CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial

Abstract

Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19^- or CD19^lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial ( NCT03233854 ) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL (n = 17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL (n = 21), 62% of patients responded with 29% CR. Relapses were CD19^-/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22^-/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency.

Fig. 1. IHC demonstrates CD19^−/lo disease postaxi-cel and quantitative flow cytometry of LBCL preaxi-cel therapy is associated with disease progression.

a, Preaxi-cel H-scores did not distinguish long-term responders and those with progression postaxi-cel (P = 0.32 by t-test, P = 1 Fisher’s exact test). Waterfall plot of CD19 IHC H-scores preaxi-cel therapy (n = 44 patients). The H-score was calculated by the percentage of positive tumor cells (0–100) × stain intensity (1–3). The dashed line denotes the H-score of 150, which was used to define antigen positivity. ND, not detectable. b, Paired CD19 H-scores preaxi-cel and at progression show significant differences (P = 0.003 by Wilcoxon signed-rank test). Using an H-score cutoff of 150, and the observed rate of CD19^−/lo progression (10 out of 16 patients), the estimated 95% binomial CI (Wilson score) for CD19^−/lo progression was 38–82%. N/A, no data point. c, Representative patients with IHC demonstrating decreased CD19 expression at the time of progression (A75, relapse H-score = 160; A62 relapse H-score = 120; A30, relapse H-score = 100; A53, relapse H-score = 0) d, Preaxi-cel site density by quantitative flow cytometry in a patient with ongoing response (A116) compared with a patient who experienced progression (A140) e, Preaxi-cel median CD19 site density by quantitative flow cytometry organized from highest (dark blue) to lowest (white) in 15 patients. Patients with lower site density were more likely to experience disease progression after axi-cel (P = 0.03 by Firth logistic regression). Based on the fit model, 3,000 molecules per cell was defined as the cutoff for CD19 positivity. f, Median site density at the time of axi-cel progression (n = 8). Four patients had a site density <3,000 molecules per cell. g, The preaxi-cel H-score did not correlate with antigen site density by quantitative flow cytometry (n = 12) (Spearman r = 0.28, P = 0.38). Source data

Fig. 2. Characterization of CAR products throughout the manufacturing process reveals compositional and phenotypic changes.

a, CD19-22-CD8.BB.z-CAR contained the CD19 FMC63 and CD22 M971 scFvs, CD8α hinge and transmembrane domains, a 4-1BB costimulatory domain and a CD3ζ domain. The unique bispecific structure shows FMC63 heavy chain proximal, followed by M971 light chain, M971 heavy chain and FMC63 light chain distal. b, CAR T manufacturing and clinical trial schema. The manufacturing schema shows the TransAct process change from old to new matrix. The clinical trial schema shows the screening, lymphodepletion, CAR T cell infusion and disease evaluation time points. LP, lumbar puncture. c, Improved culture expansion resulting from the new matrix manufacturing process compared to the old matrix (P < 0.0001, two-tailed t-test). d, Significant reduction in doubling time with the new matrix process compared to the old matrix (P = 0.0411, two-tailed t-test). e, No significant difference in transduction efficiency between old and new matrix (P = not significant (NS), two-tailed t-test). Overall average transduction efficiency was 60.1% (n = 39 individual products). f, Composition of apheresis, CD4/8-enriched and CD19-22.BB.z product over time, looking at T cell (CD3⁺CD56⁻), B cell or leukemic cell (CD20⁺), CD4⁺, CD8⁺, NKT-like (CD3⁺CD56⁺CD16⁺), NK, monocyte and neutrophil subsets. g, Phenotyping of CAR T cell product reveals a skewing toward CD4⁺ cells (n = 39 individual products). h, Comparing the fold increase from apheresis to enrichment to final product reveals the skewing toward CD4⁺ cells that occurred during culture between enrichment and final product (P < 0.0001, two-tailed t-test). i, Phenotyping of T cell memory subsets revealed an enrichment in T_SCM (P < 0.0001, two-tailed t-test) and T_CM (P < 0.0001, two-tailed t-test) cell subsets and a depletion of the T_EMRA (P < 0.0001, two-tailed t-test) subset. There was no significant change in T_N or T_EM cell subsets between enrichment and CD19-22.BB.z product. Source data

Fig. 3. CD19-22.BB.z-CAR is active in both LBCL and B-ALL.

a, Swimmer plot showing the duration of remission and ongoing responses in patients with lymphoma (n = 21). Five patients had an increasing depth of response from 1 to 3 months postinfusion b, PET scans for patient S2 showing partial remission at 1 month postinfusion with subsequent progression 6 months after infusion. c, Lymphoma disease monitoring using ctDNA. After infusion of CD19-22.BB.z for patient SL02, disease burden continued to decrease; this was coincident with prolonged persistence of CD19-22.BBZ. d, Overall survival for 21 infused patients with LBCL. e, PFS for the cohort with lymphoma. f, Swimmer plot for the cohort with B-ALL (n = 17). Two patients received a consolidative allogeneic stem cell transplant (white star) g, PET scans for patient SA8, with large bulk disease preinfusion that improved to a CR 6 months postinfusion. h, Disease monitoring of patient SA8 using cellular-based NGS with sensitivity of 10⁻⁶ demonstrates increasing disease control over time coinciding with improving PET response and ongoing persistence of CD19-22.BB.z. i, Overall survival of 17 infused patients with B-ALL. j, PFS for the cohort with B-ALL. k, Absolute number of circulating CD4 and CD8 CD19-22.BB.z CAR T cells after infusion as measured by flow cytometry (n = 38 autologous infused products). l, Number of circulating CD19-22.BB.z copies per 50 ng of genomic DNA as measured by qPCR (n = 33 autologous infused products) showing initial expansion and persistence of CD19-22.BB.z as measured at 1 and 2 months (days 35–75 postinfusion), 3 months (days 76–120) and 6 months after infusion (days 120–200). Source data

Fig. 4. CD19 negative relapse with preserved CD22 site density after CD19-22.BB.z-CAR and diminished CAR T functionality against CD22.

a, Antigen density of patient S24 demonstrating both CD19 and CD22 expression preCD19-22.BB.z (top) with loss of CD19 and preservation of CD22 at progression (bottom, green arrows). b, CD19 and CD22 assessment by conventional flow cytometry in patients with B-ALL pretreatment and postprogression demonstrates CD19 loss with CD22 preservation. c, In B-ALL, 3 of 4 patients with antigen quantification pre- and postCD19-22-CD.BB.z demonstrated loss of CD19 expression associated with preserved CD22 expression. d, CD19 and CD22 antigen density at progression (n = 11 patients) after CD19-22.BB.z, with patient S24 highlighted in green. Six patients had <1,150 CD19 molecules per cell with a median CD22 of approximately 6,000 molecules per cell. Dashed line denotes the cutoff at 3,000 CD19 molecules per cell. e, Schematic of CD19-22.BB.z bispecific CAR, displaying the loop structure. f, Histogram of CD19 and CD22 expression on NALM6 lines tested in g–j. g, ICS heatmap representing the secretion or expression of CD69, CD107a, TNF-α, IFN-γ and IL-2 from CD19-22.BB.z infusion products (n = 11 individual products) stimulated with the NALM6 tumors lines from f. The heatmap shows greater activation and secretion of cytokines with stimulation with N6-CD19 and N6-CD19/22 lines versus N6-CD22 stimulation. h, Schematic of bispecific CD19-22.BB.z versus monospecific CD22.BB.z. i, ICS stimulation of CD19-22.BB.z (n = 11) versus monospecific CD22.BB.z (n = 5) CAR products against the CD22^high cell line shows increased cytokine secretion of IL-2 and TNF-α through the monospecific CAR22.BB.z (two-tailed t-test). j, Using the single-cell IsoPlexis platform, stimulation of clinical products (n = 7 individual products) with N6-CD19 showed a higher PSI compared to N6-CD22. The CD22 scFV on the bispecific CAR had lower PSI compared to the monospecific CAR22.BB.z (n = 4 individual products) when stimulated with N6-CD22 (Mann–Whitney U-test). Source data

Extended Data Fig. 1. Model-based prediction of relapse after axi-cel.

Estimated risk of relapse for a given pre-treatment CD19 antigen density based on a fitted penalized logistic regression model.

Extended Data Fig. 2. Evaluation of pre-axi-cel antigen density.

CD19 IHC H-score and antigen density by quantitative flow cytometry in 15 patients prior to axi-cel. Spearman correlation for these values shown in 1G.

Extended Data Fig. 3. CONSORT Diagram.

Consort diagram of CD19-22-CD8.BB.z clinical trial.

Extended Data Fig. 4. CD19/22 CAR T manufacturing flowchart and product breakdown by culture days, manufacturing matrix, dose level, and disease.

a, Manufacturing schematic detailing difference in timing of TransAct washout between the Old and New Matrix. b, Table of Infusion Product Characteristics, categorized by the number of culture days. c, Table of Infusion Product Characteristics, categorized by disease cohort. d, Significant difference in product expansion between the old and new matrix at harvest (p < 0.0001, t-test, two tailed). Box plot center line corresponds to the median; hinges correspond to the 25^th and 75^th percentiles with the whiskers signifying minimum and maximum values e, No significant difference in vector copy number based on manufacturing matrix. f,g, Significant decreases in CD39+ (p = 0.012, t-test, two-tailed) and PD1+ (p = 0.0003, t-test, two-tailed) CAR T cells manufactured using the new matrix versus the old matrix. h, Significant increases in CD39+ and PD1+ and a significant decrease in CD57+ expression on CD3+ cells from CD4/8 enriched to final CAR T product (p < 0.0001, t-test, two-tailed; for all 3 tests). No difference in LAG3 expression. Source data

Extended Data Fig. 5. Adverse events summary.

Adverse events with a Grade≥3 incidence rate of 5% or higher were included. CAR specific toxicities were included regardless of incidence.

Extended Data Fig. 6. LBCL disease monitoring by ctDNA.

Patients with baseline tumor sample were assessed for a dominant clone to allow for disease tracking by cell free tumor DNA in the peripheral blood. Measurements were performed at pre-specified timepoints from pre-infusion to the time of disease progression. Source data

Extended Data Fig. 7. B-ALL disease monitoring by ClonoSeq.

Patients with baseline tumor sample were assessed for a dominant clone to allow for disease tracking by cell free tumor DNA in the peripheral blood. Measurements were performed at pre-specified timepoints from pre-infusion to the time of disease progression. Source data

Extended Data Fig. 8. In vivo CAR-T cell expansion is CD8 predominant and relates to exhaustion phenotype of CAR-T products.

a,b Peak CD19-22.BBZ cells as measured by flow cytometry compared by (a) disease type and (b) dose level. No significant difference observed. c,d. CD19-22.BB.z AUC was measured by the trapezoidal method from infusion until Day 60 post infusion.Grades 2-4 (d) CRS (p = 0.04) and (c) neurotoxicity (p = 0.03) associated with higher CD19-22.BBZ AUC. Comparisons by Wilcoxon rank sum. e, Area under the curve calculated by trapezoidal integration of CD4 and CD8 CD19-22.BB.z cells from infusion to 2 months post infusion. The AUC for CD8 was greater than that of CD4 (p = 1.2 × 10⁻⁵) f, Peak expansion of CD4 and CD8 CD19-22.BB.z-CAR T cells measured by flow cytometry. Peak CD8 CD19-22.BB.z-CAR was greater than that of CD4 (p = 6.0 × 10⁻⁶). Comparison in e and f performed by paired Wilcoxon signed rank test. g, Change in the CD4:CD8 ratio from CD19-22.BB.z infusion product to peak expansion, shows the outgrowth of CD4 cells during manufacturing is not seen in the patients at time of peak CAR expansion, where CD8 CAR T+ cells are more abundant. N = 38 patients for a-g. h,i Significantly higher percentage of CD4 CAR T cells in the infusion product express CD39 (p = 5.2 × 10⁻⁸) and PD-1 (p = 1.2 × 10⁻¹⁰) compared to CD8 CAR-T cells (Wilcoxon signed rank) (n = 34 patients). Gating on CD3+ CAR+ cells. Box plot center line corresponds to the median; hinges correspond to the 25^th and 75^th percentiles with the whiskers extending to the smallest or largest value at most 1.5 x IQR from the hinge. All tests were two-sided and not adjusted for multiple comparisons. Source data

Extended Data Fig. 9. Site density change after CD19-22.BB.z.

a, Waterfall plot of assessment of CD19 for patients with LBCL by IHC H-score pre-CD19-22.BB.z and post progression. Cutoff for CD19 positivity was 150. b, Waterfall plot of assessment of CD22 in patients with LBCL by IHC H-score pre-CD19-22-CD8.BB.z and post progression (n = 11 patients for a-b). Cutoff for CD22 positivity was 150. Source data

Extended Data Fig. 10. Functional assessment of CD19-22 scFvs by Incucyte and comparison of MFI, transduction efficiency and VCN of CD19-22.BB.z-CAR and CD22-BB.z.CAR.

a, Incucyte shows tumor killing after co-culture with CD19-22, CD19, CD22, or Mock T cells, is mediated successfully through either the CD19 scFv or CD22 scFv (n = 1 donor, technical triplicates) b, Mean fluorescence intensity measured via a fluorescently tagged CD22 molecule.c, CAR transduction efficiency and vector copy number. For b-c, n = 5 CD22-BB.z products, 11 CD19-22.BB.z. Source data