A novel and efficient CD22 CAR-T therapy induced a robust antitumor effect in relapsed/refractory leukemia patients when combined with CD19 CAR-T treatment as a sequential therapy

Abstract

Background: CD19 chimeric antigen receptor (CAR) therapy has achieved impressive success in relapsed or refractory (R/R) B-cell malignancies, but relapse due to antigen escape is increasingly appearing reported. As the expression profile of CD22 is similar to that of CD19, CD22 has become a candidate target when CD19 CAR-T therapy fails.

Methods: A novel CD22 CAR incorporating scFv derived from an HIB22 hybridoma which bound the first and second Ig-like extracellular domains of CD22 antigen was constructed. Preclinical investigation of the CD22 CAR-T therapy against B-cell malignancies was evaluated by coculturing CD22 CAR-T cells with tumor cell lines or primary blasts from patients in vitro and using a xenograft mouse model in vivo. Further clinical study of CD22/CD19 CAR-T sequential therapy was conducted in 4 R/R adult B-cell acute lymphoblastic leukemia (B-ALL) patients.

Results: The novel CD22 CAR-T treatment had specific cytotoxicity to CD22 + target cells, and the survival time of mice in the CD22 CAR-T treatment group was significantly prolonged. Furthermore, it's validated that sequential CD22/CD19 CAR-T therapy was significantly superior than single CD19 or CD22 CAR-T treatment in a relapse xenograft model. All 4 patients achieved complete remission (CR) with negative minimal residual disease (MRD), including 3 patients who had received prior CD19-related immunotherapy. The proliferation of CD19 and CD22 CAR-T cells was observed respectively in vivo, and 3 of the 4 patients experienced cytokine release syndrome (CRS); 2 of these patients had grade 1 CRS and 1 had grade 3 CRS. Long term follow-up showed that 3 of the 4 (75%) patients had sustained CR for up to 1 year. Analysis of antigen expression in the relapsed patients demonstrated that loss or diminution of CD19 and CD22 expression might cause antigen escape from CAR-T surveillance.

Conclusions: In summary, the novel CD22 CAR-T therapy was validated with antitumor effects both in vitro and in vivo. Furthermore, our study demonstrated the safety and robust efficacy of sequential CD22/CD19 CAR-T therapy in xenograft models and clinical trials, especially as the salvage treatment for R/R B-ALL patients with antigen loss or in whom anti-CD19 related immunotherapy failure failed.

Trial registration: Chinese Clinical Trial Registry (ChiCTR): ChiCTR1900025419, Supplementarily registered 26 August, 2019.

Keywords: Antigen escape; B-ALL; CAR-T; CD19; CD22; HIB22; Sequential therapy.

Fig. 1

Construction and structural characteristics of a novel CD22 CAR derived from HIB22. a Homology models of hCD22 (ECD, domains 1–3). b Homology models of HIB22 scFv. c Docking mode of HIB22 scFv with CD22(ECD, domains 1–3). Blue, hCD22 ECD epitopes; Red, HIB22 epitopes. d Sequence of hCD22 (ECD, domains 1–3). Gray background, amino acids as epitopes predicted involved in binding with hCD22 (ECD, domains 1–3). e Partial interaction mode between HIB22 scFv and hCD22 (ECD, domains 1–3). Blue, hCD22 (ECD, domains 1–3) epitopes and presentative amino acids involved in non-bond interaction. Red, HIB22 scFv epitopes and presentative amino acids involved in non-bond interaction. f Schematic of CD22 CAR construct. CD8α TM, CD8α transmembrane domain. CD3ζ, CD3 zeta domain. g Expression of CAR on T cells surface. The CD22 CARs or empty vectors were transduced to T cells to obtain corresponding CD22 CAR-T cells or control VEC-T cells. CAR expression was analyzed by FACS. h, i Collective analysis of expression of CAR h and copies number of CAR transduced to genomic DNA i from 3 donors. j SFI (specific fluorescence index) of CD22 expression on B-ALL patient samples. The SFI is calculated as follows: SFI = (MFI of specific antibody—MFI of isotype control)/MFI of isotype control, where MFI is the mean fluorescence intensity

Fig. 2

In vitro evaluation of CD22 CAR T cells. a A representative diagram of flow cytometry analysis of residual Daudi, Namalwa, Nalm-6 and K562 cells when cocultured with CAR-T cells at 0 h (upper panel) and 48 h (lower panel). b Percentage of residual Namalwa or K562 cells detected by flow cytometry after co-culturing with CAR T or VEC-T cells for 24 h at indicated E:T ratio (1:8, 1:4,1:1,4:1,8:1). c. Percentage of degranulated CAR-T cells (CD107a positive T cells/CAR positive T cells) after cocultured with Daudi, Namalwa, Nalm-6 and K562 for 24 h. d ELISA detection of IFN-γ and TNF-α in the supernatants of VEC-T or CAR-T cocultured with Daudi, Namalwa and K562 for 24 h. e Primary ALL cells were collected from 5 patients, and cocultured with CD22 CAR-T cells or vector-T cells at E:T ratio of 1:1 respectively for 48 h. The residual tumor ALL cells was defined as 7AAD⁻CD3⁻GFP⁻CD19⁺CD10⁺ by flow cytometry. f Percentage of degranulated CAR-T cells (CD107a positive T cells/CAR positive T cells) after cocultured with patient samples for 5 h

Fig. 3

In vivo validation of CD22 CAR-T and CD22/CD19 sequential CAR-T cells in wild-type Namalwa inoculated mice model. a Schematic of in vivo evaluation of CAR-T. NOD/SCID mice were challenged with 2 × 10⁶ wild type Namalwa cells on day 0, Mice in the sequential treatment group received 6 × 10⁶ CD22 CAR-T(infection efficiency 50%–60%) cells on day 4 and 6 × 10⁶ CD19 CAR-T (infection efficiency 50%–60%) cells on day 5. Mice in the CD19 CAR-T group received 6 × 10⁶ CD19 CAR-T cells on day 4 and and day 5, respectively. Mice in the CD22 CAR-T group received 6 × 10⁶ CD22 CAR-Tcells on day 4 and day 5, respectively. b. IVIS imaging of disease burden monitored by BLI at the indicated time points. c. Average radiance quantification (p/sec/cm²/sr) for Namalwa at the indicated time points. d T cell persistence in peripheral blood on day 20. (n = 5 per column) e Average body weight of two groups after CAR-T cell treatment. f Kaplan–Meier survival curves of VEC-T and CAR-T treatment groups. The P-values were determined by log-rank test. P < 0.001 when group Vector-T compared with CAR-T. N = 5–6 for VEC-T or CAR-T group

Fig. 4

In vivo validation of CD22 CAR-T and CD22/CD19 sequential CAR-T cells in Namalwa-CD19KO and CD22KO inoculated mice model. a Schematic of in vivo evaluation of CAR-T. NOD/SCID mice were challenged with a mixture of 1 × 10⁶ Namalwa-CD19KO, and 1 × 10⁶ Namalwa-CD22KO lines on day 0, Mice in the sequential treatment group received 6 × 10⁶ CD22 CAR-T(infection efficiency 50%–60%) cells on day 4 and 6 × 10⁶ CD19 CAR-T (infection efficiency 50%–60%) cells on day 5. Mice in the CD19 CAR-T group received 6 × 10⁶ CD19 CAR-T cells on day 4 and and day 5, respectively. Mice in the CD22 CAR-T group received 6 × 10⁶ CD22 CAR-Tcells on day 4 and day 5, respectively. b IVIS imaging of disease burden monitored by BLI at the indicated time points. c Average radiance quantification (p/sec/cm²/sr) for Namalwa at the indicated time points. d T cell persistence in peripheral blood of mice on day 20. (n = 5 per column) e Average body weight of two groups after CAR-T cell treatment. f Kaplan–Meier survival curves of VEC-T and CAR-T treatment groups. The P-values were determined by log-rank test. P < 0.001 when group Vector-T compared with CAR-T. N = 6 for VEC-T or CAR-T group

Fig. 5

The robust expansion of CAR-T cells and antileukemic activity of sequential CD22/CD19 CAR-T therapy. a Response and long term follow-up of each patient who received CD22/CD19 CAR-T sequential therapy. b Percentage of circulating CAR-T (Fab positive T) cells in total T cells analyzed by flow cytometry. c, d Copy numbers of CD22 CAR C and CD19 CAR D which integrated in genomic DNA per μg PBMC genomic DNA in each patient at different time points

Fig. 6

Safety profile of CD22/CD19 CAR-T therapy. a Temperature change and occurrence of fever of each patient after CD22/CD19 CAR-T cells infusion. b, c C-reactive protein (b) and ferritin (c) in circulating peripheral blood of each patient at different time points. d–g Change of cytokine levels, including IFN-γ, IL-2R, TNF-α, IL-6, and IL-10 in 4 patients after received sequential CAR-T therapy. Fold change of cytokines was calculated via the formula: Fold change of cytokines = cytokine levels at different times/cytokine levels on day -2 or day 0 before CAR-T cells infusion

Fig. 7

Analysis of CD19 and CD22 cell-surface expression in three patients at pretreatment and relapsed stage. BMMCs from patients before CAR-T cells infusion and relapsed after immunotherapy were obtained, stained with CD45, CD34, CD10, CD33, CD19 and CD22 and analyzed by flow cytometry. Leukemia blasts were gated via CD45/SSC two parameters graph and subgated in CD34 or CD10, followed by analysis of surface expression of CD19 and CD22 on gated cells. Red dots represent leukemia blasts. Black dots represent normal cells. a, b and e. Flow cytometry analysis of BMMCs samples from 3 patients at pretreatment and relapse stage, including patient 4 (a), patient 3 (b) and patient 2 (e). c. CD19 expression in patient 3 by histogram analysis at pretreatment and relapse stage indicated loss of CD19 antigen during treatment. d Histogram analysis of CD22 on BMMCs samples from patient 3 before CAR-T cells infusion and relapse stage showed CD22 downregulation