Safety and Efficacy of Intratumoral Injections of Chimeric Antigen Receptor (CAR) T Cells in Metastatic Breast Cancer

Abstract

Chimeric antigen receptors (CAR) are synthetic molecules that provide new specificities to T cells. Although successful in treatment of hematologic malignancies, CAR T cells are ineffective for solid tumors to date. We found that the cell-surface molecule c-Met was expressed in ∼50% of breast tumors, prompting the construction of a CAR T cell specific for c-Met, which halted tumor growth in immune-incompetent mice with tumor xenografts. We then evaluated the safety and feasibility of treating metastatic breast cancer with intratumoral administration of mRNA-transfected c-Met-CAR T cells in a phase 0 clinical trial (NCT01837602). Introducing the CAR construct via mRNA ensured safety by limiting the nontumor cell effects (on-target/off-tumor) of targeting c-Met. Patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 × 10⁷ or 3 × 10⁸ cells. CAR T mRNA was detectable in peripheral blood and in the injected tumor tissues after intratumoral injection in 2 and 4 patients, respectively. mRNA c-Met-CAR T cell injections were well tolerated, as none of the patients had study drug-related adverse effects greater than grade 1. Tumors treated with intratumoral injected mRNA c-Met-CAR T cells were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, all surrounded by macrophages at the leading edges and within necrotic zones. We conclude that intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors. Cancer Immunol Res; 5(12); 1152-61. ©2017 AACR.

Fig. 1. Cytotoxicity of c-Met-CAR T cells

Chromium release cytolytic activity assays of mRNA CAR T cells in two breast cancer cell lines, BT20 and TB129. Each assay was performed in duplicates and repeated in at least three independent experiments. A) Upper panel, BT20 tumor cell lysis induced by CAR T directed against c-Met (cMet.BBz, solid triangles), mesothelin (SS1.BBz, solid squares) or CD19 (CD19.BBz, solid circles) at various effector T cell:tumor cell (E:T) ratios. mRNA c-Met-CAR T cells were more effective than mRNA meso-CAR T cells in inducing CAR T cell directed tumor cell lysis. Lower panel, histograms of flow cytometry analyses of c-Met and mesothelin expression on BT20 cells as stained by antibodies against c-Met and mesothelin. B) Upper panel, TB129 tumor cell lysis induced by CAR T directed against c-Met (cMet.BBz, solid triangles), mesothelin (SS1.BBz, solid squares) or CD19 (CD19.BBz, solid circles) at various E:T ratios. mRNA c-Met-CAR T cells cytolytic activity was similar to that of mRNA meso-CAR T cells in inducing CAR T directed tumor cell lysis. Lower panel, histograms of flow cytometry analyses of TB129 cells as stained by antibodies against c-Met and mesothelin.

Fig. 2. Antitumor efficacy of mRNA c-Met-CAR T cells in NSG mice

Each experiment was performed using groups of 7–8 female and male NOD/scid/γc(^–/–) (NSG) mice and repeated in one independent experiment without the use of cyclophosphamide. Tumor growth curve as measured by bioluminescence imaging demonstrated effectiveness of multiple intratumoral injections of mRNA c-Met-CAR T cells into pre-established c-Met⁺ Click beetle green luciferase labeled tumor xenografts (SK-OV-3/luc) in controlling tumor growth in NSG mice. Red squares depicted bioluminescence fold change in tumor size of mRNA c-Met-CAR T cells treated mice; blue square depicted those of mRNA CD19-CAR T cell (allogeneic control) treated mice; and green squares depicted those of phosphate buffered saline (PBS, negative control) treated mice. Intratumoral injection time points were depicted by red arrows. Intraperitoneal (IP) injections of Cytoxan (cyclophosphamide) were given 24 hours prior to the intratumoral injections as depicted by green arrows to eliminate the previous T cells, preventing the development of graft-versus-host disease (21).

Fig 3. Histology and IHC of tumor tissue pre and post intratumoral injection of mRNA c-Met-CAR T cells from one treated patient

Histologic (H&E) and IHC evaluation of c-Met expression and T cell (CD3⁺, CD4⁺, CD8⁺) and macrophage (CD68⁺) infiltration in tumor tissues of pre-treatment, away from intratumoral injection site and at intratumoral injection site from a patient treated with 3 × 10⁸ mRNA c-Met-CAR T cells.