Intratumoral Inhibition of C5a Using Anti-C5a Monoclonal Antibody Inhibits Lymphoma Progression

Abstract

Background: The complement system can have both pro-inflammatory and immunosuppressive effects. Our preliminary studies demonstrated that C5a can limit antigen-specific responses, in part, by enhancing the induction of immunosuppressive T regulatory (Treg) cells by primary human antigen-presenting cells and malignant B cells. The current study explored the effect of intratumoral C5a on B lymphoma progression and intratumoral T cells using the A20 murine lymphoma mouse model.Methods:(A) Co-injection approach: Female Balb/C mice were subcutaneously injected with A20 B lymphoma cells mixed with either 20 μg of rat anti-mouse C5a neutralizing monoclonal antibody (mAb) or matching isotype control mAb. Tumor growth was followed and the phenotype of tumor-infiltrating lymphocytes analyzed by flow cytometry.(B) Therapeutic approach: Established subcutaneous A20 tumors (7-9 mm diameter) were treated intratumorally with 20 µg of anti-mouse C5a neutralizing mAb (considered as day 1). A second dose (20 µg) of mAb was given intratumorally on day 5. Control mice were treated similarly with matching isotype control mAb. Tumor growth was followed.Data was analyzed using standard t-test (for tumor growth and immune responses) and Log-rank (Mantel-Cox) test (for comparing survival).Results:(A) Co-injection approach: Co-injection of anti-C5a mAb at the time of tumor cell inoculation significantly delayed A20 tumor growth (Fig. 1; p<0.05) and led to prolonged survival of anti-C5a-treated tumor-bearing mice when compared to the control mice (p=0.001).Tumors from mice that were co-injected with anti-C5a mAb showed significantly higher infiltration of activated CD8+ T cells producing IFNg than the control mice (Fig. 2; p=0.034). Anti-C5a-treated mice also had a lower percentage of circulating Tregs (p=0.023) and splenic Tregs (p=0.0001) compared to control mice.(B) Therapeutic approach: Treatment of pre-established subcutaneous A20 tumors with anti-mouse C5a mAb slowed tumor growth significantly when compared to the control mice (Fig. 3; **p<0.01; *p<0.05). Tumor-bearing mice treated intratumorally with anti-C5a also showed significant improvement in the survival when compared to tumor-bearing mice treated with isotype control Ab (p=0.008).Conclusions:Intratumoral anti-C5a mAb therapy inhibits progression of B lymphoma and improves survival, likely by reducing immunosuppression. We are currently exploring the mechanisms behind C5a-promoted tumor immunosuppression. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.