Genetic modification of T cells redirected toward CS1 enhances eradication of myeloma cells

Abstract

Purpose: Our goal is to test whether CS1 could be targeted by chimeric antigen receptor (CAR) T cells to treat multiple myeloma (MM).

Experimental design: We generated a retroviral construct of a CS1-specific CAR and engineered primary human T cells expressing the CAR. We then tested the capacity of CS1-CAR T cells to eradicate human MM tumor cells in vitro, ex vivo, and in vivo using orthotopic MM xenograft mouse models.

Results: In vitro, compared with mock-transduced T cells, upon recognizing CS1-positive MM cells, CS1-CAR-transduced T cells secreted more IFN-γ as well as interleukin (IL)-2, expressed higher levels of the activation marker CD69, showed higher capacity for degranulation, and displayed enhanced cytotoxicity. Ectopically forced expression of CS1 in MM cells with low CS1 expression enhanced recognition and killing by CAR T cells. Ex vivo, CS1-CAR T cells also showed similarly enhanced activities when responding to primary MM cells. More importantly, in orthotopic MM xenograft mouse models, adoptive transfer of human primary T cells expressing CS1-CAR efficiently suppressed the growth of human MM.1S and IM9 myeloma cells and significantly prolonged mouse survival.

Conclusions: CS1 is a promising antigen that can be targeted by CAR-expressing T cells for treatment of MM.

©2014 American Association for Cancer Research.

Figures

Figure 1. Generation and expression of CS1-specific second-generation CAR

A, Schematic diagram of the Pinco-CS1-CAR retroviral construct containing a single-chain variable fragment (scFv) against CS1 linked to CD28 and CD3ζ endodomains. LTR: long terminal repeat, SP: signal peptide, VH: variable H chain, L: linker, VL: variable L chain. B, PBMCs were activated with CD3 and CD28 beads and transduced with the Pinco-CS1-CAR or Pinco construct. GFP positive cells were sorted, and cell lysates were subjected to immunoblot analysis under reducing conditions with anti-human CD3ζ primary antibody. C, Mock- or CS1-CAR-transduced T cells from healthy donors were stained with biotin-labeled goat anti-mouse Fab specific or isotype-matched control antibody, followed by streptavidin and CD3 antibody staining.

Figure 2. CS1-redirected T cells secrete more IFN-γ and IL-2 than mock T cells in response to CS1-expressing myeloma cell lines

A, Flow cytometric analysis of CS1 expression on the surface of myeloma cell lines. The four myeloma cell lines indicated were stained with PE-conjugated anti-CS1 mAb antibody (solid line) or isotype-matched control Ab (dotted line). B, Mock- or CS1-CAR-transduced healthy donor T cells (2 × 105) were cultured alone (No target) or stimulated with an equal number of myeloma cells expressing different levels of CS1 for 24 h, and the supernatants were harvested to measure IFN-γ secretion via ELISA. C, Cells were treated as in (B), and IL-2 secretion in cell-free supernatants was determined via ELISA.

Figure 3. CS1-redirected T cells preferentially eradicate myeloma cells obviously expressing CS1 protein

A, 51Cr-labeled NCI-H929, IM9, MM.1S and RPMI-8226 myeloma cells (5 × 103) were co-cultured with mock- or CS1-CAR-transduced T cells at the indicated Effector/Target (E/T) ratios for 4 h, and target lysis (51Cr release) was measured. B, Expression of the degranulation marker CD107a and the T cell activation marker CD69 on mock- or CS1-CAR-transduced T cells were evaluated by flow cytometry following 4 h co-culture with NCI-H929 cells. Compared to mock-transduced T cells, CS1-CAR-transduced T cells displayed superior degranulation and higher T-cell activation in response to CS1-expressing NCI-H929 cells. C, Mock- and CS1-CAR-transduced T cells were permeabilized for intracellular staining with mAb specific for granzyme B and perforin, and analyzed by flow cytometry.

Figure 4. Ectopic overexpression of CS1 in MM cells triggers enhanced cytotoxicity and cytokine secretion after recognition by CS1-CAR T cells

A, Flow cytometric staining for CS1 protein or IgG isotype control (dotted line) on the surface of RPMI-8226 cells overexpressing CS1 (RPMI-8226-CS1, solid heavy line) or an empty vector control (RPMI-8226-PCDH, solid light line). B, Cytotoxicity of mock-or CS1-CAR-transduced T cells against RPMI-8226-CS1 and RPMI-8226-PCDH cells. RPMI-8226-CS1 and RPMI-8226-PCDH cells were incubated with mock- or CS1-CAR-transduced T cells at indicated E/T ratios for 4 h, and specific lysis was determined using a standard 51Cr release assay. C, Mock- or CS1-CAR-transduced T cells (1 × 105) were cultured alone or stimulated with an equal number of either RPMI-8226-CS1 or RPMI-8226-PCDH cells. Cell-free supernatants from these cultures were used to determine IFN-γ secretion via ELISA. D, Supernatants from cell cultures in (C) were assayed for IL-2 secretion via ELISA.

Figure 5. CS1-CAR T cells specifically recognize and eliminate CS1-expressing human primary myeloma cells ex vivo

A, PBMCs from MM patients were activated with anti-CD3 and anti-CD28 beads and transduced with Pinco-CS1-CAR or Pinco construct (mock) as described in Methods. Cells were stained with anti-mouse Fab and anti-human CD3 antibodies. Results from 1 of 4 patients with similar data are shown. B, Flow cytometric staining for CS1 protein in CD138+ myeloma cells freshly isolated from MM patients. Results from 3 of 10 patients with similar data are shown. C, CD138+ myeloma cells in (B) were co-cultured with autologous mock- or CS1-CAR-transduced T cells in (A) at indicated E/T ratios for 4 h, and specific lysis was determined using a standard 51Cr release assay. D, Cells were treated as in (C) except that the E/T ratio was 1:1 and the incubation time was extended to 24 h, and IFN-γ secretion was determined via ELISA.

Figure 6. CS1-redirected T cells inhibit tumor growth and prolong mouse survival in an orthotopic MM.1S xenograft mouse model

A, Dorsal and ventral bioluminescence imaging of five representative mice bearing MM.1S tumors from each indicated group. NSG mice were i.v. inoculated with 8 × 106 MM.1S cells expressing luciferase (day 0). On day 7 and day 14 after inoculation, each mouse received PBS (placebo control group), 10 × 106 mock T cells (mock control group) or CS1-CAR T cells (CAR treatment group). B, Kaplan-Meier survival curve of MM.1S-bearing mice treated with PBS, mock T cells or CS1-CAR T cells.