culture medium, 10% FBS. The anti-HLA-DR and anti-CD20 RTX and OBZ IgG and IgM isotype antibodies were incubated with the cells at a concentration of 2 pg / ml.

[0057] The administration of the mAbs was carried out by alternating the anti-CD20 (RX or OBZ) and anti-HLA-DR combinations according to the description below: - IgG + IgM treatment - IgM + IgG treatment - IgM + IgM treatment Control IgG and IgM isotypes were also evaluated.

[0058] The percentage (%) of target B cells [Fig. 8A], B cell death [Fig. 8B], and the effect of control isotypes [Fig. 8C] were assessed after 6 hours (H6) of incubation by flow cytometry using an anti-CD19-AF700 antibody for specific detection of B lymphocytes and annexin V coupled to FITC for detection of dead cells (Excitation: 488 nm; Emission: 350 nm). Data were collected and analyzed using Kaluza software.

[0059] [Figure 9] represents the structure of hexameric and pentameric IgM.

[0060] Figure 10 shows the comparative anti-tumor response of hexameric and pentameric IgM by the Incucyte technique after 48 hours in 7 patients with CLL. Kinetic analysis of cytotoxicity and oxidative stress was performed using the Incucyte® Live-Cell Analysis System (Essen BioScience, Ann Arbor, ML, USA) at 37°C with 5% CO2. Peripheral blood mononuclear cells (PBMCs) were seeded in 96-well culture plates (300,000 cells / well in 200 pL of complete RPMI medium). For cell viability analysis, the medium was supplemented with 5 pM of Incucyte® Cytotox Green reagent, which specifically penetrates dead cells. Intracellular oxidative stress was quantified using the CelIROX™ Deep Red probe (5 pM). The mortality rate was calculated by cross-referencing cytotoxicity and oxidative stress signals, normalized by the detected cell surface area.Antibodies were tested in duplicate on PBMCs. Acquisitions were performed hourly for 48 to 72 hours using a 20x objective. Four images were collected per well at each time point. Phase-contrast imaging was used to monitor the morphological dynamics of cell populations during treatment.

[0061] Figure 11 presents a comparative dose-response curve analysis of B-cell depletion in three CLL patients (CLL1, CLL2, CLL3). The cytotoxicity of hexameric and pentameric IgM was evaluated as a dose effect (1–5–10 pg / ml) on PBMCs from three CLL patients. CLL cells were suspended at 1 million per milliliter in RPMI culture medium, 10% FBS. Monoclonal antibodies (mAbs) were added in increasing doses and incubated for 6 hours. The percentage of B-cell target cells after treatment was measured by flow cytometry using an anti-CD19-AF700 antibody. Data were collected and analyzed using Kaluza software.

[0062] [Figure 12] represents the comparative analysis of the bioactivity of pentameric and hexameric IgM after resistance induction: a) study of B lymphocyte depletion; b) study of cell death. The bioactivity of hexameric and pentameric anti-HLA-DR antibodies was evaluated under two culture conditions in a resistance environment, CpG oligodeoxynucleotides (0.25 pM) without or with CD40L (1 pg / ml) (n=2 to 8 CLL depending on the conditions). CLL cells were suspended at 1 million per milliliter in RPMI culture medium, 10% FBS. The mAbs were incubated with the cells at a concentration of 2 pg / ml. The percentage (%) of B target [Fig. 12a] as well as the % of B cell death [Fig. 12a]12b] were evaluated after 6 hours of incubation by flow cytometry using an anti-CD19 antibody, AF700, 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 were collected and analyzed using Kaluza software.

[0063] [Figure 13] represents the endothelial toxicity study of pentameric and hexameric IgM on: a) primary HUVEC cells in the absence or presence of PBMCs from CLL patients; b) the EA.hy926 endothelial cell line. In both experiments, cells were stimulated with 10 ng / ml of interferon gamma (IFNγ) for 24 hours to induce membrane expression of the HLA-DR protein. Endothelial toxicity was assessed on primary HUVEC (PromoCell) cells in 12-well plates (2.10 5HUVEC / well / ml) [Fig. 13a]. For direct toxicity assessment, mAbs were added at 5 pg / ml. For indirect toxicity assessment, HUVECs were co-cultured with CLL PBMCs (6 million / ml) at the time of mAb addition. After 6 hours of incubation, the PBMCs (non-adherent cells) were washed off, and then the HUVECs were detached by trypsin. Mortality was assessed by flow cytometry with annexin V / IP staining (apoptosis: annexin V+; necrosis: annexin V+ / IP+). Endothelial toxicity in the EA.hy926 cell line, derived from transformed human umbilical vein endothelial cells, was assessed in a 96-well plate (2.5.10 5EA.hy926 / ml) [Fig. 13b]. The monoclonal antibodies (mAbs) were added at 5 pg / ml in the presence of 20% human serum. The anti-HLA-DR IgG L243 antibody (Biolegend) was tested in comparison to hexameric and pentameric IgM. After 24 hours of treatment, cell viability was assessed using the MTS assay (Promega) after 1 h of incubation. The cells' ability to reduce MTS to formazan is measurable at 490 nm. This absorbance represents the mitochondrial activity induced by treatment with the monoclonal antibody in question (hexameric IgM, pentameric IgM, or anti-HLA-DR L243 antibody). One-hour incubation with 10 mM H2O2 was used as a positive control, consistent with the MTS assay. The results represent the absorbance value of the test wells less the absorbance obtained for the cell-free medium (DO 490: test - blank).

[0064] Figure 4 illustrates the study of anti-B-lymphocyte cytotoxicity induced by the IgG / IgM combination in a non-Hodgkin lymphoma model: a) Cells from patients with B-cell non-Hodgkin lymphoma (B-NHL): marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL); b) and c) The Raji cell line was used as a model for studying Burkitt lymphoma in 3D spheroid culture. Figure 14a assesses the toxicity of NHL PBMCs after 4 hours of treatment in a 96-well plate (1 million / ml) by flow cytometry using an anti-CD19-AF700 antibody with annexin V staining (apoptosis). The monoclonal antibodies (mAbs) were added at 2 pg / ml in the presence of 10% FBS. Data were collected and analyzed using Kaluza software. The results represent the % of tumor target B and the % mortality on these same cells. [Fig.14b] The toxicity of mAbs on the Raji cell line was evaluated in the 3D spheroid model in the absence and presence of T lymphocyte effectors from a healthy donor. The cells were seeded at 1.10. 4 cells per well of round-bottom ULA plates (Face 11 i tate) and cultured for 3 days, before or without the addition of T effectors (LT) at 3.10 4 Cells were added per well, and monoclonal antibodies (mAbs) were added in parallel at 2 pg / ml. Acquisition was performed using the Incucyte™ and the Spheroid Analysis Module. The histogram on the right represents the surface area of ​​the spheroids after day 4 treatment in µm 2 The results in the left histogram represent the surface area of ​​live cells after treatment on day 4 (= MRD in pm) 2), i.e., the total surface area of ​​the spheroid ("brightfield mask") minus the area of ​​cell death ("CytotoxGreen mask"). [Fig. 14c] The images corresponding to the spheroids after 4 days of treatment were obtained with the Incucyte™; the white area in the spheroids corresponds to the surface area of ​​live cells (MRD). The scale bar represents 1.3 mm.

[0065] EXAMPLES

[0066] EXAMPLE 1: MATERIALS AND METHODS

[0067] - Production of recombinant anti-HLADR IgM [24-25].

[0068] The original anti-HLA-DR monoclonal antibody was generated in mice using human cells expressing HLA-DR as the antigen. Complementary DNA (cDNA) encoding the variable domains VH and VL of this antibody was isolated from murine hybridoma (International Application WO201 0 / 145895)

[0023] . This cDNA enabled the production of this antibody in recombinant form under the IgG1 and IgM isotypes.

[0069] An anti-HLA-DR IgM monoclonal antibody in its hexameric form (12 Fab domains instead of 2 with an lgG1) was produced using the baculovirus / insect cell expression system. For this purpose, the cDNAs encoding the variable VH and VL regions 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

[0024] . 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

[0025] . The recombinant viruses were cloned by the plaque lysis method. 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, recombinant antibodies secreted during infection of Sf9 cells cultured in serum-free medium 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.

[0070] An anti-HLA-DR IgM monoclonal antibody in its pentameric form (10 Fab domains instead of 2 with an lgG1) was produced using the baculovirus / insect cell expression system. For this purpose, the cDNAs encoding the variable VH and VL regions 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

[0024] . A 3 èmeA transfer vector was generated by cloning the cDNA encoding the J chain with the following sequence: MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDI VERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQ SNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 10). Triple recombinant viruses were generated after co-transfection of Sf9 cells (ATCC CRL1711) with the three loaded transfer vectors and viral DNA according to the technology described in International Application WO2019 / 081858

[0025] . 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, recombinant antibodies secreted during infection of serum-free 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.

[0071] - CLL cells

[0072] All experiments were conducted using primary tumor cells from patients (with their informed consent), both 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 phenotypic, cytogenetic, and molecular parameters allows for correlation studies between the treatment of interest and the response to it.

[0073] - B-cell lymphomas

[0074] The experiments on lymphoma cells were performed in vitro using primary tumor B lymphocytes and a tumor cell line. The primary cells were obtained from patients with B-cell non-Hodgkin lymphoma (B-NHL): two marginal zone lymphomas (MZL) and one mantle cell lymphoma (MCL) (with their informed consent). The Raji cell line was used as a model for studying Burkitt lymphoma in the absence or presence of T lymphocytes from a healthy donor.

[0075] - Identification of residues participating in the epitope recognized by the anti-HLA-DR IgM isotype monoclonal antibody used.

[0076] 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.

[0077] While only one α chain exists (HLADRA UNIPROT P01903.DRA_HUMAN), there are numerous variants of the β chain. The work carried out to identify the epitope was performed using as a model a heterodimer composed of the α chain and the β chain HLA-DRB1 (HLADRB UNIPROT P01911,DRB1_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 raw probability of belonging to the epitope, from dark gray for the highest probability to light gray for the lowest (but still significant) probability.

[0078] - Comparison of the specificity and individuality of antigenic recognition of the IgM isotype anti-HLA-DR monoclonal antibody used with 4 commercial antibodies.

[0079] Four commercial anti-HLA-DR IgG isotype antibodies (Biolegend LN3 and L243; Beckman IM1638U and IMMU-357) coupled to FITC (fluorescein isothiocyanate) were incubated at a saturating dose with Daudi B cell line (400,000 Daudi cells per assay, in 1X PBS at 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, an anti-HLA-DR Fab'2 (derived from IgM or IgG), or nothing. After 15 minutes of incubation with the commercial antibodies, the cells were washed and analyzed by flow cytometry (Navios, Beckman; FL1 laser for FITC detection).

[0080] The results show that all 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 that is identical or overlaps with that of IMMU-357.

[0081] It should be noted that the L243 antibody (IMMU114) has been used for clinical trials [27-34],

[0082] - Immunofluorescence on a slide.

[0083] CLL cells were suspended at 2 million per milliliter in RPMI culture medium without fetal bovine serum (FBS) on 16 mm diameter glass coverslips placed in 12-well culture plates containing anti-HLA-DR IgG and IgM monoclonal antibodies coupled to FITC at 2 pg / ml. After 4–6 hours of incubation, the well contents were rinsed with phosphate buffer (1x PBS), and the cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After two washes with 1x PBS, the preparations were covered with DAPI-containing mounting medium 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.

[0084] - Bioactivity: effect of mAbs on the viability of tumor B lymphocytes and determination of the cytotoxic activity of antibodies.

[0085] CLL or NHL cells are suspended at 1 million per milliliter 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.

[0086] According to the experiments, B lymphocyte viability was compared following 3 regimens of mAb administration allowing for chronobiological analysis of the treatments:

[0087] - Synchronous IgG1 and IgM treatment

[0088] - Treatment with IgG 1 for 30 minutes followed by IgM

[0089] - Treatment with IgM for 30 minutes followed by lgG1.

[0090] To assess 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, mimicking the tumor microenvironment in vivo. Following this, the cells were treated with monoclonal antibodies (mAbs) for 6 hours at a dose of 2 pg / ml. B cell death was assessed by annexin V staining. Cell death analysis was performed after 6 hours of incubation (at H6) using flow cytometry with 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). The data is collected and analyzed using Kaluza software.Normalized cell death is calculated using this formula (I): (I) % normalized cell death = 100 - ((%T*100) / %C) with “%T”, the % of live cells in the test tube and “%C”, the % of viability in the control condition.

[0091] The cytotoxicity index, combining 3 parameters representative of cytotoxicity, is calculated according to formula (II):

[0092] (II) Cytotoxicity Index (CI) = (% MTT - % MTC) + (% BC - % BT) + (% NCC - % NCT) where % MTT and % MTC are the % total cell death in the test and control tubes (without antibodies); % BC and % BT are the % of LB cells in the control and test tubes; and % NCC and % NCT are the % of cells with normal size / structure in the test and control tubes. - Detection of MRD in chronobiology in vivo: the CLL-PDX NGS murine model.

[0093] On day 0, CLL-PDX NGS mice were intravenously (IV) injected with fresh CLL PBMCs (3><10 7cells / mice). On the first day, the mice were randomly divided into four groups (5 mice per group) and immunotherapy was performed with an IV dose of each mAb. Two experiments were conducted combining anti-CD20 IgG 1 mAbs and anti-HLA-DR IgM, first with RTX and then with 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 RTX (7.5 mg / kg) or OBZ (7.5 mg / kg) followed by IV anti-HLA-DR IgM (7.5 mg / kg) < 2 hours after IgG 1 (group 4). CLL-PDX NGS mice were sacrificed on day five after single-dose treatment at JO.

[0094] 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.

[0095] - Quantitative detection of human cells by q-PCR.

[0096] The detection of primary human B-cell tumors in mouse spleen, bone marrow, lung, and liver was based on the quantitative detection of human Alu sequences in mouse organ DNA extracts using a protocol developed by Funakoshi et al., 2017

[0035] . For DNA extraction, 100 microliters of homogenized organ in PBS were used. The QIAGEN BioSprint 15 DNA Blood & Tissue Kit (Ref: 940017) was used according to 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 α-actin primers (mp-actin) were used as housekeeping genes (forward: 5'-AAGGCCAACCGTGAAAAGAT-3' (SEQ ID NO: 8); reverse: 5'-GTGGTACGACCAGGGATAC-3' (SEQ ID NO: 9)

[0036] . 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 for acquisition monitoring 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, and a step of melting curve at 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec.A quantitative measure of amplifiable mouse DNA was obtained by amplifying the mouse p-actin genomic DNA sequence with m-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 was normalized to the relative amount of mp-actin and expressed as ACt = (Ctmp-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 - ACtreatment. 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.

[0097] - Cytotoxicity of mAbs in the spheroid lymphoma model: Raji cell line.

[0098] The evaluation of the anti-B lymphocyte cytotoxicity of mAbs in the Raji 3D lymphoma cell model was performed in spheroid culture, a model commonly used to represent in vivo tissue environments and for screening therapeutic molecules. The spheroids were created by culturing 1.10 4Raji cells were cultured in RPMI medium in ULA non-adherent plates (Facellitate) at 37°C with 5% CO2 for 72 hours. Antibodies, with or without T lymphocytes from a healthy donor (30,000 cells), were then added at 2 pg / ml. The antibodies were added to RPMI medium containing CytotoxGreen at a final concentration of 5 pM for monitoring cell death using IncuCyte™ with the 4x objective. Image analysis was performed using IncuCyte® software; the surface area of ​​spheroids (brightfield mask, brightfield acquisitions) and the surface area of ​​CytotoxGreen-positive dead cells were used to calculate the live cell surface area after treatment in pm 2 (= MRD).

[0099] EXAMPLE 2: RESULTS

[0100] - Identification of residues participating in the epitope recognized by the anti-HLA-DR IgM isotype monoclonal antibody used.

[0101] The HLA-DR molecule is composed of two subunits, alpha and beta. Based on the highest-ranked docking positions, target residues were noted according to their probability of belonging to the epitope. Residues expected to belong to the epitope are indicated on the sequence and on the target structure (Fig. 1).

[0102] They are divided into four categories based on 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 target is renumbered as a single chain. Furthermore, not all amino acids in the target chains are visible in the 3D structure. The residues predicted to belong to the epitope and the validation peptides are indicated on the target sequence.

[0103] From these residues, four main interaction regions were defined on the target. The interaction regions are indicated on the target structure. Interestingly, the anti-HLA-DR monoclonal antibody preferentially recognizes an epitope of the HLA-DR alpha chain, but also certain amino acids of the beta chain.

[0104] - Membrane clustering effect (oligomerization) of the HLA-DR epitope induced by the anti-HLA-DR monoclonal antibody of the IgM isotype and biological effects related to epitope clustering. The anti-HLADR antibody of an IgM isotype exhibits very different properties compared to IgGI of the same specificity, in particular the oligomerization of the target not observed by the anti-HLA-DR IgG agonist.

[0105] 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).

[0106] The results show that the IgG1 anti-HLA-DR isotype induces only a random and disorganized distribution of the HLA-DR epitope within the membrane of CLL B cells. In contrast, the IgM anti-HLA-DR isotype rapidly induces (< 3 hours) the grouping of HLA-DR molecules into epitope-membrane clusters specifically in areas of intercellular contact.

[0107] This oligomerization leads to the recruitment of HLA-DR within lipid rafts and the induction of numerous other new biological activities linked to this epitope clustering:

[0108] - Interaction with the cytoskeleton, reorganization of F-actin and the cytoskeleton;

[0109] - Strong colocalization of cortactin and F-actin in the membrane structures of podosomes involved in strong homotypic BB lymphocyte adhesion;

[0110] - Strong homotypic aggregation (HA) and lymphocyte clustering of B cells clearly visible in optical microscopy and real-time imaging with the Incucyte™.

[0111] - Interaction with novel signal transduction pathways inducing B lymphocyte cell death (NOX2 activation, production of oxygen free radicals and creation of lethal B cell oxidative stress, phosphorylation of JNK1 / 2, ERK1-2 and Bcl-2 proteins, Beclin 1 activation, significant intrinsic B cell production of interferon-gamma);

[0112] - All these biological events leading to the death of the tumor target B lymphocyte. 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).

[0113] - Comparative bioactivity of the anti-HLA-DR IgM isotype mAb versus anti-HLADR IgG.

[0114] Observable: treatment with the anti-HLA-DR IgM isotype induces greater anti-tumor efficacy compared to the anti-HLA-DR IgG1 isotype (Fig. 4A).

[0115] When the anti-HLA-DR monoclonal antibody (mAb) was used in the hexameric IgM isotype, a substantial and significant reduction in the viability of tumor B cells was observed. Efficacy was assessed 6 hours (H6) after exposure to a single dose of mAb. The lgG1 anti-HLA-DR isotype is less effective than IgM in inducing CLL B cell death: the lgG1 anti-HLA-DR 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 IgM anti-HLA-DR antibody (*p < 0.05; n = 36).

[0116] - Combinations of type I and type II anti-CD20 antibodies plus anti-HLA-DR mAb: the anti-HLA-DR IgM isotype mAb potentiates the antitumor activity on CLL B cells of the anti-CD20 lgG1 isotype mAb.

[0117] 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 II. 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 IgM doses of 0.5 pg / ml, demonstrating synergistic activity at low doses of IgM in vitro and in vivo (IgM dose < 2 pg / ml). This observed "low-dose IgM" synergistic activity suggests a potential pharmacokinetic and pharmacodynamic benefit in vivo.

[0118] Efficacy was assessed 6 hours (H6) after exposure to a single dose of monoclonal antibodies (mAbs). Compared to H6 controls, 2 pg / ml of anti-CD20 mAbs induced a mean normalized B-cell death rate in CLL ranging from 10 ± 1.2% for RTX (n = 36) to 28.6 ± 5.1% for OBZ (n = 17). The combination of anti-HLA-DR IgGI and anti-CD20 IgGI did not increase cellular cytotoxicity, with approximately 20% cell death (data not shown). Conversely, treatment combined with the anti-HLA-DR IgM isotype significantly increases cell death detected at H6, compared to IgG 1 anti-CD20 alone (39.14 ± 4.14% for RTX + IgM; 50.06 ± 5.18% for OBZ + IgM).

[0119] The combination of anti-CD20 IgG1 and 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 monoclonal antibodies (mAbs) relies on the amplification of the NADPH oxidase pathway via the B lymphocyte membrane protein NOX2, whose synergistic activation by IgG1 and IgM generates reactive oxygen species (ROS). ROS lead to the induction of neoplastic clonal B lymphocyte death, a B cell death independent of caspase activation. This is a regulated, caspase-independent necrotic cell death induced by ROS responsible for lethal oxidative stress, which causes endoplasmic reticulum stress, which is associated with intramitochondrial calcium transfer that we measured by the Rhod-2 probe.The extent of intramitochondrial calcium transfer from the endoplasmic reticulum via the IP3R protein is correlated with IgM treatment, and its intensity is correlated with observed B lymphocyte death. Oxidative stress and intramitochondrial calcium transfer lead to B lymphocyte mitochondrial dysfunction and lethal mitochondrial morphological damage.

[0120] We have regularly observed lethal mitochondrial lesions in electron microscopy studies, at H6 of treatment, by the combination of lgG1 mAbs then IgM: edematous bloating of all mitochondria, severe involution of mitochondrial cristae, mitochondrial rupture.

[0121] These major and lethal morphological and functional mitochondrial alterations (B lymphocyte mortality by cellular necrosis induced by ATP deficiency) constitute the property of the synergy of IgG 1 anti-CD20 and IgM anti-HLA-DR, generating B lymphocyte death by induced and regulated necrosis, preceded by edema of the mitochondria and the perinuclear membrane.

[0122] - Anti-HLA-DR x anti-CD20 combinations: IgM anti-HLA-DR monoclonal antibody potentiating the anti-tumor activity of an IgG1 or IgM anti-CD20 monoclonal antibody (derived from RTX). Impact of the tumor microenvironment and autologous serum.

[0123] SA reproduces the tumor microenvironment (soluble immunosuppressive factors and survival factors) with greater fidelity, compared to SVF.

[0124] Observed: The use of autologous serum (AS) from patients with CLL decreases the efficacy of monoclonal antibodies (mAbs) alone or in combination with IgG1 plus anti-HLA-DR IgM, compared to the use of 10% FBS (the in vitro reference serum). Importantly, the use of an anti-CD20 IgM with anti-HLA-DR IgM not only renders the combination insensitive to the inhibitory effect of AS but also increases its bioactivity under this immune resistance condition.

[0125] The bio-activity of the mAbs is evaluated at 6 hours (H6) after exposure to a single dose of antibody.

[0126] In SA, compared to the use of the SVF 10%, the levels of business interruption are as follows:

[0127] AcM RTX anti-CD20: average loss of -2.5% for IgG 1 and average loss of -6.2% for IgM anti-CD20.

[0128] IgM anti-HLA-DR: a decrease of approximately -2.7%.

[0129] For the combination of RTX IgG + anti-HLA-DR IgM: a decrease of approximately -9.5%.

[0130] A low dose (< 2 pg / ml) of anti-HLA-DR IgM induces synergistic efficacy with type I and type II anti-CD20 lgG1 mAbs, both in 10% FBS and in autologous patient serum (n = 3) (Fig. 5A).

[0131] In FBS, 10% compared to H6 controls, 2 pg / ml of RTX anti-CD20 monoclonal antibody induces a low cytotoxicity index against CLL tumor B cells, representing an increase of approximately 5.4% for RTX IgG1 and 13.8% for RTX IgM. In FBS, the addition of anti-HLA-DR IgM to anti-CD20 monoclonal antibodies significantly increases B cell death compared to IgGI and IgM anti-CD20 alone: ​​mean gain of +32.8% for RTX IgG + IgM anti-HLA-DR; mean gain of +17.6% for RTX IgM + IgM anti-HLA-DR.

[0132] In SA, the anti-B lymphocyte cytotoxicity index with the anti-CD20 RTX is approximately 3.1% for IgGI 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: an average gain of +25.6% for RTX IgG + IgM anti-HLA-DR; an average 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: an average gain of 8.6%.

[0133] 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 lgG1 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.

[0134] Comparing the normalized cell death of B cells without pretreatment with CpG ODN / CD40L, a decrease in basal cell death was observed in the presence of CpG ODN / CD40L (Fig. 5B). Pretreatment with CpG ODN and CD40L effectively resulted in 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%) was observed after a cell freeze / thaw cycle, confirming the resistance effect on B cell death induced by pretreatment with CpG ODN and CD40L (and indeed, the positive control of untreated cells in the CpG / CD40L condition).

[0135] 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 lgG1 allows the significant induction of substantial cell death in CpG / CD40L compared to the effect of anti-CD20 IgG1, without any benefit from the combination with an lgG1 (average of the conditions without CpG / CD40L with anti-CD20: 1.93%; with IgM: 6.3%). with CpG / CD40L and anti-CD20 used alone: ​​an average decrease of -0.6%, in CpG / CD40L condition with treatment with an anti-HLA-DR IgM in combination with an anti-CD20: an increase of approximately 15.9% is observed.

[0136] The bioactivity of the IgM monoclonal antibody remains unchanged and induces the same B-cell mortality threshold, even under tumor-resistant 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.

[0137] - Chronobiology of in vitro and in vivo IgM anti-HLA-DR x lgG1 anti-CD20 (OBZ) treatment.

[0138] in vitro chronobiology

[0139] The viability of B lymphocytes was analyzed chronobiologically to optimize the anti-tumor response. B lymphocyte viability was compared according to the following three regimens of monoclonal antibody (mAb) administration: - Synchronous IgG1 and IgM treatment

[0140] - Treatment with IgL for 30 minutes followed by IgM

[0141] - IgM treatment for 30 minutes followed by lgG1

[0142] Observable: we show that treatment with lgG1 followed by treatment with anti-HLA-DR IgM (after 30 minutes of exposure to IgGI) induces strong homotypic BB aggregation and significantly higher cell death, compared to other chronobiological forms, synchronous IgG1 and IgM treatment and IgM then lgG1 (Fig. 6A) (Fig. 6B).

[0143] Efficacy was assessed at 6 hours (H6) by flow cytometry after exposure to a single dose of AcMs.

[0144] A low dose (< 2 pg / ml up to 0.5 pg / ml) of anti-HLA-DR IgM led to synergistic efficacy with anti-CD20 lgG1 mAbs (RTX and OBZ) (FBS 10%), with IgG chronobiology before IgM (RTX, n = 3; OBZ, n = 4) (Fig. 6A).

[0145] For the RTX + anti-HLA-DR IgM combination, the anti-B lymphocyte cytotoxicity index is increased by 21.5% when IgG is administered before IgM (cytotoxicity index at 6 hours for IgG then IgM = 47.5% and IgM then IgG = 26%). For the OBZ + anti-HLA-DR IgM combination, the anti-B lymphocyte cytotoxicity index is increased by 11.2% when IgG is administered before IgM (cytotoxicity index at 6 hours for IgG then IgM = 78% and IgM then IgG = 66.8%).

[0146] Real-time imaging assessments with image capture on J1 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).

[0147] Evaluation of MRD: In vivo chronobiology

[0148] The goal of achieving unmeasurable residual disease is crucial 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 monoclonal antibodies (mAbs) due to the development of acquired resistance.

[0149] Observable: In vivo treatment of CLL-PDX NGS mice with a combination of RTX (or OBZ) monoclonal antibodies and anti-HLA-DR IgM induces a strong antitumor response against CLL clonal B lymphocytes. MRD analysis shows a significantly greater antitumor response induced by the combination of RTX (or OBZ) plus IgM than by RTX (or OBZ) monotherapy.

[0150] Indeed, using the PDX-CLL NGS murine model, we show that residual tumor disease remains detectable after treatment with lgG1 isotype anti-CD20 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).

[0151] 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 IgGI with a low dose of the anti-HLA-DR IgM monoclonal antibody.

[0152] The lgG1 RTX isotype combined with the anti-HLA-DR IgM isotype reduces MRD levels in the spleen by a factor of 17 in vivo. The lgG1 OBZ isotype combined with the anti-HLA-DR IgM isotype reduces MRD levels in the spleen by a factor of 2 in vivo (Fig. 7A bc and 7B bc).

[0153] The reduction in MRD was confirmed in vivo in 2 different models, in chicken embryo tumor xenograft and in mouse xenograft with PBMCs from patients with CLL (PDX-CLL NGS mouse model).

[0154] - Comparison of the biological effect of IgG / IgM combinations on cells from patients with chronic lymphocytic leukemia (CLL).

[0155] The purpose of this test is to verify whether the association of a therapeutic monoclonal antibody of the anti-CD20 IgG isotype (type I and II) with a therapeutic monoclonal antibody of the anti-HLA-DR IgM isotype (in isodose) is the rule: that of the ability of an IgM to amplify the bio-activity of an IgG.

[0156] PBMCs from patients with CLL were treated with anti-CD20 and anti-HLA-DR of IgG or IgM isotype for 6h (H6) and analyzed by flow cytometry (detection of annexin V+ labeling, indicating progressive cell death).

[0157] Two reading windows are used.

[0158] Both reflect the bioactivity of monoclonal antibodies, alone or in IgG + IgM combinations: a- The ability to deplete the tumor B lymphocyte (B) population at T6 (evaluation of tumor lysis). This is identified in Fig. 8A by the symbol: %LB. b- The ability to induce B lymphocyte cell death in cells retaining a normal size / structure (referred to as "remaining B cells") at H6 as well. Cell death is detected by annexin V+ membrane staining. It is identified in Fig. 8B by the symbol: % cell death. c- At the H6 analysis time, acquired cell death (a- complete B lymphocyte lysis and B lymphocyte depletion) and ongoing cell death (b- so-called "remaining" B lymphocytes "annexin V+") are added together to give an overall B lymphocyte cell death.

[0159] T-cell (T-cell) toxicity that could have been induced by the antibodies alone or in combination was also analyzed. This toxicity was assessed by calculating the proportion of T cells before and after antibody treatment. No T-cell toxicity was ever observed, either with IgG and IgM alone or in combination (data not shown).

[0160] Figure 8A shows that B cell depletion (% B cell) is maximal for the "anti-CD20 IgG + anti-HLA-DR IgM" combinations compared to the same combinations with inverted isotypes. The "double IgM" combinations are also effective, with the "RX IgM + HLA-DR IgM" combination resulting in a B cell reduction of 19.7% and the "OBZ IgM + HLA-DR IgM" combination resulting in a B cell reduction of 29.4%.

[0161] Figure 8B shows that cell death in the remaining B cells is highest with OBZ IgG + HLA-DR IgM, as supported by the invention. The anti-CD20 IgG + anti-HLA-DR IgM combinations exhibit higher cytotoxicity (53.61% and 75.56%) than the same combinations with the reversed combined isotypes. The latter (anti-CD20 IgM + anti-HLA-DR IgG) nevertheless induce significant mortality of 37.09% and 49.96% in the remaining B cells. The dual IgM combinations are also effective with RX IgM + HLA-DR IgM: +34.3% and OBZ IgM + HLA-DR IgM: +44.6%.

[0162] Figure 8C illustrates the importance of epitopes for antibody bioactivity using a control isotype (non-relevant isotype, either IgG or IgM) without specific target recognition. Control IgG and IgM (iso-IgG and iso-IgM) showed no effect on CLL B cells. The combination of RX IgG and iso-IgM also showed no synergistic effect. With the combination of OBZ IgG and iso-IgM, the same B cell depletion and cell death values ​​were observed as with the use of OBZ IgG alone. Therefore, there was no effect of a non-relevant isotype on the activity of type I or II anti-CD20 IgG (negative control).

[0163] These results demonstrate the importance of epitopes in IgM / IgG association. The control IgM isotype provides no benefit to anti-CD20 IgG antibodies.

[0164] - Comparison of the bio-activity of recombinant pentameric anti-HLA-DR IgM and hexameric anti-HLA-DR IgM.

[0165] By co-expression of the gene encoding the J chain and the genes encoding the heavy and light chains of IgM, a pentameric IgM was produced (Figure 9). A comparative analysis of the bioactivity of pentameric and hexameric anti-HLA-DR IgM was performed on primary B-cell tumor cells from patients with B-cell chronic lymphocytic leukemia (CLL). After 48 hours of real-time mortality monitoring using the "Incucyte" technique, the data were normalized to the control and presented as a percentage of cell viability (histogram, Fig. 10A). Cumulative data for 7 CLL patients showed no significant difference between the two IgMs. In 3 CLL patients, the pentameric IgM demonstrated superiority (Fig. 10B). The respective efficacy of pentameric and hexameric IgM appears to be patient-dependent.

[0166] In the three patients with CLL, dose-response analyses showed strong B-cell depletion efficacy of both IgM antibodies at very low doses (0.25–1 pg / ml) (Fig. 11). Pentameric IgM demonstrated superiority at these same doses. The effect was similar between the two IgM antibodies at doses of 2 or 4 pg / ml, depending on the patient. At higher doses (>4 pg / ml), inhibition of the antibody effect was observed (the dose-response effect of the IgM antibody promotes the binding of the CH4 of its Fcp to the B-cell FCMR, which is responsible for inducing a B-cell survival signal). The results thus demonstrate the superior B-cell depletion capacity of pentameric IgM; its optimum was reached at an antibody concentration of 4 pg / ml.

[0167] The effect of induced resistance on IgM bioactivity was tested in 4 to 9 experiments depending on the conditions at H6. The culture medium was supplemented with CpG-ODN (CpG oligodeoxynucleotide) in the absence or presence of CD40 ligand (CD40L). These two culture conditions allowed the induction of a cellular environment known to regulate apoptosis by inducing pro-survival signals (Granziero et al., 2001)

[0037] . Under resistance conditions, IgM showed a good effect on B lymphocyte depletion (Fig. 12a) as well as on the induction of cell death in the remaining B cells (Fig. 12b). Since cell death is a phenomenon occurring upstream of the disappearance of tumor B lymphocytes by cell lysis (= B cell depletion), the decrease in the percentage of B cells allows for a kinetic interpretation of the antibody effect.B cells that survived and maintained a normal size / structure at this time point in the study allowed for the observation of a persistent antibody effect if annexin V+ staining was observed (cells showing a biological sign of mortality and destined for cell lysis). These two parameters were analyzed at H6.

[0168] - Study of the endothelial toxicity of pentameric and hexameric anti-HLA-DR IgM.

[0169] The cytotoxicity of anti-HLA-DR IgM antibodies was evaluated on HUVEC endothelial cells and on the EA.hy926 cell line after 24 hours of stimulation with interferon gamma to induce HLA-DR protein expression (a pro-inflammatory condition). Endothelial mortality is predictive of the occurrence of vasculitis in vivo. Cytotoxicity on HUVEC cells was evaluated as a direct effect or in the presence of PBMCs from CLL patients and their respective serum to mimic physiological conditions, as well as potential indirect cytotoxicity. The results showed no toxicity of anti-HLA-DR IgM towards endothelial cells [Fig. 13a], nor induction of oxidative stress in endothelial cells (data not shown), whether or not PBMCs from CLL patients were present. Toxicity on the EA cell line...hy926, the interferon-gamma-induced HLA-DR protein, was evaluated in human serum after 24 hours of treatment with anti-HLA-DR IgM antibodies. The anti-HLA-DR IgG antibody L243 was used as a commercial control and I H2O2 as a positive control for cell death. The results showed no toxicity of our pentameric and hexameric anti-HLA-DR IgM antibodies, with functional mitochondrial metabolism identical to the control condition [Fig. 13b]. The anti-HLA-DR IgG, L243, showed a significant increase in mitochondrial activity (p = 0.003), which was not observed with the anti-HLA-DR IgM antibodies.

[0170] - Comparison of the biological effect of IgG / IgM combinations on lymphoma cells. To evaluate the toxicity of mAbs in another model of B-cell malignant evolution, bioactivity was tested on cells from patients with B-cell non-Hodgkin lymphomas (B-NHL): marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL) [Fig. 14a]. The Raji cell line was used as a model for studying Burkitt lymphoma in 3D spheroid culture [Fig. 14b] and [Fig. 14c]. In PBMCs of B-cell non-Hodgkin lymphoma (B-HL) (MZL and LM), the combination of anti-CD20 IgG1 OBZ with anti-HLA-DR IgM enhances the effect of OBZ on reducing the percentage of B-cell tumor target and on B-cell mortality of the remaining cells, after a short exposure time of 4 hours of treatment (MZL n=2 and LM n=1). These two lymphomas show similar sensitivity to IgG / IgM therapies.The evaluation of IgG / lgM combinations in the Raji spheroid model after 4 days of treatment was performed in the absence or presence of healthy donor T lymphocytes to assess the effector-mediated cell ... 2The analyses reveal that treatment with OBZ alone already leads to a reduction in MRD, indicating a loss of viability. This decrease is strongly accentuated by the synergistic combination with hexameric IgM. The presence of T cells further reinforces this effect, reflecting the involvement of T lymphocyte cellular immunity in the observed anti-B lymphocyte bioactivity. Images [Fig. 14.c] of the spheroids on day 4 of treatment allow observation of both the decrease in spheroid size and the MRD, represented by the white area. The corresponding values ​​are reported below the images, showing a decrease of 3.7 x 10⁻⁶. 6 pm 2 in the control at 0.27.10 6 pm 2 in the presence of OBZ / lgM + LT. The addition of LT allows a decrease of 0.43 x 10 6 pm 2the MRD of the OBZ / IgM. On observe also the presence of a central noise of dead cells, resulting from a spontaneous necrosis. List of references

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Claims

DEMANDS 1) Anti-HLA-DR IgM isotype monoclonal antibody. 2) Monoclonal antibody of IgM isotype anti-HLA-DR according to claim 1, wherein said antibody is in pentameric or hexameric form. 3) Monoclonal antibody of IgM isotype anti-HLA-DR according to claim 1 or 2, wherein said antibody potentiates the activity of monoclonal antibodies of lgG1 isotype anti-CD20 and / or of IgM isotype anti-CD20. 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 IgG 1 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 isotype IgG 1 is an anti-CD20 type I. 6) Pharmaceutical composition according to claim 4, wherein said at least one monoclonal antibody of isotype IgG 1 is an anti-CD20 type II. 7) Monoclonal antibody of IgM isotype anti-HLA-DR 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) Monoclonal antibody of IgM isotype anti-HLA-DR 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 malignant B lymphoid hemopathies, B phenotype non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL).

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