Sonya James

Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation

Anti-CD20 monoclonal antibodies (mAbs) are classified into type I (rituximab-like) or type II (tositumomab-like) based on their ability to redistribute CD20 molecules in the plasma membrane and activate various effector functions. To compare type I and II mAbs directly in vivo and maximize Fc effector function, we selected and engineered mAbs with the same mouse IgG(2)a isotype and assessed their B-cell depleting activity in human CD20 transgenic mice. Despite being the same isotype, having similar affinity, opsonizing activity for phagocytosis, and in vivo half-life, the type II mAb tositumomab (B1) provided substantially longer depletion of B cells from the peripheral blood compared with the type I mAb rituximab (Rit m2a), and 1F5. This difference was also evident within the secondary lymphoid organs, in particular, the spleen. Failure to engage complement did not explain the efficacy of the type II reagents because type I mAbs mutated in the Fc domain (K322A) to prevent C1q binding still did not display equivalent efficacy. These results give support for the use of type II CD20 mAbs in human B-cell diseases.

Sustained TL1A expression modulates effector and regulatory T-cell responses and drives intestinal goblet cell hyperplasia

The tumor necrosis factor (TNF) superfamily protein TNF-like 1A (TL1A) is the ligand for death receptor 3 (DR3). TL1A is induced on activated dendritic cells (DCs) and its expression has been linked to human inflammatory bowel disease. To address how TL1A might influence intestinal inflammation, we generated transgenic mice that constitutively express TL1A on DCs. TL1A transgenic mice developed striking goblet cell hyperplasia in the ileum that was associated with elevated interleukin (IL)-13 levels in the small intestine. IL-13- and IL-17-producing small intestinal lamina propria T cells were increased in TL1A transgenic mice. TL1A also enhanced regulatory T (Treg) cell turnover in vivo and directly stimulated Treg cell proliferation in vitro. The presence of TL1A attenuated the ability of Treg cells to suppress conventional T cells, an effect that required DR3 signaling in either conventional T cells or Treg cells. Our findings identify mechanisms by which chronic DR3 signaling could promote pathogenesis in inflammatory bowel disease.

Optimising anti-tumour CD8 T-cell responses using combinations of immunomodulatory antibodies

Immunostimulatory mAb as vaccine adjuvants for the treatment of cancer hold considerable potential for boosting weak responses when used against immunogenic tumours, or in combination with various other vaccines. We now show that when administered with OVA, the combination of anti‐4‐1BB mAb with anti‐CD40, anti‐OX40 or anti‐CD25 resulted in a fourfold enhancement in the antigen‐specific T‐cell response compared with anti‐4‐1BB mAb alone, with a similar enhancement in memory responses following rechallenge with OVA. Although the number of antigen‐specific T‐cells generated after treatment with each of the combinations was similar, marked functional differences were detected. In particular, anti‐4‐1BB/anti‐CD25 resulted in excellent expansion of specific CD8+ T cells but produced fewer IFN‐γ‐secreting effector cells than the other combinations. Anti‐4‐1BB/anti‐OX40 proved to be the most potent, inducing the most effective T‐cell responses in the RIPmOVA diabetes model with adoptively transferred OVA‐specific T cells, and, when given with a peptide vaccine, protecting mice against the poorly immunogenic B16‐F10 tumour. Overall the results suggest that although these combinations of mAb look promising in terms of their therapeutic potential, further functional assays are needed to compare their effects.

Immunomodulatory monoclonal antibodies combined with peptide vaccination provide potent immunotherapy in an aggressive murine neuroblastoma model

Purpose: Neuroblastoma is one of the commonest extracranial tumors of childhood. The majority of patients present with metastatic disease for which outcome remains poor. Immunotherapy is an attractive therapeutic approach for this disease, and a number of neuroblastoma tumor antigens have been identified. Here, we examine the therapeutic potential of combining immunomodulatory monoclonal antibodies (mAb) with peptide vaccination in murine neuroblastoma models. Experimental Design: Neuroblastoma-bearing mice were treated with mAb targeting 4-1BB, CD40, and CTLA-4 alone, or in combination with a peptide derived from the tumor antigen survivin (GWEDPPNDI). Survivin-specific immune response and therapeutic efficacy were assessed. Results: In the Neuro2a model, treatment of established tumor with anti-4-1BB, anti-CD40, or anti-CTLA-4 mAb results in tumor regression and long-term survival in 40% to 60% of mice. This is dependent on natural killer (NK) and CD8+ T cells and is associated with tumor CD8+ lymphocyte infiltrate. Successful therapy is achieved only if mAb is given to mice once tumors are established, suggesting dependence on sufficient tumor to provide antigen. In the more aggressive AgN2a and NXS2 models, single-agent mAb therapy provides ineffective therapy. However, if mAb (anti-CTLA-4) is given in conjunction with survivin peptide vaccination, then 60% long-term survival is achieved. This is associated with the generation of survivin-specific T-cell immunity, which again is only shown in the presence of tumor antigen. Conclusions: These data suggest that the combination of antigen and costimulatory mAb may provide effective immunotherapy against neuroblastoma and may be of particular use in the minimal residual disease setting. Clin Cancer Res; 19(13); 3545–55. ©2013 AACR.

Death receptor 3 is essential for generating optimal protective CD41 T-cell immunity against Salmonella

The TNF receptor superfamily member death receptor 3 (DR3) exacerbates Th2‐ and Th17‐cell‐mediated inflammatory and autoimmune conditions, yet no role in host defence has been reported. Here, we examined the role of DR3 during infection with Salmonella enterica serovar Typhimurium. Infection resulted in protracted expression of the DR3 ligand TL1A but not the related TNF superfamily proteins OX40L or CD30L. TL1A expression was localized to splenic F4/80+ macrophages where S. enterica Typhimurium replicates, and temporally coincided with the onset of CD4+‐cell expansion. To address the relevance of the TL1A‐DR3 interaction, we examined immune responses to S. enterica Typhimurium in mice lacking DR3. Infected DR3‐/‐ mice harboured reduced numbers of antigen‐experienced and proliferating CD4+ T cells compared with WT mice. Furthermore, the frequency of IFN‐γ+ CD4+ T cells in DR3‐/‐ mice was lower throughout the time of bacterial clearance. Importantly, bacterial clearance, which is dependent on Th1 cells, was also impaired in DR3‐/‐ mice. This defect was intrinsic to CD4+ T cells as evidenced by an increase in bacterial burden in RAG2‐deficient mice receiving DR3‐/‐ CD4+ T cells compared with WT CD4+‐cell recipients. These data establish for the first time a role for DR3 in a host defence responce.

Ligation of CD11c during vaccination promotes germinal centre induction and robust humoral responses without adjuvant

In this study, we investigated the mouse dendritic cell (DC) receptor, complement receptor 4 (CR4; CD11c/CD18), as an immunotarget for triggering humoral immunity. Comparison of antibody titres generated against a panel of 13 anti‐antigen‐presenting cell receptor monoclonal antibodies, with or without conjugated ovalbumin (OVA), revealed uniquely rapid and robust responses following CR4 targeting, with antibody titres approaching 1 : 100 000 7 days after a single dose of antigen. Furthermore, using just 100 ng OVA conjugated to anti‐CD11c Fab′, we generated anti‐OVA titres greater than those produced by a 100‐fold higher dose of OVA in complete Freund’s adjuvant at day 28. These anti‐OVA antibody titres were sustained and could be boosted further with targeted OVA on day 21. Investigations to explain this vaccine potency showed that, in addition to targeting splenic DC, anti‐CDl1c antibodies delivered a powerful adjuvant effect and could boost humoral immunity against OVA even when the OVA was targeted to other molecules on DC, such as major histocompatibility complex class II, CD11a and CD11b. However, interestingly, this adjuvant effect was lost if OVA was targeted to other cells such as B cells via CD21 or CD19. The adjuvant effect was mediated through a marked enhancement of both germinal centre and extrafollicular plasma cell formation in responding spleens. These results demonstrate that anti‐CD11c monoclonal antibody can both target antigen and act as a powerful adjuvant for rapid and sustained antibody responses. They also point to an interesting role for CR4 on DC in triggering B cells during humoral immunity.

Development and Characterization of Monoclonal Antibodies Specific for Mouse and Human Fcγ Receptors

FcγRs are key regulators of the immune response, capable of binding to the Fc portion of IgG Abs and manipulating the behavior of numerous cell types. Through a variety of receptors, isoforms, and cellular expression patterns, they are able to fine-tune and direct appropriate responses. Furthermore, they are key determinants of mAb immunotherapy, with mAb isotype and FcγR interaction governing therapeutic efficacy. Critical to understanding the biology of this complex family of receptors are reagents that are robust and highly specific for each receptor. In this study, we describe the development and characterization of mAb panels specific for both mouse and human FcγR for use in flow cytometry, immunofluorescence, and immunocytochemistry. We highlight key differences in expression between the two species and also patterns of expression that will likely impact on immunotherapeutic efficacy and translation of therapeutic agents from mouse to clinic.

Antibody Tumor Targeting Is Enhanced by CD27 Agonists through Myeloid Recruitment

Summary Monoclonal antibodies (mAbs) can destroy tumors by recruiting effectors such as myeloid cells, or targeting immunomodulatory receptors to promote cytotoxic T cell responses. Here, we examined the therapeutic potential of combining a direct tumor-targeting mAb, anti-CD20, with an extended panel of immunomodulatory mAbs. Only the anti-CD27/CD20 combination provided cures. This was apparent in multiple lymphoma models, including huCD27 transgenic mice using the anti-huCD27, varlilumab. Detailed mechanistic analysis using single-cell RNA sequencing demonstrated that anti-CD27 stimulated CD8+ T and natural killer cells to release myeloid chemo-attractants and interferon gamma, to elicit myeloid infiltration and macrophage activation. This study demonstrates the therapeutic advantage of using an immunomodulatory mAb to regulate lymphoid cells, which then recruit and activate myeloid cells for enhanced killing of mAb-opsonized tumors.

CD27 STIMULATION ENHANCES CD20 MAB THERAPY THROUGH ACTIVATION OF INNATE IMMUNITY

curative, but are poorly tolerated in adults and patients with HIV. A single‐center study of 30 patients showed that lower treatment intensity with infusional DA‐EPOCH‐R was highly curative and well tolerated in adults with sporadic or HIV‐associated BL (N Engl J Med 2013; 369:1915‐1925). To validate these results, we undertook a multicenter study of DA‐EPOCH‐R in adult BL and investigated if a risk‐ adapted approach could further reduce treatment toxicity. Methods: Patients with newly diagnosed BL, age 18 years or older and any HIV status were enrolled at 24 participating sites. Patients were considered low‐risk (LR) if they had a normal LDH, ECOGperformance status of 0 or 1, stage I or II disease, and no tumor lesion over 7 cm. All other patients were considered high‐risk (HR). LR patients received 3 cycles of DA‐EPOCH‐R without intrathecal treatment. HR patients with negative brain MRI and negative CSF cytology/ flow cytometry received 6 cycles of DA‐EPOCH‐R with MTX 12 mg IT on days 1 and 5 during cycles 3‐6 (8 total doses). HR patients with active CNS disease received 6 cycles of DA‐EPOCH‐R and concurrent MTX 12 mg IT twice weekly for 2 weeks past negative results (minimum of 4 weeks), followed by MTX 12 mg IT once weekly x 6, and MTX 12 mg IT monthly x 6. Results: 112 of 116 planned patients have been enrolled; 110 who completed at least 1 cycle of therapy are included in this analysis. Characteristics include median (range) age 48 (19‐86) years with 53 (48%) patients aged 50 years or over and 29 (26%) patients aged 60 years or over; male sex 86 (78%); stage III or IV disease 76 (69%); elevated LDH 73 (66%); CNS involvement 11 (10%); HIV positive 29 (26%). The frequency of bone marrow disease is under evaluation. Thirteen (12%) and 97 (88%) patients were classified as LR and HR, respectively. There were 6 deaths in the HR arm not attributed to disease progression/relapse: 2 deaths due to infection, and 1 death each attributed to respiratory failure, second malignancy, myocardial infarction, and unknown. All other reported toxicities were expected toxicities of DA‐EPOCH‐R.With a median follow‐up of 34 months, the progression‐free survival (PFS) for all patients beyond 10.2 months is 84.6% (95% CI: 75.6‐90.4%); time‐to‐progression (TTP) is 91.1% (95% CI: 82.8‐95.4%) and overall survival is 84.7% (95% CI: 75.4‐ 90.7%). Age (> 40y versus <40y) and HIV status did not impact survival (see table). PFS for LR patients was 100% and 82% for HR patients. Notably, only 1 patient who progressed after DA‐EPOCH‐R was successfully salvaged and is still alive. Conclusions: This multicenter study confirms that DA‐EPOCH‐R is well tolerated and highly effective in adult BL including patients 3 40 years. Abbreviated treatment with 3 cycles of DA‐EPOCH‐R is highly effective for LR disease, while the outcome of HR patients compares favorably with more intensive regimens. Further details on outcomes of patients with CNS and BM involvement will be provided at the meeting. Accrual to this study is ongoing with full accrual anticipated this year [NCT01092182]. A randomized study comparing the regimen to standard BL therapy is in progress.