Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide

Abstract

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4^CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.

Fig. 1:. Engineering lenalidomide ON- and OFF-switch controllable CAR T cells.

(A) Schematic of the OFF-switch degradable CARs and their degradation induced by lenalidomide (len) or other thalidomide analogs. The double line represents the plasma membrane with extracellular above the line and intracellular below the line. (B) Characterization of surface CAR abundance by flow cytometry using an antibody against the Myc tag (not shown in the schematic). Jurkat cells were engineered to express an anti-CD19 CAR (left) or the same with addition of the zinc finger degron from IKZF3 (Degradable CAR, right) and exposed to 1 μM lenalidomide or vehicle control overnight. UTD, untransduced. (C) Schematic of the ON-switch split CARs incorporating a lenalidomide-inducible dimerization domain composed of portions of CRBN in one subunit and IKZF3 in the other and their activation by lenalidomide or other thalidomide analogs. (D) Jurkat cells were engineered to express a conventional CAR (CAR) or a split CAR, co-cultured overnight with either K562 cells or K562 cells engineered to express CD19 and 1 μM lenalidomide or vehicle control, and the percentage of cells positive for the early activation marker CD69 was quantified by flow cytometry. Data representative of 1 of 2 independent experiments performed in technical duplicate (B) or triplicate (D). Error bars indicate mean ± SD of triplicate wells. Two-tailed t-test, #### P < 0.00005; ns, P > 0.05.

Fig. 2:. Engineering of a lenalidomide-inducible dimerization system and ON-switch split CAR.

Bioluminescence resonance energy transfer (BRET) was used to measure the association between the indicated NanoLuc luciferase and HaloTag fusion proteins in 293T cells. (A) BRET analysis of IKZF3 interaction with CRBN deletion variants. IKZF3 = amino acids (AA) 130–189 of IKZF3. NTD, N-terminal domain; LLD-N, Lon-like domain (N-terminal portion); LLD-C, Lon-like domain (C-terminal portion); TBD, thalidomide-binding domain. (B) BRET analysis of IKZF3-CRBNΔ3 incorporated into cell surface-localized fusion proteins. 1928 = FMC63 scFv – CD28 hinge, transmembrane, and costimulatory domains. CD8-CD28 = CD8 hinge and transmembrane domain and CD28 co-stimulatory domain. PD1 = PD1 transmembrane and cytoplasmic domain. LYN-CD28 = LYN myristoylation and palmitoylation motif – CD28 costimulatory domain. MG132 was included in experiments A and B. (C) BRET analysis of CD8-CD28-CRBNΔ3 and 1928-IKZF3 with or without intracellular Lys → Arg mutations (iK0). mBU, miliBRET units. Individual values and nonlinear regression are shown. Data represent one independent experiment performed in technical duplicate. Two-way ANOVA, **** P < 0.0001.

Fig. 3:. A screen of 440 hybrid zinc fingers identifies “super-degrons” targeted by subnanomolar concentrations of thalidomide analogs.

(A) Schematic for the design and screening of a hybrid zinc finger (ZF) library encoded in a eGFP-tagged protein degradation reporter lentivector that also expressed mCherry as a control for lentivector transgene expression. Jurkat cells were transduced with the lentivirus library and then exposed to various thalidomide analogs or vehicle control (DMSO). FACS was used to isolate eGFP^low cells, and next-generation sequencing (NGS) was used to quantify the relative abundance of each sequence with and without drug treatment. (B) Flow plot for Jurkat cells transduced with the eGFP-tagged zinc finger library after overnight incubation with 1 μM lenalidomide or vehicle control. (C) Fold-enrichment of sequencing read counts (lenalidomide/DMSO, scale 0 – 20) and corresponding empirical rank-sum test P values (0 – 5). Average of nine replicates is presented (library of triplicate barcoded sequences assayed with triplicate independent biologic replicates). (D) Sequence features for N- and C-terminal domains present in top candidate super-degrons. Amino acid positions with prior crystallographic evidence of side-chain interactions with pomalidomide (open circle) or CRBN (green circle) are noted. Jurkat cells were engineered to express the indicated zinc finger constructs and treated with various thalidomide analogs. (E and F) Degradation of GFP-tagged zinc fingers was assessed by flow cytometry from the vehicle control-normalized eGFP/mCherry fluorescence ratio in Jurkat cells after treatment with lenalidomide (E) or iberdomide (F). Individual values and nonlinear regression are shown. Experiment was performed once in technical duplicate. (G) Nonlinear regression was used to calculate half maximal degradation concentration (DC50) values for zinc finger degradation by each drug.

Fig. 4:. Lenalidomide ON-switch control of split CAR function.

(A) Schematic of split CAR constructs. Each split CAR is composed of the indicated antigen-binding part (part A) and the CD3z-containing part (part B). In part A, the dimerization module is encoded either by amino acids (AAs) 130–189 of IKZF3, including the minimal drug- and CRBN-interacting zinc finger (AAs 146–168) and adjacent sequences that further enhance CRBN binding (split CAR_I), or by the ZFP91-IKZF3 hybrid zinc finger, which incorporates ZFP91 AAs 450–460 in place of IKZF3 AAs 146–156 (split CAR_H). The intracellular domains of each part A are protected from CRL4^CRBN ubiquitination by Lys→ Arg “K0” substitutions. In part B, CRBNΔ3 is a deletion variant lacking the DDB1-interacting domain from AAs 194–247. CAR indicates the second generation receptor FMC63-CD28-CD3z. To detect transduced cells by flow cytometry, part A includes a P2A element followed by mCherry, and part B includes in-frame eGFP. (B) Percent of CD69-positive Jurkat CAR T cells after overnight coculture with K562-CD19 cells and a range of lenalidomide concentrations. The maximum plasma concentration for once daily 25 mg lenalidomide in multiple myeloma patients is indicated by the dotted line. (C) FACS purification of the indicated populations of primary T cells six days after transduction with lentivectors encoding parts A and B of split CAR_H. (D) Cytotoxic activity of each sorted cell population after overnight coculture with NALM6 target cells at the indicated effector:target ratios and lenalidomide or vehicle control. The comparison of AB versus UTD (untransduced) control cells is noted. (E) NALM6 cytotoxic activity of split CAR_H versus CAR and UTD. (F) Cytotoxic activity or IFN-γ concentration (measured by bead array) from the indicated CAR T cells after overnight coculture with an equal number of NALM6 cells and a range of lenalidomide concentrations. (G) CAR T cell proliferation after 5 days of coculture with NALM6 cells. For in vitro experiments, error bars indicate mean ± SD of triplicate wells in all cases except proliferation, which shows individual values and means of duplicate wells. (H) Experimental design of in vivo study. (I) Bioluminescence flux (photons/s) of whole mice in each group, indicating tumor burden. (J) Bioluminescence at representative time points. ANOVA of split CAR with versus without lenalidomide is indicated. (K) Blood T cell quantification at representative time points. t-tests of split CAR with versus without lenalidomide treatment at each timepoint are indicated. (L) Percent of human CD3+ cells in the spleen and bone marrow at day 23 (D23). For in vivo experiments, error bars indicate mean ± SD of pentuplicate groups. Data represent 1 of 2 independent experiments. Two-way ANOVA, * P < 0.05; ** P < 0.005; **** P < 0.0001; ns, P > 0.05. Two-way t-test, ## P < 0.005; ### P < 0.0005; ns, P > 0.05.

Fig. 5:. Subtherapeutic lenalidomide concentrations trigger super-degron OFF-switch degradable CARs with 4–1BB and CD3z signaling domains.

(A) Degron-containing CAR constructs. The IKZF3 degron (ID) is encoded by the fragment of IKZF3 from amino acid (AA) 130–189. The ZFP91-IKZF3 super degron (SD) incorporates ZFP91 AAs 450–460 in place of IKZF3 AAs 146–156. The mutated super-degron (mutSD) has a C452A mutation in the ZFP91 fragment that abolishes drug binding. (B) Jurkat cells expressing the indicated CARs were drug treated overnight and then analyzed by Western blot for CAR (using an antibody recognizing CD3z) and other specified targets. (C) Surface CAR abundance (detected with antibody recognizing the Myc tag) on CD4+ and CD8+ CAR T cells after overnight incubation with lenalidomide or vehicle control. UTD, untransduced. (D) Cytotoxic activity of primary human CAR T cells after overnight coculture with NALM6 target cells and lenalidomide or vehicle control. (E) Cytokine concentrations in coculture supernatants measured by bead array (9:1 CAR T:NALM6 ratio). (F) Cytotoxic activity of or IFN-γ secreted from the indicated CAR T cells after overnight coculture with an equal number of NALM6 cells and a range of lenalidomide concentrations. IFN-γ was normalized to the concentration without lenalidomide for each construct. (G) CAR T cell proliferation after 5 days of coculture with NALM6 cells. Data represent 1 of 2 independent experiments. Error bars indicate mean ± SD of triplicate wells. Individual values and means are shown for multiplexed cytokine analysis and proliferation, which were performed in duplicate. Two-way ANOVA, **** P < 0.0001; ns, P > 0.05. Two-way t-test, ## P < 0.005; ns, P > 0.05.

Fig. 6:. Reversible lenalidomide OFF-switch control of a degradable CAR in vivo.

(A) Schematic of luciferase-tagged CAR constructs. (B) Experimental design for in vivo CAR depletion analysis: NSG mice were injected intravenously with 5 × 10⁶ Jurkat cells expressing the SD or mutSD CAR reporter; after engraftment, bioluminescent imaging was performed before and after one dose of 10 mg/kg pomalidomide administered by oral gavage. (C) Luminescence 24 hours before, 6 hours after, and 24 hours after pomalidomide. Two-way t-test, ## P < 0.005 at 6 hours post, P > 0.05 at other timepoints. (D) Bioluminescence flux (photons/s) of whole mice in each group, indicating CAR protein abundance. Data represent 1 of 2 independent experiments.

Fig. 7:. Degradable CAR function and cytokine OFF-switch in vivo.

(A) Experimental design: NSG mice were injected intravenously with 1 × 10⁶ GFP+/luciferase+ JeKo-1 tumor cells. At day 0, mice were randomly assigned on the basis of tumor burden to receive 1 × 10⁶ untransduced, CAR, or SD-CAR T cells. (B) Bioluminescence of whole mice in each group, indicating tumor burden. (C) Bioluminescence flux (photons/s) at representative time points. Two-way ANOVA, ns, P > 0.05. (D, E) Percent of JeKo-1 and human CD3+ cells in the bone marrow and spleen at day 35 (D35). Error bars indicate mean ± SD of pentuplicate groups. (F) Experimental design for in vivo CAR T cell cytokine release analysis: NSG mice were injected intravenously with 1 × 10⁶ NALM6 cells. At day 0, mice were randomly assigned on the basis of tumor burden to receive 2 × 10⁶ untransduced or SD-CAR T cells. From days 3 – 5, mice received no treatment, once daily, or twice daily 30 mg/kg pomalidomide by oral gavage. On the afternoon of day 5, serum was analyzed by bead array for human cytokines. (G, H) Serum IFN-γ and IL-2 concentrations. QD, daily. BID, twice daily. Two-way t-test, # P < 0.05; ns, P > 0.05.