Preclinical Efficacy of BCMA-Directed CAR T Cells Incorporating a Novel D Domain Antigen Recognition Domain

Abstract

Chimeric antigen receptor (CAR) T-cell therapies directed against B-cell maturation antigen (BCMA) have shown compelling clinical activity and manageable safety in subjects with relapsed and refractory multiple myeloma (RRMM). Prior reported CAR T cells have mostly used antibody fragments such as humanized or murine single-chain variable fragments or camelid heavy-chain antibody fragments as the antigen recognition motif. Herein, we describe the generation and preclinical evaluation of ddBCMA CAR, which uses a novel BCMA binding domain discovered from our D domain phage display libraries and incorporates a 4-1BB costimulatory motif and CD3-zeta T-cell activation domain. Preclinical in vitro studies of ddBCMA CAR T cells cocultured with BCMA-positive cell lines showed highly potent, dose-dependent measures of cytotoxicity, cytokine production, T-cell degranulation, and T-cell proliferation. In each assay, ddBCMA CAR performed as well as the BCMA-directed scFv-based C11D5.3 CAR. Furthermore, ddBCMA CAR T cells demonstrated in vivo tumor suppression in three disseminated BCMA-expressing tumor models in NSG-immunocompromised mice. On the basis of these promising preclinical data, CART-ddBCMA is being studied in a first-in-human phase I clinical study to assess the safety, pharmacokinetics, immunogenicity, efficacy, and duration of effect for patients with RRMM (NCT04155749).

©2022 The Authors; Published by the American Association for Cancer Research.

Figures

Figure 1.

Design and origin of ddBCMA CAR, a D domain–based chimeric antigen receptor. A, A structural model of α3D (PDB 2A3, ref. 25) with the 12 randomized positions of the D domain library highlighted in blue. B, To determine whether the epitope for ddBCMA overlaps with C11D5.3 scFv on BCMA, 10 nmol/L BCMA-His was captured on the NTA Chip followed by 100 nmol/L (green) or 0 nmol/L (black) of ddBMCA-Fc injection for 120 seconds, and then followed by a 120-second injection of 100 nmol/L C11D5.3 scFv-Fc. C, Map of ddBCMA CAR expression cassette cloned into a lentiviral transfer plasmid (SP, signal peptide; F, flag tag). D, Mock or ddBCMA CAR–transduced T cells were analyzed by flow cytometry for CAR expression (using APC-conjugated anti-FLAG antibody) and binding to FITC-labeled BCMA protein. E and F, Phenotyping is shown for three individual donor T-cell lots transduced with ddNEG CAR, ddBCMA CAR, or scFv CAR. Cells were stained for CD4, CD8, CD62L, and CD45RA. Naïve or stem cell memory (Naïve/TSCM; CD62L+/CD45RA+), central memory (TCM, CD62L+/CD45RA−), effector memory (TEM; CD62L−/CD45RA−), and TEMRA (CD62L−/CD45RA+) populations were quantified for the CD4+ (E) and CD8+ (F) populations. G, Cell surface expression (percentage of CAR) and geometric mean fluorescence of CAR+ cells normalized to ddNEG CAR expression are shown for paired samples of ddBCMA CAR and scFv CAR from 7 independent T-cell lots; ***, P < 0.001; **, P < 0.01; *, P < 0.05 by paired one-way ANOVA (E and F) or paired t-test (G).

Figure 2.

ddBCMA CAR induces T-cell signaling and lysis of a panel of BCMA-positive tumor cell lines. A, Jurkat reporter cells transfected with ddBCMA CAR or scFv CAR were cultured at a 1:1 ratio with the indicated cell lines for 5 hours, followed by luciferase substrate addition and measurement of luciferase activity. Average relative luminescence units (RLUs) signal is shown with individual data points (n = 2) from a representative experiment performed a total of four times. B, Cell lysis experiments were performed with titrated dilutions of ddNEG, ddBCMA, and scFv CAR transduced T cells cultured with 40,000 cells of the indicated cell lines. Calculated the percentage of lysis is shown as mean ± s.e.m. (n = 3). B, ***, P < 0.001; **, P < 0.01 for both ddBCMA CAR and scFv CAR compared with ddNEG CAR by two-way ANOVA.

Figure 3.

Measures of T-cell activation from ddBCMA CAR engagement with target cells. A and B, IFNγ and IL2 cytokines were measured from 16 hours cocultures of ddNEG, ddBCMA, or scFv CAR-transduced T cells and indicated cell lines at 1:1 E:T ratio. Cytokine concentration measurements are shown as mean with individual data points (n = 2) from a representative experiment repeated five times with three different donors. C, CD107a surface staining and D. intracellular IFNγ was measured from T cells after 5 hours of coculture with the indicated cell lines. Percentage of positive staining of the CD3+FLAG+ population is shown as average ± s.e.m. (n = 3) from a representative experiment repeated five times with two different donors. E, CD3+ T-cell counts were measured by flow cytometry after 4 days coculture with the indicated cell lines; shown as average ± s.e.m. (n = 3) from a representative experiment repeated twice. F, Binding of soluble BCMA (sBCMA) to the indicated T cells was assessed via flow cytometry G. 4-1BB and CD69 double-positive T cells were quantified by flow cytometry after incubation with H929 target cells or sBCMA, as shown (n = 3; representative of two experiments). H, Cell lysis experiments were performed with titrated T cells cultured in the presence of 40,000 H929 cells and the indicated concentration of sBCMA. The calculated percent lysis is shown as mean ± s.e.m. (n = 3). I and J, IFNγ and IL2 cytokines were measured from supernatants collected in H. Red dotted line indicates upper limit of quantification from standard curve for IL2 ELISA. Data in H–J are from a representative experiment repeated with three different donors. C–E, ***, P < 0.001 for indicated target cells compared with T cells alone or H929 BCMA–/– and compared with ddNEG CAR. G, ***, P < 0.001; **, P < 0.01; *, P < 0.05 by two-way ANOVA. H–J, ***, P < 0.001 comparing all ddBCMA CAR conditions with all ddNEG conditions; #, P < 0.05 for 0 versus 0.5 μg/mL sBCMA; $, P < 0.05 for 0 versus 1.5 μg/mL sBCMA. All statistics calculated by two-way ANOVA with Tukey's correction for multiple comparisons.

Figure 4.

Long-term in vitro expansion and in vivo efficacy of ddBCMA CAR T cells. A–C, ddNEG, ddBCMA, and scFv CAR T cells were subjected to four rounds of stimulation with H929 GFP/luc cells and assessed throughout for proliferation (A), cytotoxic function (B), and cytokine production (C). Data shown are calculated from triplicate cultures maintained in parallel and are representative of three independent experiments. D and E, Xenografts were established for 37 days in NSG mice by intravenously injection of 2 × 106 U266 cells, followed by single intravenously infusion of 1.5 × 106 CAR+ T cells (day 0). Tumor burden, as measured by luminescence signal, was monitored by IVIS imaging as shown in D and quantified in E. ***, P < 0.001; **, P < 0.01; *, P < 0.05 calculated by one-way ANOVA in A–C and two-way ANOVA in D and E with Tukey's correction for multiple comparisons.

Figure 5.

ddBCMA CAR T-cell in vivo activity is dose dependent. Xenografts were established in NSG mice for 23 days following intravenous injection of 2 × 106 MM.1S cells, followed by single intravenous infusion of CAR+ T cells (day 0). Tumor burden, as measured by luminescence signal, was monitored by IVIS imaging as shown in A and quantified in B. B, Statistical significance (P < 0.05) is shown for comparison with HBSS (*), ddNEG CAR ($), and 0.5 × 106 ddBCMA CAR treatment groups (#). Circulating cells isolated from blood on day 14 after CAR T-cell infusion were analyzed for total GFP+ (i.e., circulating tumor cells) count (C), CAR+ cell count (D), percentage of CAR positive in the hCD45+/CD3+ population (E), and for CD4:CD8 ratio (F). C–F, All quantification is representative of data gathered from 150 μL of a total volume of 175 μL and are shown as average ± s.e.m. (n = 4–5). *, P < 0.05; **, P < 0.01; ***, P < 0.001, for indicated comparisons calculated by one-way ANOVA.