Bispecific CAR-T cells targeting both CD19 and CD22 for therapy of adults with relapsed or refractory B cell acute lymphoblastic leukemia

Abstract

Background: Despite the impressive complete remission (CR) induced by CD19 CAR-T cell therapy in B-ALL, the high rate of complete responses is sometimes limited by the emergence of CD19-negative leukemia. Bispecific CAR-modified T cells targeting both CD19 and CD22 may overcome the limitation of CD19-negative relapse.

Methods: We here report the design of a bispecific CAR simultaneous targeting of CD19 and CD22. We performed a phase 1 trial of bispecific CAR T cell therapy in patients with relapsed/refractory precursor B-ALL at a dose that ranged from 1.7 × 106 to 3 × 106 CAR T cells per kilogram of body weight.

Results: We demonstrate bispecific CD19/CD22 CAR T cells could trigger robust cytolytic activity against target cells. MRD-negative CR was achieved in 6 out of 6 enrolled patients. Autologous CD19/CD22 CAR T cells proliferated in vivo and were detected in the blood, bone marrow, and cerebrospinal fluid. No neurotoxicity occurred in any of the 6 patients treated. Of note, one patient had a relapse with blast cells that no longer expressed CD19 and exhibited diminished CD22 site density approximately 5 months after treatment.

Conclusion: In brief, autologous CD19/CD22 CAR T cell therapy is feasible and safe and mediates potent anti-leukemic activity in patients with relapsed/refractory B-ALL. Furthermore, the emergence of target antigen loss and expression downregulation highlights the critical need to anticipate antigen escape. Our study demonstrates the reliability of bispecific CD19/CD22 CAR T cell therapy in inducing remission in adult patients with relapsed/refractory B-ALL.

Trial registration: ClinicalTrials.gov identifier: NCT03185494.

Keywords: B-ALL; Bispecific; CAR; CD19; CD22; Immunotherapy; Tumor antigen escape.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Preclinical evaluation of bispecific CD19/CD22 CAR T cells. a Schematic of the bispecific CD19/CD22 CAR structure. b Cytotoxicity of CAR T cells against target cells carrying luciferase reporter gene evaluated by luminescent assay, after co-culturing with tumor cells for 4 h at the indicated E:T ratios, with CAR T cells. c Cytokine production by anti-CD19 CAR-, anti-CD22 CAR-, and bispecific CAR-expressing T cells co-incubated with K562, K562-CD19, K562-CD22, and K562-CD19CD22 cell lines. Bars represent mean + SD of replicate samples. Data are representative of three independent experiments performed with CAR T cells from three separate donors. A two-tailed, unpaired two-sample t test was used for statistical analysis

Fig. 2

Expansion and persistence of bispecific CAR T cells and tumor response in patients treated with bispecific CAR T cells. a Treatment response of each patient after bispecific CAR T cell treatment and the duration of response. Ongoing remission is marked by a black arrow. Patient number is shown to the left. b The presence of CD19/CD22 CAR T cells in the peripheral blood as assessed by quantitative real-time polymerase chain reaction (PCR) assay. Genomic DNA was isolated from samples of whole blood samples collected at serial time points before and after cell infusion. of the y axis.. c The results of flow cytometry analysis showing in vivo expansion of bispecific CAR T cells in the peripheral blood and bone marrow of representative patient 5, who achieved a MRD negative. Both the x and y axes are log10 scales. MRD, minimal residual disease

Fig. 3

Serum cytokines, inflammatory markers, and B cell markers before and after bispecific CD19/CD22 CAR T cell therapy. a Serum levels of cytokines and inflammatory markers measured at the indicated time points after cell infusion. Analytes with a fold change greater than or equal to three are indicated and plotted as relative changes from the baseline (determined on day 0 before infusion). Increases in the levels of cytokines, including interleukin-1β, GM-CSF, interleukin-2, interleukin-6, and interleukin-10, tumor necrosis factor α, and interferon gamma, were observed in the three patients. b Serum ferritin and C-reactive protein concentrations. c Circulating blasts and low levels of nonmalignant B cells in patient 5 peripheral blood at day 1, followed by CAR T cell expansion coincident with the clearance of leukemia and nonmalignant B cells by day 20; the CD19/CD22 CAR T cells disappeared and B cell recovery occurred by day 100. Green dots show leukemic blasts (CD19+CD34+); red dots represent normal B cells

Fig. 4

Persistent fevers in patients with acute lymphoblastic leukemia after infusion with bispecific CAR T cells. Changes in body temperature after cell infusion, including the maximum temperature per 24-h period in the six patients. The green line marks the minimum temperature for a fever (38 °C)

Fig. 5

Changes in CD19 and CD22 cell surface expression in relapsed patient baseline and at the time of relapse. Bone marrow samples were obtained from patients 1 and 2 before cell infusion and at the time of relapse. Mononuclear cells isolated from the bone marrow samples were stained for CD45, CD34, CD19, and CD22. After gating on live cells, the blast gate (CD45+side scatter [SSC] low) was subgated on CD34+ cells, and histograms were generated for CD19 and CD22 expression. a CD19 and CD22 expression of B-ALL tumor cells in patient 1 from the initial diagnostic BM sample and the relapsed sample. b CD19 and CD22 expression of B-ALL tumor cells in patient 4 from the initial diagnostic BM sample and the relapsed sample. c Non-transduced (NT) T cells from the leukapheresis product or CAR T cells from the end-of-product formulated cells were incubated with the relapsed B-ALL tumor cells from patients 1, 2, and 4. Effectors were incubated with tumor cells at a 50:1 effector/target ratio for 4 h. Supernatants were harvested and analyzed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). d Changes in the CD19 cell surface expression in patient 2 between the baseline time point and the time of relapse. e Direct Sanger sequencing performed with patient 2 cDNA. Mutations in exon 2 of CD19 were found in the relapse samples from patient 2 and were predicted to result in a truncated protein. f Emergence of leukemia cell populations with CD22 expression different from that of the cells harvested before treatment. g The change in CD22 cell surface expression in patients 1, 2, and 4