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, an animal (rat) 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 rat 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 therapies have shown increased efficacy.

Fig. 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.

Fig. 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.

Fig. 3

(A) 7.16.4 generated statistically significant responses on B16/neu growth compared to B16 and isotype (IgG2a) controls. The bar shows statistical significance between the two groups of particular interest: (1) B16/neu + 7.16.4 and (2) Naïve (wild-type) B16 + 7.16.4. (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. Numbers at risk are shown. Mice that achieved complete clinical response (cure) were euthanized at day 90.

Fig. 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 (NK1.1) inhibited and essentially reversed the anti-tumor effects of 7.16.4 on both tumor growth (A) and survival (B). In (A), the bar shows statistical significance between the two groups of particular interest: (1) mice that received 7.16.4, which had the lowest tumor growth and (2) mice that received 7.16.4 + NK1.1, which showed similar growth to the negative control group and the NK1.1 control. In (B), complete response was again obtained for 4/12 mice treated with 7.16.4 alone. These mice were euthanized at day 90 post-tumor inoculation. Resected tumors from mice treated with NK1.1 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 NK1.1 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).

Fig. 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 in vivo. 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 (mice euthanized at day 90). In (A), the bar shows statistical significance between the two groups of particular interest: (1) naïve B16 + pLenti6.3 c-erb-B2 and (2) naïve B16 + pLenti6.3 c-erb-B2 + 7.16.4.