Article Near-Infrared Photoimmunotherapy Combined with CTLA4 Checkpoint Blockade in Syngeneic Mouse Cancer Models Yasuhiro Maruoka, Aki Furusawa , Ryuhei Okada, Fuyuki Inagaki , Daiki Fujimura, Hiroaki Wakiyama , Takuya Kato, Tadanobu Nagaya, Peter L. Choyke and Hisataka Kobayashi * Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA; [email protected] (Y.M.); [email protected] (A.F.); [email protected] (R.O.); [email protected] (F.I.); [email protected] (D.F.); [email protected] (H.W.); [email protected] (T.K.); [email protected] (T.N.); [email protected] (P.L.C.) * Correspondence: [email protected] Received: 4 August 2020; Accepted: 11 September 2020; Published: 14 September 2020 Abstract: Near infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that induces necrotic/immunogenic cell death. It employs a monoclonal antibody (mAb) conjugated to a photo-absorber dye, IRDye700DX, which is activated by NIR light. Tumor-targeting NIR-PIT is also at least partly mediated by a profound immune response against the tumor. Cytotoxic T-lymphocyte antigen-4 (CTLA4) is widely recognized as a major immune checkpoint protein, which inhibits the immune response against tumors and is therefore, a target for systemic blockade. We investigated the effect of combining tumor-targeted NIR-PIT against the cell-surface antigen, CD44, which is known as a cancer stem cell marker, with a systemic CTLA4 immune checkpoint inhibitor in three syngeneic tumor models (MC38-luc, LL/2, and MOC1). CD44-targeted NIR-PIT combined with CTLA4 blockade showed greater tumor growth inhibition with longer survival compared with CTLA4 blockade alone in all tumor models. NIR-PIT and CTLA4 blockade produced more complete remission in MOC1 tumors (44%) than NIR-PIT and programmed cell death protein 1 (PD-1) blockade (8%), which was reported in our previous paper. However, the combination of NIR-PIT and CTLA4 blockade was less effective in MC38-luc tumors (11%) than the combination of NIR-PIT and PD-1 blockade (70%). Nonetheless, in many cases ineffective results with NIR-PIT and PD-1 blockade were reversed with NIR-PIT and CTLA4 blockade. Keywords: near infrared photoimmunotherapy; immune checkpoint blockade; CTLA4; CD44; monoclonal antibodies 1. Introduction Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that induces highly specific cell death in targeted tumor cells. It employs a monoclonal antibody (mAb) conjugated to a silica-phthalocyanine photoabsorbing dye, IRDye700DX (IR700) [1,2]. This antibody-IR700 conjugate is administered intravenously, and after a suitable time to allow for target binding, the tumor is exposed to 690 nm NIR light which activates IR700 [3]. NIR-PIT induces stress on the cellular membrane through photoinduced ligand-release of IR700, leading to impaired membrane function, rapid volume expansion with cell membrane rupture and extrusion of the cell contents into the extracellular space [4,5]. NIR-PIT has an important advantage over other cancer therapies, and induces highly selective necrotic/immunogenic cell death to tumor cells with minimal damage to adjacent normal cells unlike most other conventional cancer therapies that induce apoptotic cell death in both cancer and normal cells [6,7]. A phase III clinical trial of NIR-PIT using the epidermal growth factor receptor (EGFR) -targeted antibody-IR700 conjugate, Vaccines 2020, 8, 0528; doi:10.3390/vaccines8030528 www.mdpi.com/journal/vaccines Vaccines 2020, 8, 0528 2 of 14 cetuximab-IR700, in patients with inoperable head and neck cancer is underway (https://clinicaltrials.gov/ ct2/show/NCT03769506). CD44 is a well-known marker of cancer stem cells [8] and anti-CD44-mAb-IR700 NIR-PIT induces effective tumor killing in CD44-expressing syngeneic mouse models [9–11]. Recently, the combination of CD44-targeted NIR-PIT and programmed cell death protein 1 (PD-1) immune checkpoint blockade was reported to induce massive tumor cell death and innate priming of polyclonal, cancer antigen-specific T-cell responses in several cancer models, resulting in complete tumor rejection and the generation of immunologic memory [10]. However, in some cancer models, CD44-targeted NIR-PIT combined with PD-1 blockade did not show benefits over CD44-targeted NIR-PIT alone. Cytotoxic T-lymphocyte antigen-4 (CTLA4) is a widely recognized immune checkpoint protein, which inhibits the immune response against tumors [12–14], down-regulating T cell activation in response to engagement of the T cell receptor. CTLA4 is expressed transiently on activated T cells, peaking at 2–3 days after initial activation [15], and is always strongly expressed on regulatory T cells (Tregs). Blockade of the interaction between CTLA4 and its ligands, CD80/86 using anti-CTLA4 mAbs can reduce Treg populations leading to increase in effector T cells, thus enhancing anti-tumor immunity [16,17]. CTLA4 and PD-1 blockade, although both intended to enhance anti-tumor immunity, produce distinct immunoregulatory patterns that vary in effectiveness with each tumor model. Therefore, we hypothesized that NIR-PIT combined with CTLA4 blockade could achieve different and perhaps complementary therapeutic effects from NIR-PIT combined with PD-1 blockade. The purpose of this study was to investigate the in vivo therapeutic efficacy of the combination of CD44-targeted NIR-PIT and CTLA4 blockade in several syngeneic mouse models of cancer. 2. Results 2.1. Comfirmation of CD44 Expression as a Target for NIR-PIT and In Vitro Tumor Cell Killing of CD44-Targeted NIR-PIT in MC38-luc, LL/2, and MOC1 Tumor Cells To confirm CD44 expression as a target for NIR-PIT and tumor cell killing effects by CD44-targeted NIR-PIT, flow cytometry and in vitro CD44-targeted NIR-PIT were performed in the three different types of tumor cells (Figure1A–E). Flow-cytometric analysis revealed high fluorescent signal intensity in MC38-luc, LL/2, and MOC1 cells after exposure to anti-CD44-mAb-IR700 (Figure1A,B). This signal was completely reversed in the presence of excess unconjugated anti-CD44-mAb, verifying binding specificity (Figure1A,B). Based on incorporation of propidium iodide (PI), the percentage of cell death increased in a light dose dependent manner in MC38-luc, LL/2, and MOC1 cells (Figure1C–E). There was no significant cytotoxicity associated with NIR light alone in the absence of anti-CD44-mAb-IR700 and with anti-CD44-mAb-IR700 alone without NIR light (Figure1C–E). In MOC 1 cells, relatively high percentage of cells were dead even in untreated samples. This was probably because hash handling was needed to detach MOC1 cells from the plate with a cell scraper due to the strong adhesion. 2.2. Comparison of CD44 Expression among MC38-luc, LL/2, and MOC1 Tumors To compare differences in CD44 expression in vivo among the three different types of tumors, size-matched tumors were assessed by means of immunohistochemical (IHC) staining (Figure2A). CD44 expression was lower in MOC1 tumor tissues than MC38-luc and LL/2 tumors, which has also been demonstrated by flow cytometry analysis in the previous study [10]. The previous study also showed low fluorescence intensity of anti-CD44-mAb-IR700 in MOC1 tumors with quantitative analysis [10]. These suggest that tumor accumulation of anti-CD44-mAb-IR700 1 day after injection as a target antigen is predicted to be lower in MOC1 tumors and direct cell killing by CD44-targeted PIT is expected to be less efficient compared with MC38-luc or LL/2 tumors. We also examined distribution of T cells in the tumor microenvironment of three tumor types. Multiplex IHC showed all three types of tumor had infiltration of CD8 T cells, non-regulatory CD4 T cells and Tregs (Figure2A). MOC1 tumor had significantly more non-regulatory CD4 T cells in the stroma, also, although statistically not Vaccines 2020, 8, 0528 3 of 14 significant, MOC1 had tendency to have smaller number of CD8 T cells and Tregs infiltrating into the tumor (Figure2B). 2.3. Efficacy of CD44-Targeted NIR-PIT Combined with CTLA4 Blockade for MC38-luc Tumor The NIR-PIT regimen and imaging protocol are depicted in Figure3A. In the NIR-PIT treated groups, anti-CD44-mAb-IR700 was administrated one day before the first light exposure (day 1), and the NIR-light − was exposed over the following two days; 50 J/cm2 on day 0 and 100 J/cm2 on day 1 (24 and 48 h after anti-CD44-mAb-IR700 injection, respectively). IR700 fluorescence signal in tumors decreased due to dispersion of fluorophore from dying cells and partial photo-bleaching (Figure3B). To investigate tumor-killing efficacy after NIR-PIT, bioluminescence imaging (BLI) was performed before and after NIR-PIT up to day seven (Figure3E). In most mice luciferase activity decreased immediately after NIR-PIT and then gradually increased over the ensuing days (Figure3E). This implies a large amount of initial cancer cell killing, followed by slower outgrowth of cells not originally killed by NIR-PIT. The effect of the combination group was comparable with that of CD44-targerted NIR-PIT in the BLI images. In all the treated groups, BLI after treatment was significantly lower at all time points after the initial light exposure than in the control group (p < 0.05, Dunnett’s test) (Figure3F). However, there were no significant differences in luciferase activity between CTLA4 mAb alone and combination group and between CD44-targeted NIR-PIT alone and combination groups. In acute phase after treatment, there was no evidence of the success of the combination therapy compared with the single regimens. Tumor volumes in all the treated groups were significantly smaller 2, 5, 7, and 10 days after NIR-PIT compared with the control group (p < 0.05, Dunnett’s test) (Figure3G).