A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer

Abstract

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer.

Figure 1

In vitro characterization of CR2Fc. CR2Fc binds to C3-opsonized cells and increases Fc opsonization by amplifying its own ligand. (A) CR2Fc binding: EL4 cells sensitized with anti-GD2 14G2a mAbs were incubated with NMS and 20 μg/mL AlexaFluor-488–labeled CR2Fc added during serum incubation or after serum incubation (after washing). CR2Fc binding in the absence of mAb sensitization (CR2Fc only) was also determined. (B) CR2Fc-enhanced complement activation: EL4 cells were incubated in 5% or 50% NMS, with 14G2a and CR2Fc as indicated, and C3a levels in supernatant determined by Western blot and densitometry. (C) CR2Fc-enhanced complement-dependent EL4 lysis: EL4 cells were incubated in 5% or 50% normal rat serum, and with 14G2a and CR2Fc as indicated. (D) CR2Fc-enhanced complement-dependent B16 lysis: B16 cells were incubated in 50% normal rat serum, and with anti-gp75 TA99 mAb and CR2Fc as indicated. Cell lysis determined by measuring lactate dehydrogenase release. Data are mean ± SEM; n = 3.

Figure 2

CR2Fc enhances the therapeutic outcome of mAb immunotherapy. Survival (A) and number of liver micrometastases at day 19 (B) of mice challenged with EL4 cells and treated with 14G2a mAb alone or in combination with CR2Fc. Long-term survival is significantly improved with mAb and CR2Fc treatment over mAb immunotherapy alone. Survival data are combined from 2 independent experiments (n = 14), each of which yielded similar results. (B) Data are mean ± SEM; n = 4 or 5. (C) Survival of mice challenged with B16 cells and treated with TA99 mAb alone or in combination with CR2Fc; n = 5.

Figure 3

CR2Fc enhances mAb therapy in SCID beige mice. Survival of SCID beige mice challenged with EL4 cells and treated with 14G2a alone or in combination with CR2Fc. Long-term survival of mice treated with mAb and CR2Fc is significantly extended compared with mice treated with mAb therapy alone; n = 8 or 9.

Figure 4

mAb and CR2Fc treatment is not protective in FcγR-deficient mice. Survival of FcγR-deficient mice challenged with EL4 and treated with 14G2a alone or in combination with CR2Fc. In the absence of Fcγ-activating receptors, the protective effect of mAb and CR2Fc treatment is ablated in EL4-challenged mice; n = 7 or 8.

Figure 5

Macrophage depletion ablates mAb and CR2Fc-mediated protection. Survival of macrophage/monocyte-depleted mice challenged with EL4 and treated with 14G2a alone or in combination with CR2Fc. There was no difference in survival between any of the treatment groups in macrophage-depleted mice. Mice not depleted of macrophages (control) had significantly improved long-term survival, consistent with data shown in Figure 2A; n = 6 to 9.

Figure 6

mAb and CR2Fc treatment does not increase overall survival in C6-deficient mice. Survival of C6-deficient mice challenged with EL4 and treated with 14G2a alone or in combination with CR2Fc. mAb therapy with or without CR2Fc did not provide long-term protection in the absence of C6, although it did significantly (P < .05) prolong survival time. CR2Fc did not enhance the effect of mAb therapy in C6-deficient mice; n = 4 to 6.

Figure 7

CR2Fc treatment does not induce an anti-EL4 IgM or IgG response. IgM (A), IgG (B), or IgG2a (C) was measured in the serum at day 0 and on day 19 after tumor challenge by a flow cytometry-based method. No significant differences were detected in IgM response between groups (A). Increased anti-EL4 IgG was seen in mAb and mAb + CR2Fc-treated mice (B), but only IgG2a isotype was elevated (C), which is the isotype of the treatment mAb.