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

Abstract

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.

©2013 AACR.

Figures

Figure 1. Anti-4-1BB mAb provides effective therapy in Neuro2a neuroblastoma model

A/J mice inoculated s.c. with 2 × 106 Neuro2a cells on day 0 received 0.5 mg anti-4-1BB mAb or 200 μl PBS i.p on A, days 9 and 12 or B, days 3 and 6. Anti-4-1BB treatment increased survival in mice treated on days 9 and 12 (p = 0.0018), but not with treated on days 3 and 6. C, Long term survivors from (A) re-challenged with tumor (2 × 106 cells s.c) 180 days after initial inoculation showed increased survival compared with naïve age-matched controls (p = 0.0004). Data represent examples of at least two experiments, where n = 5 mice/group.

Figure 2. Anti-4-1-BB immunotherapy is dependent on CD8+ T and NK cells

A, Neuro2a inoculated A/J mice received anti-4-1BB or 200 μl PBS i.p. on days 9 and 12. Mice also received anti-CD4, anti-CD8 or anti-asialo GM1 antibodies. Anti-4-1-BB immunotherapy was abrogated after CD8+ T-cell (p = 0.0215) or NK-cell depletion (p = 0.0228), but was retained after CD4+ depletion (p = 0.5203). B, Neuro2a tumors were excised from mice 8 days after mAb treatment. Tumors from anti-4-1BB mAb treated mice showed evidence of CD8+ and NK cell infiltrates. X10 magnification, scale bar is equal to 200 μm. Data represents examples of at least three experiments, where n = 5 mice/group.

Figure 3. Single agent mAbs are not therapeutic in aggressive neuroblastoma models

A/J mice inoculated s.c. with 2 × 106 A, Neuro2a, B, AgN2a or C, NXS2 cells received 0.5 mg of anti-4-1BB, anti-CD40 or anti-CTLA-4 or 200 μl isotype control i.p. on days 9 and 12. These mAb were therapeutic in the Neuro2a model (p = 0.0006, 0.0012 & 0.0006, respectively), but not in the AgN2a (p = 0.1258, 0.8361 & 0.1034, respectively) or NXS2 (p = 7726, 0.0635 & 0.6108, respectively) models. Data represent examples of at least three experiments, where n = 5 mice/group.

Figure 4. Combining anti-CTLA-4 mAb with survivin peptide vaccination provides effective therapy in the aggressive AgN2a and NXS2 models

A, PCR determined that survivin expression (normalized to expression in brain) in normal A/J mouse tissue was low and in the malignant neuroblastoma cell lines was high. Data represents average of three experiments, with error expressed as SEM. B, A/J mice inoculated s.c. with 2 × 106 AGN2a (top panels) or NXS2 (bottom panels) on day 0 received 50 μg IFA emulsified survivin (GWEPDDNPI) or control (SIINFEKL) peptide vaccine i.d. day 3 and 0.5 mg of mAb or 200 μl isotype control i.p. on day 3 and day 6. In the AgN2a and NXS2 models combining GWEPDDNPI-peptide with anti-CTLA-4 mAb resulted in increased survival (p < 0.05). C, Anti-CTLA-4/GWEPDDNPI-peptide immunotherapy was abrogated after CD8+ T-cell (p = 0.0110) or NK-cell depletion (p = 0.0307) but was retained after CD4+ depletion (p = 0.4405). D, Long term survivors from (B) re-challenged with tumor (2 × 106 cells s.c) 120 days after initial inoculation showed increased survival compared with naïve age-matched controls (p = 0.0007). Data represent examples of three experiments, where n = 5 mice/group.

Figure 5. Anti-CTLA4 and GWEPDDNPI peptide immunotherapy generates tumor directed cytotoxicity

Splenocytes from NXS2 inoculated mice receiving anti-CTLA-4/GWEPDDNPI-peptide immunotherapy as previously described were harvested after 35 days and re-stimulated alongside splenocytes from naïve mice with GWEPDDNPI-pulsed, irradiated splenocytes for 6 days. Target-cells labeled with 51Cr were incubated with splenocytes (effectors) at 50:1, 10:1 and 1:1 effector:target (E:T) ratio for 5 hr under tissue culture condition. A, Splenocytes from mice treated with anti-CTLA-4/GWEPDDNPI peptide but not from naïve mice demonstrated lytic activity against NXS2 and AgN2a tumors (p < 0.05). B, NXS2 cell lysis was not enhanced when the NXS2 cells were pulsed with a control or GWEPDDNPI peptide. C. Fourteen days after therapy, CSFE-labeled peptide-pulsed splenocytes were adoptively transferred into NXS2 inoculated mice treated with control peptide, GWEPDDNPI, anti-CTLA-4 alone or anti-CTLA-4/GWEPDDNPI-peptide. After 24 hrs peptide-specific killing was observed only with anti-CTLA-4/GWEPDDNPI treatment, as determined by the T:NT ratio. D. Splenocytes from (C) re-stimulated as described above, were incubated with unlabeled NXS2 target cells at a 50:1 E:T ratio for 5 hr in the presence of brefeldin A under tissue culture conditions. Using intracellular flow cytometry, IFN-γ production was detected in CD8+ T-cells in splenocytes from anti-CTLA-4/GWEPDDNPI treated mice only following in vitro re-stimulation with GWEPDDNPI. Data represent examples of at least three experiments, where n = 3 mice/group. Error is expressed as SEM. * p < 0.05, ** p < 0.01, n.s. not significant.

Figure 6. Combined therapy with anti-CTLA4 mAb and GWEPDDNPI peptide requires tumor to generate anti-survivin T-cell immunity

A, Assessment using CTL killing assays demonstrated that splenocytes from naïve mice or mice treated with anti-CTLA-4/GWEPDDNPI-peptide without tumor for 35 days showed negligible lytic activity against splenocytes or neuroblastoma cells. Data represents an example of 2 experiments with 4 mice / group, error is expressed as SEM. B, A/J mice treated for 14 days with control peptide, GWEPDDNPI-peptide or anti-CTLA-4 alone or anti-CTLA-4/GWEPDDNPI-peptide in the absence of NXS2 cells did not show in vivo peptide-specific killing after 24 hr following transfer of CFSE-labeled, peptide-pulsed splenocytes. C, A/J mice received GWEPDDNPI or control peptide i.d. emulsified in IFA on day -7 and 0.5 mg of anti-CTLA-4 or 200 μl PBS i.p. on day -7 and -3. Mice were then inoculated on day 0 with 2 × 106 NXS2 cells s.c. Anti-CTLA-4/GWEPDDNPI peptide prophylactic treatment did not result in significant long term survival. Data in (B) and (C) represents examples of three experiments, where n = 5 mice/group. Error is expressed as SEM.