Developing a membrane-proximal CD33-targeting CAR T cell

Abstract

Background: CD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking.

Methods: We developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR T cell derived from a high-affinity binder obtained through membrane-proximal fragment immunization in humanized mice.

Results: We found that immunization exclusively with the membrane-proximal domain of CD33 is necessary for identification of membrane-proximal binders in humanized mice. Compared with clinically validated lintuzumab-based CAR T cells targeting distal CD33 epitopes, 3P14HLh28Z showed enhanced in vitro functionality as well as superior tumor control and increased overall survival in both low antigen density and clinically relevant patient-derived xenograft models. Increased activation and enhanced polyfunctionality led to enhanced efficacy.

Conclusions: Showing for the first time that a membrane-proximal CAR is superior to a membrane-distal one in the setting of CD33 targeting, our results demonstrate the rationale for targeting membrane-proximal epitopes with high-affinity binders. We also demonstrate the importance of optimizing CAR T cells for functionality in settings of both low antigen density and clinically relevant patient-derived models.

Keywords: Adoptive cell therapy - ACT; Chimeric antigen receptor - CAR; Immunotherapy; Leukemia; T cell.

Figure 1

Identification of a membrane-proximal CD33 antibody. (A) Heatmap showing relative binding of selected antibodies to human full-length CD33, CD33-IgC, and U937 CD33-expressing tumor lines. (B) Schematic representation of retroviral vector encoding 5′ and 3′ long terminal repeats sequence (LTR), truncated EGFR, linked with a T2A element to a fully human CD33-targeted single-chain variable fragment, human CD28 hinge, transmembrane, and intracellular domains (CD28), and human zeta chain signaling domain (z chain). A Myc-tag is included for detection of the chimeric antigen receptor (CAR). (C) Summary data of retroviral transduction efficiency of CAR constructs as determined by flow cytometry using cetuximab. P values determined by repeated measures one-way analysis of variance. Data are a mean±SEM of three biological replicates conducted in three independent experiments; ns, non-significant. (D) Representative flow cytometry plot demonstrating CAR expression following human T cell transduction, detected with fluorescently labeled Cetuximab and Myc-Tag-specific antibodies. MFI, mean fluorescence intensity.

Figure 2

Membrane-proximal targeting chimeric antigen receptor (CAR) T cells demonstrate enhanced functionality and proliferative capacity in vitro. (A) Flow cytometry histograms of CD33 expression on acute myeloid leukemia (AML) cells detected with isotype control or fluorescently labeled CD33-specific antibody. (B) Quantitative geometric mean fluorescence intensity (MFI) of CD33 expression on AML cells detected with either isotype control or fluorescently labeled CD33-specific antibody. (C) 24-hour D-luciferin assay demonstrating lysis of CD33-expressing tumor cells (n=4 biological replicates; ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05 by two-way analysis of variance (ANOVA)). Data are a mean±SEM of four biological replicates. (D) 96-hour D-luciferin assay demonstrating lysis of CD33 expressing tumor cells (n=4 biological replicates; ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05 by two-way ANOVA). Data are a mean±SEM of four biological replicates. (E) Quantification of flow cytometric analysis demonstrating enhanced proliferation by membrane-proximal CD33 targeting CAR T cell in the presence of U937-CD33^high tumor cells at E:T ratios of 1:2.5 (left) or 1:10 (right) (n=3 biological replicates; ****p<0.0001 by two-way ANOVA at day 21). Arrows indicate when additional target cells were added. Data are a mean±SEM of three biological replicates.

Figure 3

Membrane-proximal targeting chimeric antigen receptor (CAR) T cells are characterized by a unique activation profile in vitro. (A) 24-hour cytokine secretion profile of Tc1/Th1 cytokines when co-cultured with U937-CD33^high, OCiAML3-CD33^low, U937-CD33^IgC, and U937-CD33^KO tumor as detected by human 12-plex Luminex panel (n=3; ****p<0.0001; *p<0.05 by Dunnett’s multiple comparison tests against H195DEL control). Data are a mean±SEM of three biological replicates conducted in three independent experiments. (B) Quantification of flow cytometric analysis showing CAR T cell intracellular production of Tc1/Th1 activation cytokines when cultured with U937-CD33^high tumor (n=4; ****p<0.0001; **p<0.01; *p<0.05 by two-way analysis of variance (ANOVA); ns, non-significant). Data are a mean±SEM of four biological replicates conducted in three independent experiments. (C) Qualitative representation of CD4⁺CAR⁺ and CD8⁺CAR⁺ Tc1/Th1 activation cytokines secretion. Cytokines evaluated were IFN-γ, IL-2, granzyme B, and TNF-α. (D) Quantification of flow cytometric analysis showing CD4⁺ CAR T cell intracellular production of Tc1/Th1 activation cytokines when cultured with U937-CD33^high tumor (n=4; ****p<0.0001, *p<0.05 by two-way ANOVA). Data are a mean±SEM of four biological replicates conducted in three independent experiments. Cytokines evaluated were IFN-γ, IL-2, granzyme B, and TNF-α. (E) Quantification of flow cytometric analysis showing CD8⁺ CAR T cell intracellular production of Tc1/Th1 activation cytokines when cultured with U937-CD33^high tumor (n=4; ***p<0.001; **p<0.01; *p<0.05 by two-way ANOVA). Data are a mean±SEM of four biological replicates conducted in three independent experiments. Cytokines evaluated were IFN-γ, IL-2, granzyme B, and TNF-α. (F) Quantification of flow cytometric analysis demonstrating CAR T cell activation status 7 days post-antigen stimulation with U937-CD33^high tumor (n=4; ****p<0.0001 by two-way ANOVA). Data are a mean±SEM of four biological replicates conducted in three independent experiments. (G) Qualitative representation of flow cytometric analysis comparing CAR T cell activation profiles. Data are pooled from four biological replicates conducted in three independent experiments.

Figure 4

Membrane-proximal CD33-targeting chimeric antigen receptor (CAR) T cells provide enhanced survival in xenograft mouse model. (A) Schematic diagram of in vivo experimental setup. NCG mice were inoculated with U937-CD33^high tumor and subsequently treated with CAR T cells. (B) Survival of NCG mice bearing U937-CD33^high tumors and treated with titrated doses of CAR T cells (n=5; **p<0.01, *p<0.05). P values for survival determined by log-rank Mantel-Cox test, with 95% CI. (C) Tumor regression of NCG mice inoculated with U937-CD33^high tumor and subsequently treated with 5.0×10⁵ of CAR T cells. (D) Bioluminescence over time of U937-CD33^high in tumor-bearing NCG mice treated with 5.0×10⁵ CAR T cells. (E) Schematic diagram of in vivo experimental setup. NCG mice were inoculated with OCiAML3-CD33^low tumor and subsequently treated with CAR T cells. (F) Survival of NCG mice inoculated with OCiAML3-CD33^low tumors and treated with 5.0×10⁵ CAR T cells (n=5; *p<0.05). P values for survival determined by log-rank Mantel-Cox test, with 95% CI. (G) Tumor regression of NCG mice bearing OCiAML3-CD33^low tumors and treated with 5.0×10⁵ CAR T cells. (H) Bioluminescence over time of OCiAML3-CD33^low in tumor-bearing NCG mice treated with 5.0×10⁵ CAR T cells.

Figure 5

Membrane-proximal CD33-targeting chimeric antigen receptor (CAR) T cells decrease tumor burden in patient-derived acute myeloid leukemia (AML) xenograft model. (A) Flow cytometry histograms of CD33 expression on AML60B patient sample detected with isotype control or fluorescently labeled CD33-spcific antibody. (B) Quantitative geometric mean fluorescence intensity (MFI) of CD33 expression on AML60B patient sample detected with either isotype control or fluorescently labeled CD33-specific antibody. (C) Schematic diagram of experimental setup of a patient-derived xenograft model. NCG mice were inoculated with patient-derived AML blasts and treated with allogeneic CAR T cells. Bone marrow aspirates were analyzed 28 days post tumor inoculation. (D) Quantification of flow cytometric analysis demonstrating decreased tumor burden in mice treated with membrane-proximal CD33-targeting CAR T cells (n=6; *p<0.05 by unpaired t-test). (E) Survival of NCG mice-bearing AML60B patient-derived tumor and treated with membrane-distal or membrane-proximal CAR T cells (n=6; **p<0.01; *p<0.05). P values for survival determined by log-rank Mantel-Cox test, with 95% CI.