Fig 2: Complete responder mice, previously treated with anti-PD-1 and photoimmunotherapy, mount tumor-specific memory immune responses. Mice that had achieved a complete response (n = 7) after dual treatment with anti-PD-1 and photoimmunotherapy were re-challenged with syngeneic CT26-EphA2 cells in the contralateral hind flank, followed by 4T1 cells in the axilla (A). Naïve mice (n = 10) were inoculated as control. Tumor growth was measured to determine rejection (B & C). The resulting tumor growth curves after rechallenge are representative of at least two replicate experiments performed by different personnel

Fig 3: Combining photoimmunotherapy with checkpoint inhibition elicits enhanced anticancer responses. Mice were implanted with CT26-EphA2 tumors, then treated with anti-EphA2-IR700 alone, or EphA2 photoimmunotherapy. Anti-PD-1 was administered to indicated groups at days 4, 6, 8, and 11 post-inoculation, and tumor growth was measured from days 18 to 22, and graphed here through day 18 (A). Final tumor measurements: anti-PD-1 vs EphA2 photoimmunotherapy + anti-PD-1, p = 0.0001; EphA2 photoimmunotherapy vs EphA2 photoimmunotherapy + anti-PD-1, p < 0.05 based on two-way ANOVA and Tukey test. The percent of complete responses (calculated by number of CRs/total animals in treatment group) as defined as undetectable tumors at study end were determined (B). Activation patterns among intratumoral lymphocytes were determined by flow cytometry at day 21, among mice that had palpable tumors. Tumors with limiting volume were pooled to achieve sufficient cell yield for analysis (C). Mice were implanted with tumors (n = 15 per group) and received saline, anti-EphA2-IR700 without illumination, anti-PD-1 monotherapy (days 4, 6, 8, and 11 post implant), or anti-CD8 (days 4 and 7 for CD8 T cell depletion) and evaluated for tumor growth through day 18 post implant. Tumor-bearing mice also received photoimmunotherapy treatment (as described above, n = 12), photoimmunotherapy treatment with anti-PD-1 (n = 11), or photoimmunotherapy treatment with anti-PD-1 and anti-CD8 (n = 9), and evaluated for tumor growth through day 18. Final tumor measurements: EphA2 photoimmunotherapy + anti-PD-1 vs. EphA2 photoimmunotherapy + anti-PD-1 + anti-CD8, p < 0.01 based on two-way ANOVA and Tukey test (D). MCA205-EphA2 cells were inoculated into the hind flanks of mice. Mice were then treated with saline, anti-PD-1 alone, EphA2 photoimmunotherapy, or EphA2 photoimmunotherapy combined with anti-PD-1 antibodies, as indicated (n = 10 mice per group). Anti-PD-1 was administered beginning on day 7 and continuing 3 times per weeks until study end. Tumor growth (E) and survival (F) were measured through days 33 and 63, respectively. Final tumor measurements: anti-PD-1 vs EphA2 photoimmunotherapy + anti-PD-1, p < 0.0001; EphA2 photoimmunotherapy vs EphA2 photoimmunotherapy + anti-PD-1, n.s. based on two-way ANOVA and Tukey test (E). The tumor growth curves shown are representative of at least two replicate experiments performed by different personnel

Fig 4: Characterization of an immunocompetent mouse tumor model for cancer-targeted photoimmunotherapy. Flow cytometry analysis demonstrated stable expression of mouse EphA2 on the surface of CT26 mouse colon carcinoma cells after viral transduction (A). CT26-EphA2 cells were treated with increasing concentrations of anti-EphA2-IR700 followed by illumination at indicated doses, demonstrating that EphA2 photoimmunotherapy results in cell killing in a light- and conjugate- dose-dependent manner in vitro (B). Anti-EphA2-IR700 accumulates in CT26-EphA2 tumors in mice after systemic administration, as detected by fluorescence imaging (C). Mice were implanted with CT26-EphA2 tumors, then treated with anti-EphA2-IR700 and light or only anti-EphA2-IR700 without light, as reference. Tumor growth was inhibited in the anti-EphA2-IR700 plus light group (n = 15 per group). Final tumor measurements: saline vs EphA2 photoimmunotherapy, p < 0.01; EphA2 vs EphA2 photoimmunotherapy, p < 0.0001 based on two-way ANOVA and Tukey test. The tumor growth results shown are representative of several replicate experiments performed by different personnel. (D). The ability of EphA2 photoimmunotherapy to elicit vaccinal effects as compared to known ICD-inducing or non-ICD-inducing conditions was determined. Cells killed with cisplatin, doxorubicin, or anti-EphA2-IR700 plus light were allowed to achieve 50% cell death, then were implanted into the right hind flank of naïve mice. Seven days after implant with dying cells, mice were challenged with viable CT26-EphA2 cells in the opposite hind flank and tumor growth was measured for 21 days (E)

Fig 5: Photoimmunotherapy plus anti-PD-1 treatment expands peripheral tumor-specific CD8 + T cells. At day 14 after photoimmunotherapy, splenocytes from mice bearing CT26-EphA2 tumors that had been treated with either anti-EphA2-IR700, anti-EphA2-IR700 plus anti-PD-1, EphA2 photoimmunotherapy, or EphA2 photoimmunotherapy plus anti-PD-1, were harvested and expanded in the presence of the antigen AH1, which is expressed on the surface of CT26 cells, and co-cultured with CT26 cell monolayers in vitro at the indicated effector:target ratios. The AH1 antigen-specific cytotoxic potential of primed splenocytes was measured by Lactic Acid Dehydrogenase (LDH) release assay