Endogenous CD28 drives CAR T cell responses in multiple myeloma

Abstract

Recent FDA approvals of chimeric antigen receptor (CAR) T cell therapy for multiple myeloma (MM) have reshaped the therapeutic landscape for this incurable cancer. In pivotal clinical trials B cell maturation antigen (BCMA) targeted, 4-1BB co-stimulated (BBζ) CAR T cells dramatically outperformed standard-of-care chemotherapy, yet most patients experienced MM relapse within two years of therapy, underscoring the need to improve CAR T cell efficacy in MM. We set out to determine if inhibition of MM bone marrow microenvironment (BME) survival signaling could increase sensitivity to CAR T cells. In contrast to expectations, blocking the CD28 MM survival signal with abatacept (CTLA4-Ig) accelerated disease relapse following CAR T therapy in preclinical models, potentially due to blocking CD28 signaling in CAR T cells. Knockout studies confirmed that endogenous CD28 expressed on BBζ CAR T cells drove in vivo anti-MM activity. Mechanistically, CD28 reprogrammed mitochondrial metabolism to maintain redox balance and CAR T cell proliferation in the MM BME. Transient CD28 inhibition with abatacept restrained rapid BBζ CAR T cell expansion and limited inflammatory cytokines in the MM BME without significantly affecting long-term survival of treated mice. Overall, data directly demonstrate a need for CD28 signaling for sustained in vivo function of CAR T cells and indicate that transient CD28 blockade could reduce cytokine release and associated toxicities.

Keywords: CAR T cells; CD28; co-stimulation; metabolism; multiple myeloma; tumor microenvironment.

Figure 1:. Endogenous CD28 blockade impairs hBCMABBζ CAR T cell anti-myeloma efficacy.

(A) Schematic of second-generation retroviral CAR construct used to generate BCMA targeted human CAR T cells. (B) CAR T cell cytotoxic activity during a 24-hr. co-culture with luciferase-tagged MM.1S (left) or U266 (right) myeloma cells ± abatacept. Data are shown as mean ± SD and representative of hBCMABBζ CAR T cells generated from 3 healthy donors. *p<0.05 by two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. (C) Diagram of experimental setup used to evaluate hBCMABBζ CAR T + abatacept therapy in a human MM xenograft model. NSG mice were intravenously inoculated with 1 × 10⁶ MM.1S-luc myeloma cells on day −28 and treated with CAR T cells on day 0. Mice received 200 μg abatacept 3x/week beginning the day before infusion and continuing through endpoint. Tumor burden was monitored by IVIS bioluminescent imaging (BLI) 2x/week. (D) Representative bioluminescent images of MM.1S bearing mice on specified days following CAR T cell infusion. (E) Tumor burden expressed as relative photon flux measured by BLI from MM.1S-luc bearing mice treated with hBCMABBζ CAR T or control T cells ± abatacept (200 μg, 3x/week). Each line represents an individual mouse (n = 7 mice per CAR T cell treated group). (F) Kaplan – Meier analysis of survival of hBCMABBζ CAR T or control T cells ± abatacept (200 μg, 3x/week) treated MM.1S-luc bearing mice (n = 8 – 12 mice per CAR T cell treated group). Median survival of hBCMABBζ CAR T treated mice was >100 days post CAR T cell infusion vs. 55 days for hBCMABBζ CAR T + abatacept treated mice. ****p<0.0001 by log-rank Mantel-Cox test. (G) Tumor burden expressed as relative photon flux measured by BLI from MM.1S high tumor burden mice (inoculated on day −35) treated with hBCMABBζ CAR T or control T cells ± abatacept (200 μg, 3x/week). Each line represents an individual mouse (n = 4 mice per CAR T cell treated group). (H) Kaplan – Meier analysis of survival of hBCMABBζ CAR T or control T cells ± abatacept (200 μg, 3x/week) treated MM.1S-luc high tumor burden mice (n = 4 mice per group). Median survival of hBCMABBζ CAR T treated mice was 80 days vs. 45 days for hBCMABBζ CAR T + abatacept treated mice. ***p<0.0001 by log-rank Mantel-Cox test.

Figure 2:. CD28^iKO hBCMAmBBmζ CAR T cells are functionally impaired in vivo.

(A) Schematic depicting the process of manufacturing mouse CD28 knockout (CD28^iKO) hBCMAmBBmζ CAR T cell. (B) Surface CD28 protein expression on CD28^fl/fl versus CD28^iKO mouse T cells during CAR T cell manufacture. (C) Median fluorescent intensity (MFI) of CD28 measured by flow cytometry at the conclusion of CD28^fl/fl versus CD28^iKO mouse CAR T manufacture. Data shown as mean ± SD from 10+ independent experiments. *p<0.05, ****p<0.0001 by one-way ANOVA. (D) CD28^fl/fl versus CD28^iKO hBCMAmBBmζ CAR T cell cytotoxic activity during 24 hr. co-culture with luciferase-tagged 5TGM1^hBCMA mouse myeloma cells. Cell viability was assessed by luciferase assay. Data are shown as mean ± SD and representative of at least 3 independent experiments. *p<0.05, **p<0.01 by two-way ANOVA with Tukey’s multiple comparison test. (E) Heatmap representation of culture supernatant mouse cytokine concentrations measured by multiplexed Luminex assays at the conclusion of a 24-hr. co-culture of hBCMAmBBmζ CD28^fl/fl or CD28^iKO CAR T cells with 5TGM1^hBCMA myeloma cells. Log₂ transformed cytokine concentrations represent the mean of 4 independent experiments. (F) Diagram of experimental setup used to evaluate CD28^fl/fl versus CD28^iKO hBCMAmBBmζ CAR T cell therapy in a mouse MM xenograft model. RAG2^−/− mice were inoculated intravenously with 2 × 10⁶ 5TGM1^hBCMA-luc cells on day −14 and treated with CD28^fl/fl or CD28^iKO CAR T cells on day 0. Tumor burden was monitored by IVIS bioluminescent imaging (BLI) 2x/week through endpoint. (G) Tumor burden expressed as relative photon flux measured by BLI from 5TGM1^hBCMA-luc bearing mice treated with CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T or control T cells. Each line represents an individual mouse (n = 6 mice per CAR T cell treated group). (H) Kaplan – Meier analysis of survival of CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cell or control T cell treated 5TGM1^hBCMA-luc bearing mice (n = 6 mice per CAR T cell treated group). Median survival of CD28^fl/fl hBCMAmBBmζ CAR T treated mice was 38 days post-CAR T cell infusion vs. 24 days for CD28^iKO hBCMAmBBmζ CAR T treated mice. *p<0.05, **p<0.01, ***p<0.001 by log-rank Mantel-Cox test. (I) Heatmap representation of cytokine levels in the MM BME 7 days following infusion of CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells into 5TGM1^hBCMA-luc bearing mice. Bilateral hind limbs were harvested, and BM was flushed into 15 μL PBS for multiplexed cytokine analysis. Log₂ transformed cytokine concentrations represent the mean of 3 mice per group.

Figure 3:. Perturbation of oxidative metabolism and redox homeostasis in stimulated CD28^iKO hBCMAmBBmζ CAR T cells.

(A) Representative Seahorse Glycolysis Stress Test performed 24 hr. after stimulation of CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells with 5TGM1^hBCMA myeloma cells. Connected data points represent mean extracellular acidification rate (ECAR) ± SD of four technical replicates at indicated time points during a representative experiment that was repeated at least 3 times. Dashes indicate the timing of glucose, oligomycin (Oligo) and 2-deoxyglucose (2-DG) injection. (B) Quantified rates of glycolysis (left), glycolytic capacity (middle), and glycolytic reserve (left) in CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells ± 24-hr. stimulation with 5TGM1^hBCMA myeloma cells. Bars represent mean ± SD, dots represent independent experiments. ns = not significant by one-way ANOVA. (C) Representative Seahorse Mito Stress Test performed 24 hr. after stimulation of CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells with 5TGM1^hBCMA myeloma cells. Connected data points represent mean oxygen consumption rate (OCR) ± SD of four technical replicates at indicated time points during a representative experiment that was repeated at least 3 times. Dashes indicate the timing of oligomycin (Oligo), FCCP, and rotenone (Rot) + antimycin A (Ant. A) injection. (D) Quantified basal OCR (right), uncoupled maximal respiration (middle), and spare respiratory capacity (SRC) expressed as percent of basal OCR (left) in CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells ± 24 hr. stimulation with 5TGM1^hBCMA myeloma cells. Bars represent mean ± SD, dots represent independent experiments. *p<0.05, **p<0.01 by one-way ANOVA. (E) Ratio of NADH to NAD⁺ ratios in CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells prior to 4-OHT mediated CD28 deletion or ± 24 hr. stimulation with 5TGM1^hBCMA myeloma cells. Bars represent mean ± SD from 3 independent experiments. *p<0.05 by paired Student’s t test. (F) Diagrams depicting enzymes of central carbon metabolism that interconvert NADH and NAD⁺. (G) Relative expression of mRNAs coding enzymes depicted in (F) in CD28^iKO versus CD28^fl/fl hBCMAmBBmζ CAR T cells following 24 hr. stimulation with 5TGM1^hBCMA myeloma cells. Bars represent mean log₂ transformed ΔΔCt values ± SD, dots technical replicates pooled from 3 independent experiments. Tbp and Actb were used as endogenous controls. ****p<0.0001 by two-way ANOVA.

Figure 4:. Diminished in vivo expansion of 4-BB co-stimulated CD28^iKO CAR T cells.

(A) Expansion of CD28^fl/fl versus CD28^iKO hBMCAmBBmζ CAR T cells over the course of manufacturing. ns = not significant by two-way ANOVA. (B) Expression of the proliferation marker Ki-67 in CD28^fl/fl versus CD28^iKO hBCMAmBBmζ CAR T cells following 24 hr. stimulation with 5TGM1 target cells as assessed by flow cytometric analysis. ns = not significant by one-way ANOVA. (C) Diagram of experimental setup. 5TGM1^hBCMA bearing RAG2^−/− mice were treated with CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells (4D and 4E) or MM.1S bearing NSG mice were treated with hBCMABBζ CAR T cells ± 200μg abatacept on days −1, 1, 3, 5, 7 (4F) and euthanized 7 days post adoptive transfer for blood collection and hind limb BM harvest. (D) CAR T cell frequency assessed by flow cytometry in bone marrow (BM, left) and peripheral blood (right) one week after adoptive transfer of CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells into 5TGM1^hBCMA myeloma bearing mice. Bars represent mean ± SD, dots indicate individual mice (n = 3–6 mice per group). **p<0.01, ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test. (E) CD28 surface protein expression on BM-infiltrating CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells one week after adoptive transfer into 5TGM1^hBCMA myeloma bearing mice. (F) CAR T cell frequency assessed by flow cytometry in bone marrow (BM, left) and peripheral blood (right) one week after adoptive transfer of hBCMABBζ CAR T cells into MM.1S myeloma bearing mice ± abatacept. CAR T population identified by surface staining and analyzed by flow cytometry. Bars represent mean ± SD, dots indicate individual mice (n = 4–5 mice per group). ***p<0.001 by two-way ANOVA with Tukey’s multiple comparison test. (G) Representative IVIS bioluminescence images of T-lux luciferase expressing hBCMAmBBmζ CAR T cells ± abatacept (200μg, 3x/week) on day 7 or day 15 after infusion. (H) Quantification of photon flux by IVIS imaging within the hind limb region of interest (ROI) over a four-week period following T-lux hBCMAmBBmζ CAR T cell infusion ± abatacept (200μg, 3x/week) into 5TGM1^hBCMA myeloma bearing mice. Dots represent individual mice (n = 3 mice per group), lines are best-fit sigmoidal curves, and significance was determined by mixed effects modeling.

Figure 5:. Transient CD28 blockade limits inflammatory cytokines in the MM BME without affecting survival of hBCMAmBBmζ CAR T cell treated mice.

(A) Diagram of experimental setup. MM.1S bearing NSG mice were treated with hBCMABBζ CAR T cells ± 200μg abatacept on days −1, 1, 3, 5, 7 (5B and 5C) or 5TGM1^hBCMA bearing RAG2^−/− mice were treated with CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells (5D and 5E) and euthanized 7 days post adoptive transfer for hind limb BM harvest. (B) Myeloma burden assessed by flow cytometry for human CD138⁺ cells in the bone marrow of MM.1S bearing mice treated as described in 5A. Bars represent mean ± SD, dots indicate individual mice. **p<0.01 by one-way ANOVA, ns = not significant. (C) Heatmap representation of human cytokine levels in the MM BME 7 days after treatment of MM.1S bearing mice with hBCMABBζ CAR T cells ± abatacept. Bilateral hind limbs were harvested, and BM was flushed into 15 μL PBS for multiplexed cytokine analysis. Log₂ transformed cytokine concentrations represent the mean of 3–6 mice per group. (D) Heatmap representation of murine cytokine levels in the MM BME 7 days after treatment of 5TGM1^hBCMA bearing mice with hBCMAmBBmζ CAR T cells ± abatacept. Bilateral hind limbs were harvested, and BM was flushed into 15 μL PBS for multiplexed cytokine analysis. Log₂ transformed cytokine concentrations represent the mean of 2–3 mice per group. (E) Bar graphs showing concentrations of murine IP-10 (left) and IL-12 (right) in the MM BME 7 days after treatment of 5TGM1^hBCMA bearing mice with CD28^fl/fl hBCMAmBBmζ CAR T cells ± abatacept versus CD28^iKO hBCMAmBBmζ CAR T cells. Bars represent mean ± SD of 2–3 biological replicates. **p<0.01, ***p<0.001 by one-way ANOVA. (F) Diagram of experimental setup. MM.1S bearing NSG mice were treated with hBCMABBζ CAR T cells ± 200μg abatacept on days −1, 1, 3, 5, 7 and tumor burden was monitored by bioluminescent imaging 2x/week until endpoint. (G) Kaplan – Meier analysis of survival of hBCMABBζ CAR T ± transient abatacept or mock transduced T cell treated MM.1S-luc bearing mice (n = 4–5 mice per CAR T cell treated group). Median survival of hBCMABBζ CAR T treated mice was >100 days post CAR T cell infusion vs. 97 days for hBCMABBζ CAR T + transient abatacept treated mice. Statistical significance was determined by log-rank Mantel-Cox test.