T Cells Genetically Modified to Express an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor Cause Remissions of Poor-Prognosis Relapsed Multiple Myeloma

Abstract

Purpose Therapies with novel mechanisms of action are needed for multiple myeloma (MM). T cells can be genetically modified to express chimeric antigen receptors (CARs), which are artificial proteins that target T cells to antigens. B-cell maturation antigen (BCMA) is expressed by normal and malignant plasma cells but not normal essential cells. We conducted the first-in-humans clinical trial, to our knowledge, of T cells expressing a CAR targeting BCMA (CAR-BCMA). Patients and Methods Sixteen patients received 9 × 106 CAR-BCMA T cells/kg at the highest dose level of the trial; we are reporting results of these 16 patients. The patients had a median of 9.5 prior lines of MM therapy. Sixty-three percent of patients had MM refractory to the last treatment regimen before protocol enrollment. T cells were transduced with a γ-retroviral vector encoding CAR-BCMA. Patients received CAR-BCMA T cells after a conditioning chemotherapy regimen of cyclophosphamide and fludarabine. Results The overall response rate was 81%, with 63% very good partial response or complete response. Median event-free survival was 31 weeks. Responses included eradication of extensive bone marrow myeloma and resolution of soft-tissue plasmacytomas. All 11 patients who obtained an anti-MM response of partial response or better and had MM evaluable for minimal residual disease obtained bone marrow minimal residual disease-negative status. High peak blood CAR+ cell levels were associated with anti-MM responses. Cytokine-release syndrome toxicities were severe in some cases but were reversible. Blood CAR-BCMA T cells were predominantly highly differentiated CD8+ T cells 6 to 9 days after infusion. BCMA antigen loss from MM was observed. Conclusion CAR-BCMA T cells had substantial activity against heavily treated relapsed/refractory MM. Our results should encourage additional development of CAR T-cell therapies for MM.

Trial registration: ClinicalTrials.gov NCT02215967.

Figures

Fig 1.

Chimeric antigen receptor (CAR)–B-cell maturation antigen (BCMA) T cells have antimyeloma activity. (A) Waterfall plot with each bar representing the maximum percentage change of the myeloma marker for each patient. We compared pretreatment and nadir post-treatment values of involved free κ or λ light chains or the intact monoclonal antibody (M protein). The intact immunoglobulin M protein was used if it was measurable. If the M protein was not measurable, free light chains were used. (B) Event-free survival in weeks for patients treated with 9 × 106 CAR+ T cells/kg. Median event-free survival was 31 weeks. Seven patients were censored. One patient was censored because he refused to come to appointments starting 5 weeks after CAR-BCMA infusion. The other six patients were censored for being in an ongoing response at last follow-up. Only five censor marks are present because two patients each were censored at 17 and 26 weeks after CAR-BCMA infusion, and the censor marks are too close to be distinguished. Events were all progressive multiple myeloma except for patient 13, in whom the event was initiation of new multiple myeloma therapy without clear progression of myeloma. (C) Before CAR-BCMA T-cell infusion, patient 14 had a large left abdominal soft tissue plasmacytoma, as seen on computed tomography imaging. (D) This abdominal mass significantly decreased in size 4 weeks after CAR-BCMA T-cell infusion. (E) The mass continued to decrease in size 9 weeks after CAR-BCMA T-cell infusion. (F) The mass was not appreciable on computed tomography 55 weeks after CAR-BCMA T-cell infusion. (G) Patient 14’s λ light chains decreased quickly after CAR-BCMA T-cell infusion. Twenty-five weeks after CAR-BCMA T-cell infusion, λ light chains began to increase, but the ratio of λ to κ light chains remained normal, indicating recovery of normal plasma cells. (H) Patient 14 received intravenous immunoglobulin infusions for hypogammaglobulinemia after CAR-BCMA T-cell infusion, as indicated by the gold arrows. His IgG, IgA, and IgM subsequently all increased. He has not received intravenous immunoglobulins since 13 weeks after CAR-BCMA T-cell infusion. (I) Plasma cells were evaluated in bone marrow core biopsies by CD138 immunohistochemistry (IHC) staining before CAR-BCMA treatment and 2 months after CAR-BCMA T-cell infusion. The percentage of plasma cells decreased in all nine evaluable patients. P = .0039 by Wilcoxon matched-pairs signed rank test. Only nine of 16 patients were evaluable for plasma cell eradication by IHC, because three patients had levels of bone marrow myeloma too low to evaluate by IHC pretreatment, three patients did not have a post-treatment bone marrow biopsy because of lack of clinical response, and one patient did not have a post-treatment bone marrow biopsy because of patient noncompliance.

Fig 2.

Chimeric antigen receptor (CAR)–B-cell maturation antigen (BCMA) T cells eradicated bone marrow myeloma, and serum BCMA declined with myeloma elimination. (A) Patient 1’s bone marrow biopsy before infusion of CAR-BCMA T cells demonstrated 20% to 25% involvement with multiple myeloma by CD138 staining. The multiple myeloma cells expressed BCMA. (B) Eleven weeks after CAR-BCMA T-cell infusion, Patient 1’s bone marrow biopsy showed no evidence of multiple myeloma by CD138 or BCMA staining. (C) Patient 1’s κ light chains decreased quickly after CAR-BCMA T-cell infusion and remained undetectable 38 weeks after CAR-BCMA T-cell infusion. (D) BCMA can be detected in patient serum. Patients with responses of partial response, very good partial response, and stringent complete response had substantially decreased levels of serum BCMA after CAR-BCMA T-cell infusion. P < .001 for the comparison of before treatment and after treatment. (E) Serum BCMA levels remained stable or only slightly decreased in patients with responses of stable disease or progressive disease after CAR-BCMA T-cell infusion (P = .75, not significant). For D and E, before treatment samples were collected just before the start of conditioning chemotherapy. After treatment samples were collected 26 to 39 days after CAR T-cell infusion, except for patient 20, whose sample was collected 15 days after CAR T-cell infusion, and patient 23, whose sample was collected 14 days after CAR T-cell infusion. Statistical comparison was by the Wilcoxon matched-pairs signed rank test. (F) Patient 21 had a myeloma response of very good partial response. Thirteen weeks after CAR-BCMA infusion, the patient had myeloma progression with increased κ light chains and progression of bone lesions. Simultaneously, serum BCMA increased. (G) Compared with patients experiencing less than grade 3 cytokine-release syndrome (CRS), patients experiencing grade 3 or 4 CRS had a significantly higher percentage of bone marrow plasma cells before CAR-BCMA T-cell infusion. P = .04 by Mann-Whitney test. H and E, hematoxylin and eosin.

Fig 3.

Many cytokines were prominently increased after anti-BCMA CAR (CAR-BCMA) T cell infusions, and peak CAR cell levels were associated with anti-myeloma responses. (A) Serum levels of 19 cytokines were determined at multiple time points after CAR-BCMA infusion for all patients. For each cytokine, the peak fold-increase over pretreatment cytokine levels was calculated. The peak fold-increase is shown as a blue dot, and the median peak fold-increase for each cytokine is shown as a gold bar. (B-D) The peak absolute serum levels of each of 19 cytokines were compared between patients with a maximum CRS grade of 3 or 4 at any time point versus patients with a maximum CRS grade less than 3 at all time points. The three cytokines with the lowest P value for the comparison were (B) IL-15, (C) IL-10, and (D) IL-8. For these comparisons, all 16 patients were included. Comparison of peak cytokine levels was made by the Mann-Whitney test, and correction for multiple comparisons was performed with the Bonferroni-Dunn test. (E and F) The percentage of peripheral blood mononuclear cell that were CAR+ cells was determined by quantitative polymerase chain reaction, and the absolute number of CAR+ cells/μL of blood was determined by multiplying the percentage of CAR+ cells by the sum of blood lymphocytes plus monocytes/μL. The time courses of CAR+ cell levels in the blood of each patient obtaining responses of stringent complete response, very good partial response, or partial response (responders) were determined. The patients are divided among two graphs to allow viewing of the lines for each patient. Day 0 is the day of CAR-BCMA infusion. (G) The time courses of CAR+ cell levels in the blood of the three patients with treatment outcomes of stable disease or progressive disease (nonresponders) were determined by quantitative polymerase chain reaction. The y-axis scale is the same as in (A) to emphasize the lower level of blood CAR+ cells in nonresponders. (H) Peak blood CAR+ cell levels were higher in responders versus nonresponders. (P = .013, Mann-Whitney test; n = 16). GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; TNF, tumor necrosis factor.

Fig 4.

After infusion, CD8+ highly differentiated T cells dominate the chimeric antigen receptor (CAR)–B-cell maturation antigen (BCMA) T-cell population, and multiple myeloma (MM) cells can lose BCMA expression after anti-BCMA CAR T cells (CAR-BCMA) T-cell infusions. (A) The ratio of CD4+ to CD8+ CAR-BCMA T cells decreased after infusion (P < .001). For A, data were from CD4 versus CD8 flow cytometry plots gated on CD3+CAR+ lymphocytes. For A through F, infusion means a cell sample obtained just before infusion, and ex vivo means cells from a patient blood sample obtained 6 to 9 days after infusion. For A through F, all comparisons were made by the Wilcoxon matched-pairs signed rank test (n = 14). Patients 4 and 20 are not included, because their postinfusion blood CAR+ cell levels were too low for accurate flow cytometry analysis. (B) The fraction of CAR+ T cells expressing KLRG-1 increased significantly on CAR-BCMA–expressing T cells after infusion. P < .001 for the comparison of CD4+ infusion versus ex vivo CAR-BCMA T cells. P < .001 for the comparison of CD8+ infusion versus ex vivo CAR-BCMA T cells. (C) The fraction of CAR+ T cells expressing CD57 increased significantly on CAR-BCMA–expressing T cells after infusion. P = .004 for the comparison of CD4+ infusion versus ex vivo CAR-BCMA T cells. P < .001 for the comparison of CD8+ infusion versus ex vivo CAR-BCMA T cells. (D) The fraction of CAR+ T cells expressing PD1 increased significantly on CAR-BCMA–expressing T cells after infusion. P < .001 for the comparison of CD4+ infusion versus ex vivo CAR-BCMA T cells. P < .001 for the comparison of CD8+ infusion versus ex vivo CAR-BCMA T cells. For B through D, data are from plots gated on either CD4+CD3+ lymphocytes or CD8+CD3+ lymphocytes. (E and F) After infusion, the fraction of CAR+ T cells that had a phenotype consistent with either naïve or central memory (CM) T cells decreased, and the fraction of CAR+ T cells with a phenotype consistent with either effector memory (EM) T cells or effector memory RA (TEMRA) T cells increased. Naïve was defined as C-C chemokine receptor 7 (CCR7)+ CD45RA+. CM was defined as CCR7+ CD45RA-negative. EM was defined as CCR7-negative CD45RA-negative. TEMRA was defined as CCR7-negative CD45RA+. These markers were evaluated on CD3+ lymphocytes that expressed CAR-BCMA and either CD4 (E) or CD8 (F). For CD4+ T cells, there were statistically significant differences between infusion and ex vivo Naïve + CM (P < .001) and infusion and ex vivo EM + TEMRA (P < .001). For CD8+ T cells, there were statistically significant differences between infusion and ex vivo Naïve + CM (P < .001) and infusion and ex vivo EM + TEMRA (P < .001). (G) Surface expression of BCMA was measured by flow cytometry before CAR-BCMA therapy. Wide variability in BCMA expression between patients was observed. There was a nonstatistically significant trend (NS by Mann-Whitney test) toward higher BCMA T-cell expression, as measured by antibody binding capacity assay, among patients obtaining responses of stringent complete response, very good partial response, or partial response (responders) compared with patients with outcomes of stable disease or progressive disease (nonresponders). (H) Patient 11 had an MM response of VGPR. For all plots, red indicates MM cells, green indicates normal plasma cells, and purple indicates B cells. Before CAR-BCMA T-cell infusion, the MM population expressed high levels of BCMA. Fifty-six weeks after CAR-BCMA T-cell infusion, the small number of MM cells that were present lacked BCMA expression. Sixty-eight weeks after CAR-BCMA T-cell infusion, the MM cells displayed mixed BCMA expression, with some cells negative for BCMA expression. Multiple myeloma cells were defined as CD138+ CD38+ CD19-negative CD81-negative cells. Normal plasma cells were defined as CD138+ CD38+ CD19+ CD81+ cells. B cells were defined as CD138-negative CD19+ CD20+ cells.