REDIRECTING T CELLS WITH CHIMERIC ANTIGEN RECEPTORS TO TARGET CD123+ LEUKEMIA

ABSTRAC REDIRECTING T CELLS WITH CHIMERIC ANTIGEN RECEPTORS TO TARGET CD123+ LEUKEMIA Radhika Thokala, Ph.D* Advisory Professor: Dean Anthony Lee, M.D, Ph.D CD123 or interleukin receptor alpha (IL-3Rα) is expressed on hematological malignancies such as acute myeloid leukemia (AML) and some acute lymphoblastic leukemia (ALL). Significantly, CD123 is over-expressed on leukemic stem cells (LSCs) compared to normal hematopoietic stem cells and thus targeting this tumor- associated antigen (TAA) provides the potential to prevent relapse. The prototyical chimeric antigen receptor (CAR) is fashioned by combining the variable light (V L) and heavy (VH) as a scFv derived from a single monoclonal antibody (mAb) specific for the TAA. We describe a new approach for generating CD123-specific CARs generating a chimeric scFv that is made up of the VL and VH harvested from two mAbs that are each specific for CD123. The hypothesis is VL and VH from different antibodies to the same TAA can be recombined to form unique binding domains that retain antigen specificity but may have altered binding characteristics. This non-homologous recombination of antibody binding domain may be used to select CAR for optimal anti-tumor characteristics, such as increasing the therapeutic index. The chimeric scFvs were derived by fusing the VL and VHchains derived from mAbs 26292, 32701, 32703, 32716 specific to CD123. Sleeping Beauty (SB) was employed as a non-viral gene transfer s ystem to stably express 2nd generation CARs in T cells derived from peripheral blood mononuclear cells (PBMC). The CARs were co-expressed with inducible Caspase 9 (iCaspase9) for conditional ablation of T cells in case of off-target toxicities. The SB plasmids coding for two CARs (transposons) activated T cells via chimeric CD28 with CD3-zeta and CD137 with CD3-zeta were electroporated into PBMC. Following electrotransfer of the SB system the genetically modified T cells were preferentially propagated on activating and propagating cells (AaPC) designated as Clone 1-CD123. The AaPC were derived from K562 cells genetically modified to co-express co-stimulatory molecules (CD86 and CD137L), a membrane bound cytokine (IL-15 fused to IL-15Rα), and the TAAs CD123 and CD19. CAR+ T cells specifically produced IFN-γ and lysed CD123+ leukemic cell lines and primary AML patient samples, but did not lyse D123neg tumor cells. The addition of a chemical dimerizer to activate iCaspase9 resulted in destruction of genetically modified T cells. Both populations of CAR+ T cells produced and eliminated leukemic tumors in vivo. We observed no difference in the anti-tumor effects whether the CARs triggered T cells via CD28 or CD137. These studies suggest that CD123 can be targeted by CAR+ T cells and that the hybrid arrangement of VL and VH maintained specificity for CD123

Redirecting Specificity of T cells Using the <i>Sleeping Beauty</i> System to Express Chimeric Antigen Receptors by Mix-and-Matching of V_L and V_H Domains Targeting CD123⁺ Tumors

<div><p>Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a V_H and V_L from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using V_H and V_L chains derived from different CD123-specific mAbs to generate a panel of CAR⁺ T cells. While all CARs exhibited specificity to CD123, one V_H and V_L combination had reduced lysis of normal hematopoietic stem cells. This CAR’s <i>in vivo</i> anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR⁺ T cells. These data help support the use of CD123-specific CARs for treatment of CD123⁺ hematologic malignancies.</p></div

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations.

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire

Comparison of costimulatory domains for the treatment of AML using CD123-specific CAR⁺ T cells in a murine model.

<p><b>(A)</b> Schematic of the TF1 xenograft model. 2.5 × 10⁶ TF1-<i>effLuc</i>-mKate cells were injected intravenously into NSG mice on day 0. On Day 5, tumor engraftment was quantified using non-invasive bioluminescence imaging (BLI), and mice were randomly divided into 3 groups: untreated (control), CD123-CD28-treated, or CD123-CD137-treated. CAR-treated mice were given infusions of T cells followed by IL-2 treatment and BLI on day 5, 11 and 20. Untreated mice received no T cells. <b>(B)</b> BLI images of mice display an overlay of luciferase activity, using the color scale shown on the right, displayed over the white-light image of the mice. <b>(C)</b> Histograms represent the luciferase activity measured by BLI for each group (** p < 0.01). <b>(D)</b> Kaplan-Meier curves display the survival analysis of xenograft mice treated with CD123-specific CAR T cells (** p < 0.01).</p

Immunophenotype of CAR⁺ T cells with CD28 or CD137 costimulatory domains.

<p>(A) Flow analysis of memory markers on CD3⁺CAR⁺ T cells. Flow cytometry histograms are representative images from one of three donors tested for each memory marker vs. CD45RA (Y axis). (B) Expression of CD4, CD8, and CD56 is shown as in (A) (C) Histograms represent the percentage of CAR⁺ T cells expressing each memory or exhaustion marker (mean ± SEM, n = 3). (D) Histograms represent the percentage of CAR⁺ T cells in each subset, based on flow cytometry phenotype: T_Naïve (CD45RA⁺, CD62L⁺, CD95^-, CCR7⁺), T_EMRA (CD45RA⁺, CD62L^neg, CD95^neg, CCR7^neg), T_EM (CD45RA^neg, CD62L^neg, CD95⁺, CCR7^neg) and T_CM (CD45RA^neg, CD62L⁺, CD95⁺, CCR7⁺) in CD123-CD28 CAR⁺ T cells (black bars) and CD123-CD137 CAR⁺ T cells (grey bars) (n = 3). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159477#pone.0159477.g002" target="_blank">Fig 2E, 2F and 2G</a> display the quantitation of mRNA transcripts of lymphocyte genes expressed in CAR T cells as analyzed by non-enzymatic digital multiplex array. (E) Transcriptional profile of activation-, co-stimulation- and exhaustion-related genes. (F) Transcriptional profile of genes associated with differentiation phenotype and memory stage (G) Transcriptional profile of genes for cytokine receptors and markers associated with effector function.</p

Efficacy of CD123-specific CAR⁺ T cells for the treatment of B-ALL in a murine model.

<p><b>A)</b><i>In vitro</i> lysis of B-ALL cell lines by CD123-specific CAR⁺ T cells measured with a 4 hour chromium release assay <b>(B)</b> Schematic of the RCH-ACV B-ALL xenograft model. The experimental design is similar to that shown in 5A, but T cells and cytokines were given on days 7, 14 and 21, with imaging weekly. <b>(C)</b> BLI imaging of the CAR-treated and untreated groups on day 28. Images are displayed as in 5B. <b>(D)</b> Luciferase activity measured by BLI in the CAR-treated group compared with the untreated group. <b>(E)</b> Kaplan-Meier curves display the survival analysis of xenograft mice treated with CD123-specific CAR T cells compared with untreated mice. ** p < 0.01.</p

Production and testing of CD123-specific CARs with chimeric scFvs.

<p><b>(A)</b> Schematic diagrams of conventional and chimeric scFv specific for CD123. CARs 1 to 4: CD123-specific CARs generated by fusing V_L and V_H chains of mAbs specific to CD123. CARs 5–9: Chimeric scFvs created by mix-and-matching V_L and V_H chains. The scFvs of CARs 1–9 were fused to the signaling domains of CD28 and CD3ζ via CD8α hinge and TM domains. CAR-10 was derived by fusing the chimeric scFv from CAR 6 to the CD3ζ and CD28 endo-domains via the IgG4 hinge and CD28 TM domains. <b>(B)</b> Expansion kinetics of CARs 1–4 (left) and CARs 5–10 (right) over a period of 28 days from day 1 following electroporation of SB CAR plasmids. Data are pooled from 3 donors; graph displays mean ± SEM <b>(C)</b> CAR expression on Day 21 after electroporation. CAR expression was detected by CD123 recombinant protein fused to Fc followed by serial staining with fluorescence-labeled anti-Fc and anti-CD3 antibodies. <b>(D)</b> <i>in vitro</i> lysis of CD123⁺ target cells Nalm 6, TF1, 293T-parental cells, CD123-transfected 293T cells, and 123^neg by CAR⁺ T cells. Histograms represent the mean ± SEM, n = 3. <b>(E)</b> CAR⁺ T cell killing of BM-derived target cells. Mononuclear cells were isolated from normal human bone marrow samples and sorted for expression of lineage markers into lineage-positive (Lin⁺) and lineage-negative (Lin^neg) groups. The latter presumable reflects the HSC pool. The BM-derived cells were then labeled with PKH-26 and incubated with CAR⁺ T cells for 2 days before vitality was assessed by flow cytometry. The percent lysis compared to controls is shown. Histograms represent the mean ± SEM of 3 replicates. (F) Interferon-γ release by CAR⁺ T cells after exposure to CD123. Day 28 CD123-specific CAR⁺ T cells were incubated for 24 hours with Nalm-6 cells (CD123⁺), 293T cells (CD123^neg), or alone, then the supernatant tested for cytokine expression using Biolegend plex Th1 cytokine capture beads, measured by flow cytometry. Results for IFN-γ are shown; other cytokines were not detectible over background. Histograms represent mean ± SEM for 2 replicates from 2 different experiments.</p

<i>in vitro</i> lysis of AML tumor cell lines and primary AML samples.

<p><b>(A)</b> Overlay histograms display the flow cytometric analysis of CD123 expression on AML cell lines MV4-11, Molm-13, TF1, OCI-AML3, EL4-Parental and EL4-CD123. Isotype control is shown in grey, and specific staining by the unfilled black line. <b>(B)</b> Specific lysis of CD123-CD28 and CD123-CD137 CAR⁺ T cells against AML cell lines EL4, CD123^neg OCI-Ly19, MV4-11, TF1, EL4-CD123, Molm-13, and OCI-AML3 assessed with a 4 hour chromium release assay. Histograms represent mean ± SEM, n = 3 <b>(C)</b> Flow cytometric analysis of CD123 expression on primary AML samples used in the co-culture assay depicted in <b>(D)</b>. Lysis of PKH-26 labeled primary AML cells by CD123-CD28 or CD123-CD137 CAR T cells at 1:1 ratio for 72 hours. CD19-specific CAR⁺ T cells were used as a negative control.</p

<i>Sleeping Beauty</i> Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations

<div><p>T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19⁺ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two <i>Sleeping Beauty</i> transposons were constructed with 2^nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR⁺ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1⁺ tumors. Moreover, such cells could eliminate ROR1⁺ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR⁺ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.</p></div

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells’ efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells. Keywords: glioblastoma, chimeric antigen receptor, CAR, IL-13Rα2, minibody, canine, immune checkpoint blockade, PD-1, CTLA-4, TIM-