Suresh Veeramani

Depletion of the C3 component of complement enhances the ability of rituximab-coated target cells to activate human NK cells and improves the efficacy of monoclonal antibody therapy in an in vivo model

Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced natural killer (NK)-cell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK-cell activation when it was preincubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or nonmalignant ascites was used as surrogates for extravascular fluid, suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted before mAb treatment in a syngeneic murine model of lymphoma. Survival of lymphoma-bearing mice after treatment with CVF plus mAb and with a human C3 derivative with CVF-like functions (HC3-1496) plus mAb was both superior to that of mAb alone. These studies show that complement depletion enhances NK-cell activation induced by rituximab-coated target cells and improves the efficacy of mAb therapy in a murine lymphoma model.

Rituximab infusion induces NK activation in lymphoma patients with the high-affinity CD16 polymorphism

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity involving FcγRIIIa (CD16) likely contributes to the clinical efficacy of rituximab. To assess the in vivo effects of CD16 polymorphisms on rituximab-induced NK activation, blood was evaluated before and 4 hours after initiation of the initial dose of rituximab in 21 lymphoma subjects. Rituximab induced NK activation and a drop in circulating NK-cell percentage in subjects with the high-affinity [158(VF/VV)] but not the low-affinity [158(FF)] CD16 polymorphism. There was no correlation between NK-cell activation or NK-cell percentage and polymorphisms in CD32A, C1q, or CH50. We conclude that NK activation occurs within 4 hours of rituximab infusion in subjects with the high-affinity CD16 polymorphism but not those with the low-affinity CD16 polymorphism. This finding may help explain the superior clinical outcome seen in the subset of high-affinity CD16 polymorphism lymphoma patients treated with single-agent rituximab.

Complement in Monoclonal Antibody Therapy of Cancer

Monoclonal antibodies (mAb) have been used as targeted treatments against cancer for more than a decade, with mixed results. Research is needed to understand mAb mechanisms of action with the goal of improving the efficacy of currently used mAbs and guiding the design of novel mAbs. While some mAb-induced tumor cell killing is a result of direct effects on tumor cell signaling, mAb opsonization of tumor cells also triggers activation of immune responses due to complement activation and engagement of antibody receptors on immune effector cells. In fact, complement has been shown to play an important role in modulating the anti-tumor activity of many mAb through complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and through indirect effects by modulating the tumor microenvironment. Complement activity can have both agonistic and antagonistic effects on these processes. How the balance of such effects impacts on the clinical efficacy of mAb therapy remains unclear. In this review, we discuss the mAbs currently approved for cancer treatment and examine how complement can impact their efficacy with a focus on how this information might be used to improve the clinical efficacy of mAb treatment.

Chemoimmunotherapy for relapsed/refractory and progressive 17p13‐deleted chronic lymphocytic leukemia (CLL) combining pentostatin, alemtuzumab, and low‐dose rituximab is effective and tolerable and limits loss of CD20 expression by circulating CLL cells

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) patients with purine analog refractory disease or TP53 dysfunction still have limited treatment options and poor survival. Alemtuzumab‐containing chemoimmunotherapy regimens can be effective but frequently cause serious infections. We report a Phase II trial testing the efficacy and tolerability of a short‐duration regimen combining pentostatin, alemtuzumab, and low‐dose high‐frequency rituximab designed to decrease the risk of treatment‐associated infections and to limit the loss of CD20 expression by CLL cells. The study enrolled 39 patients with progressive CLL that was either relapsed/refractory (n = 36) or previously untreated with 17p13 deletion (17p13−) (n = 3). Thirteen (33%) patients had both 17p13− and TP53 mutations predicted to be dysfunctional, and eight patients had purine analog refractory CLL without TP53 dysfunction. Twenty‐six (67%) patients completed therapy, with only five (13%) patients having treatment‐limiting toxicity and no treatment‐related deaths. Twenty‐two (56%) patients responded to treatment, with 11 (28%) complete responses (four with incomplete bone marrow recovery). Median progression‐free survival was 7.2 months, time to next treatment was 9.1 months, and overall survival was 34.1 months. The majority of deaths (82%) were caused by progressive disease, including transformed diffuse large B‐cell lymphoma (n = 6). Correlative studies showed that low‐dose rituximab activates complement and natural killer cells without a profound and sustained decrease in expression of CD20 by circulating CLL cells. We conclude that pentostatin, alemtuzumab, and low‐dose high‐frequency rituximab is a tolerable and effective therapy for CLL and that low‐dose rituximab therapy can activate innate immune cytotoxic mechanisms without substantially decreasing CD20 expression. Am. J. Hematol. 89:757–765, 2014.

Abstract 5022: Generation of T regulatory-cell specific RNA aptamers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: RNA aptamers are small RNA oligonucleotides that bind to molecular targets in a manner analogous to antibodies. Theoretically, RNA aptamers have an advantage over antibodies in that large numbers of aptamers can be selected, sequenced and synthesized inexpensively in the laboratory. Here, we report a novel strategy for generating human T regulatory cell (Treg)-specific RNA aptamers. Methods: Human Treg-specific RNA aptamers were developed using whole cell systematic evolution of ligands by exponential enrichment (SELEX). Briefly, a diverse library of 2′fluoro-modified RNA sequences (∼1012) were transcribed from DNA templates containing random 20-mer regions flanked by SEL-2 specific constant sequences. Eight rounds of SELEX were conducted using mononuclear cells from a different donor for each round. Cells were separated into Tregs (CD4+CD25high) and non-Treg CD4 cells (CD4+CD25low) by magnetic cell sorting using a CD4+CD25+ human Treg isolation kit. The aptamer library was pre-cleared using non-Treg CD4 cells. Unbound aptamers were incubated with Tregs from the same donor. Tregs were washed, lysed and Treg-associated aptamers amplified by RT-PCR with SEL2 primers, transcribed and prepared for the next round. Aptamers that bound to human Tregs were collected after each round and sequenced using Illumina-based high-throughput sequencing. Results SELEX was used to successfully enrich for Treg-specific RNA aptamers. Between 2,800,000 and 3,900,000 aptamers were sequenced per round. The number of unique reads decreased from 3,391,279 (initial library) to 594,206 (round 8). At the end of SELEX, 36 unique sequences were found in >1000 copies per million reads. The prevalence of most of these aptamers went up with each round of SELEX. For example, the aptamer designated as Tr-6 was not detected in the unselected pool of aptamers, but 220 copies were found after round 2, 1631 after round 4, and 4832 after round 8. After all 8 rounds, the top 36 sequences represented 25% of the total aptamer population. Twenty-four aptamers were selected for further evaluation based on copy number, consistent enrichment after each round (indicating aptamers that bound to Tregs from each individual donor), bioinformatic properties that predict an aptamer with effective binding, and separation into different sequence and structure families, from which representative sequences were selected. Four aptamers have been tested for their ability to bind to Treg and CD4+ non-Tregs. All bind more extensively to Treg than to CD4+ non-Tregs. Conclusion and future directions: A large number of human Treg-specific RNA aptamers was selected using whole cell SELEX. Ongoing studies are assessing the antigen specificity of the selected aptamers and whether they cross-react with Tregs from other species. The long term goal is to use these aptamers as in vitro diagnostic agents and therapeutically to reduce Treg activity, thereby enhancing the anti-tumor immune response. Citation Format: Suresh Veeramani, Sue E. Blackwell, William H. Thiel, Paloma H. Giangrande, George J. Weiner. Generation of T regulatory-cell specific RNA aptamers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5022. doi:10.1158/1538-7445.AM2015-5022

Abstract 5393: C5a complement enhances generation of APC-induced Foxp3+ T regulatory cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: The complement system has complex interactions with the cellular immune system. Preliminary studies demonstrated that presence of complement significantly enhances the ability of malignant B cells and professional antigen-presenting cells to induce T regulatory (Treg) cells in vitro. The current study was designed to identify the specific complement component involved and to evaluate if complement depletion improves active immune responses through inhibition of Treg activity. Methods: Enriched CD4+ T cells from healthy human donors were co-cultured in vitro with primary myeloid dendritic cells (mDCs) in the presence of purified active complement proteins or unmodified human serum. The effect of serum treated with agents that block C5a generation, such as soluble complement receptor 1 (sCR1) that inhibits C5 convertase formation, or cobra venom factor (CVF) that enzymatically depletes C3 and C5, was also evaluated. Following culture, the percent of CD4+ T cells expressing Treg phenotype (CD3+CD4+CD25highFoxp3+) was evaluated by flow cytometry. In vivo, the effect of C5a inhibition on active immunization was also assessed following immunization of C57Bl/6 mice with a single, subcutaneous injection of ovalbumin (100 μg/mice) with or without agents that block C5a generation. Development of anti-ovalbumin antibody and anti-ovalbumin T cell responses were analyzed by ELISA and flow cytometry. Results: In vitro (human system): 1. In the presence of purified C5a protein, primary mDCs induced significantly more Tregs in co-cultured CD4+ T cells. Fewer Tregs were seen in the absence of complement, or with purified C1q, C3a, C3b or C5 proteins [p0.05 for Media vs other proteins]. 2. Serum C5a depletion or inhibition of C5a generation by anti-human C5a or sCR1 treatments decreased serums ability to enhance mDC-induced Tregs [Intact serum vs depleted serum, p<0.05]. In vivo (murine system): 1. Circulating CD4+ T cells with T regulatory phenotype (Foxp3+) were significantly lower in mice injected with Ova plus CVF or sCR1 than mice injected with Ova alone [p=0.021 (CVF), 0.016 (sCR1)]. 2. Inhibition of C5a generation by sCR1 or CVF at the site of ovalbumin immunization resulted in higher anti-ovalbumin serum IgM levels [Ova alone vs Ova plus CVF or sCR1 – p=0.047 or p-0.014, respectively] and higher ovalbumin-specific CD8+ T cells in draining lymph nodes [Ova alone vs Ova plus CVF or sCR1 - p=0.048 or 0.041, respectively]. Conclusion: C5a complement plays a key role in the induction of Tregs induced by professional APCs. Inhibition of C5a generation at the site of immunization results in increased antigen-specific active immune responses. Ongoing studies are exploring the mechanisms responsible for the effect of C5a on APC-induced Treg generation. The data from this study has potential clinical applications, such as enhancing active immune responses against a broad variety of less immunogenic antigens, including cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5393. doi:1538-7445.AM2012-5393

Abstract LB-152: Complement promotes induction of Foxp3+ T regulatory cells by B lymphoma cells and professional antigen-presenting cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction : The complement system has complex interactions with the cellular immune system. Malignant B cells have been reported to enhance T regulatory (Treg) cell activity. However, the effect of complement on the Treg cells is not known. The objectives of the current study were to evaluate whether complement can enhance Treg induction by either malignant B cells or professional antigen-presenting cells (APCs) and to analyze its role on the development of active immune responses. Methods : Peripheral blood mononuclear cells (PBMCs) or enriched CD4+ T cells from healthy donors were co-cultured with Raji B lymphoma cells or enriched myeloid dendritic cells (mDCs). These cells were treated with either unmodified autologous human serum, as a source of complement, or serum from which complement had been inhibited by heat-inactivation (HI), cobra-venom factor (CVF) or soluble complement receptor 1 (sCR1) treatment. Co-cultures to which purified C5a protein was added were also evaluated. Following additional incubation, percent CD4+ T cells expressing the Treg phenotype (CD3+CD4+CD25highFoxp3+) was evaluated by flow cytometry. Treg function was determined by analyzing their inhibitory effect on mitogen-stimulated CD8+ T cells. To assess the in vivo effect of complement on the development of an active immune response, C57Bl/6 mice were immunized with single, subcutaneous injection of a model antigen, ovalbumin (100 μg/mice), mixed with complement-inhibiting agents and anti-ovalbumin antibody response was monitored by ELISA. Results 1. Raji B lymphoma cells induced significantly higher percentages of Tregs in co-cultured PBMCs in the presence of unmodified human serum (p=0.038) when compared to HI or CVF or sCR1-treated human serum (p=0.037, 0.022 and 0.0002, respectively). 2. Treg induction was also increased when CD4+ T cells were co-cultured with mDCs in the presence of serum complement or purified C5a protein (p=0.002, p=0.039, respectively). 3. Treg function, as indicated by suppression of granzyme B production by mitogen-treated CD8+ T cells, was reduced by complement depletion. 4. In vivo , local complement inhibition at the site of immunization resulted in significantly higher anti-ovalbumin IgM responses (inhibition vs no inhibition – p<0.05). Conclusion : Serum complement, including C5a, promotes induction of Treg cells by B lymphoma cells and APCs, such as mDCs, which can be reduced by inhibition of serum complement. In vivo complement inhibition enhances active immune responses. These studies suggest a immune-regulatory role for complement proteins in lymphoma and that local complement depletion could be used as a novel strategy to boost active immune responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-152. doi:10.1158/1538-7445.AM2011-LB-152