Logic-Gated ROR1 Chimeric Antigen Receptor Expression Rescues T Cell-Mediated Toxicity to Normal Tissues and Enables Selective Tumor Targeting

Abstract

Many potential targets for CAR-T cells in solid tumors are expressed in some normal tissues, raising concern for off-tumor toxicity. Following lymphodepletion, CAR-T cells targeting the tumor-associated antigen ROR1 lysed tumors in mice but induced lethal bone marrow failure due to recognition of ROR1⁺ stromal cells. To improve selectivity, we engineered T cells with synthetic Notch (synNotch) receptors specific for EpCAM or B7-H3, which are expressed on ROR1⁺ tumor cells but not ROR1⁺ stromal cells. SynNotch receptors induced ROR1 CAR expression selectively within the tumor, resulting in tumor regression without toxicity when tumor cells were segregated from, but not when co-localized with, normal ROR1⁺ cells. This strategy, thus, permits safe targeting of tumors that are sufficiently separated from normal cells.

Keywords: B7-H3; ROR1; T cells; chimeric antigen receptor; combinatorial antigen recognition; immunotherapy; logic gating; mesenchymal stem cell; synthetic Notch receptors; toxicity.

Figure 1.. ROR1 CAR-T cells induce lethal toxicity in mice pre-conditioned with radiation.

(A) Map of retroviral constructs used to generate ROR1 CAR and control T cells. TM = transmembrane. hIgG4 = human immunoglobulin G4. scFv = single chain variable fragment. (B) Percent change in body weight in BALB/c mice (left) and representative flow cytometric plots showing frequency of CD8⁺CD45.1⁺ donor T cells of live cells and frequency of CD8⁺tCD19⁺ cells of CD45.1⁺ donor T cells 7 days post-transfer (right). (C) Percent change in body weight in BALB/c mice. n=4 mice per group. Two-way ANOVA with Tukey’s post-test (ROR1 CAR-T vs control T at Day 7, 9, 11: p<0.00001). (D) Representative flow cytometric plots showing expression of tCD19 transduction marker and PD-1 on CD8⁺CD45.1⁺ donor T cells 4 days post-transfer (left) and summary of absolute number and PD-1 median fluorescence intensity (MFI) on CD45.1⁺CD8⁺tCD19⁺ control or ROR1 CAR-T cells 4 days post-transfer (right). n=4 mice per group. Unpaired Student’s two-way t-test (Abs #: spleen, p=0.0004; BM, p=0.0002; PD-1: spleen, p=0.0007; BM, p=0.0007; blood, p=0.000008). (E) White blood cell (WBC), lymphocyte, red blood cell (RBC), and platelet (PLT) counts in peripheral blood. n=4 mice per group. Two-way ANOVA with Tukey’s post-test (ROR1 CAR-T vs control T: Lymphocytes Day 14, p<0.005; RBC Day 10,14, p<0.00001; PLT Day 14, p<0.005). (F) Representative H&E stains of spleen and femur (BM) 14 days post-transfer (left) and histopathology scoring of control or ROR1 CAR-T cell-treated tissues (right). n=4 mice per group. Two-way ANOVA with Sidak post-test (Spleen Day 8,10,14, p<0.00001; BM Day 10, p=0.004; BM Day 14, p<0.00001). Data are representative of 5 independent experiments. All data are presented as the mean values ± SEM. See also Table S1 and Figures S1-S2.

Figure 2.. ROR1 CAR-T cell-mediated toxicity is dependent on degree of lymphodepletion and dose of ROR1 CAR-T cells.

(A) Percent change in body weight in BALB/c mice lymphodepleted (LD) as indicated. n=4 mice per group. Two-way ANOVA with Tukey’s post-test (ROR1 CAR-T vs control T: 500 R Day 7, 9, p<0.00001; 200 mg/kg cyclophosphamide (Cy) Day 7, p=0.0004; 200 mg/kg Cy Day 9, p<0.00001). (B) Frequency of CD8⁺CD45.1⁺tCD19⁺ ROR1 CAR-T cells in peripheral blood of BALB/c mice receiving 100 R, 500 R, 100 mg/kg Cy, or 200 mg/kg Cy for LD. n=4 mice per group. (C-E) Percent change in body weight (C), frequency (D) and PD-1 and TIM3 expression (E) on CD8⁺CD45.1⁺tCD19⁺ donor T cells in peripheral blood of BALB/c mice irradiated 500 R and treated as indicated. n=4 mice per group. Two-way ANOVA with Tukey’s post-test (1 M CAR-T vs control T: Day 7, p=0.0003; Day 9,18, p<0.00001; 0.5 M CAR-T vs control T: Day 20, p<0.00001; Day 24, p=0.0005; Day 27, p=0.0044). Data are representative of 2 independent experiments. All data are presented as the mean values ± SEM. See also Figure S3.

Figure 3.. ROR1 CAR-T cell-mediated toxicity is dependent on ROR1 expression in non-hematopoietic cells.

(A) Percent change in body weight (left), RBC count in peripheral blood (middle), and representative pictures of spleens 9 days post-transfer (right) from B6 Ror1^fl/fl (WT) or EIIα-Cre⁺Ror1^fl/fl (KO) mice treated with 500 R and control or ROR1 CAR-T cells. n=3 mice per group. Left: two-way ANOVA with Tukey post-test (WT+CAR-T vs. KO+CAR-T: Day 7,8,9, p<0.00001). Middle: one-way ANOVA with Tukey post-test (p=0.0003). (B) Percent change in body weight (left), RBC count in peripheral blood (middle), and representative pictures of spleens 15 days post-transfer (right) from WT>WT or ROR1-KO>WT BM chimeric mice treated with 500 R and control or ROR1 CAR-T cells. n=3 mice per group. Left: two-way ANOVA with Tukey post-test (WT>WT CAR-T vs. KO>WT CAR-T: n.s. = not significant). Middle: one-way ANOVA with Tukey post-test. (C) Percent change in body weight (left), RBC count in peripheral blood (middle), and representative pictures of spleens 40 days post-transfer (right) from WT>WT or WT>ROR1-KO BM chimeric mice treated with 500 R and control or ROR1 CAR-T cells. n=3 mice per group. Left: two-way ANOVA with Tukey post-test (WT>WT CAR-T vs. WT>KO CAR-T: Day 7, p=0.00013; Day 9, p=0.0045; Day 10,11, p<0.00001). Middle: one-way ANOVA with Tukey post-test (p=0.0044). Data are representative of 2 independent experiments. All data are presented as the mean values ± SEM.

Figure 4.. Bone marrow stromal cells express ROR1 and are targeted by ROR1 CAR-T cells.

(A) qPCR analysis of Ror1 expression in sorted BM progenitors (left) and BM stromal cells (right) from BALB/c mice left untreated or irradiated 500 R 48 hr prior to euthanasia. Ror1 expression was normalized to Actb expression and expressed relative to Ror1 expression in E10.5 embryos. OBL = osteoblasts. MSC = mesenchymal stem cells. EC = endothelial cells. n=3 mice per group. (B) ELISA analysis of IFNγ production by CD8⁺ control or ROR1 CAR-T cells co-cultured for 48 hr with primary MSC derived from WT or ROR1-KO femurs and expanded in vitro for 14 days. Data are summarized from 3 independent experiments. Unpaired two-way Student’s t-test (p=0.0041). (C) Absolute number of cells in BM flushed from femurs and tibia of control or ROR1 CAR-T cell-treated mice 9 days post-transfer (left) and number of MSC, pre-B cell, granulocyte/macrophage, erythrocyte, and megakaryocyte colonies formed after culture of BM cells from femurs of control or ROR1 CAR-T cell-treated BALB/c mice collected 9 days post-transfer (right). n=3 mice per group. Unpaired two-way Student’s t-test (# BM cells, p=0.0004; MSC, p=0.0004; Pre-B, p=0.0023). (D) ELISA analysis of IFNγ production by CD8⁺ control or ROR1 CAR-T cells co-cultured for 48 hr with CD45⁺ or CD45⁻splenic cells. Data are summarized from 3 independent experiments. Unpaired two-way Student’s t-test (p=0.0049). (E) qPCR analysis of Ror1 expression in splenic subpopulations sorted from BALB/c mice left untreated or irradiated 500 R 48 hr prior to euthanasia. n.d. = not detected. Data are summarized from 3 independent experiments. All data are presented as the mean values ± SEM.

Figure 5.. SynNotch EpCAM-inducible ROR1 CAR-T cells selectively target EpCAM⁺ROR1⁺ cells but not EpCAM⁺ROR1⁻ or EpCAM⁻ROR1⁺ cells in vitro.

(A) Representative flow cytometric analysis of EpCAM expression on 4T1 tumor cells, MSC, osteoblasts (OBL), CD45⁻Ter119⁻PDGFRβ⁺ spleen cells, and CD45⁻Ter119⁻VE-cadherin⁺ spleen cells from BALB/c mice. Percent lysis of the indicated ⁵¹Cr-labeled tumor cells after 6 hr (top) or 24 hr (bottom) of co-culture with CD8⁺ untransduced (black), ROR1 CAR-T cells (gray), or EpCAM-inducible ROR1 CAR-T cells (open circles). (C) Intracellular cytokine analysis of tCD19 CAR marker and IFNγ expression in the indicated T cells co-cultured with tumor cells in the presence of Brefeldin A for the last 6 hr of culture. Data are representative of two independent experiments. See also Figures S4 and S5.

Figure 6.. SynNotch EpCAM-inducible ROR1 CAR-T cells rescue toxicity to ROR1⁺ normal tissues while maintaining activity against ROR1⁺ tumors.

(A) Percent change in body weight in BALB/c mice inoculated with 4T1-mROR1 tumors and treated as indicated. n=6–12 mice per group. Two-way ANOVA with Tukey post-test (ROR1-CAR vs. EpCAM-inducible ROR1 CAR: Day 4, p<0.0001; Day 7, p<0.0001; Day 10, p=0.0002). (B) RBC and PLT counts from peripheral blood of tumor-bearing mice treated as indicated. n=6–12 mice per group. Two-way ANOVA with Tukey post-test (ROR1-CAR vs. EpCAM-inducible ROR1 CAR: RBC Day 4,8,15,22:, p<0.0001; PLT Day 8 p=0.0114; PLT Day 15, 22, p<0.0001). (C) Representative pictures of spleens 10 days post-transfer from tumor-bearing BALB/c mice treated as indicated. (D) Tumor volume in BALB/c mice treated as indicated. n=6–12 mice per group. Two-way ANOVA with Tukey post-test (ROR1-CAR vs. UT: Day 14, p<0.0001; Day 17, p<0.0001; EpCAM-inducible ROR1 CAR vs. UT: Day 14, p<0.0001; Day 17, p<0.0001; ROR1-CAR vs EpCAM-inducible ROR1 CAR: n.s). (E) Survival of mice treated as indicated. n=6–12. Log-rank Mantel-Cox test (UT vs. ROR1 CAR, p=0.0003; UT vs. EpCAM-inducible ROR1 CAR, p=0.0016; ROR1 CAR vs. EpCAM-inducible ROR1 CAR, p=0.0001). (F) Representative flow cytometric analysis of ROR1 CAR and PD-1 expression on CD8⁺CD45.1⁺ donor T cells from mice treated with Cy and the indicated T cells 10 days post-transfer. (G) Summary of frequency of ROR1 CAR⁺, CAR⁺Ki67⁺, and CAR⁺PD-1⁺ T cells of CD8⁺CD45.1⁺ donor T cells from mice treated with Cy and the indicated T cells 10 days post-transfer. n=6–12 mice per group. Unpaired two-way Student’s t-test (Frequency: spleen, p<0.0001; BM, p<0.0001; PD-1: spleen, p=0.0004; BM, p=0.0003; Ki67: spleen, p<0.0001; BM, p<0.0001). Data are summarized from 2 independent experiments. All data are presented as the mean values ± SEM.

Figure 7.. SynNotch CD19-inducible ROR1 CAR-T cells are unable to rescue toxicity to ROR1⁺ normal tissues in the presence of circulating and bone marrow-resident CD19⁺ROR1⁺ Raji tumors.

(A) Map of lentiviral constructs encoding CD19-Gal4VP64 synNotch Receptor, UAS inducible R11 ROR1 CAR, and constitutive R11 ROR1 CAR. (B) Raji tumor bioluminescence over time in NSG mice irradiated 250 R and treated with the indicated primary human T cells. n=6–7 per group. Two-way ANOVA with Tukey post-test (UT vs. ROR1 CAR-T: Day 13, 20, p<0.0001; UT vs. CD19-inducible ROR1 CAR-T: Day 13, 20, p<0.0001). (C) Summary of tEGFR CAR marker and PD-1 expression on the indicated donor CD8⁺CD45⁺ T cells in peripheral blood. n=4–7 per group. Two-way ANOVA with Tukey post-test (CAR: UT vs. ROR1 CAR, Day 3, 7, p<0.0001; ROR1 CAR vs. CD19-inducible ROR1 CAR, Day 3, p<0.0001; Day 7, n.s. PD-1: UT vs. ROR1 CAR, Day 3, 7, p<0.0001; ROR1 CAR vs. CD19-inducible ROR1 CAR, Day 3, p<0.0001; Day 7, n.s.). (D) Survival of tumor-bearing mice irradiated 250 R and treated with the indicated T cell groups. n=6–7 per group. Log-rank Mantel-Cox test (UT vs. ROR1 CAR, p=0.0007; UT vs. CD19-inducible ROR1 CAR, p=0.0007; ROR1 CAR vs. CD19-inducible ROR1 CAR, n.s.). Data are representative of 2 independent experiments. All data are presented as the mean values ± SEM. See also Figure S6.

Figure 8.. Primary human synNotch B7-H3-inducible ROR1 CAR-T cells rescue toxicity to ROR1⁺ normal tissues while maintaining activity against human ROR1⁺ tumors.

(A) Percent change in body weight in NSG mice implanted with MDA-MB-231 tumors, irradiated 250 R and treated with the indicated primary human T cells. n=4 mice per group. Two-way ANOVA with Tukey post-test (ROR1-CAR vs. EpCAM-inducible ROR1 CAR: Day 13, 14, 19, p<0.0001). (B) RBC and PLT counts from peripheral blood of tumor-bearing mice treated as indicated 20 days post-T cell transfer. n=4 mice per group. Two-way ANOVA with Tukey post-test (RBC: ROR1 CAR vs. EpCAM-inducible ROR1 CAR, p=0.0426; UT vs. ROR1 CAR, p=0.0418. PLT: ROR1 CAR vs. EpCAM-inducible ROR1 CAR, p=0.0117; UT vs. ROR1 CAR, p=0.0442). (C and D) Tumor bioluminescence (C) and survival (D) of NSG mice treated as indicated. n=4 mice per group. Two-way ANOVA with Tukey post-test (ROR1 CAR vs. UT: Day 13, p=0.0167; Day 20, p<0.0001. EpCAM-inducible ROR1 CAR vs. UT: Day 13, p=0.0029; Day 20, p<0.0001. ROR1 CAR vs EpCAM-inducible ROR1 CAR: n.s). (E) Representative flow cytometric analysis of ROR1 CAR and Ki67 expression on CD8⁺CD45⁺ donor T cells from NSG mice irradiated 250 R and given indicated T cells 22 days post-transfer. (F) Summary of frequency of ROR1 CAR⁺, Ki67⁺, and PD-1⁺ T cells of CD8⁺CD45⁺ donor T cells from mice irradiated 250 R and given indicated T cells 22 days post-transfer. n=4 mice per group. Unpaired two-way Student’s t-test (Frequency: spleen, p<0.00001; BM, p<0.00001; tumor, n.s. Ki67: spleen, p<0.0001; BM, p<0.0001; tumor, n.s. PD-1: spleen, p<0.0001; BM, p<0.0001; tumor, n.s.). Data are representative of 2 independent experiments. All data are presented as the mean values ± SEM. See also Figures S7 and S8.