Construction of c-erb-B2 lentivirus vector and confirmation of transfected wild-type B16 (B16/neu).
Successful construction of the c-erb-B2 lentivirus (Fig. 1A) and transfection into naïve (wild-type) B16 melanoma cells were confirmed via q-PCR (Fig. 1B), Western blot (Fig. 1C), and flow cytometry (Fig. 1D). Original Western blots with a positive neu control using the c-erb-B2 expressing mouse cell line MMC (derived from mammary cells of female BALB/c mice) are included in the supplementary materials (Supplementary Fig. 1). Figure 1C shows a cropped version of this Western blot, which highlights the c-erb-B2 expression in our newly created c-erb-B2-expressing cell line compared to naïve B16. Similar to human melanoma, naïve B16 showed only approximately 1% endogenous c-erb-B2 (neu) expression on flow cytometry. This was significantly increased with pLenti6.3 c-erb-B2 transfection to over 95% surface expression of c-erb-B2. The c-erb-B2-expressing B16 was designated “B16/neu.” We also confirmed via gene sequencing that the c-erb-B2 insert represented the oncogenic variant of neu, as opposed to the wild-type phenotype (Fig. 1E).14,15 Expression of the oncogenic c-erb-B2 variant was important for our model in order to better replicate human breast cancer, where oncogenic HER2 overexpression is the target of anti-HER2 therapies.16
Anti-neu monoclonal antibody (7.16.4) has no effect on B16/neu in vitro.
Compared to naïve B16, the newly created B16/neu cell line showed increased growth and cell viability in vitro as measured by the Cyquant assay. This was observed throughout all time points up to 96 hours. When the mouse anti-rat c-erb-B2 monoclonal antibody 7.16.4 was added to the cell culture at increasing dose concentrations, there was no significant effect on B16/neu growth in vitro (Fig. 2). As expected, there was also no effect from 7.16.4 on naïve (non-c-erb-B2-expressing wild-type) B16.
Interestingly, the absence of effect of 7.16.4 on B16/neu was contrary to our group’s previous investigation of 7.16.4 in c-erb-B2 + mouse breast cancer (using the neu + MMC cell line).17 However, naïve B16 does not normally express oncogenic c-erb-B2 (as shown in Fig. 1D), and its oncogenicity is driven by other melanoma-specific mutations.18,19 Thus, neutralization of B16/neu growth by 7.16.4 in vitro was not expected.
Anti-neu 7.16.4 generates in vivo responses against B16/neu.
To test our hypothesis that 7.16.4 could generate anti-tumor responses against B16/neu in vivo, B16/neu tumors were grown orthotopically within the dorsal skin of both male and female C57BL/6 mice. Although prior studies including our own have not demonstrated sex-based differences in B16 growth,20,21 we accounted for possible sex-based differences in the setting of the newly transfected c-erb-B2, which is most often associated with breast cancer cells in female subjects. Equal numbers of male and female mice were used for each experiment. Experiments were completed in triplicate to assess for reproducibility, and data were pooled for analysis.
For tumor subcuticular inoculation, 1 x 105 B16/neu cells were injected in 0.1 ml of PBS with a 23-gauge needle. Treatments were initiated when tumors reached 5 mm3 in their longest dimension (approximately 7–10 days post-inoculation). As controls, naïve B16 at the same tumor inoculation dosage was included. The antibody 7.16.4 was administered intraperitoneally (ip) at 400 µg in 0.2 ml of PBS every 3 days until mice reached the predefined endpoints listed in the Methods. As an antibody control, isotype IgG2a was used at the same frequency and dosage ip (400 µg). Tumor measurements were taken every 5–6 days.
Figure 3 shows the tumor growth (A) and survival analysis (B) for the control and experimental groups. As expected, there was no effect of 7.16.4 on naïve B16. There was slower tumor growth observed with the B16/neu controls (PBS or isotype) compared to naïve B16, but these differences were not statistically significant (p = 0.08 for PBS control, p = 0.09 for isotype control). When compared to isotype controls, B16/neu tumors treated with 7.16.4 showed statistically significant decreased tumor growth (p = < 0.000001) and improved survival with 5/12 (41.2%) tumors showing complete tumor response and regression (p = < 0.0001). Mice that obtained complete tumor response, of which 2 were female and 3 were male, did not have tumor volumes that exceeded 10 mm3 during the course of the experiment. This suggested that tumor growth kinetics beyond 10 mm3 was sufficient to overcome the anti-tumor effects of 7.16.4. Antibody therapy was stopped 60 days after tumor inoculation, and the mice with complete response showed long-term survival (over 90 days) and were then humanely euthanized.
Anti-neu 7.16.4 responses are mediated by NK-cell antibody-dependent cell-mediated cytotoxicity.
Whereas 7.16.4 did not demonstrate a neutralizing effect on B16/neu in vitro (as shown in Fig. 2), we hypothesized that the mechanism by which in vivo c-erb-B2 expression facilitated anti-tumor responses from 7.16.4 (as shown in Fig. 3) was obtained by NK-cell antibody-dependent cell-mediated cytotoxicity (ADCC). To test our hypothesis, we included anti-NK cell monoclonal antibody (NK 1.1) at 200 µg injected every 3 days ip for C57BL/6 mice bearing B16/neu tumors. The NK 1.1 injections started 1 week before B16/neu inoculation to deplete NK cells prior to tumor growth and 7.16.4 treatment.
The addition of NK 1.1 inhibited the effect of 7.16.4 on B16/neu tumors (Fig. 4). Mice treated with NK 1.1 and 7.16.4 or NK 1.1 alone (control) showed similar growth rates (Fig. 4A) and survival (Fig. 4B) to the negative (PBS) controls. Equal numbers of male and female mice were used, and experiments were completed in triplicate. Again, mice treated with 7.16.4 only demonstrated superior statistically significant responses for both tumor growth (p = < 0.000001 compared to 7.16.4 + NK 1.1) and survival with 37.5% achieving complete clinical response and long-term survival (p = 0.0289).
Whole tumors were removed from mice treated with NK 1.1 +/- 7.16.4 and from non-responders that were treated with 7.16.4. Resected tumors were sectioned and analyzed by IHC for NK cells. Mice treated with 7.16.4 only (7.16.4 control) showed a qualitatively significant higher number of stained NK cells within the harvested B16/neu tumors (Fig. 4C) compared to mice treated with NK 1.1 +/- 7.16.4 (Fig. 4D), which showed essentially no NK cells within the tumor parenchyma. This provided pathological confirmatory evidence that NK 1.1 effectively inhibited NK cell activity via NK cell depletion, and that the in vivo anti-tumor mechanism of action of 7.16.4 was NK-cell ADCC. IHC for NK cells was also performed on normal (non-tumor bearing) C57BL/6 splenic tissue as a control, showing expectedly high levels of NK cells within the spleen parenchyma (Fig. 4E).
In vivo transfection of naïve B16 melanoma tumors with c-erb-B2 lentivirus combined with 7.16.4 generates anti-tumor responses.
The presented experiments have thus far utilized B16/neu that was developed in vitro using our c-erb-B2 lentivirus (pLenti6.3 c-erb-B2). To better model human melanoma that does not normally express high levels of HER2/neu, we tested the in vivo injection of naïve B16 tumors with pLenti6.3 c-erb-B2. We hypothesized that in vivo transfection of naïve B16 tumors with pLenti6.3 c-erb-B2 would generate c-erb-B2 as a neoantigen target for 7.16.4 and allow for the repurposing of anti-c-erb-B2 (7.16.4) therapy.
To obtain the optimal dose of in vivo pLenti6.3 c-erb-B2, we tested a linear set of viral doses from 6 x 105 pfu to 1 x 107 pfu. These doses were injected into naïve B16 tumors when they grew to a minimum of 5 mm3. Virus was injected every 3 days. Aliquots of the different viral dosages were delivered in 50 µl PBS. At smaller tumor sizes (5–10 mm3), 1–2 injections into the tumor were performed. As tumors grew beyond 10 mm3, 3–5 injections were performed to cover as much of the tumor volume as possible. Virus injections were performed for a total of 4 intratumoral inoculations (or 12 days).
In vivo surface expression of c-erb-B2 as a neoantigen target for 7.16.4 was quantified by flow cytometry after resecting and processing the tumors. c-erb-B2 expression using this approach was shown to be dose dependent, with the highest dose (1 x 107 pfu) yielding approximately 45% successful expression of c-erb-B2 in vivo. This was the selected dose of pLenti6.3 c-erb-B2 used in this set of experiments. As a virus control, normal or “blank” pLenti6.3 was used at the same intratumoral dose and schedule.
For C57BL/6 mice bearing naïve B16 (non c-erb-B2-expressing) tumors, 7.16.4 (400 µg ip) was added to pLenti6.3 c-erb-B2 after the first 2 doses (6 days) of virus (either pLenti6.3 control or pLenti6.3 c-erb-B2). Virus and 7.16.4 injections were given every 3 days until endpoints were met. Similar to our prior experiments, equal numbers of male and female mice were used, and experiments were completed in triplicate, again to meets standard for rigor and reproducibility.
Figures 5A and B show the tumor growth and survival data, respectively. Similar to our experiments with B16/neu (derived in vitro) and 7.16.4, the in vivo inoculation of naïve (non-c-erb-B2) B16 tumors generated c-erb-B2 as a neoantigen target for 7.16.4. Naïve B16 tumors treated with pLenti6.3 c-erb-B2 and 7.16.4 showed statistically significant responses compared to isotype controls. These responses were demonstrated by decreased tumor growth (p = < 0.000001 ) and improved long-term survival (p = 0.0015). Similar to our experiments with B16/neu derived in vitro, our in vivo approach yielded a 41.2% complete clinical response (5/12 mice, 3 female and 2 male). These mice showed long-term survival to over 90 days. Again, tumors in these mice did not progress beyond 10 mm3 during the treatment period.