Preclinical Evaluation of B7-H3-Specific Chimeric Antigen Receptor T cells for the Treatment of Acute Myeloid Leukemia

Abstract

Purpose:

The development of safe and effective CAR T cell therapy for acute myeloid leukemia (AML) has largely been limited by the concomitant expression of most AML-associated surface antigens on normal myeloid progenitors and by the potential prolonged disruption of normal hematopoiesis by the immunotargeting of these antigens. The purpose of this study was to evaluate B7-H3 (B7-homolog 3) as a potential target for AML-directed CAR T cell therapy. B7-H3, a coreceptor belonging to the B7 family of immune checkpoint molecules, is overexpressed on the leukemic blasts of a significant subset of patients with AML and may overcome these limitations as a potential target antigen for AML-directed CAR-T therapy.

Experimental Design:

B7-H3 expression was evaluated on AML cell lines, primary AML blasts, and normal bone marrow progenitor populations. The anti-leukemia efficacy of B7-H3-specific CAR T cells (B7-H3.CAR-Ts) was evaluated using in vitro coculture models and xenograft models of disseminated AML, including patient-derived xenograft models. The potential hematopoietic toxicity of B7-H3.CAR-Ts was evaluated in vitro using colony formation assays and in vivo in a humanized mouse model.

Results:

B7-H3 is expressed on monocytic AML cell lines and on primary AML blasts from patients with monocytic AML, but is not significantly expressed on normal bone marrow progenitor populations. B7-H3.CAR-Ts exhibit efficient antigen-dependent cytotoxicity in vitro and in xenograft models of AML, and are unlikely to cause unacceptable hematopoietic toxicity.

Conclusions:

B7-H3 is a promising target for AML-directed CAR-T therapy. B7-H3.CAR-Ts control AML and have a favorable safety profile in preclinical models.

INTRODUCTION

Acute myeloid leukemia (AML) accounts for approximately 80% acute leukemias in adults and has an incidence of at least 20,000 cases per year in the United States (1). Despite advances in risk stratification, optimization of chemotherapy regimens, improvement in stem cell transplantation techniques, and the advent of multiple targeted therapies, prognosis remains poor. These clinical findings emphasize the need for novel treatment strategies (1). Adoptive cellular therapy with chimeric antigen receptors (CAR) T cells has emerged as a highly effective form of immunotherapy for B-cell acute lymphoblastic leukemia (ALL) and certain other B-cell malignancies (2); however, safe and effective CAR T cell therapy for AML remains elusive. The success of CAR T cell (CAR-T) therapy in B-cell malignancies is predicated on the tolerability of the protracted B-cell aplasia that often accompanies CD19-directed CAR-T therapy. In contrast, prolonged disruption of normal myelopoiesis (especially neutropenia) would not be tolerable, and the development of CAR-T therapy for AML has been largely limited by the fact that most AML-associated surface antigens are also expressed on normal myeloid progenitor cells (3).

While it is difficult to envision the identification of cell surface antigens that are absent on all normal myeloid progenitors and yet expressed on all subtypes of AML, it seems plausible that some antigens absent on early myeloid lineage cells may be preferentially overexpressed in certain AML subtypes. We have identified B7-H3 as one such candidates. B7-H3 (B7-homolog 3, or CD276) is a coreceptor belonging to the B7 family of immune checkpoint molecules. Its structure is that of a 110-kDa type I transmembrane protein with four Ig-like domains, a transmembrane domain, and a short cytoplasmic tail (4). The receptor for B7-H3 has yet to be definitively identified (5,6), and the nature of its signaling also remains controversial (7). Although B7-H3 mRNA is expressed in many normal tissues, corresponding protein expression is limited (7). In multiple human cancers, however, B7-H3 protein is overexpressed. Examples include various solid tumors (glioblastoma, lung, breast, colon, pancreatic, renal, ovarian, prostate, and melanoma) as well as a subset of patients with AML (7,8). B7-H3 expression in AML appears to be higher in AML with a monocytic immunophenotype (8), which is often associated with aggressive clinical features (9,10). To evaluate the potential safety of B7-H3 as a target for CAR-T therapy, we have previously performed a comprehensive evaluation of B7-H3 expression in normal human tissues and demonstrated a lack of major organ toxicity in a syngeneic tumor model (11). We describe here the pre-clinical assessment of B7-H3.CAR-Ts as a possible therapy for a subset of patients with AML and our evaluation of the potential hematopoietic toxicity of this approach.

METHODS

Cell lines and human samples.

De-identified cryopreserved samples of peripheral blood and bone marrow aspirate from patients with newly diagnosed AML were obtained through the UNC Tissue Procurement Facility (UNC Hospital, Chapel Hill, NC). Primary AML blasts for use in patient-derived xenograft (PDX) models were obtained from the Public Repository of Xenografts (PRoXe, Dana-Farber Cancer Institute, Boston, MA). See supplemental methods for additional details, descriptions of other cell lines used, and tissue culture conditions.

CFSE assays.

T cells were labeled with 1.5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) as previously described (12), followed by coculture with irradiated B7-H3⁺ or B7-H3^- cell lines. After 5-days, T cell proliferation was assessed via flow cytometry analysis.

Generation of retroviral vectors and activated B7-H3.CAR-Ts.

The B7-H3-specific single-chain variable fragment from the murine monoclonal antibody, 376.96 (13), was cloned from the 376.96 mouse hybridoma as previously described (11). Generation of retroviral vectors and activated B7-H3.CAR-Ts has been previously described (11,14,15) and is summarized further in the supplemental methods.

Flow cytometry.

Flow cytometry data acquisition was performed using a BD FACSCanto II or BD FACSFortessa flow cytometer and BD Diva software (BD Biosciences). Data analysis was performed using FlowJo software versions 9.3 and 10.5 (Treestar, Ashland, OR). See supplemental methods for a list of monoclonal antibodies used.

In vitro analysis of B7-H3.CAR-T activity against AML cell lines.

The activity of B7-H3.CAR-Ts was evaluated in coculture models with B7-H3⁺ AML cell lines or primary patient-derived monocytic AML blasts. For cell lines, 0.5 × 10⁶ tumor cells were plated in a 24-well plate in 2 mL of media (without any exogenous cytokines added). For primary AML blasts, 1 × 10⁶ cells from cryopreserved bone marrow aspirates were plated in a 24-well plate. Control T cells or B7-H3.CAR-Ts were then added at a 1:5 effector to target (E:T) ratio. Twenty-four hours later, 500μL of co-culture supernatant was collected and the concentration of interferon-γ and interleukin-2 was measured by ELISA (R&D Systems, Minneapolis, MN). On day 4–7 of co-culture, cells were collected, and the percentage of T cells and residual tumor cells was assessed via flow cytometry. Monocytic cell lines and primary AML blasts were identified via staining for CD33 and T cells via staining for CD3.

Colony formation assay of normal hematopoietic progenitors.

CD34⁺ cells were isolated via magnetic microbead (Miltenyi Biotec, Auburn, CA) selection from umbilical cord blood units obtained from healthy donors. CD34⁺ cells were coincubated with B7-H3.CAR-28, CD19.CAR-28, or control T cells (derived from donor PBMCs) at an E:T ratio of 10:1 (T cell concentration of 5×10⁵/mL) for 6-hours after which the coculture was diluted 1:11 (without depletion of T cells) in methylcellulose-containing medium supplemented with recombinant cytokines (MethoCult H4434 Classic, STEMCELL Technologies) and plated in duplicate as previously described (16). After two weeks in culture, granulocyte-macrophage progenitor (CFU-GM) and common myeloid progenitor (CFU-GEMM) colonies were scored in a blinded fashion using a high-quality inverted microscope.

Xenograft mouse models using AML cell lines.

For mouse xenograft models, monocytic AML cell lines (THP1 and OCI-AML2) were transduced with a retroviral vector encoding the eGFP firefly luciferase (eGFP-FFluc) gene (17). For the THP1 model, a single-cell clone was first selected based on high eGFP expression and high in vitro firefly luciferase (FFluc) activity (18). eGFP-FFluc-THP1 or FFluc-OCI-AML2 cells were injected into the lateral tail vein of 6- to 8-week-old female NOD scid IL-2Rγ^−/− (NSG) mice. Seven days later, mice were injected intravenously with CAR T cells. Further details of each experiment are described below. Tumor growth was monitored via bioluminescence imaging using an IVIS lumina II in vivo imaging system (PerkinElmer) (16). Mice were monitored and euthanized according to UNC-IACUC standards and/or when the luciferase signal (total flux) reached a pre-determined threshold (1 × 10¹⁰ photos/sec for eGFP-FFLuc-OCI-AML2 model and 1 × 10¹¹ photos/sec for eGFP-FFluc-THP1 model due increased FFluc intensity compared with eGFP-FFLuc-OCI-AML2).

Patient-derived xenograft models.

Patient-derived AML blasts were obtained from PRoXe (samples DFAM-6855 and CBAM-44728) and expanded in vivo in NSG mice. For the first model (DFAM-6855) AML blasts were injected intravenously into 6- to 8-week-old female NSG mice and engraftment was monitored via weekly flow cytometry analysis of peripheral blood CD45⁺CD33⁺ cells. When the majority of mice had >20% peripheral blood AML blasts, they were randomized and injected intravenously with either B7-H3.CAR-28 or control T cells. Eight days later, all mice were sacrificed and flow cytometry was used to quantify AML blasts and T cells in blood, bone marrow, and spleen. In an additional experiment, blasts were retrovirally transduced to express the fusion protein eGFP-FFLuc, expanded in vivo in NSG mice, sorted by flow cytometry based on eGFP expression, expanded again in vivo, and then similarly injected into female NSG mice. After 4-days, mice were randomized and injected intravenously with either B7-H3.CAR-28 or CD19.CAR-28 T cells. Tumor growth was assessed via bioluminescence imaging using an IVIS lumina II in vivo imaging system (PerkinElmer) (16) unless otherwise indicated. Mice were monitored and euthanized according to UNC-IACUC standards. In a second model (CBAM-44728), AML blasts were expanded in vivo and then injected intravenously in 6- to 8-week-old female NSG mice. After 4-days, mice were injected intravenously with either B7-H3.CAR-28 or CD19.CAR-28 T cells. All mice were sacrificed on day 63 after T cell injection and flow cytometry analysis was used to quantify AML blasts and CAR-Ts in the bone marrow, spleen, and blood.

Humanized mouse models.

Hematopoietic stem cells were isolated from human fetal liver tissue specimens (obtained from medically indicated or elective pregnancy termination) and were injected intrahepatically into 2- to 6-day-old NSG mice (19). Engraftment of human leukocytes was monitored in the peripheral blood beginning at 6 weeks. Following reconstitution of 40–60% human leukocytes, two mice were sacrificed and magnetic microbeads (Miltenyi Biotec) were used to isolate human T cells (CD4⁺ or CD8⁺ cells) from the murine spleens. After activation with CD3 and CD28 antibodies, a subset of T cells was retrovirally transduced to generate B7-H3.CARs as described above. B7-H3.CAR-28 or control T cells were then injected intravenously into the remaining mice at days 0 and 16. After the initial T cell injection, mice were monitored regularly for the development of graft versus host disease (GvHD) or systemic inflammatory response syndrome (SIRS). Levels of circulating human CD45⁺CD3⁺, CD45⁺CD19⁺, CD45⁺CD33⁺, and CD45⁺CD14⁺ cells were monitored via flow cytometry on a weekly basis. Mice were sacrificed on day 40 and flow cytometry was used to evaluate these populations in the bone marrow and spleen, as well as levels of human CD34⁺CD38⁺ and CD34⁺CD38^- cells in the bone marrow.

RESULTS

B7-H3 is expressed on monocytic AML cell lines and expression compares favorably to other potential targets for AML-directed CAR-T therapy.

Human monocytic AML cell lines (THP1, U937, OCI-AML2, and OCI-AML3) were stained with the B7-H3-specific mAbs, 376.96 and 7–517, demonstrating high expression of B7-H3 (Fig. 1A). In addition to staining with the B7-H3-specific mAb 7–517, the same cell lines, as well as a B7-H3^- acute promyelocytic leukemia cell-line (HL-60), and healthy donor peripheral blood monocytes (CD3^-CD19^-CD20^-CD56^-HLA-DR⁺CD14⁺) were stained with mAbs specific for other recently identified candidate surface antigens for AML-directed CAR-T therapy (ADGRE2, CCR1, CD70, LILRB2, and CLEC12A) (20). As shown in Fig. 1B, the lack of B7-H3 expression on normal human monocytes and high expression on monocytic AML cell lines is similar to the expression pattern of CD70, whereas ADGRE2, CCR1, LILRB2, and CLEC12A were all found to be expressed on normal monocytes and had variable expression on monocytic AML cell lines.

Figure 1. B7-H3 is expressed on monocytic AML cell lines and primary AML blasts from patients with monocytic AML, but not on bone marrow progenitor cells.

(A) Expression of B7-H3 in four human monocytic AML cell lines stained with the 376.96 or 7–517 mAbs (black lines) versus isotype control (gray fill) as assessed by flow cytometry. (B) Expression of B7-H3 and other potential target antigens for AML-directed CAR-T therapy (20) as assessed by flow cytometry, on five human AML cell lines and on classical CD14⁺CD16^– peripheral blood monocytes from normal donors. (C) Representative flow cytometry plots demonstrating B7-H3 expression on primary AML blasts. (D) B7-H3 expression on primary AML blasts from patients (n=10) with monocytic AML stained with 376.96 or 7–517 mAbs (boxplot indicates median and interquartile range). (E) Expression of monocytic and myeloid markers (as assessed by flow cytometry) on the AML blasts and lymphocytes from the same cohort of patient samples. (F,G) Expression of B7-H3 as assessed by flow cytometry staining with 7–517 mAb on the indicated hematopoietic progenitor populations in normal bone marrow. (F) Gating strategy. (G) Staining in the indicated populations with B7-H3 mAb 7–517 (black) versus isotype control (gray fill). (H,I) Expression of B7-H3 as assessed by flow cytometry staining with mAb 376.96 on the indicated hematopoietic progenitor populations in normal bone marrow. (H) Gating strategy. (I) Staining in the indicated populations with B7-H3 mAb 376.96 (black) versus secondary antibody alone (gray fill). THP1 is shown as positive staining control. MEP, megakaryocyte-erythroid progenitors. CMP, common myeloid progenitors. GMP, granulocyte-macrophage progenitors. HSC, hematopoietic stem cells. MPP, multipotent progenitor cells. Error bars indicate standard error of the mean (SEM).

B7-H3 is expressed on the surface of primary AML blasts from patients with monocytic AML but not in normal bone marrow progenitor populations.

We obtained deidentified cryopreserved pre-treatment bone marrow aspirates (n=10) from patients with monocytic AML (see Fig. 1E for expression of myeloid and monocytic markers and see Suppl. Table 1 for clinical annotation associated with these samples) and demonstrated that blast populations (identified based on CD45-SSC gating, Fig. 1C) had positive staining with the B7-H3-specific mAbs, 376.96 and 7–517 (Fig. 1D). B7-H3 expression varied across samples, with some samples displaying uniformly high B7-H3 expression.

With the caveat that B7-H3 is likely regulated at the post-transcriptional level (7), we also analyzed a previously published RNA-seq dataset (21) including 200 patients with untreated, de novo AML. B7-H3 expression was higher in patients with monocytic AML (French-American-British [FAB] M5) (p<0.0001) and patients with acute promyelocytic leukemia (FAB M3 or documented PML-RARA fusion; p<0.0001 and p=0.0004, respectively), whereas B7-H3 expression was lower in FAB M2 AML (p<0.0001) (Suppl. Fig 1A,C…D). B7-H3 expression was higher in patients with mutations in NPM1 (p=0.0224), and lower in patients with mutations in IDH1/IDH2 (p=0.0460), CEBPA (p<0.0001), and WT1 (p=0.0001) (Suppl. Fig 1E). There was also a positive correlation of B7-H3 expression with CD33 (Suppl. Fig 1F) and CD11b (Suppl. Fig 1G) expression, and a negative correlation between the expression of B7-H3 and CD34 (Suppl. Fig 1H). When stratified by B7-H3 Z-score of <0 and ≥0, the median overall survival (mOS) was 26.3 and 11.8 months, respectively, although the difference was not significant (p=0.2005) (Suppl. Fig 1I). When acute promyelocytic leukemia was excluded, mOS was 21.5 and 9.9 months (p=0.0432) (Suppl. Fig 1J). In a separate dataset including samples from 562 patients with both newly diagnosed and previously treated AML (22) we again found significantly higher B7-H3 expression in patients with FAB M3 (p=0.0026) and M5 (p=0.0119) AML, and lower expression in FAB M2 AML (p=0.0018) (Suppl. Fig 1K). In the same dataset (22), we found higher B7-H3 expression in patients with relapsed versus untreated AML (p=0.0281) (Suppl. Fig 1L).

We also evaluated the expression of B7-H3 on normal myeloid progenitor populations using cryopreserved normal bone marrow aspirates. Bone marrow aspirates were analyzed by flow cytometry using the gating strategy shown in Fig. 1F to identify the following hematopoietic progenitor populations: megakaryocyte-erythroid progenitors (MEPs^-), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), hematopoietic stem cells (HSCs), and multipotent progenitor cells (MPPs). We demonstrated no significant staining with the B7-H3-specific mAb 7–517 on any of these populations (Fig 1G). Similarly, using the gating strategy shown in Fig 1H, we evaluated the CD45⁺Lineage^-CD34⁺CD38⁺ and CD45⁺Lineage^-CD34⁺CD38^- populations, and demonstrated no significant staining with the B7-H3-specific mAb 376.96 (Fig 1I).

B7-H3.CAR-Ts target B7-H3⁺ AML cell lines and primary AML samples.

Utilizing the single chain variable fragment (scFv) from the B7-H3 mAb 376.96 (13,23), we previously generated retroviral constructs encoding B7-H3.CAR-28 (incorporating the CD28 endodomain) and B7-H3.CAR-BB (incorporating the 4–1BB endodomain) (11). Following activation with CD3/CD28, donor peripheral blood mononuclear cells (PBMCs) were retrovirally transduced with the constructs above to generate B7-H3.CAR-Ts (Fig. 2A). CD19.CAR-28-Ts were used as a control. Median (range) transduction efficiencies of B7-H3.CAR-28, B7-H3.CAR-BB, and CD19.CAR-28 among eight representative donors were 77% (66–87%), 70% (55–87%), and 73% (65–84%), respectively (Fig. 2B). The anti-leukemia efficacy of B7-H3.CAR-Ts was evaluated in co-culture assays. B7-H3.CAR-Ts, but not control T cells, eliminated B7-H3⁺ cell lines (OCI-AML2, OCI-AML3, THP1, and U937) (Fig. 2C,D). In the same experiments, we demonstrated significant levels of interferon-γ (IFNγ) (Fig. 2E) and interleukin-2 (IL2) (Fig. 2F) in the media harvested, following a 24-hour incubation, from co-cultures with B7-H3.CAR-Ts, but not control T cells. To further assess antigen specificity of B7-H3.CAR-Ts, we stained B7-H3.CAR-Ts with CFSE and demonstrated B7-H3-specific proliferation of B7-H3.CAR-Ts (but not control T cells) in coculture with B7-H3-positive cell lines (THP1, U937) but not B7-H3-negative cell lines (Kasumi, HL60) (Fig. 2G).

Figure 2. B7-H3.CAR-Ts target B7-H3⁺ AML cell lines.

(A) Representative flow cytometry histograms indicating transduction efficiency of donor T cells with the indicated CAR constructs (empty fill) compared with non-transduced T cells (gray fill). (B) Transduction efficiencies of donor T cells (n=8) with each of the indicated CAR constructs. (C) Representative flow cytometry plots of OCI-AML2, OCI-AML3, THP1, or U937 cell lines (CD3^-CD33⁺) co-cultured with B7-H3.CAR-Ts or control T cells (CD3⁺CD33^-), and (D) percent CD33⁺ tumor cells after 5–7 days in co-culture (n=3–5 donors). For all cell lines, p<0.0001 for comparison of B7-H3.CAR-Ts to control T cells (one-way ANOVA with Holm-Sidak test adjusted p value). (E,F) Summary of the concentration of IFNγ (E) and IL2 (F) in the co-culture media after 24 hours as measured by ELISA (n=3–5). (G) Representative CFSE dilution assay demonstrating proliferation of B7-H3.CAR-Ts (dashed lines), compared to control T cells (solid lines with gray fill), when co-cultured with THP1 or U937 cells (B7-H3⁺). No significant proliferation was observed when T cells were co-cultured with Kasumi or HL60 cells (B7-H3^-). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Holm-Sidak test adjusted p value. Error bars indicate SEM.

To assess the activity of B7-H3.CAR-Ts against primary AML blasts, we co-cultured B7-H3.CAR-Ts derived from healthy donors with primary AML blasts obtained from patients with monocytic AML. When co-cultured at a 1:5 effector:target ratio for 48 hours, B7-H3.CAR-Ts but not control T cells, displayed significant cytotoxicity against primary monocytic AML blasts (Fig. 3A,B). We also demonstrated significant release of IFNγ and IL2 by B7-H3.CAR-Ts as measured in the media harvested from co-cultures following a 24-hour incubation (Fig. 3C,D). The B7-H3 expression for the same patient samples is shown in Fig 1D. B7-H3.CAR-Ts appeared to have more cytotoxic activity against AML blasts with higher B7-H3 expression, although this difference was not statistically significant (Suppl. Fig 3). Given the shorter incubation time for these co-cultures as compared with the co-cultures with AML cell lines shown in Fig. 2C…F, we demonstrated that under co-culture conditions comparable to those used in Fig. 3A…D and Fig. 3H…K, healthy donor-derived B7-H3.CAR-Ts (n=3) exhibited similar cytotoxicity (p<0.0001) against the AML cell line, THP1 (Fig. 3M).

Figure 3. B7-H3.CAR-Ts target primary AML blasts from patients with monocytic AML.

(A) Representative flow cytometry plots of primary AML blasts (CD3^-CD33⁺) co-cultured with donor-derived B7-H3.CAR-Ts or control T cells (CD3⁺CD33^-). (B) Percent CD33⁺ cells after 48-hours in co-culture of primary AML blasts (n=10) with donor-derived (n=2–3 donors) B7-H3.CAR-Ts or control T cells (p<0.0001 for one-way ANOVA of means of replicates; p<0.0001 for comparisons of both B7-H3.CAR-28 and B7-H3.CAR-BB to control T cells using Holm-Sidak’s multiple comparison test). (C, D) Summary of the concentration of IFNγ (C) (p=0.0059, one-way ANOVA of means of replicates; p=0.0206 comparison of B7-H3.CAR-28 to control T cells and p=0.0082 for comparison of B7-H3.CAR-BB to control T cells, Holm-Sidak’s multiple comparison test) and IL2 (D) (p=0.0031, one way ANOVA of means of replicates; p=0.0156 for comparison of B7-H3.CAR-28 to control T cells and p=0.0064 for comparison of B7-H3.CAR-BB to control T cells, Holm-Sidak test adjusted p value) in the co-culture media after 24 hours as measured by ELISA (n=10 patient samples, T cells from 2–3 donors). (E) B7-H3 expression, based on staining with the B7-H3 mAbs 376.96 (performed for 3 of the 4 samples) and 7–517, on primary AML blasts used for autologous cocultures summarized in panels H-K. (F) Representative flow cytometry histograms (data included in panel G) indicating transduction efficiency of bone marrow-derived T cells (from AML patients) with the indicated CAR constructs (empty fill) compared with non-transduced T cells (gray fill). (G) Transduction efficiencies of patient bone-marrow-derived T cells used in panels H-L. (H) Representative flow cytometry plots of primary AML blasts (data included in panel I) co-cultured with autologous patient-derived B7-H3.CAR-Ts, CD19.CAR-Ts, or control T cells (CD3⁺CD33^-). Coculture of OCI-AML3 cells with the same CAR T cells is shown as a control. (I) Percent CD33⁺ cells after 48-hours in co-culture of primary AML blasts (n=4) with autologous B7-H3.CAR-Ts or control T cells. (J,K.) IFNγ (J) and IL2 (K) in the media of the co-culture experiments shown in H-I, as measured by ELISA after 24 hours (n=4, p-values as shown). (L) Percent CD33⁺ cells after co-culture of U937 cells (CD33⁺) with AML patient-derived (n=3) B7-H3.CAR-Ts or control T cells for 48 hours. (M) Percent CD33⁺ cells after co-culture of THP1 cells (CD33⁺) with healthy donor-derived B7-H3.CAR or control T cells for 48-hours. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Holm-Sidak test adjusted p value. Error bars indicate SEM.

We utilized cryopreserved bone marrow aspirates from patients (n=4) with AML with variable B7-H3 expression (Fig. 3E) to generate B7-H3.CAR-Ts and activated control T cells. Median (range) transduction efficiencies of B7-H3.CAR-28 and B7-H3.CAR-BB were 56% (49–68%) and 56% (46–59%), respectively (Fig. 3F,G). Utilizing a second cryopreserved bone marrow aspirate sample from the same patient, we demonstrated that autologous B7-H3.CAR-28-Ts (p=0.0085) and B7-H3.CAR-BB-Ts (p=0.0085) both displayed significant cytotoxicity against primary AML blasts when co-cultured at a 1:5 E:T ratio for 48 hours (Fig. 3H,I). Based on clinical annotation, the median blast percentage of the bone marrow aspirates used for these co-cultures was 86% (range 78–92%). For co-culture analysis, blasts were identified as CD45⁺/CD33⁺ cells. Release of IFNγ and IL2 by B7-H3.CAR-Ts and control T-cells was measured in the media harvested from co-cultures following a 24-hour incubation period. IFNγ and IL2 release was detected from B7-H3.CAR-Ts and was mostly undetectable in the media of control T-cells (p=0.0750 and p=0.1524 for IFNγ and IL2, respectively; Fig. 3J`,K). Under identical coculture conditions, we also demonstrated the ability of AML patient-derived (n=3) B7-H3.CAR-28-Ts (p=0.0049) and B7-H3.CAR-BB-Ts (p=0.0049) to efficiently kill the AML cell line, U937 (Fig 3L).

B7-H3.CAR-Ts show antitumor activity in xenograft mouse models of disseminated AML.

We evaluated the in vivo efficacy of B7-H3.CAR-Ts in two murine models of disseminated AML as described above. In the first model, eGFP-FFLuc-OCI-AML2 cells (2 × 10⁶ cells/mouse) were injected intravenously into NSG mice on day −7, followed by injection (1 × 10⁷ cells/mouse) of B7-H3.CAR-28-Ts (n=10), B7-H3.CAR-BB-Ts (n=10), or CD19.CAR-28-T (n=7) on days 0 and +20. Leukemia proliferation was assessed via weekly bioluminescence imaging. Tumor proliferation in mice treated with B7-H3.CAR-Ts was significantly delayed, as compared to mice treated with CD19.CAR-28-Ts (p<0.0001) (Fig 4A…D). We elected to perform a second CAR-T injection in this model because of data from prior experiments in which a higher eGFP-FFLuc-OCI-AML2 dose (5 × 10⁶ cells/mouse) was injected intravenously in NSG mice at day −7, followed by a single injection (1 × 10⁷ cells/mouse) of B7-H3.CAR-28-Ts, B7-H3.CAR-BB-Ts, or CD19.CAR-28-Ts (n=5 per group) at day 0 (Suppl. Fig 4A…D). In this experiment, we observed a significant prolongation of survival (p=0.0008) in B7-H3.CAR-T-treated mice, but all mice ultimately relapsed prior to day 40. We hypothesized that this may have been related to the kinetics of the tumor cell growth compared with CAR-T cell expansion in vivo, and that a second CAR-T cell injection could potentially overcome this limitation. In a pilot experiment, we also observed that mice initially engrafted with eGFP-FFLuc-OCI-AML2 cells on day −7 (5 × 10⁶ cells/mouse), followed by CD19.CAR-T injection (1 × 10⁷ cells/mouse) on day 0, tumor growth could be controlled after injection of B7-H3.CAR-28-Ts (1 × 10⁷ cells/mouse) on day 20 (Suppl. Fig 4E…G).

Figure 4. B7-H3.CAR-Ts control leukemia growth in xenograft models.

(A-D) OCI-AML2 cells labeled with eGFP-FFLuc were injected intravenously into NSG mice on day −7 followed by injection of B7-H3.CAR-Ts (n=10 mice per construct, T cells from 2 donors) or CD19.CAR-Ts (n=8 mice, 2 donors) on days 0 and 20. (A) Representative tumor bioluminescent images at the specified time points. (B) Experimental schema. (C) Tumor bioluminescence intensity (BLI) of each mouse plotted over the course of the experiment. (D) Kaplan-Meier survival curves indicating time to death or documented BLI greater than specified threshold. (E-H) THP1 cells labeled with eGFP-FFLuc were injected intravenously into NSG mice on day −7 followed by injection of B7-H3.CAR-Ts (n=10 mice per construct, T cells from 2 donors) or CD19.CAR-Ts (n=8 mice, 2 donors) on day 0. (E) Representative tumor bioluminescent images at the specified time points. (F) Experimental schema. (G) Tumor BLI of each mouse plotted over the course of the experiment. (H) Kaplan-Meier survival indicating time to death or documented BLI greater than specified threshold. For (D) and (H), p-values indicate log-rank test.

In the second model, eGFP-FFLuc-expressing THP1 cells (5 × 10⁶ cells/mouse) were similarly injected intravenously into NSG mice on day −7, followed by intravenous injection (1 × 10⁷ cells/mouse) of B7-H3.CAR-Ts (n=10 mice per construct) or CD19.CAR-28-Ts (n=8 mice). Tumor growth was similarly assessed via weekly bioluminescence imaging. Both B7-H3.CAR-28-Ts and B7-H3.CAR-BB-Ts, but not CD19.CAR-28-Ts, effectively controlled the growth OCI-AML2 cells in vivo (p=0.0004) (Fig 4E…H). Surface B7-H3 expression was found to be preserved on eGFP-FFluc-THP1 cells isolated from mice which relapsed after treatment with B7-H3.CAR-Ts (Suppl. Fig 2A,B).

B7-H3.CAR-Ts show antitumor activity in a patient-derived xenograft model of AML.

To evaluate the activity of B7-H3.CAR-Ts against human primary AML blasts, we utilized patient-derived xenograft mouse models of AML. For these experiments due to the limited availability of tumor cells, we conducted the experiment using only B7-H3.CAR-Ts carrying the CD28 costimulation. For the first PDX model, we initially confirmed B7-H3 expression on primary AML blasts from sample DFAM-6855 (99.1% B7-H3 positive using mAb 7–517, Fig 5A) and confirmed the in vitro activity of B7-H3.CAR-28-Ts against these cells (Fig 5B). NSG mice were then injected (1 × 10⁶ cells/mouse) on day −21 with primary AML blasts (Fig 5C). After confirmation of engraftment (Fig 5D), B7-H3.CAR-Ts (n=7) or control T cells (n=5) were injected (1 × 10⁷ cells/mouse) on day 0. Mice were sacrificed on day 8 and peripheral blood, bone marrow, and spleens were harvested (Fig 5C). Compared with mice treated with control T cells, we demonstrated significantly decreased huCD45⁺CD33⁺ AML blasts in the peripheral blood (p=0.0016) and spleen (p=0.007) (Fig 5E). Also, compared with control mice, we demonstrated significantly increased CD8⁺ T cell expansion in the spleens of B7-H3-CAR-28-T treated mice (Fig 5F).

Figure 5. B7-H3.CAR-Ts control leukemia growth in a patient-derived xenograft model of AML.

(A) Expression of B7-H3 on patient-derived AML blasts based on staining with mAb 7–517 (black lines) versus isotype control (gray fill). (B) Patient-derived AML blasts were co-cultured in vitro with B7-H3.CAR or control T cells. (C) Unlabeled patient-derived AML blasts were injected into the lateral tail veins of 6- to 8-week-old NSG mice on day −21, followed by injection of B7-H3.CAR-28 or control T cells at day 0. (D) Engraftment in all mice was confirmed via peripheral blood flow cytometry analysis on day 0 (representative flow cytometry plots on left). (E) On day 8, all mice were sacrificed, and the CD45⁺CD33⁺ cells in blood, bone marrow, and spleen, were quantified by flow cytometry. (F) CD4⁺ and CD8⁺ cells were quantified in blood and spleen. (G) Patient-derived AML blasts were labeled with eGFP-FFLuc, expanded in vivo, then injected intravenously into 6- to 8-week-old NSG mice on day −4, followed by injection of B7-H3.CAR-Ts (n=7 mice) or CD19.CAR-Ts (n=3 mice) on day 0. (H) Representative tumor bioluminescent images at the specified time points. (I) Tumor bioluminescence intensity (BLI) of each mouse plotted over time. For days 14, 17, 21, and 26, p<0.0001, t-test of BLI with Holm-Sidak test adjusted p values. (J) Kaplan-Meier survival curves indicating time to death, p=0.0027, log-rank test. Patient-derived AML blasts are from sample DFAM-6855 (PRoXe). For panels D, E, and F, groups were compared using unpaired and nonparametric Mann Whitney tests with two-tailed p-values as shown. Error bars indicate SEM.

In an additional experiment, the same patient-derived AML blasts were transduced to express eGFP-FFLuc and were injected intravenously (1 × 10⁶ cells/mouse) into NSG mice on day −4, followed by injection (1 × 10⁷ cells/mouse) of B7-H3.CAR-Ts (n=7 mice) or CD19.CAR-Ts (n=3 mice) on day 0 (Fig 5G). Leukemia proliferation was assessed via weekly bioluminescence imaging. Compared with CD19.CAR-T-treated mice, B7-H3.CAR-T-treated mice experienced significantly delayed tumor growth (p<0.0001) (Fig 5H,I) and significantly prolonged survival (p=0.0027) (Fig 5J). B7-H3 expression was also found to be preserved on patient-derived AML blasts recovered from the spleen after relapse following treatment with B7-H3.CAR-28-Ts (Suppl. Fig 2E).

In a second PDX model, we used patient-derived AML blasts from sample CBAM-44728 as described above. After confirming B7-H3 expression (Suppl. Fig 5B), blasts were injected intravenously (8 × 10⁵ cells/mouse) into NSG mice on day −4, followed by intravenous injection (1 × 10⁷ cells/mouse) of B7-H3.CAR-28-Ts (n=6 mice) or CD19.CAR-Ts (n=5 mice) on day 0 (Suppl. Fig 5C). All mice were sacrificed on day 63, followed by analysis of patient-derived AML blasts and CAR-Ts in the bone marrow, spleen, and/or blood. AML blasts were significantly decreased (undetectable in 4/6 mice; two with ≤ 100 blasts/femur) in the bone marrow of B7-H3.CAR-28-T compared with CD19.CAR-T-treated mice (p=0.0043) and undetectable in the spleens of B7-H3.CAR-28-T-treated mice (p=0.0022) (Suppl. Fig 5D). Both B7-H3.CAR-28-Ts and CD19.CAR-Ts were detectable in the blood and/or spleens of all mice (Suppl. Fig 5E).

B7-H3.CAR-Ts do not exhibit toxicity against normal myeloid progenitor populations in vitro and in vivo in a humanized mouse model.

To evaluate the potential toxicity of B7-H3.CAR-Ts against normal myeloid progenitor populations, we first utilized an in vitro colony forming unit (CFU) assay. Umbilical cord-blood derived CD34⁺ cells were co-incubated with B7-H3.CAR-28, CD19.CAR-28, or control T cells derived from donor PMBCs (n=4 cord-blood donors, each co-cultured with T cells from 2 PBMC donors) followed by culture in semisolid methylcellulose medium. No significant differences were observed in the numbers of granulocyte-macrophage progenitor (CFU-GM), common myeloid progenitor (CFU-GEMM), or total colonies (p=0.75, p=0.73, and p=0.96, respectively) (Fig. 6A).

Figure 6. B7-H3.CAR-Ts do not exhibit unacceptable toxicity against normal myeloid progenitor populations.

(A) Human umbilical cord blood derived CD34⁺ cells were coincubated with B7-H3.CAR-Ts, CD19.CAR-Ts, or control T cells, followed by transfer to semisolid medium for two weeks, after which granulocyte-macrophage progenitor (CFU-GM) and common myeloid progenitor (CFU-GEMM) colonies were scored. No significant differences were observed between groups for either colony type (one-way ANOVA). (B-H) Humanized mouse model to evaluate effects of B7-H3.CAR-Ts on normal hematopoiesis. (B) Schema demonstrating generation of humanized mouse model. NSG mice injected intrahepatically with human fetal liver-derived CD34⁺ cells. Following engraftment, subset of mice (n=2) were sacrificed. Human CD3⁺ (huCD3⁺) cells were isolated from murine spleens and used to generate CAR-Ts. (C) Flow cytometry histograms indicating transduction efficiency of B7-H3.CAR-28-Ts and CD19.CAR-28-Ts. (D) Flow cytometry analysis of THP1 (B7-H3⁺) or BV173 (B7-H3^-) cells cocultured with B7-H3.CAR-28 or control T cells (generated as shown in panel B) for 4 days. (E) Humanized mice shown in panel B were injected intravenously with 5 × 10⁶ (day 0) and 10 × 10⁶ (day 16) B7-H3.CAR-28 (n=5); CD19.CAR-28 (n=5), or control (n=4) T cells (normalized for transduction efficiency). Percent (top) and absolute numbers (bottom) of human CD3⁺, CD14⁺, and CD33⁺ cells in the peripheral blood of humanized mice are shown at the indicated time-points. Vertical dotted lines indicate injections of B7-H3.CAR-28, CD19.CAR-28, or control T cells as described in the text. (F) Human CD3⁺, CD14⁺, and CD33⁺ cells in murine spleens at the conclusion of the experiment (G) Human CD3⁺, CD14⁺, and CD33⁺ cells in murine bone marrow (H) Human CD34⁺CD38⁺ and CD34⁺CD38^- populations in murine bone marrow. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Holm-Sidak test adjusted p value. Error bars indicate SEM.

To assess the effects of B7-H3.CAR-Ts on hematopoietic progenitors, we utilized a murine model of normal human hematopoiesis. Human fetal liver-derived CD34⁺ cells were engrafted in NSG mice (n=16) as described above. When the median human CD45⁺ cell percentage was 47%, two mice were sacrificed and human T-cells were isolated and used to generate B7-H3.CAR-28-Ts and CD19-CAR-28-Ts (transduction efficiencies 69% and 79%, respectively; Fig. 6B…C). B7-H3.CAR-28-T function was confirmed via in vitro co-culture assay with the THP1 (B7-H3⁺) and BV173 (B7-H3^-) cell lines (Fig 6D). The remaining mice (n=14) were injected intravenously with either B7-H3.CAR-28 (n=5), CD19.CAR-28 (n=5), or control (n=4) T cells (5 × 10⁶ cells/mouse) at days 0 and 16. Human CD3⁺, CD33⁺, and CD14⁺ cells in the peripheral blood were assessed at baseline and every 7–9 days thereafter. Compared with mice treated with CD19.CAR-28 and control T cells, mice treated with B7-H3.CAR-28-Ts had no significant decreases in the levels of circulating CD14⁺ and CD33⁺ cells (Fig 6E). Mice were sacrificed at the conclusion of the experiment and spleen and bone marrow were evaluated via flow cytometry. Compared with mice treated with control T cells, absolute numbers of CD14⁺ and CD33⁺ cells were moderately decreased, but not eliminated, in the spleens of B7-H3.CAR-28-T treated mice (p=0.0196 and 0.0359, respectively) (Fig. 6F). No significant differences in the level of CD14⁺ and CD33⁺ cells were detected in the bone marrow (Fig. 6G).

Myeloid progenitor populations were subsequently evaluated in the bone marrow by gating on live, huCD45⁺, lineage^- (CD3^-CD14^-CD16^-CD19^-CD20^-CD56^-), CD34⁺CD38⁺ and CD34⁺CD38^- populations. Compared with mice treated with control T cells, there was a significant decrease in the percentage of CD34⁺CD38⁺ cells in mice treated with both B7-H3.CAR-28-Ts (p=0.0169). However, this effect did not appear to be specific to B7-H3.CAR-Ts as a similar decrease in this population was seen in mice treated with CD19.CAR-28-Ts (p=0.0206); as discussed below, we hypothesize that this is related to nonspecific CAR-T activation (Fig. 6H). Apparent decreases in the percentage of CD34⁺CD38^- cells in B7-H3.CAR-28-T and CD19.CAR-28-T treated mice were not statistically significant (p=0.0705; Fig. 6H).

DISCUSSION

We have previously generated B7-H3-specific CAR T cells, demonstrated anti-tumor efficacy in several solid tumor models, confirmed the absence of significant B7-H3 expression in most normal human tissues, and, taking advantage of the cross-reactivity B7-H3.CAR-Ts with the murine form of B7-H3, demonstrated a lack of evident toxicities in a syngeneic tumor model, further supporting the potential clinical translation of B7-H3.CAR-Ts (11). In the current study, we investigated the potential of B7-H3 as a clinically relevant target antigen for AML-directed CAR T cell therapy. We have demonstrated that B7-H3.CAR-Ts could represent a safe and effective therapy for a subset of patients with AML, and that such therapy is unlikely to cause unacceptable hematopoietic toxicity.

In addition to direct anti-tumor effects, it is also possible that B7-H3.CAR-Ts could augment adaptive immune responses in AML (7,24,25). B7-H3 expression on tumor cells, and on myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment, is likely to play an immunosuppressive role, and may drive immune escape in multiple cancer types (7,24–27). Solid tumor patients treated with an anti-B7-H3 monoclonal antibody were shown to have significant post-treatment increases in T cell receptor clonality (26). B7-H3 and other costimulatory ligands (e.g. PD-L1 and CD80) are also upregulated in AML patients who relapse following allogeneic stem cell transplantation, suggesting that B7-H3 may contribute to impaired allorecognition by donor T cells in this setting (28).

B7-H3 is thus a promising therapeutic target for human cancers and multiple clinical trials are evaluating anti-B7-H3 monoclonal antibodies, including a bispecific antibody targeting B7-H3 and CD3 (7,27–34). In the present study, we have provided efficacy and additional safety data to support the clinical assessment B7-H3.CAR-Ts as a treatment for AML. Our data suggest that B7-H3.CAR-Ts could potentially overcome the prolonged hematopoietic toxicity that has limited the development of CAR-Ts targeting certain other AML-associated antigens (35,36). In the humanized mouse model described above, compared with mice treated with control T cells, we observed a similar decrease in myeloid progenitor populations in mice treated with both B7-H3.CAR-Ts and CD19.CAR-Ts. We suspect that this is explained by nonspecific activation of the CAR-Ts rather than antigen-specific toxicity against myeloid progenitors. Although plasma cytokine levels were not measured in this experiment, in a similar humanized mouse model, our group previously demonstrated significantly increased levels of IFNγ, TNFα, and IL6 in the plasma of mice treated with CD19.CAR-Ts as compared to control T cells (37). Our data, together with the lack of reported myeloid toxicity in patients infused with a B7-H3-specific monoclonal antibody (33), suggest that targeting B7-H3 will be associated with a better safety profile compared with other pan-myeloid markers. Nonetheless, for patients enrolled in a phase I clinical study of B7-H3.CAR-Ts, it would be recommended to have the option of hematopoietic stem cell rescue if needed.

Although we did not observe B7-H3 antigen loss in cells isolated from relapsed mice in either our xenograft mouse models using AML cell lines or in the PDX models, it remains to be seen whether antigen-escape will limit the clinical efficacy of B7-H3.CAR-Ts in AML patients. It is also possible that increased adaptive immune responses could overcome this potential limitation. If antigen escape does occur, B7-H3 could still represent an attractive potential target antigen for use in a combinatorial approach. Finally, we recognize that the current study also does not fully characterize the expression of B7-H3 on AML leukemic stem cell (LSC) populations. AML LSCs are most well-defined in the ~75% of AML samples that are positive for CD34, and typically reside in the CD34⁺CD38^- population of AML blasts. In patients with CD34^- AML, the majority of LSCs are thought to reside in the CD34^- population; however LSCs in this subset of AML are less well-studied (30). Indeed, recent studies evaluating potential LSC targets for CAR T cell therapy in AML have focused only on the CD34⁺CD38^- blast population (32). We have found that most B7-H3⁺ AML is negative for CD34 (which is consistent with a monocytic immunophenotype), and it is thus more challenging to reliably evaluate the LSC expression of B7-H3. It is notable, however that B7-H3.CAR-Ts demonstrated in vitro cytotoxicity against patient-derived AML samples that were also shown to have the ability to reliably engraft in NSG mice. Nonetheless, in the absence of serial transfer experiments in vivo, it is not possible to definitively conclude whether there is differential activity of B7-H3.CAR-Ts against LSCs versus healthy hematopoietic progenitor cells.

The optimal choice of co-stimulatory domain for CAR T cell therapy remains unclear, and likely differs depending on factors related to the tumor growth kinetics, target antigen characteristics, and the scFv used. CD28-containing CARs are known to activate faster, produce larger magnitude changes in protein phosphorylation, and to lead to a more effector T cell-like phenotype as compared with 4–1BB-containing CARs (31). Prior studies suggest that 4–1BB-containing CARs may be better able to maintain effector functions in the setting of chronic antigen stimulation (31). Reduced levels cytokine production with 4–1BB-containing CARs may also correlate with a lower potential for CRS or neurotoxicity (31) and the only FDA-approved CAR T cell therapy for ALL incorporates the 4–1BB signaling domain. Prior work by our group has shown in a pancreatic adenocarcinoma model that 4–1BB containing B7-H3.CAR-Ts expressed lower levels of PD-1 and showed greater anti-tumor efficacy against pancreatic ductal adenocarcinoma (PDAC) cell lines transduced to constitutively express PD-L1 (11). However, the kinetics of PDAC tumor growth is clearly different than that of AML, and these advantages may not necessarily translate to the treatment of AML. In this study, most experiments were designed to evaluate B7-H3.28-CAR-Ts and B7-H3.BB-CAR-Ts in parallel. Although there was a trend towards greater efficacy of B7-H3.CAR-28-Ts, our data generally suggest similar efficacy between the two constructs, both in vitro and in mouse xenograft models. Any potential advantage of B7-H3.CAR-28-Ts is likely explained by the rapid kinetics of AML cell line expansion in the model systems used. While the optimal co-stimulatory domain remains unclear and may depend in part on the burden of disease at the time of CAR-T infusion, we have elected to pursue B7-H3.CAR-28-Ts for initial clinical evaluation in AML.

In conclusion, our data supports further evaluation of B7-H3 as a target for AML-directed CAR T cell therapy, demonstrates that B7-H3.CAR-Ts are able to control the proliferation of B7-H3⁺ AML both in vitro and in xenograft models, and with the limitations discussed above, suggests that B7-H3.CAR-Ts are unlikely to cause significant hematopoietic toxicity.

KEY POINTS.

• B7-H3 is expressed on a substantial subset of AML and is a potential target for AML-directed chimeric antigen receptor T cell therapy

• B7-H3-directed CAR T cells are effective in preclinical models of AML and are unlikely to cause unacceptable hematopoietic toxicity.

STATEMENT OF TRANSLATIONAL RELEVANCE.

Because of its high expression on malignant cells and its restricted distribution in normal tissues, B7-H3 is an attractive target for antibody-based cancer immunotherapy. We have previously generated B7-H3-specific CAR T cells (B7-H3.CAR-Ts), demonstrated anti-tumor efficacy in several solid tumor models, and demonstrated lack of evident toxicities in a syngeneic tumor model. Our current results suggest that B7-H3 is expressed on a subset of AML, but is not detectable on normal bone marrow progenitor populations. We have demonstrated that B7-H3.CAR-Ts exhibit efficient antigen-dependent cytotoxicity in vitro and in xenograft models of AML, and are unlikely to cause unacceptable hematopoietic toxicity. B7-H3 is thus a viable target antigen for AML-directed CAR T cell therapy. Our results support the clinical development of B7-H3.CAR-Ts for the treatment of patients with relapsed/refractory B7-H3-positive AML.