CD4/CD8 T-Cell Selection Affects Chimeric Antigen Receptor (CAR) T-Cell Potency and Toxicity: Updated Results From a Phase I Anti-CD22 CAR T-Cell Trial

Abstract

Purpose: Patients with B-cell acute lymphoblastic leukemia who experience relapse after or are resistant to CD19-targeted immunotherapies have limited treatment options. Targeting CD22, an alternative B-cell antigen, represents an alternate strategy. We report outcomes on the largest patient cohort treated with CD22 chimeric antigen receptor (CAR) T cells.

Patients and methods: We conducted a single-center, phase I, 3 + 3 dose-escalation trial with a large expansion cohort that tested CD22-targeted CAR T cells for children and young adults with relapsed/refractory CD22+ malignancies. Primary objectives were to assess the safety, toxicity, and feasibility. Secondary objectives included efficacy, CD22 CAR T-cell persistence, and cytokine profiling.

Results: Fifty-eight participants were infused; 51 (87.9%) after prior CD19-targeted therapy. Cytokine release syndrome occurred in 50 participants (86.2%) and was grade 1-2 in 45 (90%). Symptoms of neurotoxicity were minimal and transient. Hemophagocytic lymphohistiocytosis-like manifestations were seen in 19/58 (32.8%) of subjects, prompting utilization of anakinra. CD4/CD8 T-cell selection of the apheresis product improved CAR T-cell manufacturing feasibility as well as heightened inflammatory toxicities, leading to dose de-escalation. The complete remission rate was 70%. The median overall survival was 13.4 months (95% CI, 7.7 to 20.3 months). Among those who achieved a complete response, the median relapse-free survival was 6.0 months (95% CI, 4.1 to 6.5 months). Thirteen participants proceeded to stem-cell transplantation.

Conclusion: In the largest experience of CD22 CAR T-cells to our knowledge, we provide novel information on the impact of manufacturing changes on clinical outcomes and report on unique CD22 CAR T-cell toxicities and toxicity mitigation strategies. The remission induction rate supports further development of CD22 CAR T cells as a therapeutic option in patients resistant to CD19-targeted immunotherapy.

Trial registration: ClinicalTrials.gov NCT02315612.

Figures

FIG 1.

Cytokine and inflammatory markers, chimeric antigen receptor (CAR) expansion, and toxicity profiling. (A) Comparison of peak ferritin across all 5 doses explored. (B) Comparison of peak values of ferritin between those who received CD4/CD8 T-cell selection (CD4/8-TCS) v CD3/CD28-enriched CAR T-cell products. (C and D) Comparison of peak values of interleukin 6 (IL-6) and IL-1B between those who received CD4/8-TCS v CD3/CD28-enriched CAR T-cell products. (E and F) CAR T-cell expansion shown for all patients in the first 30 days, separated by dose level (DL) and as assessed by absolute CAR T cells on the basis of percent absolute lymphocyte count that was CAR T-cell positive at the various time points, DL1-TCS had limited data at earlier time points. (E) Peak CAR T-cell expansion for all participants by DL in the first 30 days as determined by quantitative polymerase chain reaction (PCR). P not significant. (F) DL3 did not have any samples available for PCR analysis.

FIG 2.

Manifestations of hemophagotcytic lymphohistiocytosis (HLH)–like toxicities and use of anakinra in participant 33. (A) Bone marrow (BM) biopsy stained with hematoxylin and eosin (HE; original magnification ×200) that shows normocellular marrow with increased blasts. (B) CD79a and (C) CD10 immunohistochemical stains highlight increased B lymphoblasts. (D) BM biopsy (HE original magnification ×200) shows hypocellular marrow with decreased trilineage hematopoiesis and increased macrophages. (E) CD163 immunohistochemical stain highlights hemophagocytic macrophages. (F) BM aspirate stained with modified Giemsa shows hemophagocytic macrophages. (G) BM biopsy (HE original magnification ×200) shows normocellular marrow with trilineage hematopoiesis with no evidence of leukemia or hemophagocytosis. (H) BM aspirate stained with Giemsa (original magnification ×500) shows progressive trilineage hematopoiesis. (I) Clinical course demonstrates separation in time from onset of cytokine release syndrome (CRS) to HLH/macrophage activation syndrome (MAS)–like manifestations. CAR, chimeric antigen receptor.

FIG 3.

Response and outcomes after CD22 chimeric antigen receptor (CAR) T-cell infusion. (A) Waterfall plot of best response after CD22 CAR T cell. Participants were stratified by dose level (DL) and cytokine release syndrome (CRS) grade. Note that participants 26 and 46 represent the same patient. The participant was initially treated at DL2, achieved an MRD-negative complete remission (CR) and proceeded to hematopoietic stem-cell transplantation (HSCT), but subsequently experienced relapsed approximately 1 year post-HSCT. After relapse, the participant was re-enrolled as a new participant and had a new apheresis and new product manufactured and was treated at DL1 T-cell selection (TCS), achieved an MRD-negative CR, and proceeded to a second HSCT. Participants 5 and 35 similarly also represent the same patient. Participant 5 was treated at DL1 and was a nonresponder. The participant subsequently underwent allogeneic HSCT with a new donor after additional alternative therapy, with subsequent relapse. This participant underwent a new apheresis and had a new product manufactured at DL2 and achieved a CR. (B) CD22 site density stratified by those who attained MRD-negative CR v those who did not. (C) CD22 site density compared pretreatment with the CD22 site density in those with residual disease or at the time of relapse. (D) Duration in continuous remission among those who achieved CR. Shown are the duration of remission and time of relapse stratified by antigen negative/dim relapse or antigen-positive relapse. Two participants were re-enrolled as new participants as clarified in (A). (E) Relapse-free survival (RFS) from time of infusion for those who achieved remission. (F) Overall survival (OS) stratified by those who proceeded to HSCT (red line) v those who did not (blue line), using a landmark analysis of 126 days after CAR infusion. P = .045. (*) Participant 26 experienced relapsed with antigen-positive disease and was re-enrolled as a new participant with a new product infused and went directly to a second HSCT. (^) Participant 37 received a second infusion for antigen-positive relapse and remains in an ongoing remission at approximately 9 months postinfusion (data not shown). TRM, treatment-related mortality; subject 54 died from complications of transplant; subject 16 died from sepsis following CD22 CAR T cells and did not proceed to transplant.

FIG 3.

Response and outcomes after CD22 chimeric antigen receptor (CAR) T-cell infusion. (A) Waterfall plot of best response after CD22 CAR T cell. Participants were stratified by dose level (DL) and cytokine release syndrome (CRS) grade. Note that participants 26 and 46 represent the same patient. The participant was initially treated at DL2, achieved an MRD-negative complete remission (CR) and proceeded to hematopoietic stem-cell transplantation (HSCT), but subsequently experienced relapsed approximately 1 year post-HSCT. After relapse, the participant was re-enrolled as a new participant and had a new apheresis and new product manufactured and was treated at DL1 T-cell selection (TCS), achieved an MRD-negative CR, and proceeded to a second HSCT. Participants 5 and 35 similarly also represent the same patient. Participant 5 was treated at DL1 and was a nonresponder. The participant subsequently underwent allogeneic HSCT with a new donor after additional alternative therapy, with subsequent relapse. This participant underwent a new apheresis and had a new product manufactured at DL2 and achieved a CR. (B) CD22 site density stratified by those who attained MRD-negative CR v those who did not. (C) CD22 site density compared pretreatment with the CD22 site density in those with residual disease or at the time of relapse. (D) Duration in continuous remission among those who achieved CR. Shown are the duration of remission and time of relapse stratified by antigen negative/dim relapse or antigen-positive relapse. Two participants were re-enrolled as new participants as clarified in (A). (E) Relapse-free survival (RFS) from time of infusion for those who achieved remission. (F) Overall survival (OS) stratified by those who proceeded to HSCT (red line) v those who did not (blue line), using a landmark analysis of 126 days after CAR infusion. P = .045. (*) Participant 26 experienced relapsed with antigen-positive disease and was re-enrolled as a new participant with a new product infused and went directly to a second HSCT. (^) Participant 37 received a second infusion for antigen-positive relapse and remains in an ongoing remission at approximately 9 months postinfusion (data not shown). TRM, treatment-related mortality; subject 54 died from complications of transplant; subject 16 died from sepsis following CD22 CAR T cells and did not proceed to transplant.

FIG 4.

Impact of CD4/CD8 T-cell selection (TCS) on the starting apheresis product. (A) Shown are three examples of products manufactured using the CD4/CD8 selection method. Flow cytometry plots show that participants 32 and 40 had elevated frequencies of CD19/CD22 B cells in their starting apheresis. Participant 43 had elevated frequencies of CD14 monocytes and CD56 natural killer (NK) cells, which precluded generation of a CD19 chimeric antigen receptor (CAR) T-cell product elsewhere. Upon selection, all these products showed high T-cell purities (> 85%). In participants 32 and 40, ≥ 45% of the cells in the starting apheresis product were CD22-expressing B cells, and approximately 35% of the cells were T cells. The final product displayed high transduction efficiencies as measured by protein L, and all showed high fold expansion (FE). (B) Shown is a direct comparison between two different manufacturing methods, CD3/CD28 enrichment v CD4/CD8 selection. A single apheresis product was cryopreserved into multiple aliquots and used to start the manufacturing processes. Participant 25 exhibited very high frequencies of monocytes and NK cells in the apheresis material. Upon CD3/CD28 enrichment, the final product did not transduce or expand (upper right). CD4/CD8 selection, however, showed that the product could be recovered to a high level of T-cell purity postselection (bottom left) and exhibited both high transduction efficiencies and FE of the final product (bottom right). FE was calculated by dividing the final total cell number by the starting cell number on the day of transduction (day 2). (C-G) Comparison of the CD22 CAR T-cell product across manufacturing strategies. (C) Transduction efficiency and (D) FE were assessed in patient samples that had undergone manufacturing using elutriation (n = 6) or CD3/CD28 enrichment (n = 19) and compared with CD4/CD8 selection (n = 26). The CD3 percentage was evaluated in the (E) postapheresis starting material and in the post-CD3/CD28 enrichment or (F) CD4/CD8 TCS product. (G) CD3 T-cell recovery was calculated for available samples postenrichment or postselection.

FIG 4.

Impact of CD4/CD8 T-cell selection (TCS) on the starting apheresis product. (A) Shown are three examples of products manufactured using the CD4/CD8 selection method. Flow cytometry plots show that participants 32 and 40 had elevated frequencies of CD19/CD22 B cells in their starting apheresis. Participant 43 had elevated frequencies of CD14 monocytes and CD56 natural killer (NK) cells, which precluded generation of a CD19 chimeric antigen receptor (CAR) T-cell product elsewhere. Upon selection, all these products showed high T-cell purities (> 85%). In participants 32 and 40, ≥ 45% of the cells in the starting apheresis product were CD22-expressing B cells, and approximately 35% of the cells were T cells. The final product displayed high transduction efficiencies as measured by protein L, and all showed high fold expansion (FE). (B) Shown is a direct comparison between two different manufacturing methods, CD3/CD28 enrichment v CD4/CD8 selection. A single apheresis product was cryopreserved into multiple aliquots and used to start the manufacturing processes. Participant 25 exhibited very high frequencies of monocytes and NK cells in the apheresis material. Upon CD3/CD28 enrichment, the final product did not transduce or expand (upper right). CD4/CD8 selection, however, showed that the product could be recovered to a high level of T-cell purity postselection (bottom left) and exhibited both high transduction efficiencies and FE of the final product (bottom right). FE was calculated by dividing the final total cell number by the starting cell number on the day of transduction (day 2). (C-G) Comparison of the CD22 CAR T-cell product across manufacturing strategies. (C) Transduction efficiency and (D) FE were assessed in patient samples that had undergone manufacturing using elutriation (n = 6) or CD3/CD28 enrichment (n = 19) and compared with CD4/CD8 selection (n = 26). The CD3 percentage was evaluated in the (E) postapheresis starting material and in the post-CD3/CD28 enrichment or (F) CD4/CD8 TCS product. (G) CD3 T-cell recovery was calculated for available samples postenrichment or postselection.