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Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells

Abstract

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19 disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials (NCT02588456 and NCT02650414) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

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Fig. 1: Clinical trial of CAR22 T cells in adults and children with relapsed ALL.
Fig. 2: scFv linker influences CAR surface membrane activity.
Fig. 3: CAR clustering leads to antigen-independent signaling.
Fig. 4: Functional characterization of CAR22-engineered T cells.
Fig. 5: Development of a new CD22 CAR with potent preclinical activity.

Data availability

All requests for raw and analyzed preclinical data and materials will be promptly reviewed by the University of Pennsylvania to determine if they are subject to intellectual property or confidentiality obligations. Patient-related data not included in the paper were generated as part of clinical trials and may be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement. Sequences for the m971 CAR are publicly available under patent no. PCT/US2013/060332. Sequences for the CD22-f2 CAR are the private property of Novartis. The raw data for Supplementary Figs. 1, 2d–l, 3d–f and 4d–l are located in the Supplementary Dataset. Source data are provided with this paper.

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Acknowledgements

We thank F. Chen and N. Koterba for technical assistance with the cytokine quantification assays and J. Schug for assistance with the RNA sequencing. The pediatric trial was supported by the CHOP Immunotherapy Frontier Program, in addition to support from the Emily Whitehead Foundation, V Foundation and Curing Kids Cancer (S.A.G.). The preclinical research was supported by the Society of Immunotherapy for Cancer Holbrook Kohrt Immunotherapy Translational Fellowship (N.S.); a Breakthrough Bike Challenge Buz Cooper Scholarship (N.S.); Stand Up To Cancer (SU2C) Innovative Research Grant no. SU2C-AACR-IRG 12-17 (D.M.B.; SU2C is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C); National Institutes of Health grant no. R01GM104867 (J.K.B.); a Cancer Research Institute Irvington Fellowship (N.H.R.); National Heart, Lung, and Blood Institute grant no. HL125018 and National Institute of Allergy and Infectious Diseases grant nos. AI124769, AI129594 and AI130197 (D.L.); Japan Agency for Medical Research and Development grant no. P20am0101108 (D.M.S.); NCI grant no. K08CA194256 (S.G.); an American Society of Hematology Scholar Award, NCI grant nos. 1K99CA212302 and R00CA212302 (M.R.); University of Pennsylvania-Novartis Alliance (S.G. and C.H.J.); and NCI grant nos. 1P01CA214278 and R01CA226983 (C.H.J.).

Author information

Authors and Affiliations

Authors

Contributions

N.S., B.E., D.M.B., O.S., P.R., K.D.C., Y.G.L., R.P., I.C., A.S., S.L.H., A.P., L.Z., L.P., B.G., M. Ramones, D.A.C., J.P., S.F.L., N.H.R., J.K.B., F.C., M.D., M.A.M., T.L., D.L., D.M.S., C.H.J., S.L.M., S.G. and M. Ruella designed, performed and oversaw the research. X.M.L. performed the biostatistical analysis on the RNA sequencing. N.V.F. was principal investigator of the adult clinical trial. S.A.G. was principal investigator of the pediatric clinical trial. R.M.Y. and J.L.B. provided significant intellectual contribution to the design and research. N.S., S.G. and M. Ruella wrote the manuscript. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Nathan Singh or Marco Ruella.

Ethics declarations

Competing interests

M.R. and S.G. hold patents related to CART22. C.H.J. has received grant support from Novartis and has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania. C.H.J. is also a scientific founder and holds equity in Tmunity Therapeutics. S.A.G. has received support from Novartis, Servier and Kite and serves as a consultant, member of the scientific advisory board or study steering committee for Novartis, Cellectis, Adaptimmune, Eureka, TCR2, Juno, GlaxoSmithKline, Vertex, Cure Genetics, Humanigen and Roche. B.E., L.Z., L.P., A.P., B.G., M. Ramones and J.B. are employees of Novartis. S.F.L. has received grant support from Novartis, Tmunity and Cabaletta and has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania; he has acted as a consultant for Kite/Gilead. S.L.M. has served as a member of advisory boards or steering committees for Novartis and Kite. All other authors declare no competing interests.

Additional information

Peer review information Nature Medicine thanks Aude Chapuis, Kristen Hege, Qian Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1

CONSORT structure of clinical trials of CD22 CAR T cells in adults and children.

Extended Data Fig. 2 Concentration of serum cytokines in patients treated with CAR22-long T cells.

Adult patient 1 had grade 3 CRS with mean GCSF, IL-6 and MCP-1 greater than all other patients (denotated with an *), and significantly higher levels of IL-10, IFNγ, IL-1Ra, IL-2R, IL-8, MIP-1α and VEGF than at least 3 other patients.

Extended Data Fig. 3 Re-expansion of CART19 cells in patients treated with CART22.

a–d, CART19 and CART22 composition in apheresis and CART22 products in a, pediatric patient #2 and b, adult patient #2, and expansion of CART19 and CART22 in c, pediatric patient #2 and d, adult patients #2 over time after infusion of CART22. e-i, Evaluation of peripheral blood from adult patient 2 at peak CART19 expansion (day 21). CD3 + peripheral blood mononuclear cells (PBMCs) from a healthy donor were used as either e, negative, untransduced control or f, CAR19 and CAR22 dual-engineered positive control. g, CD3 + PBMCs, h, CD3 + CD4 + PBMCs, and i, CD3 + CD8 + PBMCs from adult patient 2.

Extended Data Fig. 4 CART22 phenotypes over time.

a, Cell growth during T cell engineering. Expression of b, PD-1, c, LAG3, and d, Tim3. *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Statistics reflect differences between CAR22-short and long T cells. Statistics reflect differences between CAR22-short and long T cells.

Source data

Extended Data Fig. 5

a, b, Fluorescent microscopy of T cells engineered with GFP-linked a, CAR19-short or b, CAR19-long constructs. c-f, CAR22-short T cells form more effective immune synapses. c, % of cells forming CART:tumor cell immune conjugates after 5 and 15 minutes of co-culture. d, phosphorylated CD3ζ (three independent experiments, n = 820 measurements per group) and e, phosphorylated Zap70 (n = 180 measurements per group) in CAR T cells engaged with Nalm6. f, Quantification of CAR T cell mobility in mice engrafted with Nalm6, as measured by % of cells moving within an intravital imaging field (n = 5 per group).

Source data

Extended Data Fig. 6 CART22-short cells are primed for activation.

a, Difference in phospho-peptide quantity in stimulated CAR22-short compared to CAR22-long T cells. Proteins with >1.5-fold difference are shown. b, Upregulated transcriptional programs in CAR22-short compared to CAR22-long T cells than have been stimulated by CD22-coated beads for 18 hours. c, Volcano plot of differentially expressed transcripts in CAR22-short compared to CAR22-long T cells.

Source data

Extended Data Fig. 7

a, Measurement of activation-induced cell death by Annexin-V expression. Anti-tumor activity of an in vitro b, treatment or c, stress model of T cells bearing either a short or long CAR composed of the m971 scFv with the CD28 co-stimulatory domain. *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Statistics reflect differences between CAR22-short and long T cells.

Source data

Extended Data Fig. 8 Functional characterization of CAR33-enginereed T cells.

a, Growth of CD33 + Molm14 acute myeloid leukemia cells and b, CAR33-bearing T cells during in vitro co-culture. Quantification of c, IFNγ, d, IL-2 and e, TNFα by CART33 cells during an in vitro co-culture with Molm14 cells. *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Statistics reflect differences between CAR33-short and long T cells.

Source data

Extended Data Fig. 9 Functional characteristics of CAR19-engineered T cells.

Anti-tumor activity in an in vitro a, treatment or b, stress model of T cells bearing either a short or long CD19-targeted CAR. c, Anti-tumor effect of long or short CART19 cells in a xenograft model of Nalm6 ALL (representative of two individual experiments, n=5 mice per condition; see Supplementary Figure 3 for individual animal responses from this experiment and the replicate experiments). Quantification of d, IFNγ, e, IL-2 and f, TNFα by CART33 cells during an in vitro co-culture with Molm14 cells. *P<0.05, **P<0.001, ***P<0.0001, ****P<0.00001. Statistics reflect differences between CAR19-short and long T cells.

Source data

Extended Data Fig. 10 Anti-tumor activity of all four CD22 CAR T cells in a xenograft model of SEM ALL.

n = 5 mice per condition; see Supplementary Figure 5 for individual animal responses.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, NCT02588456 Clinical Protocol, NCT02650414 Clinical Protocol, NCT02650414 DSMP.

Reporting Summary

Supplementary Table 1

Additional clinical data for NCT02588456 and NCT02650414.

Supplementary Table 2

Full toxicity reporting.

Supplementary Video 1

Supplementary Data 1

Source data for supplementary figures.

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Statistical source data.

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Singh, N., Frey, N.V., Engels, B. et al. Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells. Nat Med 27, 842–850 (2021). https://doi.org/10.1038/s41591-021-01326-5

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