Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma

Abstract

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies.

Copyright© Ferrata Storti Foundation.

Figures

Figure 1.

CD38-CAR construct and CD38-CART-cell phenotype. (A) Schematic overview of the CD38-CAR construct. The CD38-scFv sequence is based on three different antibody sequences (028, 056 and 026, see also Online Supplementary Table S1), with CD8a as a transmembrane domain and 4-1BB and CD3ζ as intracellular domains. (B) CAR expression on the cell surface of healthy donor T cells was determined by binding of biotinylated protein L to the scFv domain (left panel), stained with phycoerythrin-labeled streptavidin. The results for CD38-CART cells generated with CAR056, representative of all three CAR, are shown. The expression of surface markers CD4 and CD8 (right panel) was determined by fluorescence-labeled monoclonal antibodies. (C) The expansion of mock and CD38-CART cells after transduction (left panel) and after the second round of stimulation (right panel; new stimulation set at “0”). (D) The relative 3H-thymidine uptake (left panel) of mock and CD38-CART cells after 72 h stimulation with the CD38+ MM cell line UM9: responder ratio of 3:1. Error bars represent mean + SEM, n=3. The results are expressed as relative stimulation index, compared to mock, and considered significant if the stimulation index is ≥3. The cytokine secretion (right panel) from mock and CD38-CART cells stimulated with αCD3/CD28 beads or the MM cell line UM9. The cytokine secretion was measured with a flow cytometry-based CBA kit (BD) in the cell-free supernatants after 24 h of stimulation. The graph shows the secretion of interferon (IFN)-γ, tumor necrosis factor (TNF) and interleukin (IL)-2. Secretion of IL-4, -5 and -10 was below the detection limits. These data are not therefore shown in this figure. Similar results were obtained in two independent assays.

Figure 2.

Efficacy of CD38-CART cells at lysing MM cell lines. In 24 h cytotoxicity assays, three different types of CD38-CART cells were tested against two MM cell lines with different CD38 expression levels: (A) U266, a CD38− cell line, (B) UM9, a CD38+ cell line. Effector:target ratios are indicated. Target cells per well were 10,000 MM cells. Closed circles (●) indicate mock cells and open squares, triangles and diamonds (□, ◊, △) indicate the CAR028, 056 and 026 constructs, respectively. Error bars indicate mean ± SD. (C) Correlation between mean fluorescent intensity (MFI) of CD38 on target cells and consequential CD38-CAR specific lysis. CD38-CART cells (CAR056) were co-cultured with leukemic cell lines and allogeneic healthy donor peripheral blood mononuclear cells. The resulting lysis in a 3:1 ratio was determined with bioluminescence imaging or flow cytometry, minus the spontaneous lysis caused by mock T cells. Open circles (○) indicate MM cell lines (LME-1, UM9, MM1.S, U266, L363 and UM3), triangles (▲) indicate AML (HEL, MOLM13), T lymphoblast (CEM) and Burkitt lymphoma (Daudi), and closed circles (●) indicate healthy immune cells (T=T cells, B=B cells, NK=NK cells, Mo=monocytes, C=CEM, H=HEL, M=MOLM13, D=Daudi), Error bars represent mean ± SEM of duplicate measurements.

Figure 3.

Efficacy of CD38-CART cells generated from healthy individuals at lysing primary MM cells. (A) Bone marrow-derived mononuclear cells from three MM patients, all three refractory to lenalidomide and bortezomib, and bone marrow mononuclear cells from two AML patients were co-incubated with no, mock- or CD38-CART cells generated from healthy peripheral blood mononuclear cells for 16 h. Closed circles (●) indicate mock and open squares (□) indicate CAR056T cells (representative of all CAR). The graphs depict the resulting lysis of CD138+/CD38+ cells (MM) or CD13+/CD7+/CD45dim/CD38+ cells (AML1, moderate CD38 expression) and CD33+/CD133+/CD45dim/CD38+ cells (AML2, high CD38 expression) in three effector:target cell ratios. The percent lysis in these flow cytometry assays was calculated as described in the Methods section. (B) Efficacy of CD38-CART cells generated from a MM patient: CAR expression on the cell surface of the patient’s T cells was determined by flow cytometry with protein L staining (see also Figure 1). (C) Bone marrow-derived mononuclear cells from the MM patient were co-incubated with autologous mock- or CD38-CART cells for 16 h. The graph depicts resulting lysis of CD138+/CD38+ cells at two ratios, determined in flow cytometry-based assays.

Figure 4.

Tumor growth in mock- and CD38-CART-cell-treated mice. (A) Analysis of CD38-CART cells after 2 weeks of in vitro culture, with fluorescence-labeled mono -clonal antibodies for CD45RA and CD62L and CD38. (B) Leukocyte transmigration assay, in which mock and CART cells were cultured in a transwell system in the inserts with human umbilical vein endothelial cells, which were activated with tumor necrosis factor (TNF)-α. Spontaneous TNFα-induced transmigration was compared to active migration induced by 10% human serum in the lower compartment. % migrated cells = [Relative Fluorescence Units (RFU) of cells in lower compartment / RFU of total cells in both compartments] * 100%. (C) Analysis of tumor load in mice by quantification of bioluminescent imaging measurements. Each group contained six mice, each harboring six scaffolds. Results are mean tumor load (cpm/cm2) of six mice per group. Closed circles (●) indicate mock cells and open squares (□) indicate CAR056 cells. The error bars represent mean + SEM, n=6. The differences between groups were analyzed after week 6 using an unpaired Student T test, P<0.0001 (D) Bioluminescent imaging of mice on the right side; mice were implanted with fully humanized bone marrow stromal cell scaffolds each coated with 1×106 UM9-GFP-Luc tumor cells. At 7, 9 and 13 days after implantation, mice were injected intravenously with 20×106 mock or CD38-CART cells. (E) Representative immunohistochemistry figure: remaining tumors were stained with CD38 and CD138 antibody, T = tumor, sc = scaffold.

Figure 5.

The impact of CD38-CART cells on non-malignant hematopoietic cells in bone marrow and outgrowth of hematopoietic cell lineages. (A) Bone marrow mononuclear cells from three MM patients were co-incubated with none, mock- or CD38-CART cells for 16 h. The graphs depict the resulting lysis of the total or the CD38+ fractions of CD3+ (T cells), CD56+ (mainly natural killer cells), CD14+ (monocytes) and CD19+ (B cells) subsets at three effector:target rations, determined with flow cytometry and calculated as described in the Methods section. Results are from three individual experiments combined. Closed circles (●) indicate mock cells and open squares (□) indicate CAR056 cells. Error bars represent mean ± SEM, n=3. (B) CD34+ fraction of bone marrow mononuclear cells from healthy donors was co-incubated with none, mock- or CD38-CART cells for 4 h at different target:effector cell ratios before being transferred into the semisolid hematopoietic progenitor cell culture medium. After incubation, cells were analyzed by flow cytometry for surviving CD34+ cells with CD38 expression. The graphs depict the resulting lysis of the total or the CD38+ fraction of CD34+ cells. Closed circles (●) indicate mock cells and open squares (□) indicate CAR056 cells. (C) After 14 days of culture in plastic dishes, colony-forming unit-monocytes (CFU-M), and CFU-granulocytes (CFU-G) were visible. (D) The numbers of CFU-M and CFU-G colonies were determined microscopically. Results of a representative experiment are shown as mean ± SD.

Figure 6.

Dimerizer AP20187-mediated elimination of the iCasp9 suicide gene-transduced CD38-CAR T cells. (A) Flow cytometry histogram plots: CD38-CAR T cells that were additionally transduced with the iCasp9-GFP construct. The upper panel shows the untreated cells: 50% GFP+; the lower panel shows the cells treated with 100 nM dimerizer AP20187 (B/B). (B) Lysis of the UM9 cell line by iCasp9-transduced CD38-CAR T cells that were untreated or treated with the dimerizer. The significant reduction of GFP+ cells (A) is a consequence of cell death activated by the dimerizer B/B. Note (in B) the decrease in cytolysis is proportional to the elimination of the suicide gene-transduced cells (50% of all CAR+ cells in (A). The residual cytolysis is thus caused by the CAR+ cells that were not transduced with iCasp9 n=2, mean ± SD. (C) CD38-CART iCasp9-GFPhigh sorted cells. The upper panel shows the untreated cells 100% GFP+; the lower panel shows the cells treated with 100 nM dimerizer B/B. (D) Lysis of the UM9 cell line by iCasp9high-CD38-CART cells that were untreated or treated with the dimerizer. Closed circles (●) indicate mock cells and open diamonds (◊) and triangles (△) indicate CAR056 without and with B/B, respectively. n=2, mean ± SD.