Systematic preclinical evaluation of CD33-directed chimeric antigen receptor T cell immunotherapy for acute myeloid leukemia defines optimized construct design

Abstract

Background: Successful development of chimeric antigen receptor (CAR) T cell immunotherapy for children and adults with relapsed/refractory acute myeloid leukemia (AML) is highly desired given their poor clinical prognosis and frequent inability to achieve cure with conventional chemotherapy. Initial experiences with CD19 CAR T cell immunotherapy for patients with B-cell malignancies highlighted the critical impact of intracellular costimulatory domain selection (CD28 vs 4-1BB (CD137)) on CAR T cell expansion and in vivo persistence that may impact clinical outcomes. However, the impact of costimulatory domains on the efficacy of myeloid antigen-directed CAR T cell immunotherapy remains unknown.

Methods: In this preclinical study, we developed six CAR constructs targeting CD33, a highly expressed and validated AML target, comprised of one of three single-chain variable fragments with CD3ζ and either CD28 or 4-1BB costimulatory domains. We systematically compared the preclinical in vitro and in vivo efficacy of T cells lentivirally transduced with CD33 CAR constructs (CD33CARTs) against human AML.

Results: We observed potent in vitro cytokine production and cytotoxicity of CD33CARTs incubated with human CD33+ AML cell lines, as well as robust in vivo antileukemia activity in cell line and childhood AML patient-derived xenograft (PDX) models. Gemtuzumab-based CD33CARTs were unexpectedly toxic in vivo in animal models despite observed in vitro anti-leukemia activity. CD28-based CD33CARTs consistently induced more robust inhibition of leukemia proliferation in AML cell line and PDX models than did 4-1BB-based CD33CARTs. A 'best-in-class' lintuzumab-CD28/CD3ζ CAR construct was thus selected for clinical translation.

Conclusions: CD33 is a critical antigen for potential immunotherapeutic targeting in patients with AML. Based on this rigorous preclinical evaluation, our validated clinical grade lintuzumab-CD28/CD3ζ CD33CART immunotherapy is now under evaluation in a first-in-child/first-in-human phase 1 clinical trial for children and adolescents/young adults with relapsed/refractory AML.

Trial registration number: clinicaltrials.gov; NCT03971799.

Keywords: adoptive; chimeric antigen; hematologic neoplasms; immunotherapy; pediatrics; receptors; translational medical research.

Figure 1

CD33 chimeric antigen receptor construct (CD33CART) design and validation. (A) Schema of second-generation CD33-redirectedCAR constructs. Single-chain variable fragments (ScFvs) were derived from commercially available CD33 antibodies gemtuzumab ozogamicin (hP67.6; green), lintuzumab (SGN-33; light blue), and M195 (dark blue) and paired with a CD8 hinge or CD28 transmembrane (28tm) domain linker, 4-1BB or CD28 costimulatory endodomain, and a CD3ζ endodomain (designated BBz and 28z, respectively). CD123 CAR T cells with a 32 716 ScFv were used as a positive control for some studies. (B) Flow cytometry analysis of protein L expression demonstrating transduction efficiency of T cells transduced with gemtuzumab-based (gem), lintuzumab-based (lin), and M195-based CD33 CAR constructs or CD123 CAR constructs. (C) Flow cytometry quantification of CD33 site density (molecules/cell) on AML cell lines (MOLM14, MV4;11, THP-1, U937) and childhood acute myeloid leukemia (AML) patient-derived xenograft (PDX) models (JMML117, AML290) used for in vitro and/or in vivo testing of CD33CARTs. CD123 site density is shown as a comparator. The B-ALL cell line NALM-6 was used as a CD33-negative control.

Figure 2

Robust in vitro anti-leukemia activity of CD33 chimeric antigen receptor constructs (CD33CARTs). (A) CD33CART or CD123CART (positive acute myeloid leukemia (AML) killing control) cells were co-incubated with target-positive AML cells in vitro in a 1:1 ratio as indicated for 18 hours, and IL-2 and IFN-γ levels were quantified by ELISA in the culture supernatant. NALM-6 was again used as a negative control cell line. Experiments were performed in triplicate (technical replicates) using two normal T cell donors (biologic replicates). Data are displayed as mean±SD. (B) Cell-Titer Glo viability assays of human AML cell lines co-incubated with CD33CARTs or CD123CARTs in 1:1 effector: T cell show marked inhibition of cell growth over time with all tested CAR T cell products using two normal T cell donors. The B-ALL cell line NALM-6 was used as a CD33-negative control. T cell transduction efficiency ranged from 41% to 94% (mean 75%) depending on the CAR construct used. 28z=CD28/CD3ζ endodomains, BBz=4–1BB/CD3ζ endodomains. Cytokine and viability assays were performed in technical triplicates with biologic duplicates (T cell donor number 1 and number 2). Mean data are displayed for each condition with SD also shown for the viability assays. Data were analyzed by two-way ANOVA with multiple comparisons analysis of column means. All six CD33CARTs induced statistically significant killing of CD33 +AML cell lines at day 4 versus no T cell or GFP-transduced ((-)TC) negative controls (p<0.0001 for all comparisons), but were not statistically different from one another.

Figure 3

CD33 chimeric antigen receptor construct (CD33CART) inhibits acute myeloid leukemia (AML) proliferation in vivo. (A) Luciferase-transduced MOLM14 cells (1×10⁶) were injected intravenously via tail vein into NSG mice on day 0. Once engraftment was documented by bioluminescent imaging (BLI) on day 7, cohorts of three mice were randomized to intravenous treatment with saline, GFP-transduced T cells ((-)TC), or one of the CD33CARTs as designated (5×10⁶ total cells/mouse; red arrow). Mice were followed by weekly BLI and euthanized when a predetermined maximal radiance level of 1×10⁶ photons/s/cm²/sr was detected, indicative of leukemia progression. All three gemtuzumab-based (gem), lintuzumab-based (lin), and M195-based CD33CARTs demonstrated some anti-leukemia activity in vivo with complete inhibition of MOLM14 proliferation observed only in the 4-1BB/CD3ζ (BBz) construct-containing T cells versus partial activity with CD28/CD3ζ(28z) versions. (B) Graphic representation of summary BLI radiance data was performed in Prism. 28z, CD28/CD3ζ endodomains, BBz, 4–1BB/CD3ζ endodomains.

Figure 4

CD33 chimeric antigen receptor construct (CD33CART) inhibits leukemia proliferation in vivo in childhood acute myeloid leukemia (AML) patient-derived xenograft models. Busulfan-conditioned NSGS mice engrafted with primary AML cells (tertiary PDX models (A) JMML117 and (B) AML290) were randomized (n=5 mice/cohort) to intravenous treatment with saline, irrelevant target GFP-transduced T cells ((-)TC), or one of the CD33CARTs as designated (5×10⁶ total cells/mouse). CD33CART transduction efficiency ranged from 19.9% to 88.3% for these experiments with information detailed in online supplemental table 1. Mice were followed by weekly flow cytometric quantification of human CD33+/CD45 +AML and CD3+ CAR T cells in peripheral blood. Animals were euthanized at 2 or 4 weeks after treatment depending on rate of leukemia progression in control animals or >15% wt loss. AML cells and T cells were quantified in murine bone marrow and/or spleens as above. Superior leukemia clearance was again seen with the lintuzumab-based CD33CART with a CD28/CD3ζendodomain (lin-28z). *P<0.05, **p<0.01, ***p<0.001 by ANOVA with Dunnett post-test for multiple comparisons of treatment group versus saline control (black bars). Absence of symbol indicates lack of statistical significance.

Figure 5

In vivo exhaustion potential of lintuzumab-based CD33 chimeric antigen receptor constructs (CD33CARTs) in an acute myeloid leukemia (AML) xenograft model. (A) Luciferase-expressing MOLM14 xenograft mice were created as in figure 3. Cohort of five mice were treated with one 4×10⁶ dose of GFP-transduced T cells ((-)TC), lintuzumab-CD28/CD3ζ (lin-28z), or lintuzumab-4-1BB/CD3z (lin-BBz) CD33CART intravenous on day 4 (red arrow), then followed by bioluminescent imaging. CD33CART transduction efficiency ranged from 19.9% to 88.3% for these experiments with information detailed in online supplemental table 1. (B) Representative dot plots show flow cytometric detection of percentages of human CD3+ CD33 CART cells in harvested end-study murine spleens at day 26 following T cell treatment (upper panels). T cell quantification (lower panels) detected greater numbers in lin-BBz-treated versus lin-28z-treated animals. (C) Representative dot plots show flow cytometric analysis of CD4/CD8 T cell profiles (upper panels) with ratio calculation (lower panel) in end-study murine spleens. (D) Similar percentages of cell surface CD33 CAR positivity (bound to recombinant CD33-Fc fusion protein) were detected in CD4+ T cells, but not CD8+ T cells, when comparing lin-28z and lin-BBz CD33CARTs. (E) Naïve (N), effector memory (EM), and effector memory re-expressing CD45RA (TEMRA) human T cells in murine spleens at the day 26 end-study timepoint were further characterized by CCCR7+ CD45RA+, CCR7-CD45RA-, and CCR7-CD45RA+ flow cytometric profiling, respectively. Representative plots for CD4+ and CD8+CD33CARTs are shown (upper panels) and quantification of the different populations within the CD4+ (left) and CD8+ (right) subsets are shown. (F) Exhaustion phenotypes of CD33CARTs were evaluated as a function of PD1 and PD1/LAG3 expression profiles and representative dot plots are presented with %cells designated numerically for each gated quadrant (top). Quantification of PD1+ and PD1+/LAG3 +CD4+ and CD8+ T cells in murine spleens at the day 26 end-study timepoint are shown for lin-28z versus lin-4BBz CD33CART treatments. Paired data were analyzed by Student’s t-test with *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Figure 6

Minimal bystander toxicity of clinical CD33 chimeric antigen receptor construct (CD33CART) product. (A) 1×10⁵ GFP-transduced T cells ((-)TC) or clinical-grade lintuzumab-CD28/CD3ζ (lin-28z) cells were co-incubated in a 1:1 ratio with induced pluripotent stem cell lines of the designated normal tissue types for 18 hours. IFN-γ levels were quantified by ELISA in the culture supernatant as in figure 2. MOLM14 cells were used as a CD33 +positive control. No appreciable cytokine production was detected in any of the normal tissue conditions.

Figure 7

Preclinical validation of anti-leukemia activity of clinical-grade CD33 chimeric antigen receptor construct (CD33CART). The lintuzumab-based CD33CART with a CD28/CD3ζendodomain (lin-28z) was selected for further development and evaluation in a phase 1 clinical trial. (A) Transduction efficiency of clinical grade lin-28z CD33CART was 30.3% for this experiment. (B) Luciferase-transduced MOLM14 cells (1×10⁶) were injected intravenous into NSG mice on day 0. Animals were assessed by bioluminescent imaging (BLI) and then randomized to treatment with GFP-transduced T cells ((-)TC) or clinical-grade lin-28z CD33CART cells (5×10⁶ total cells/mouse) on day 3 (red arrow). Mice were followed by weekly BLI and euthanized when a predetermined maximal radiance level of 1×10¹⁰ photons/s/cm²/sr was detected Complete inhibition of leukemia proliferation was again observed with CD33CART.