Christopher E. Dahle

A polymorphism in the complement component C1qA correlates with prolonged response following rituximab therapy of follicular lymphoma.

Purpose: Complement may play a role in the clinical response to rituximab and other monoclonal antibody–based therapies of cancer. The purpose of this study was to explore the relationship between the C1qA[276] polymorphism and the clinical response to rituximab in patients with follicular lymphoma. Experimental Design: Genotyping for C1qA[276A/G] was done in 133 subjects with follicular lymphoma treated with single-agent rituximab, and correlation with clinical response was done using Cox regression analysis. Results: Prolonged remission was observed among subjects that responded clinically to rituximab therapy and were carriers of the A allele compared with homozygous G subjects. Homozygous G subjects had a time to progression of 282 days, whereas A-allele carriers had a time to progression of 708 days [hazard ratio, (HR), 2.5; 95% confidence interval (95% CI), 2.0-3.1; P = 0.02]. Among subjects who achieved complete remission, homozygous G subjects had a time to progression of 250 days, whereas A-allele carriers had a time to progression of 1,118 days (HR, 4.5; 95% CI, 4.1-4.8, P = 0.04). The difference persisted after controlling for CD32 and CD16 polymorphisms. In patients who responded to rituximab used as first-line agent, a linear trend was observed among the C1qA[276] genotypes, with homozygous A subjects achieving complete response at a higher rate compared with heterozygous or homozygous G subjects. Conclusions: Our findings indicate that polymorphisms in the C1qA gene may affect the clinical response and duration of response to rituximab therapy of follicular lymphoma. These results could have direct implications on designing antibodies with improved efficiency and enhance our understanding of the role of complement in monoclonal antibody therapy.

APC Stimulated by CpG Oligodeoxynucleotide Enhance Activation of MHC Class I-Restricted T Cells

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.

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.

Potent antigen-specific immune responses stimulated by codelivery of CpG ODN and antigens in degradable microparticles

CpG ODN stimulates a TH1 response through its receptor Toll-like receptor 9 (TLR9). TLR9 is a receptor that is found intracellularly. Microparticles are efficiently internalized by dendritic cells (DCs) and macrophages and would thus be an ideal delivery vehicle for CpG ODN to reach its target site thereby enhancing the TH1 response to an antigen also encapsulated in the microparticle. Here, we show that careful control over fabrication parameters can produce biodegradable microparticles with predictable size distributions, surface morphology, and shape. Entrapment efficiencies of the model antigen OVA ranged from 19% to 23% with an average loading of 10 μg/mg of microparticles. For CpG ODN, these values were 33% to 35%, which corresponded to an average loading of 8.5 μg/mg of microparticles. The microparticles release CpG ODN and OVA in a burst followed by sustained release profile. At the highest concentration of microparticles incubated with a pure DC cell line, 92% of DCs had internalized microparticles by 16 hours, confirming that DCs efficiently take up the microparticles. Microparticles are capable of inducing DC maturation as determined by up-regulation of CD80 and CD86 markers. Although the presence of CpG ODN in the microparticles did not impact on the phenotype of the DCs, it was necessary for DCs to induce activation of antigen-specific T cells as indicated by interferon-γ production. Microparticles entrapping both antigen and CpG ODN induced significantly higher amounts of anti-OVA antibody production than other preparations such as the soluble OVA and CpG ODN (P<0.01) and stimulated stronger IgG2a production than delivery of microparticles entrapping antigen alone. We conclude that coencapsulating immunostimulatory CpG ODN and antigen in degradable microparticles is an effective approach to enhancing development of a TH1 immune response.

The pattern of clinical breast cancer metastasis correlates with a single nucleotide polymorphism in the C1qA component of complement

Complement is one of primary defense mechanisms against intravascular microorganisms and could play a role in the immune response to malignancy and hence its clinical behavior. We evaluated if the sole coding polymorphism of C1qA associates with outcome in patients with breast carcinoma. Genotyping for C1qA[276A/G] was performed in 63 breast cancer subjects with localized tumor and compared with that in 38 breast cancer subjects with metastasis. Established risk factors for clinical outcome were considered and evaluated in multivariable analysis. Breast cancer subjects with heterozygous or homozygous C1qA[276G] genotype had a higher rate of metastasis than subjects with the homozygous C1qA[276A] genotype [hazard ratio (HR) 2.4, 95% confidence interval (CI) 1.1–4.1]. This association was stronger when only metastatic sites associated with hematogenous spread, i.e., to the bone, liver, and brain, were considered (HR 3.5, 95% CI 1.4–5.6) and remained statistically significant after adjustment for the number of positive lymph nodes, estrogen receptor status, and progesterone receptor status. There was no statistical difference in the C1qA[276A/G] allelic distribution between all subjects with breast cancer and controls. These results suggest there could be an association of a single nucleotide polymorphism at position 276 of the C1qA component of complement with breast cancer metastasis to sites linked to hematogenous spread of disease. The C1qA polymorphism associated with decreased distant metastasis has also been correlated with an increased incidence of subcutaneous systemic lupus and C1q deficiencies, suggesting that an altered immune response may play a role in the observed association.

ISOLATING RNA FROM CLINICAL SAMPLES WITH CATRIMOX-14 AND LITHIUM CHLORIDE

RNA is a highly informative molecule that has great potential as a target for diagnostic studies. This potential can be reached only when reliable methods for isolating RNA are available in the clinical environment. Cationic surfactants lyse cells and precipitate nucleic acids. We have described a novel cationic surfactant (tetradecyltrimethylammonium oxalate, Catrimox‐14™), which is particularly effective in precipitating RNA from cells and which can be applied to clinical specimens. We examine the utility of a method of recovering RNA from the surfactant‐nucleic acid precipitate in which 2 M lithium chloride is used to extract the DNA and surfactant from the precipitate; RNA (being insoluble in lithium chloride solution) remains in the pellet.

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

Brachyimmunotherapy (Combination Brachytherapy and Immunotherapy) Enhances Development of a Tumor Antigen-Specific CD8 Response

vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more efficient for eradication of WT1expressing tumor cells that had been implanted into mice before vaccination (a ‘‘therapeutic’’ model) compared to WT1 peptide vaccination alone. A intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTSs) and led to reject WT1-expressing tumor cells. Diseasefree survival of mice immunized with BCG-CWS and WT1 peptide was 31% and significantly longer in comparison with that of mice immunized with WT1 peptide alone (p , 0.05). Furthermore WT1-specific CTLs were ignorant of normal self-cells that expressed WT1 at physiological levels. These results showed that BCG-CWS, which was known to enhance innate immunity, could also enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.