Expression of c-erb-B2 oncoprotein as a neoantigen strategy to repurpose anti-neu antibody therapy in a model of melanoma

Abstract

In this study, we tested a novel approach of "repurposing" a biomarker typically associated with breast cancer for use in melanoma. HER2/neu is a well characterized biomarker in breast cancer for which effective anti-HER2/neu therapies are readily available. We constructed a lentivirus encoding c-erb-B2 (the animal homolog to HER2/neu). This was used to transfect B16 melanoma in vitro for use in an orthotopic preclinical mouse model, which resulted in expression of c-erb-B2 as a neoantigen target for anti-c-erb-B2 monoclonal antibody (7.16.4). The c-erb-B2-expressing melanoma was designated B16/neu. 7.16.4 produced statistically significant in vivo anti-tumor responses against B16/neu. This effect was mediated by NK-cell antibody-dependent cell-mediated cytotoxicity. To further model human melanoma (which expresses <5% HER2/neu), our c-erb-B2 encoding lentivirus was used to inoculate naïve (wild-type) B16 tumors in vivo, resulting in successful c-erb-B2 expression. When combined with 7.16.4, anti-tumor responses were again demonstrated where approximately 40% of mice treated with c-erb-B2 lentivirus and 7.16.4 achieved complete clinical response and long-term survival. For the first time, we demonstrated a novel strategy to repurpose c-erb-B2 as a neoantigen target for melanoma. Our findings are particularly significant in the contemporary setting where newer anti-HER2/neu antibody-drug candidates have shown increased efficacy.

Figures 1

(A) pLenti6.3 c-erb-B2 construct showing the insertion of the rat c-erb-B2 (neu) gene into the lentivirus DNA. The c-erb-B2-expressing B16 melanoma cell line was designated B16/neu. c-erb-B2 expression by B16/neu was confirmed by qPCR (B), Western blot (C), and flow cytometry (D), which altogether showed high levels (>95%) of c-erb-B2 transcription and surface expression by the newly created B16/neu cell line. In contrast, naïve or wild-type B16 showed only about 1% c-erb-B2 expression. (E) The oncogenic variant of c-erb-B2 was inserted into pLenti6.3 and confirmed through gene sequencing, which demonstrated a valine to glutamine mutation. The oncogenic variant differs from wild-type c-erb-B2, which contains valine at this position.

Figure 2

Naïve B16 and B16/neu were grown in vitro, and cell viability was assessed via the Cyquant assay. Overall, B16/neu showed higher cell viability at all time points compared to naïve B16. When the anti-c-erb-B2 monoclonal antibody 7.16.4 was added in increasing doses to B16/neu, no statistically significant effects were observed on cell growth at any of the tested doses.

Figure 3

(A) 7.16.4 generated statistically significant responses on B16/neu growth compared to B16 and isotype (IgG2a) controls. (B) Survival was also significantly improved with 5/12 (41.2%) of C57BL/6 mice bearing B16/neu tumors achieving a complete clinical response and long-term survival.

Figure 4

We hypothesized that 7.16.4 generated anti-tumor responses via NK-cell antibody-dependent cell-mediated cytotoxicity. The addition of anti-NK cell monoclonal antibody (NK 1.1) inhibited and essentially reversed the anti-tumor effects of 7.16.4 on both tumor growth (A) and survival (B). Resected tumors from mice treated with NK 1.1 showed little to no presence of NK cells on immunohistochemistry (C). In contrast, tumors resected from non-responders treated with only 7.16.4 showed a significantly higher number of stained NK cells on tumor sections (D), providing pathological evidence that NK 1.1 effectively eliminated NK cells from infiltrating into B16/neu tumors. IHC staining of NK cells within naïve spleens (obtained from non-tumor bearing C57BL/6 mice) was used as a control (E).

Figure 5

Naïve (wild-type) B16 tumors were inoculated with pLenti6.3 c-erb-B2 to generate c-erb-B2 as a neoantigen target for 7.16.4. The addition of 7.16.4 to pLenti6.3 c-erb-B2 resulted in statistically significant decreased tumor growth (A) and improved long-term survival (B), again with 41.2% of mice showing complete clinical response.