Jonathan Back

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies

BackgroundCD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies.MethodsGBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401.ResultsGBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization.ConclusionThese results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Abstract C164: Preclinical pharmacological characterization of GBR 401, a new monoclonal antibody directed against human CD19.

GBR 401 is a humanized monoclonal antibody directed against domain 2 of human B-lymphocyte antigen CD19 and displays a number of mechanisms which can specifically target and kill B cells. Glenmark is developing this product as a B-cell depleting agent for B cell malignancies. CD19 is the archetypal B cell marker and is used in the diagnosis of B cell lymphomas/ leukemias by the identification of abnormal B cell numbers and/or populations. Expression of CD19 is found in the majority of B cell lineage malignancies including, but not limited to non-Hodgkin9s lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia. Given its role as a signaling molecule, an antibody directed against CD19 might be predicted to affect the progression of lymphomas and leukemias. GBR 401 is a humanized IgG1 which can specifically kill CD19 bearing cells with very efficient in vitro antibody-dependent cellular cytotoxicity (ADCC) and rapid-onset apoptosis-inducing abilities. It also can inhibit the proliferation of CD19+ tumor cells but has little to no complement dependent cytotoxicity (CDC), at least, in vitro. The in vivo anti-B cell activity of GBR 401 was evaluated using human peripheral blood mononuclear cell (huPBMC) graft models in SCID mice. GBR 401 significantly depleted human B cells in grafted mice at levels down to 0.1mg/kg compared to an isotype control. GBR 401 also produced approximately 50% depletion or EC50 at 0.02mg/kg. These in vitro and in vivo pharmacology data provide a rationale for the clinical testing of GBR 401 in patients with CD19+hematologic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C164.

Abstract B122: Humanized SCID mouse as tool for evaluation of B-cell-targeting therapies.

Background: Antibody strategies are of huge importance in targeted therapies drug development. However, most of the time, antigen is specific of human species with bad cross-reactivity with mouse antigen, leading to difficulties to apply regular mouse models. The “gold” model for the preclinical evaluation of antibody strategy should have cells expressing human antigen, to analyze Fab binding activity but should also contain human immune effector cells in order to be able to test the antibody Fc mediated activity (Antibody Dependent Cell Cytotoxicity, ADCC). We here propose the use of CB17-SCID mouse strain having the ability to exhibit complement activation, useful to test the CDC (Complement Dependent Cytotoxicity) activity of antibody. This strain of mice supports the engraftment of human peripheral blood mononuclear cells (hPBMCs), containing human effector cells as NK cells and moncoytes, responsible of ADCC activity. In our case, the target of interest is expressed on human B lymphocytes, whatever the tumoral or healthy origin. The engrafted hPBMCs contained both the NKs as effector cells but also the human B cells as target cells. Methods: Whole body irradiated (D-3) female CB17-SCID mice were treated twice (Q7D×2) (starting at D-2) with subcutaneous injections of mouse NK cell-depleting antibody TM-Beta 1 and received an intraperitoneal injection of freshly prepared hPBMCs at D0. At D11 mice received a single intravenous injection of trastuzumab used as negative control, of new B cell targeting antibody or rituximab used as positive control (10 mice per group). At D15, mice were sacrificed to collect spleen. Single cell suspensions were prepared from each collected spleen, labeled with mCD45, hCD45, hCD19 and hCD20 antibodies before FACS analysis. Absolute cell number as well as percentage of human B cells were calculated. Statistical comparisons were performed using Mann-Whitney U test after having discarded outlier values by Dixon Q test. Results: All mice injected with hPBMCs were found humanized at the time of sacrifice. Percentage of human leukocytes in mouse spleen ranged from 0.6 to 59.4% with a mean value of 12.9 ± 12.5 % and a median value of 8.3 %. Considering the human B-cell percentage, only 3 out of 80 values were considered as outliers. In the trastuzumab treated group (10 mg/kg), the spleen contained about 6 % of human B cells (within the human leukocytes, i.e. hCD45+). When the mice were treated with rituximab at 2 mg/kg, a significant decrease in the percentage of human B cells was evidenced in the mouse spleen, with about 1.5% of human B cells. A dose-dependent decrease in human B cells was also observed in mouse spleen when mice were treated with the new B-cell targeting antibody (1.4 ± 1.1 % and 3.8 ± 2.2 % of human B cells for 10 mg/kg and 0.02 mg/kg respectively). The decrease starts to be significant from 0.1 mg/kg of the new B-cell targeting antibody. Conclusions: These results show that this humanized mouse model is suitable to evaluate B-cell targeting therapies. Humanization level as well as sensitivity to treatment are compatible with dose-response analysis and then offer the possibility to use this model for benchmarking of human B-cell targeting therapy tested on human target in the presence human effectors. Moreover, this model could be applied to other targets expressed on human immune cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B122.