Berengere Vire

The lymph node microenvironment promotes B-cell receptor signaling, NF-κB activation, and tumor proliferation in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B lymphocytes, involves blood, bone marrow, and secondary lymphoid organs such as the lymph nodes (LN). A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations, but its contribution in vivo remains ill-defined. To elucidate the effects of tumor-host interactions in vivo, we purified tumor cells from 24 treatment-naive patients. Samples were obtained concurrently from blood, bone marrow, and/or LN and analyzed by gene expression profiling. We identified the LN as a key site in CLL pathogenesis. CLL cells in the LN showed up-regulation of gene signatures, indicating B-cell receptor (BCR) and nuclear factor-κB activation. Consistent with antigen-dependent BCR signaling and canonical nuclear factor-κB activation, we detected phosphorylation of SYK and IκBα, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL, indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation, quantified by the expression of the E2F and c-MYC target genes and verified with Ki67 staining by flow cytometry, was highest in the LN and was correlated with clinical disease progression. These data identify the disruption of tumor microenvironment interactions and the inhibition of BCR signaling as promising therapeutic strategies in CLL. This study is registered at http://clinicaltrials.gov as NCT00019370.

Rapid clearance of rituximab may contribute to the continued high incidence of autoimmune hematologic complications of chemoimmunotherapy for chronic lymphocytic leukemia

Rituximab is an effective treatment for autoimmune cytopenias associated with chronic lymphocytic leukemia. Despite the incorporation of rituximab into fludarabine-based chemotherapy regimens, the incidence of autoimmune cytopenias has remained high. Inadequate rituximab exposure due to rapid antibody clearance may be a contributing factor. To test this hypothesis, we measured serum rituximab levels in patients treated with fludarabine and rituximab (375 mg/m2). All patients had undetectable rituximab trough levels by the end of cycle 1, and one-third had undetectable levels already on Day 6 of cycle 1. Although rituximab trough levels increased progressively with each cycle, only by cycle 4 did the median trough level exceed 10 ug/mL. The median half-life of rituximab during cycle 1 was 27 hours, compared to 199 hours during cycle 4 (P<0.0001). There was a significant inverse correlation between the rituximab half-life in cycle 1 and the degree of tumor burden (P=0.02). Two patients who were identified as having subclinical autoimmune hemolysis prior to therapy were given additional doses of rituximab during the initial cycles of therapy and did not develop clinically significant hemolysis. One patient who developed clinically significant hemolysis during therapy was given additional rituximab doses during cycles 3–5 and was able to successfully complete his treatment. In conclusion, rituximab is cleared so rapidly during the initial cycles of therapy for chronic lymphocytic leukemia that most patients have only transient serum levels. More frequent dosing of rituximab may be required to prevent autoimmune complications in at-risk patients (clinicaltrials.gov identifier:00001586).

Rituximab: Therapeutic Benefit! Vitamin R?

On November 26th, 1997, a little-known monoclonal antibody called rituximab was approved by the US Food and Drug Administration for the treatment of relapsed/refractory non-Hodgkin lymphoma (NHL). Over a decade later, rituximab has become one of the biggest therapeutic advancements in the treatment of lymphoid malignancies, redefining the standard of care for a vast majority of B-cell neoplasms. Both as a single agent and in combination with cytotoxic chemotherapy, rituximab has significantly improved response rates and progression-free survival in a variety of lymphoid malignancies, as well as overall survival and cure rates in aggressive NHL. Rituximab has also demonstrated considerable utility in a number of autoimmune hematologic and rheumatologic diseases, and is increasingly being turned to as a well-tolerated, relatively safe, and often less invasive alternative to traditional therapies for these conditions. In fact, rituximab has demonstrated safety and activity in so many diseases that it has been nicknamed “vitamin R”! All joking aside, as much as the use of rituximab has enhanced treatment options, there are still many unanswered questions and opportunities for further improvement remain aplenty. Despite a decade of experience, rituximab has preserved a certain “magical” quality. First of all, there is the experience that it can be safely added to virtually any treatment. Who would have ever thought that a drug which essentially obliterates an entire arm of the immune system for extended periods of time, could be as safe as rituximab has proven to be? Furthermore, rituximab can be combined with virtually any existing treatment strategy without significantly increased toxicity. –In this regard we may in fact have been spoiled and other monoclonal antibodies or targeted agents may not necessarily be so safe and unproblematic to integrate into existing treatment regimens. In addition to rituximab’s safety profile, uncertainties about its mechanism of action, controversies about optimal dosing, and unique and still only partially appreciated aspects of its pharmacokinetics add to the “magic”. With regard to mechanism, we know that rituximab binds to the large extracellular loop of CD20 on the surface of B-cells and depletes them. We know that cell death can occur through complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and (in some experimental systems) direct signaling. We still do not know, however, exactly how these mechanisms interface and affect each other within different tissue compartments, or how important each mechanism is individually within the context of a given disease. We also do not know whether we are maximizing efficacy and minimizing drug resistance with the current standard dose of 375mg/m2. With regard to efficacy, a recent study using a murine lymphoma model demonstrated a clear association between high tumor burden and both low post-infusion rituximab serum levels and inferior response,1 a finding that raises the possibility that we may in fact be underdosing some patients with high burdens of disease by using a dose that is adjusted only for body surface area. With regard to drug resistance, recent attention has been paid to “CD20 shaving”, a process whereby rituximab/CD20 immune complexes on malignant B cells are removed by FcγR-expressing effector cells, essentially rendering a significant portion of residual disease CD20-negative and thus refractory to subsequent rituximab treatment.2 It is thought that saturation/exhaustion of B-cell clearance mechanisms may lead to CD20 shaving. Given concerns over both insufficient dosing in the presence of high tumor burden and mechanisms of drug resistance related to bolus dosing, it may come as no surprise that some investigators have explored massively increased doses of rituximab to increase efficacy,3,4 while others have studied a metronomic approach of frequent low doses of rituximab to avoid CD20 loss.5,6 A more complete understanding of the in vivo pharmacokinetics and pharmacodynamics of rituximab may pave the way to even greater efficacy than currently possible. Time and well designed studies will tell. This edition of Seminars begins with detailed discussions of the CD20 molecule and the mechanisms of action of and resistance to rituximab, followed by reviews of its use in low-grade lymphomas, high-grade lymphomas, CLL, and autoimmune hematologic disease. These reviews provide a comprehensive overview of the clinical use of rituximab to date, as well as food for thought regarding some of the most pertinent unanswered questions regarding its use. Attention is then devoted to the phenomenon of “late-onset” rituximab-associated neutropenia, followed by a review of rituximab-associated infections. Lastly, we are given an exciting glimpse into the future with a discussion of novel anti-CD20 antibodies that hold the potential for even greater efficacy. As John F. Kennedy once said in regards to scientific progress, “The greater our knowledge increases, the greater our ignorance unfolds.” Rituximab, a product of remarkable advances in biomedicine, has become a highly effective instrument in the treatment of hematologic diseases. At the same time it has exposed areas of ignorance, which have stimulated new discoveries that will further improve treatment options for our patients.

Abstract 1490: Potentiating immunotherapy by targeting complement deposited on cancer cell surfaces

Treatment of lymphoid malignancies with anti-CD20 antibodies (mAbs) can be frustrated by the loss of cell surface CD20 through trogocytosis, creating “escape variants” that are no longer sensitive to the anti-CD20 mAb. In patients with chronic lymphocytic leukemia (CLL) treated with the anti-CD20 mAb ofatumumab, we observed that these CD20 escape variants carried covalently bound C3d complement fragments and that these C3d opsonized CLL cells persisted for weeks in circulation. Therefore, we hypothesized that C3d is a neoantigen that could be exploited to re-target cells that have escaped from anti-CD20 mAb therapy. To target complement opsonized cells we generated a human IgG1 mouse chimera mAb specific to C3d that is not competed by full length C3 in serum. To test whether targeting C3d can eliminate escape variants after anti-CD20 therapy, we collected blood samples from CLL patients before (day 1) and 24 hours after administration of ofatumumab (day 2). As expected, CLL cells on day 2 had lost CD20 expression and could neither bind, nor be killed by ofatumumab. In contrast, the anti-C3d mAb did not bind CLL cells obtained pre-treatment but bound cells obtained on day 2 with high affinity (kD = 6.7nM) and were effectively killed through CDC, NK cell mediated ADCC, and phagocytosis. Importantly, non B lymphocytes were neither bound nor killed by the anti-C3d mAb, consistent with the highly targeted and selective deposition of C3d on CD20+ cells by ofatumumab. Interestingly, when C3d opsonized CLL was exposed repetitively to anti-C3d mAb ex vivo, the amount of cell bound C3d and the fraction of cells killed increased with successive rounds of treatment consistent with an auto-amplification of C3d targeting. We tested the efficacy of a chimerized anti-C3d mAb in two mouse models. First, we transferred PBMCs obtained from CLL patients on day 2 of ofatumumab treatment (containing the C3d opsonized CD20 escape variants) into NSG mice and three days later injected either isotype control mAb (trastuzumab) or anti-C3d mAb. One injection of anti-C3d mAb effectively reduced tumor burden in both peripheral blood (from 42.5 to 0.59 CLL cells/ul of blood; p We conclude that targeting C3d deposited on cancer cells can eliminate antigen escape variants and potentiate complement fixing antibodies. Citation Format: Elizabeth J. Carstens, Martin Skarzynski, Vicent Butera, Margaret Lindorfer, Berengere Vire, Mohammed Farooqui, Christoph Rader, Ronald Taylor, Adrian Wiestner. Potentiating immunotherapy by targeting complement deposited on cancer cell surfaces. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1490.

Monoclonal antibodies targeting cell surface deposited complement fragment C3d potentiate cancer immunotherapy and eliminate antigen loss variants

Treatment-induced loss of targeted cell surface antigens through trogocytosis or internalization reduces efficacy of monoclonal antibody (mAb) therapy of cancer. However, cells that escape therapy mediated by complement-fixing mAbs carry covalently deposited complement activation fragments on their cell surfaces, in particular C3d. We hypothesized that cell-associated C3d constitutes a neoantigen that could be exploited to selectively retarget cells escaping from therapeutic mAbs. We generated an anti-C3d IgG1 human/mouse chimeric mAb specific for human C3d that is not competed by full-length C3 in human serum. We then set out to provide proof for the concept that complement-targeting mAbs can retarget cancer cells that survive mAb therapy. For this purpose, we used cells from chronic lymphocytic leukemia (CLL) patients that had substantially reduced CD20 levels due to in vivo treatment with the anti-CD20 mAb ofatumumab (OFA). The chimeric anti-C3d mAb bound cell surface C3d on these CLL cells ex vivo (KD = 6.7nM), and mediated complement-dependent, and antibody-dependent cellular cytotoxicity and phagocytosis in vitro. CLL cells opsonized by C3d in vivo and reacted with the anti-C3d mAb in vitro were further C3d opsonized, resulting in an amplification that enhanced anti-C3d mAb binding capacity and killing of target cells. In vivo, the anti-C3d mAb was effective in reducing tumor growth and extending survival in a mantle cell lymphoma xenograft mouse model. This complement-targeting mAb also depleted human primary CLL cells in the blood and spleens of xenografted NSG mice. Our results identify anti-C3d mAbs as a means to circumvent antigen loss by specifically and potently augmenting the therapeutic efficacy of complement-fixing mAbs.