CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia.
ABSTRACT: Immunotherapy with CD123-specific T-cell engager proteins or with T cells expressing CD123-specific chimeric antigen receptors is actively being pursued for acute myeloid leukemia. T cells secreting bispecific engager molecules (ENG-T cells) may present a promising alternative to these approaches. To evaluate therapeutic potential, we generated T cells to secrete CD123/CD3-bispecific engager molecules. CD123-ENG T cells recognized primary acute myeloid leukemia (AML) cells and cell lines in an antigen-dependent manner as judged by cytokine production and/or tumor killing, and redirected bystander T cells to AML cells. Infusion of CD123-ENG T cells resulted in regression of AML in xenograft models conferring a significant survival advantage of treated mice in comparison to mice that received control T cells. At high effector to target ratios, CD123-ENG T cells recognized normal hematopoietic stem and progenitor cells (HSPCs) with preferential recognition of HSPCs from cord blood compared to bone marrow. We therefore introduced the CD20 suicide gene that can be targeted in vivo with rituximab into CD123-ENG T cells. The expression of CD20 did not diminish the anti-AML activity of CD123-ENG T cells, but allowed for rituximab-mediated ENG-T cell elimination. Thus, ENG-T cells coexpressing CD20 suicide and CD123 engager molecules may present a promising immunotherapeutic approach for AML.
Project description:The development of engineered T cells to treat acute myeloid leukemia (AML) is challenging due to difficulty in target selection and the need for robust T-cell expansion and persistence. We designed a T cell stimulated to kill AML cells based on recognition of the AML-associated surface marker CLEC12A, via secretion of a CLEC12AxCD3 bispecific "engager" molecule (CLEC12A-ENG). CLEC12A-ENG T cells are specifically activated by CLEC12A, are not toxic to hematopoietic progenitor cells, and exhibit antigen-dependent AML killing. Next, we coupled stimulation of T-cell survival to triggering of a chimeric IL7 receptor with an ectodomain that binds a second AML-associated surface antigen, CD123. The resulting T cells, identified as CLEC12A-ENG.CD123IL7Rα T cells, demonstrate improved activation upon dual target recognition, kill AML, and exhibit antitumor activity in xenograft models. Enhanced T-cell activation conferred by CD123.IL7Rα was dependent both on recognition of the CD123 target and on IL7Rα-mediated downstream signaling. Expression of a chimeric IL7R targeted to a second tumor-associated antigen (TAA) should improve T-cell activity not only against hematologic malignancies, but perhaps against all cancers.
Project description:Novel therapies are needed for effective treatment of AML. In the relapsed setting, prognosis is very poor despite salvage treatment with chemotherapy. Evidence suggests that leukemic stem cells (LSCs) cause relapse. The cell surface receptor CD123 is highly expressed in blast cells and LSCs from AML patients and is a potential therapeutic target. CD123 cross-over dual-variable domain T-cell engager (CD123-CODV-TCE) is a bispecific antibody with an innovative format. One arm targets the CD3εδ subunit of T-cell co-receptors on the surface of T cells, while the other targets CD123 on malignant cells, leading to cell-specific cytotoxic activity. Here, we describe the preclinical activity of CD123-CODV-TCE. CD123-CODV-TCE effectively binds to human and cynomolgus monkey CD3 and CD123 and is a highly potent T-cell engager. It mediates T-cell activation and T-cell-directed killing of AML cells in vitro. In vivo, CD123-CODV-TCE suppresses AML tumor growth in leukemia xenograft mouse models, where it achieves an effective half-life of 3.2 days, which is a significantly longer half-life compared to other bispecific antibodies with no associated Fc fragment. The in vitro safety profile is as expected for compounds with similar modes of action. These results suggest that CD123-CODV-TCE may be a promising therapy for patients with relapsed/refractory AML.
Project description:Despite significant progress over the last few decades in the treatment of acute myeloid leukemia (AML), there still remains a major unmet medical need for this disease. Immunotherapy approaches for redirecting pan CD3<sup>+</sup> T cells to target leukemia blasts have shown limited efficacy in clinical trials and often accompanied with severe toxicity in AML patients. We designed an alternative engager molecule (Anti-TRGV9/anti-CD123), a bispecific antibody that can simultaneously bind to the Vγ9 chain of the Vγ9Vδ2<sup>+</sup> γδ T cell receptor and to AML target antigen, CD123, to selectively recruit Vγ9<sup>+</sup> γδ T cells rather than pan T cells to target AML blasts. Our results suggest that prototypic bispecific antibodies (a) selectively activate Vγ9<sup>+</sup> γδ T cells as judged by CD69 and CD25 surface expression, and intracellular Granzyme B expression, (b) selectively recruit Vγ9<sup>+</sup> γδ T cells into cell-cell conjugate formation of γδ T cells with tumor cells indicating selective and effective engagement of effector and target tumor cells, and (c) mediate γδ T cell cytotoxicity (in vitro and in vivo) against tumor antigen-expressing cells. Collectively, these findings suggest that selectively redirecting Vγ9<sup>+</sup> γδ T cells to target AML blasts has a potential for immunotherapy for AML patients and favors further exploration of this concept.
Project description:We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA-electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti-CD123-4-1BB-CD3? T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML.
Project description:Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer, early events of tumorigenesis have already taken place. Similarly, development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article, we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model, human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1), referred to as NPM1c. Following engraftment into immunodeficient mice, transduced HSPCs give rise to human myeloid leukemia, whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML, with CD123+ leukemic stem or initiating cells (LSC), resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics.
Project description:Although the CD20-targeted monoclonal antibody rituximab (RTX) has revolutionized the therapeutic landscape for B-cell malignancy, relapsed and refractory disease due to RTX resistance continue to constitute major challenges, illustrating the need for better therapies. Here, we apply drug-free macromolecular therapeutics (DFMT) that amplifies CD20 cross-linking to enhance apoptosis in RTX-resistant cells. Bispecific engager (anti-CD20 Fab' conjugated with oligonucleotide1) pretargets CD20 and the deletion of Fc-region minimizes its premature endocytosis in resistant cells that rapidly internalize and consume CD20/RTX complexes. Second-step delivery of multivalent polymeric effector (linear copolymer conjugated with multiple copies of complementary oligonucleotide 2) simultaneously hybridizes multiple CD20-bound engagers and strengthens CD20 ligation. Moreover, the restoration of CD20 expression by the pretreatment of cells with a polymer-gemcitabine conjugate, a CD20 expression enhancer, unleashes the full potential of DFMT in the CD20-deficient resistant cells. Hence, amplification of CD20 cross-linking is achieved by (1) the enhancement of surface CD20 accessibility, (2) the increase in CD20 expression, and (3) multimeric CD20 binding, which ultimately translates into the amplified activation of a wide range of innate apoptotic responses. We demonstrated that the altered molecular signaling pathway that originally results in RTX resistance could be circumvented and compensated by other DFMT-augmented pathways. Of note, our preliminary data provide proof-of-concept that CD20 cross-linking amplification emerges as an important strategy for overcoming RTX resistance.
Project description:Several immune checkpoint molecules and immune targets in leukemic cells have been investigated. Recent studies have suggested the potential clinical benefits of immuno-oncology (IO) therapy against acute myeloid leukemia (AML), especially targeting CD33, CD123, and CLL-1, as well as immune checkpoint inhibitors (e.g., anti-PD (programmed cell death)-1 and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) antibodies) with or without conventional chemotherapy. Early-phase clinical trials of chimeric antigen receptor (CAR)-T or natural killer (NK) cells for relapsed/refractory AML showed complete remission (CR) or marked reduction of marrow blasts in a few enrolled patients. Bi-/tri-specific antibodies (e.g., bispecific T-cell engager (BiTE) and dual-affinity retargeting (DART)) exhibited 11-67% CR rates with 13-78% risk of cytokine-releasing syndrome (CRS). Conventional chemotherapy in combination with anti-PD-1/anti-CTLA4 antibody for relapsed/refractory AML showed 10-36% CR rates with 7-24 month-long median survival. The current advantages of IO therapy in the field of AML are summarized herein. However, although cancer vaccination should be included in the concept of IO therapy, it is not mentioned in this review because of the paucity of relevant evidence.
Project description:The past decade witnessed the rapid development of adult B-lineage acute lymphoblastic leukemia (ALL) treatment. Beyond the development of chemotherapy regimens, immunotherapy is starting a new era with unprecedented complete remission (CR) rate. Targeting B-lineage-specific surface markers such as CD19, CD20, CD22, or CD52, immunotherapy has been demonstrating promising clinical results. Among the immunotherapeutic methods, naked monoclonal antibodies (mAbs), antibody-drug conjugate (ADC), bispecific T cell engager (BiTE), and chimeric antigen receptor (CAR) T cells are the main types. In this review, we will examine the emerging preclinical and clinical development on (1) anti-CD20 naked mAbs rituximab, ofatumumab, and obinutuzumab; (2) anti-CD19 ADCs SAR3419 and SGN-CD19A and anti-CD19 BiTE blinatumomab; (3) anti-CD22 naked mAb epratuzumab and anti-CD22 ADC inotuzumab ozogamicin; (4) anti-CD52 naked mAb alemtuzumab; and (5) anti-CD19 CAR T cells. We will discuss their efficacy, adverse effects, as well as future development.
Project description:The outcome for advanced stage hepatocellular carcinoma (HCC) remains poor, highlighting the need for novel therapies. Genetically modified mesenchymal stem cells (MSCs) are actively being explored as cancer therapeutics due to their inherent ability to migrate to tumor sites. We reasoned that MSCs can be genetically modified to redirect T cells to Glypican-3 (GPC3)<sup>+</sup> HCC, and genetically modified these with viral vectors encoding a GPC3/CD3 bispecific T cell engager (GPC3-ENG), a bispecifc T cell engager specific for an irrelevant antigen (EGFRvIII), and/or costimulatory molecules (CD80 and 41BBL). Coculture of GPC3<sup>+</sup> cells, GPC3-ENG MSCs, and T cells resulted in T cell activation, as judged by interferon ? (IFN?) production and killing of tumor cells by T cells. Modification of GPC3-ENG MSCs with CD80 and 41BBL was required for antigen-dependent interleukin-2 (IL-2) production by T cells and resulted in faster tumor cell killing by redirected T cells. In vivo, GPC3-ENG MSCs ± costimulatory molecules had antitumor activity in the HUH7 HCC xenograft model, resulting in a survival advantage. In conclusion, MSCs genetically modified to express GPC3-ENG ± costimulatory molecules redirect T cells to GPC3<sup>+</sup> tumor cells and have potent antitumor activity. Thus, further preclinical exploration of our modified approach to GPC3-targeted immunotherapy for HCC is warranted.