Fig 2: Neutralization activity of C14mab. (a) C14mab inhibits HyIL-6 induced VEGF expression in C33A. VEGF protein expression was evaluated by ELISA in the culture supernatant of C33A cells after incubation under treatment with HyIL-6 (50 ng/ml) in the presence or absence of the indicated concentrations of C14mab. The culture media were collected for the determination of VEGF levels. Data expressed as mean ± SD, n = 4. * Statistically significant difference from the positive control; P < .05. ** Statistically significant difference from the positive control; P < .01 (b) Wildtype naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for four days in the presence of TGF-ß1 (2 ng/ml) and anti-IL-2 (10 ug/ml). HyIL-6 (50 ng/ml) mediated signaling was blocked in cultures using indicated concentrations of C14mab, tocilizumab, and olokizumab. IL-17A levels in culture supernatants were determined by ELISA (R&D Systems). Values represent the mean ± SD: n = 3. ** Statistically significant differences from the positive control; P < .01. (c) BALB/c mice were injected with either C14mab (0.1 mg/ml), DPBS, or isotype control antibody (0.1 mg/kg). After 1 hr, mice were challenged with recombinant human HyIL-6 (1 ug). After an additional 6 hrs, whole blood was collected. SAA levels were determined from serum by using the mouse SAA ELISA. (d) C14mab dose-dependently decreases HyIL-6 induced SAA levels in mice. BALB/c mice were injected with the indicated dose of C14mab. After 1 hr, mice were challenged with recombinant human HyIL-6 (1 ug). After an additional 6 hrs, whole blood was collected. SAA levels were determined from serum by using the mouse SAA ELISA. Data expressed as mean ± SD, n = 5. ** Statistically significant differences from the positive control; P < .01. Fig (a) Histogram of HyIL-6 induced VEGF expression with various indicated concentration of C14mab. Highest VEGF expression noted with 10(-3) concentration of C14mab. Fig (b) Three histograms of IL-17A levels with HyIL-6 mediated signaling blockage in indicated concentrations of C14mab, Tocilizumab, and Olokizumab. Fig. (c) Histogram of IL-6 induced SAA levels in mice with indicated injected concentrations of C14mab and isotype control antibody. Fig (d) Histogram of IL-6 induced SAA levels in mice with indicated injected concentration of C14mab.

Fig 3: Anti-EphA10 antibody therapy increases the infiltration and activity of CTLs.A and B, the effect of anti-EphA10 antibody in tumor growth of BALB/c-SCID (A) and BALB/c (B) mice bearing orthotopic injected 4T1 tumor. Mice (n = 6 per group) were treated with anti-EphA10 mAb (clone #4) or IgG control. The gray box and arrows indicate the duration and time of treatment. Error bar represents mean ± SEM. Data were analyzed by two-way ANOVA analysis. ∗∗∗p < 0.001. C, the gating strategy used for flow cytometric analysis of (D–G). Cells were analyzed by forward scatter/side scatter parameters and Zombie Violet− staining to determine total live events, in which various cell populations were identified on the basis of marker expression: CD45+ (immune cell population), CD3+/CD4−/CD8+ (CD8 T cells), and granzyme B+ (GrB) in CD8 T cells. D–G, flow cytometry analysis of the amount of CD45+/CD3+ Pan-T cell (D), the amount of CD3+/CD4+/CD8- T cell (E), the amount of CD8+/GrB+ CTLs (F), and percentage of CD8+/GrB+ CTLs in CD3+ cells (G) in 4T1 tumor region under IgG control or anti-EphA10 #4 antibody treatment (300 μg/mouse). n = 6 mice per group. Data were analyzed by student t test analysis. 0.01 < ∗p < 0.05. CTL, cytotoxic T lymphocyte; EphA10, Ephrin receptor A10; IgG, immunoglobulin G; mAbs, monoclonal antibodies; TNBC, triple-negative breast cancer.