Therapeutic Activity Of Rituximab Research Articles

Hemolysis and Free Heme Impact Therapeutic Activity of Anti-CD20 Antibody

Hemolysis is a process of destruction of erythrocytes that can occur in different hereditary or acquired pathologies. It is accompanied by release of hemoglobin and heme, which both manifest potent pro-oxidative and pro-inflammatory activities. If released in large quantities hemoglobin and heme can directly contribute to systemic inflammation and tissue injury in hemolytic diseases. Moreover, it was demonstrated that heme binds to a significant fraction (ca. 10 %) of human antibodies (Abs) and this binding can result in considerable functional changes. Thus, heme-bound human IgG acquire reactivity towards a large set of unrelated proteins, including autoantigens. In a previous study we observed that 14 % of a repertoire of 117 clinical-stage therapeutic mAbs are able to bind heme and as a result they acquired autoreactivity and polyreactivity. Among these Abs some are specific for CD20 and used in lymphoma therapy (e.g. Rituximab). Importantly, therapeutic mAbs can be administered in patients with ongoing hemolysis. Thus, it is of critical importance to decipher molecular mechanism and functional consequences of interaction of heme with therapeutic mAbs and understand how the therapy of lymphoma can be affected by underling hemolysis and heme release. The present study has the following objective: to elucidate the effect of heme on the therapeutic activity of anti-CD20 therapy in in vitro and in vivo models of B cell lymphoma. Effect of heme on anti-CD20 therapeutic Abs was assessed by i) binding assays - ELISA, western blot; protein microarrays; absorbance spectroscopy; surface plasmon resonance, and ii) molecular modeling. The impact of heme on functional and therapeutic activity of Rituximab was analyzed by i) site-directed mutagenesis; ii) flow cytometry; iv) in vitro and in vivo models of human B cell lymphoma. Our data reveled molecular and functional details about the interaction of heme with Rituximab and other anti-CD20 Abs. Thus, we characterized the interaction of Abs with heme, and identified in case of Rituximab the putative binding site for heme that overlapped with antigen binding site. By using a protein array with >9800 human proteins, we identified the target proteins of heme-bound Rituximab. A mouse model of human B cell lymphoma revealed that the therapeutic activity of Rituximab was compromised following exposure to cell free heme under condition of acute intravascular hemolysis. Our study delineates that a fraction of currently used therapeutic mAbs show high sensitivity to heme and their therapeutic activity can be compromised under hemolytic conditions. This finding suggests that therapeutic interventions that reduce free heme and /or intravascular hemolysis should precede therapy with these Abs. Moreover, we identified sequence features of Abs that determine the sensitivity to heme. This finding can help to reduce sensitivity of heme of therapeutic Abs by protein engineering.

Abstract 2768: Potentiating the action of antibody-dependent cell-mediated cytotoxicity with immunocytokines

Abstract Intact antibodies in the IgG format typically display only moderate in vivo activity against solid tumor masses, when their mechanism of action relies on antibody-dependent cellular cytotoxicity (ADCC). The therapeutic activity is mainly limited by the lack of immune effector cells (and most notably NK cells) in the neoplastic mass. Our group had previously reported that the therapeutic activity of Rituximab and other intact immunoglobulins could be strongly potentiated by the antibody-based targeted delivery of interleukin-2 (IL2) to the tumor environment. The combination regimen induced complete remissions of established localized lymphomas and provided long-lasting protection from disseminated lymphoma, in mouse models that featured functional NK cells. In this study, we examined the ADCC activity of TA99, a monoclonal antibody which specifically recognizes gp75, a conserved tumor-associated antigen over-expressed in both murine and human melanoma cells. In a preventive setting TA99 in murine IgG2a format completely inhibited the formation of lung metastases in mice, which had been injected intravenously with B16F10 melanoma cells. However, when the B16F10 melanoma tumor was grown subcutaneously in immunocompetent mice, the ADCC activity was not potent enough to eradicate the pre-established tumors and only a modest tumor growth inhibition was observed. A microscopic tissue distribution analysis revealed that TA99-IgG2a had selectively accumulated in the tumor mass compared to normal organs and that NK cells within the neoplastic lesions were scarce. In order to increase NK cell density at the tumor site and potentiate ADCC activity, we developed fusion proteins of the TA99 antibody with murine TNFα, IL2 and IL12. The antibody-cytokine fusion proteins were used alone and in combination with TA99-IgG2a. Combination treatment led to a marked increase of anti-cancer activity against established subcutaneous tumors. Our findings indicate that the targeted delivery of certain pro-inflammatory cytokines to the tumor site potentiates the anti-cancer properties of intact antibodies, enhancing their ability to induce ADCC. Citation Format: Patrizia Murer, Jonathan D. Kiefer, Louis Plüss, Dario Neri. Potentiating the action of antibody-dependent cell-mediated cytotoxicity with immunocytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2768.

The Peripheral Blood Lymphocyte to Monocyte Ratio At Diagnosis Is a Potent Outcome Predictor in Diffuse Large B-Cell Lymphoma Treated with R-CHOP: A Long-Term Analysis On 973 Patients Receiving Chemotherapy with or without Rituximab

AbstractAbstract 1553▪▪This icon denotes a clinically relevant abstract Background and AimThe number and type of lymphocytes and monocytes/macrophages detectable in the peripheral blood and the lymph nodes of patients with Hodgkin and non-Hodgkin lymphomas has been recently extensively investigated and interesting results indicate they may possibly affect the pathogenesis and prognosis of these diseases. Recent results indicate that the lymphocyte/monocyte ratio (LMR), when assessed at diagnosis by a simple automatic blood count, may predict the clinical outcome of diffuse large B cell lymphoma patients (DLBCL) treated with the R-CHOP chemotherapy program (Li Z-M et al.: PLoS ONE 7(7):2012). The main objective of our study was to evaluate whether: a) the prognostic value of LMR could be confirmed either in patients treated with CHOP and Rituximab (R-CHOP) as well as in those treated with CHOP alone and b) the LMR could improve the prognostic profile as defined by the International Prognostic Index (IPI). Patients and MethodsWe retrospectively reviewed the clinical outcome of 973 DLBCL patients treated (549 with R-CHOP) and regularly followed at our institutions from 1984 to 2012. The median age of this patients cohort was 61 years (range, 18–86), the Male/Female ratio 55% and the median follow up 44 months (range, 2 – 330). According to the IPI score, 61% of patients were in the low (0–2) risk group, while 39% were in the intermediate or high-risk groups (3–5). A receiver operating characteristic (ROC) curve analysis was used to illustrate in our data set the best cut off values of peripheral blood lymphocyte absolute count (ALC), monocyte absolute count (AMC) or LMR. The relationship between IPI and the LMR was analyzed by the Fisher exact test. Univariate analysis to evaluate differences between variables was performed by the log rank. A multivariate analysis was performed by Cox proportional-hazards models. ResultsA preliminary ROC curve analysis performed on all patients (treated with or without rituximab) failed to identify any meaningful relationship between Overall Survival (OS) and the ALC, the AMC or the LMR. However, when the same analysis was restricted to patients treated with R-CHOP, we could confirm not only the positive correlation between ALC and AMC and OS, but most importantly that a LMR value >2.6 is the most sensitive (70%) and specific (53%) cut off to predict the OS. Within the R-CHOP treated cohort (N=549), we further investigated the relationships between LMR and the most relevant clinical features measured at diagnosis. Patients with a LMR ≤ 2.6 (52%) had a worst ECOG PS (p= 0.000), a higher LDH level (P= 0.000), a higher IPI (p= 0.000) and more frequently they were male (p= 0.02) and had an advanced Ann Arbor disease stage (p= 0.002). On the contrary, no statistical correlation was observed with age and the presence of extranodal sites. The proportion of patients achieving a complete response or a very good partial response was 95% in patients with a LMR >2.6 and 87% for those with a LMR ≤ 2.6 (p=0.018). More interestingly, among patients failing to achieve CR the proportion of those with a LMR ≤ 2.6 was 79% as compared to 21% among those with a LMR >2.6. The Event Free Survival of patients with a LMR >2.6 was significantly better when compared to those with a LMR ≤ 2.6 (71% vs. 59% at 5 years, p= 0.01) while no difference was observed for the Disease Free Survival. By univariate and multivariate analysis we could show that, similarly to a high IPI, a LMR ≤ 2.6 strongly predict a poor OS (p= 0.0000) (Figure 1). In addition, we could demonstrate a strong interaction between IPI and LMR since patients with a high IPI and LMR ≤ 2.6 are characterized by a very poor prognosis when compared to all the others (p= 0.000). ConclusionsOur results confirm that a LMR ≤ 2.6 when assessed at diagnosis by a simple automatic blood count is not only a strong predictor of poor survival but it may help to better define a very poor prognostic subgroup in R-CHOP treated DLBCL. This novel prognostic marker is irrelevant when applied to patients receiving chemotherapy alone, giving further support to the notion that lymphocytes and/or monocytes play a crucial role on the therapeutic activity of Rituximab. [Display omitted] Disclosures:Rambaldi:Hoffman-La Roche: Consultancy, Honoraria. Ladetto:Hoffman-La Roche: Consultancy, Honoraria. Gianni:Hoffman-La Roche: Consultancy, Honoraria. Tarella:Hoffman-La Roche: Consultancy, Honoraria.