Anti-CD19 CAR T cells with high-dose melphalan and autologous stem cell transplantation for refractory multiple myeloma

Abstract

Background: Multiple myeloma is usually fatal due to serial relapses that become progressively refractory to therapy. CD19 is typically absent on the dominant multiple myeloma cell population but may be present on minor subsets with unique myeloma-propagating properties. To target myeloma-propagating cells, we clinically evaluated autologous T cells transduced with a chimeric antigen receptor (CAR) against CD19 (CTL019).

Methods: Subjects received CTL019 following salvage high-dose melphalan and autologous stem cell transplantation (ASCT). All subjects had relapsed/refractory multiple myeloma and had previously undergone ASCT with less than 1 year progression-free survival (PFS).

Results: ASCT + CTL019 was safe and feasible, with most toxicity attributable to ASCT and no severe cytokine release syndrome. Two of 10 subjects exhibited significantly longer PFS after ASCT + CTL019 compared with prior ASCT (479 vs. 181 days; 249 vs. 127 days). Correlates of favorable clinical outcome included peak CTL019 frequency in bone marrow and emergence of humoral and cellular immune responses against the stem-cell antigen Sox2. Ex vivo treatment of primary myeloma samples with a combination of CTL019 and CAR T cells against the plasma cell antigen BCMA reliably inhibited myeloma colony formation in vitro, whereas treatment with either CAR alone inhibited colony formation inconsistently.

Conclusion: CTL019 may improve duration of response to standard multiple myeloma therapies by targeting and precipitating secondary immune responses against myeloma-propagating cells.

Trial registration: Clinicaltrials.gov identifier NCT02135406.

Funding: Novartis, NIH, Conquer Cancer Foundation.

Keywords: Cancer immunotherapy; Hematology; Oncology.

Conflict of interest statement

Conflict of interest: ALG has received research funding from Novartis and consulting fees from Medimmune, Novartis, and Kite Pharma. ADC has received consulting fees from GlaxoSmithKline, Celgene, Janssen, Takeda, Bristol-Myers Squibb, and research support from Bristol-Myers Squibb. BMW has received research funding from Janssen and Prothena and consulting fees from Novartis and Alnylam. MVM has received consulting fees from Novartis, Juno, and Kite Pharma. BLL, MCM, MVM, and CHJ are inventors on patents in the field of cell and gene therapy held by the University of Pennsylvania (patents are listed at the end of the supplemental material). WTH, SFL, JJM, SNC, IK, BLL, MCM, and CHJ have received research funding from Novartis. BLL has received personal fees from GE Healthcare and Brammer Bio that are unrelated to the submitted work and is a cofounder and holds equity in Tmunity Therapeutics. PAM has received consulting fees from Kite Pharma. Competing interests of authors from the University of Pennsylvania are managed in accordance with University policies.

Figures

Figure 1. Subject flow diagram.

Figure 2. Clinical response and residual disease characterization in subjects 1 and 5.

Trend in serum monoclonal protein concentration (M-spike) and total serum IgA in subjects 1 and 5 before and after ASCT + CTL019. (A) Subject 1. (B) Subject 5. CTX, 96-hour continuous-infusion cytotoxic chemotherapy; HDM, high-dose melphalan; RT, radiation therapy; DARA, daratumumab. (C and D) Flow cytometry to detect rare multiple myeloma plasma cells in bone marrow of subjects 1 (C) and 5 (D) at time of disease progression. Analysis is shown after gating to exclude CD3+ T cells and CD14+ monocytes. Rare CD38+CD45– plasma cells are gated (left) and shown to have kappa-restricted intracellular immunoglobulin light chain expression (right), consistent with a monoclonal population. (E) CD19 expression on rare multiple myeloma plasma cells (blue) is compared to CD19 on non-neoplastic plasma cells (CD38hiCD45+, green) and non-neoplastic B cells (CD19+CD20+, red) in bone marrow samples from subjects 1 (top) and 5 (bottom).

Figure 3. Correlation of progression-free survival versus peak CTL019 frequency in bone marrow (BM).

Rho and P values were determined using Spearman’s correlations. Numbers refer to subjects 1 and 5.

Figure 4. Anti-Sox2 immune responses after ASCT + CTL019.

(A) Left panel shows anti-Sox2 antibody concentrations before and after CTL019 + ASCT as measured by ELISA in serum diluted 1:400. Subject 6 was excluded from anti-Sox2 antibody analysis due lack of post-ASCT samples. Middle panel shows serum anti-Sox2 antibody concentrations in subject 1 after dilution of serum to 1:800 (red) and 1:1,200 (green). Right panel shows serum anti–tetanus toxoid antibody concentrations before and after CTL019 + ASCT; this analysis was restricted to subjects with samples available from 4 or more time points after ASCT. Data points indicate means, and error bars indicate range, of triplicate experiments after background subtraction. (B) Percentage of T cells proliferating as assessed by CFSE dilution in response to Sox2 peptide mixes 1-2 (left) or 3-4 (right) from subjects’ PBMCs collected before ASCT or at various time points after ASCT. Results are presented for all T cells (top row), CD8+ T cells (middle row), and CD4+ T cells (bottom row), after subtraction of percentages proliferating in unstimulated (vehicle-only) cultures. Subjects 2 and 10 were excluded from this analysis due to insufficient sample. (C) Subject 1 CFSE dilution in PBMCs collected prior to ASCT (top) and at 6 (middle) and 9 (bottom) months after ASCT in response to stimulation with Sox2-derived peptide mixes (mixes 1-2 or mixes 3-4), negative control stimulation with vehicle only, CEF peptides (peptides derived from cytomegalovirus, Epstein-Barr virus, or influenza virus), or positive control stimulation with PHA. (D) Reactivity in subject 1 PBMCs obtained 12 months after ASCT against individual overlapping Sox2 peptides from mix 3.

Figure 5. In vitro colony formation of bone marrow aspirate obtained from multiple myeloma patients not treated on our clinical trial following ex vivo incubation with CAR T cells against CD19 and/or BCMA, CAR T cells against mesothelin (meso CAR T), or nontransduced T cells.

Results are normalized to untreated (no T cells) control and presented for each individual patient (A) and in aggregate by treatment condition (B). Groups were compared using Kruskal-Wallis test. P values are reported from Dunn’s test after Sidak’s correction for multiple comparisons.