Chimeric antigen receptor T cells self-neutralizing IL6 storm in patients with hematologic malignancy

Abstract

IL6 is one of the most elevated cytokines during chimeric antigen receptor (CAR) T cell cytokine release syndrome (CRS), and IL6R blockade by Tocilizumab has successfully relieved the most life-threatening aspects of CRS in patients. In addition, latest studies demonstrated the essential role of IL1 in driving CART induced neurotoxicity in mouse models. Here we present a clinical investigation (ChiCTR2000032124; ChiCTR2000031868) of anti-CD19 and anti-BCMA CART (41BBζ) secreting an anti-IL6 scFv and IL1 receptor antagonist (IL1RA) in treating patients with hematologic malignancy. Our results revealed that IL6 and IL1B were maintained at low levels without significant elevation during CRS, rendering Tocilizumab dispensable. Moreover, treated patients did not show neurotoxicity during CRS and exhibited mild to moderate CRS. Notably, we observed high rate of complete response (CR) and significant CART expansion during treatment. In sum, we conclude that CART-secreting anti-IL6 scFv and IL1RA could self-neutralize IL6 storm and maintain low levels of IL1B during CART therapy to minimize IL6- and IL1-associated cytokine toxicity and neurotoxicity without impairing therapeutic efficacy.

Fig. 1. Design of CART for secreting IL6 and IL1 antagonists.

a The schematic illustration of CART-secreted anti-IL6 scFv and IL1RA in blocking IL6 and IL1 signalings during CRS of CART therapy. b The design of CAR construct and the sequences of aIL6 scFv and IL1RA. c Comparison of gene delivered expression of different scFv targeting IL6 or IL6R in blocking IL6 signaling and the scFv was consisting of variable regions derived from 1-Sarilumab, 2-Sirukumab, 3-Siltuximab or 4-Tocilizumab. Optimization of co-expressing IL1RA with aIL6 for inhibiting IL1 signaling. Construct 1 includes, from N-terminus to C-terminus, T2A linker, scFv derived from Sirukumab, P2A linker, and IL-1RA (T2A-Sir-P2A-IL1RA). Construct 2 contains, from N-terminus to C-terminus, T2A linker, scFv antibody, (G4S)3 linker, and IL1RA (T2A-Sir-(G4S)3-IL1RA). Construct 3 contains, from N-terminus to C-terminus, scFv antibody, (G4S)3 linker, IL1RA, and T2A linker (Sir-(G4S)3-IL1RA-T2A). Triplicate was included in the test. d Anti-tumor efficacy of anti-CD19 CART-aIL6/Fc (n = 8) and CART-Fc (n = 7) in Xenograft model and quantification of CART-secreted aIL6/Fc in D6 serum. The representative results of two independent experiments were presented. Student’s t-test was conducted, and * denotes statistical significance of P < 0.05. ns means no significance.

Fig. 2. CART expansion, cytokine changes, and CRS in the patients.

Patients (as numbered in Table 1) with refractory or relapsed ALL, CLL, Lymphoma, or MM were enrolled and treated with anti-CD19 or anti-BCMA CART secreting anti-IL6 scFv and IL1RA. After CART infusion, patients were monitored for clinical signs of CRS and examined for clinical response of tumor remission. a Changes of IFNG, IL6, IL1RA, IL1B, and CAR vector copies in blood. b Plot of IFNG peak levels versus the IL6 concentration at the time of IFNG peak during CRS. c Plot of IFNG peak levels versus the IL1B concentration at the time of IFNG peak during CRS. d The relationship between CRS gradings and IFNG peaks, or IL6, IL1B, CRP, and Ferritin levels at the time of IFNG peaks during CRS.

Fig. 3. Changes of body temperature in patients treated with CART-aIL6/IL1RA.

Patients (as numbered in Table 1) with refractory or relapsed ALL, CLL, Lymphoma, or MM were enrolled and treated with anti-CD19 or anti-BCMA CART secreting anti-IL6 scFv and IL1RA. After CART infusion, patients were monitored for recording body temperature to assess grading of CRS.

Fig. 4. Changes of CRP and Ferritin in patients treated with CART-aIL6/IL1RA.

Patients (as numbered in Table 1) with refractory or relapsed ALL, CLL, Lymphoma, or MM were enrolled and treated with anti-CD19 or anti-BCMA CART secreting anti-IL6 scFv and IL1RA. After CART infusion, patients were monitored for recording CRP and Ferritin levels to assess grading of CRS.

Fig. 5. Long term survival of patients (as numbered in Table 1) treated with CART-aIL6/IL1RA.

Patients with refractory or relapsed ALL, CLL, Lymphoma or MM were enrolled and treated with anti-CD19 or anti-BCMA CART secreting anti-IL6 scFv and IL1RA. After CART infusion, patients were examined for clinical response of tumor remission, and followed up for evaluating long-term survival. CR complete response, PR partial response, NR no response, Ag⁺ relapse antigen positive relapse, Ag^– relapse antigen negative relapse.

Fig. 6. Sustained high level of serum IL6 was associated with neurotoxicity in the patient (#1 in Table 1) after typical grade 1 CRS ended.

a The changes of body temperature after infusion. b Levels of CRP and Ferritin in peripheral blood. c Changes of IFNG versus IL6. d Changes of IFNG vs IL1B. e Changes of IL1RA vs IL6. f Kinetics of CAR vector copies vs IL1RA changes. g Changes of GM-CSF and IL10. h Changes of IL2, IL4, IL17A, and TNFA.