Immunotherapy Of Acute Myeloid Leukemia Research Articles

Dendritic Cell-Based Immunotherapy of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a type of blood cancer characterized by the uncontrolled clonal proliferation of myeloid hematopoietic progenitor cells in the bone marrow. The outcome of AML is poor, with five-year overall survival rates of less than 10% for the predominant group of patients older than 65 years. One of the main reasons for this poor outcome is that the majority of AML patients will relapse, even after they have attained complete remission by chemotherapy. Chemotherapy, supplemented with allogeneic hematopoietic stem cell transplantation in patients at high risk of relapse, is still the cornerstone of current AML treatment. Both therapies are, however, associated with significant morbidity and mortality. These observations illustrate the need for more effective and less toxic treatment options, especially in elderly AML and have fostered the development of novel immune-based strategies to treat AML. One of these strategies involves the use of a special type of immune cells, the dendritic cells (DCs). As central orchestrators of the immune system, DCs are key to the induction of anti-leukemia immunity. In this review, we provide an update of the clinical experience that has been obtained so far with this form of immunotherapy in patients with AML.

Anthracycline-based consolidation may determine outcome of post-consolidation immunotherapy in AML

Consolidation chemotherapy in acute myeloid leukemia (AML) aims at eradicating residual leukemic cells and mostly comprises high-dose cytarabine with or without the addition of anthracyclines, including daunorubicin. Immunogenic cell death (ICD) may contribute to the efficacy of anthracyclines in solid cancer, but the impact of ICD in AML is only partly explored. We assessed aspects of ICD, as reflected by calreticulin expression, in primary human AML blasts and observed induction of surface calreticulin upon exposure to daunorubicin but not to cytarabine. We next assessed immune phenotypes in AML patients in complete remission (CR), following consolidation chemotherapy with or without anthracyclines. These patients subsequently received immunotherapy with histamine dihydrochloride (HDC) and IL-2. Patients who had received anthracyclines for consolidation showed enhanced frequencies of CD8+ TEM cells in blood along with improved survival. We propose that the choice of consolidation therapy prior to AML immunotherapy may determine clinical outcome.

Recent Advances in Immunotherapy for Acute Myeloid Leukemia --Review

Currently, the traditional chemotherapeutic drugs for acute myeloid leukemia (AML) showed significant curative efficacy, including the improving long-term prognosis and the life quality of the patients, however the traditional chemotherapeuatic drugs showed the some limitatious in the aspects of enhancing the complete remission rate of newly diagnosed AML patients, overcoming the relapse after remission,as well as the primary and secondary drug-resstance to chemotherapeutics. In order to improve the long-term prognisis of patients, the immuno therapy will the best choice for these patients. This review sammarizes the main current advance of immunotherapy of AML at home and abroad, such as antibody-drug conjugate(ADC), bispecific T cell engager (BiTE), chimeric type of antigen receptor T cell (CAR-T) therapy, checkpoint inhibitors; dendritic cell vaccination and peptide vaccines; natural killer cell (NK) therapy and so on.

The HLA-B −21 dimorphism impacts on NK cell education and clinical outcome of immunotherapy in acute myeloid leukemia

AbstractNatural killer (NK) cell function is regulated by inhibitory receptors, such as the family of killer immunoglobulin-like receptors (KIRs) and the NKG2A/CD94 heterodimer. These receptors recognize cognate HLA class I molecules on potential target cells, and recent studies imply that an HLA-B dimorphism at position −21 in the gene segment encoding the leader peptide dictates whether NK cell regulation primarily relies on the KIRs or the NKG2A/CD94 receptor. The impact of this HLA-B dimorphism on NK cell–mediated destruction of leukemic cells or on the course of leukemia is largely unknown. In a first part of this study, we compared functions of NK cells in subjects carrying HLA-B −21M or 21T using interleukin-2 (IL-2)–activated NK cells and leukemic cells from patients with acute myeloid leukemia (AML). Subjects carrying HLA-B −21M harbored better-educated NKG2A+ NK cells and displayed superior capacity to degranulate lytic granules against KIR ligand-matched primary leukemic blasts. Second, we aimed to define the potential impact of HLA-B −21 variation on the course of AML in a phase 4 trial in which patients received IL-2–based immunotherapy. In keeping with the hypothesis that 21M may be associated with improved NK cell functionality, we observed superior leukemia-free survival and overall survival in −21M patients than in −21T patients during IL-2–based immunotherapy. We propose that genetic variation at HLA-B −21 may determine the antileukemic efficacy of activated NK cells and the clinical benefit of NK cell–activating immunotherapy.

Chimeric Antigen Receptor (CAR) T Cell Therapy in Acute Myeloid Leukemia (AML).

Despite high response rates after initial chemotherapy in patients with acute myeloid leukemia (AML), relapses occur frequently, resulting in a five-year-survival by <30% of the patients. Hitherto, allogeneic hemotopoietic stem cell transplantation (allo-HSCT) is the best curative treatment option in intermediate and high risk AML. It is the proof-of-concept for T cell-based immunotherapies in AML based on the graft-versus-leukemia (GvL)-effect, but it also bears the risk of graft-versus-host disease. CD19-targeting therapies employing chimeric antigen receptor (CAR) T cells are a breakthrough in cancer therapy. A similar approach for myeloid malignancies is highly desirable. This article gives an overview on the state-of-the art of preclinical and clinical studies on suitable target antigens for CAR T cell therapy in AML patients.

Abstract A044: Mutated NPM1 as target for immunotherapy of acute myeloid leukemia

Abstract The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin (NPM1) gene. Mutated NPM1 is an attractive target for immunotherapy, since it is an essential driver gene and 4 base pair frameshift insertions in exon 12 occur in 30-35% of AML, resulting in a novel C-terminal alternative reading frame of 11 amino acids. By searching in the HLA class I ligandome of primary AML, we identified multiple peptides derived from mutated NPM1. For one of these peptides, i.e., HLA-A*02:01-presented CLAVEEVSL, we searched for specific T-cells in AML patients and healthy individuals using peptide-MHC tetramers. Tetramer-positive CD8 T-cell clones were isolated and analyzed for reactivity against primary AML with mutated NPM1. From one selected clone with superior antitumor reactivity, we isolated the T-cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01-positive AML with mutated NPM1 in vitro after retroviral transfer to CD8 and CD4 T-cells. In vivo antitumor efficacy of TCR-transduced CD8 and CD4 T-cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing mutated NPM1. These data show that mutated NPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML with mutated NPM1 by TCR gene transfer in a co-receptor independent fashion. Immunotherapy targeting mutated NPM1 may therefore contribute to treatment of AML. Citation Format: Dyantha I. van der Lee, Rogier M. Reijmers, M. Willy Honders, Renate S. Hagedoorn, Rob. M. de Jong, Michel G.D. Kester, Dirk M. van der Steen, Arnoud H. de Ru, Christiaan Kweekel, Inge Jedema, Hendrik Veelken, Mirjam M. Heemskerk, Peter A. van Veelen, J.H. Frederik Falkenburg, Marieke Griffioen. Mutated NPM1 as target for immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A044.

Antigenic Targets for the Immunotherapy of Acute Myeloid Leukaemia.

One of the most promising approaches to preventing relapse is the stimulation of the body’s own immune system to kill residual cancer cells after conventional therapy has destroyed the bulk of the tumour. In acute myeloid leukaemia (AML), the high frequency with which patients achieve first remission, and the diffuse nature of the disease throughout the periphery, makes immunotherapy particularly appealing following induction and consolidation therapy, using chemotherapy, and where possible stem cell transplantation. Immunotherapy could be used to remove residual disease, including leukaemic stem cells from the farthest recesses of the body, reducing, if not eliminating, the prospect of relapse. The identification of novel antigens that exist at disease presentation and can act as targets for immunotherapy have also proved useful in helping us to gain a better understand of the biology that belies AML. It appears that there is an additional function of leukaemia associated antigens as biomarkers of disease state and survival. Here, we discuss these findings.

Epigenetic Drug Treatment Induces Presentation of New Class of Non-Exonic, Cryptic Neoantigens in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a 5-year overall survival rate of less than 30% which causes over 10,000 deaths per year in the United States. Treatment options for this disease increasingly include epigenetic drugs, such as hypomethylating agents (e.g. decitabine) or histone deacetylase (HDAC) inhibitors (e.g. pracinostat) which can function via direct cytotoxic mechanisms and also through altered differentiation of AML blasts; immunomodulatory effects like reactivation and presentation of cancer testis antigens in context of human leukocyte antigen (HLA) complexes have been reported as well, which may result in clearance of cells via the adaptive immune system. However, the landscape of immunogenic T cell epitopes induced by these drugs might be even broader than reported since standard analyses only consider exonic protein sequences and do not take into account typically untranslated genomic regions.Recently, it has been shown that single and combination treatment of decitabine and pracinostat can induce cryptic transcription start sites in generally epigenetically repressed solitary long-terminal repeats (LTRs) of the LTR12C family which give rise to novel mRNAs and resulting protein variants. We hypothesized that the intronic parts of these gene products might provide a source of cryptic T cell epitopes with high immunogenic potential, which are induced through epigenetic drug treatment.To test this hypothesis, we treated 5 different AML cell lines (HL-60, U937, OCI-AML02, MOLM13, AML14) with (1) DMSO, (2) 500 nM decitabine or (3) a combination of 500 nM decitabine and 100 nM pracinostat for 72 hours to induce transcription of non-annotated transcription start sites. Subsequently, HLA class I complexes were immunopurified and peptides presented by these complexes isolated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The activation of silenced genes by epigenetic drug treatment with either decitabine alone or the combination treatment yielded increases of about two-fold in the identified unique HLA ligands. This increase in peptide identifications also led to improved detection of cancer testis antigen-derived epitopes, as has been reported before. Intriguingly, by adding LTR12C derived sequences stretching from the published GATA2 specific binding site until the next genomic exon to the peptide search analyses we were able to identify several cryptic peptides from 4 out of 5 AML cell lines derived from these usually untranscribed genomic regions. The identifications were exclusively dependent on previous treatment with either decitabine alone or in combination with pracinostat. Though the immunogenicity of these HLA ligands has not been determined yet, we assume that due to their genetically repressed state in untreated cells, these new peptide sequences represent a new class of neoepitopes, with potential to be novel targets of existing T cells within patients or after augmentation by other immunotherapies.In summary, we demonstrated for the first time the induced presentation of epitopes from normally untranscribed LTR12C regions through epigenetic drug treatment and therefore provide a previously undescribed source of potential targets for immunotherapy in AML. DisclosuresScheinberg:Eureka: Consultancy; Ensyce: Consultancy.

Myd88/CD40 Costimulation to Enhance the Antitumor Activity of CD123-ENG T-Cells for the Immunotherapy of AML

Background: Acute myeloid leukemia (AML) represents a treatment challenge due to its poor prognosis and high associated morbidity and mortality rates. Thus, new therapies are needed. CD123-ENG T-cells are cells genetically modified to secrete bispecific antibodies that recognize CD3 (T-cells) and CD123 (AML). Preclinical studies show that these cells recruit bystander T-cells to kill CD123+ blasts in vitro and in vivo. However, CD123-ENGs are unable to maintain sequential killing capability of CD123+ targets. To overcome this limitation, we have devised an approach that provides inducible Myd88/CD40 costimulation activated by a chemical inducer of dimerization (CID). This strategy should increase the persistence, expansion and anti-AML activity of CD123-ENG T-cells.Methods: We generated a retroviral vector encoding a CD20 safety switch, CD123-ENG, and the inducible costimulatory molecule MyD88.CD40 (iMC) linked by 2A sequences (CD20.2A.CD123-ENG.2A.iMC). We used a vector specific for CD19 (CD20.2A.CD19-ENG.2A.iMC) and non-transduced (NT) cells as controls for non-specific effects of the iMC construct. We genetically modified T-cells using a retroviral transduction protocol and evaluated their effector function +/- CID. We assessed the effector function of transduced T-cells using standard immunological assays and a flow cytometry based sequential killing assay.Results: We successfully generated CD20.CD123-ENG.iMC T-cells, which maintained a transduction efficiency above 50% throughout our study period. We performed coculture and cytotoxicity assays using NT, CD20.CD123-ENG, CD20.CD19-ENG.iMC and CD20.CD123-ENG.iMC T-cells +/-CID as effectors and MOLM13 (CD123+), Kg1a (CD123+) and K562 (CD123-) as targets. Cocultures were performed +/- CID. CD20.CD123-ENG.iMC T-cells maintained CD123 antigen specificity, as evidenced by cytotoxicity and cytokine assays. CD20.CD123-ENG.iMC T-cells + CID secreted increased IL-2 and IFN-γ in the presence of CD123+ targets (KG1a and MOLM13) when compared to baseline and to CD20.CD123-ENG T-cells. In addition, CD20.CD123-ENG.iMC T-cells + CID displayed enhanced sequential killing capabilities at a 1:1 ratio, compared to CD20.CD123-ENG T-cells.Conclusion: CD20.CD123-ENG.iMC T-cells recognize and kill CD123+ AML blasts in an antigen dependent manner. In addition, control ENG.iMC T-cells have no antitumor activity, indicating that activation of Myd88/CD40 does not induce nonspecific AML blast killing. CD20.CD123-ENG.iMC T-cells have improved effector function in the presence of CID as judged by increased cytokine production and their ability to sequentially kill CD123+ target cells in vitro. Based on our promising in vitro experiments, we have initiated in vivo studies in AML xenograft models, which are currently in progress. DisclosuresLeen:Marker: Equity Ownership.

Recent advance in Immunotherapy of Acute Myeloid Leukemia

Recent advance in Immunotherapy of Acute Myeloid Leukemia

Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells

ABSTRACTA number of agents designed for immunotherapy of Acute Myeloid Leukemia (AML) are in preclinical and early clinical development. Most of them target a single antigen on the surface of AML cells. Here we describe the development and key biological properties of a tri-specific agent, the dual-targeting triplebody SPM-2, with binding sites for target antigens CD33 and CD123, and for CD16 to engage NK cells as cytolytic effectors. Primary blasts of nearly all AML patients carry at least one of these target antigens and the pair is particularly promising for the elimination of blasts and leukemia stem cells (LSCs) from a majority of AML patients by dual-targeting agents. The cytolytic activity of NK cells mediated by SPM-2 was analyzed in vitro for primary leukemic cells from 29 patients with a broad range of AML-subtypes. Blasts from all 29 patients, including patients with genomic alterations associated with an unfavorable genetic subtype, were lysed at nanomolar concentrations of SPM-2. Maximum susceptibility was observed for cells with a combined density of CD33 and CD123 above 10,000 copies/cell. Cell populations enriched for AML-LSCs (CD34pos and CD34pos CD38neg cells) from 2 AML patients carried an increased combined antigen density and were lysed at correspondingly lower concentrations of SPM-2 than unsorted blasts. These initial findings raise the expectation that SPM-2 may also be capable of eliminating AML-LSCs and thus of prolonging survival. In the future, patients with a broad range of AML subtypes may benefit from treatment with SPM-2.

Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML

Targeted immunotherapy in acute myeloid leukemia (AML) is challenged by the lack of AML-specific target antigens and clonal heterogeneity, leading to unwanted on-target off-leukemia toxicity and risk of relapse from minor clones. We hypothesize that combinatorial targeting of AML cells can enhance therapeutic efficacy without increasing toxicity. To identify target antigen combinations specific for AML and leukemic stem cells, we generated a detailed protein expression profile based on flow cytometry of primary AML (n = 356) and normal bone marrow samples (n = 34), and a recently reported integrated normal tissue proteomic data set. We analyzed antigen expression levels of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk and leukemic stem cells at initial diagnosis (n = 302) and relapse (n = 54). CD33, CD123, CLL1, TIM3 and CD244 were ubiquitously expressed on AML bulk cells at initial diagnosis and relapse, irrespective of genetic characteristics. For each analyzed target, we found additional expression in different populations of normal hematopoiesis. Analyzing the coexpression of our six targets in all dual combinations (n = 15), we found CD33/TIM3 and CLL1/TIM3 to be highly positive in AML compared with normal hematopoiesis and non-hematopoietic tissues. Our findings indicate that combinatorial targeting of CD33/TIM3 or CLL1/TIM3 may enhance therapeutic efficacy without aggravating toxicity in immunotherapy of AML.

Immunosenescence and Immunotherapy in Elderly Acute Myeloid Leukemia Patients: Time for a Biology-Driven Approach.

Acute myeloid leukemia (AML) is a disease, which mainly affects the elderly population. Unfortunately, the prognosis of patients aged >65 years is dismal, with 1-year overall survival approaching 10% with conventional therapies. The hypothesis of harnessing the immune system against cancer, including leukemia, has been postulated for a long time, and several clinical attempts have been made in this field. In the last years, we increased our knowledge about the interplay between AML and immune cells, but no major improvement has been translated, up to now, from bench to bedside. However, the outstanding results coming from the modern immuno-oncology trials with new drugs have granted a new interest for immunotherapy in AML. Accordingly, the elderly population represents an ideal target, given the low percentage of patients eligible for allogeneic stem cell transplant. With that in mind, in the era of immunotherapy, we consider immunosenescence as the optimal background to start investigating a biology-driven approach to AML therapy in the elderly. By taking into account the physiological age-related changes of immune response, more personalized and tailored use of the new drugs and strategies harnessing the immune system against AML, has the potential to increase their efficacy and impact on clinical outcomes.

Current status of immunotherapy in acute myeloid leukemia

Current status of immunotherapy in acute myeloid leukemia

Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) Tcells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR Tcells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR Tcells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.

Advances in immunotherapy for acute myeloid leukemia.

Evasion of the host immune system is a key mechanism to promote malignant progression. Therapeutically targeting immune pathways has radically changed the treatment paradigm for solid and lymphoid tumors but has yet to be approved for myeloid malignancies. Here, we summarize the most recent advances in immunotherapy for acute myeloid leukemia. Topics reviewed here include adoptive cellular approaches (chimeric antigen receptor-T cells, natural killer and other immune cells), checkpoint inhibitors (anti-PD-1/PD-L1, anti-CTLA-4 and TIM-3) and vaccines (WT-1, HLA-A2 and hTERT). Emphasis is placed on agents with clear evidence of tumor-specific immune responses and/or clinical activity in early-phase trials. Despite concerns regarding heterogeneous antigen expression and cytokine release syndrome, immunotherapy remains a highly promising strategy for acute myeloid leukemia, particularly transplant-ineligible patients and minimal residual disease states.

Pilot Study on Mass Spectrometry-Based Analysis of the Proteome of CD34⁺CD123⁺ Progenitor Cells for the Identification of Potential Targets for Immunotherapy in Acute Myeloid Leukemia.

Targeting of leukemic stem cells with specific immunotherapy would be an ideal approach for the treatment of myeloid malignancies, but suitable epitopes are unknown. The comparative proteome-level characterization of hematopoietic stem and progenitor cells from healthy stem cell donors and patients with acute myeloid leukemia has the potential to reveal differentially expressed proteins which can be used as surface-markers or as proxies for affected molecular pathways. We employed mass spectrometry methods to analyze the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia. As a reference, CD34+CD123+ normal hematopoietic progenitor cells from five healthy, granulocyte-colony stimulating factor (G-CSF) mobilized stem cell donors were analyzed. In this Tandem Mass Tag (TMT) 10-plex labelling–based approach, 2070 proteins were identified with 171 proteins differentially abundant in one or both cellular compartments. This proof-of-principle-study demonstrates the potential of mass spectrometry to detect differentially expressed proteins in two compartment fractions of the entire proteome of leukemic stem cells, compared to their non-malignant counterparts. This may contribute to future immunotherapeutic target discoveries and individualized AML patient characterization.

Allogeneic Human Double Negative T Cells as a Novel Immunotherapy for Acute Myeloid Leukemia and Its Underlying Mechanisms.

Purpose: To explore the potential of ex vivo expanded healthy donor-derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients.Experimental Design: Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry-based in vitro killing assays and xenograft models against AML patient blasts and healthy donor-derived hematopoietic cells. Mechanism of action was investigated using antibody-mediated blocking assays and recombinant protein treatment assays.Results: Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples in vitro, and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor-unrestricted manner. Importantly, allogeneic DNTs do not attack normal hematopoietic cells or affect hematopoietic stem/progenitor cell engraftment and differentiation, or cause xenogeneic GVHD in recipients. Mechanistically, DNTs express high levels of NKG2D and DNAM-1 that bind to cognate ligands preferentially expressed on AML cells. Upon recognition of AML cells, DNTs rapidly release IFNγ, which further increases NKG2D and DNAM-1 ligands' expression on AML cells. IFNγ pretreatment enhances the susceptibility of AML cells to DNT-mediated cytotoxicity, including primary AML samples that are otherwise resistant to DNTs, and the effect of IFNγ treatment is abrogated by NKG2D and DNAM-1-blocking antibodies.Conclusions: This study supports healthy donor-derived allogeneic DNTs as a therapy to treat patients with chemotherapy-resistant AML and also reveals interrelated roles of NKG2D, DNAM-1, and IFNγ in selective targeting of AML by DNTs. Clin Cancer Res; 24(2); 370-82. ©2017 AACR.

Update on Immunotherapy in AML and MDS: Monoclonal Antibodies and Checkpoint Inhibitors Paving the Road for Clinical Practice.

In the past few years, our improved understanding of the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) has led to remarkable advances in the development of novel therapeutic approaches for these diseases. This chapter summarizes the available clinical data with immune-based therapeutic modalities in AML and MDS, focusing on monoclonal antibodies, T cell engager antibodies, chimeric antigen receptor (CAR)-T cells, and checkpoint blockade via blockade of PD-1/PD-L1 or CTLA4. Numerous clinical trials are currently ongoing in patients with AML and MDS, both in the frontline and relapsed refractory setting. Given the natural diversity of AML blasts, it became apparent that the best responses would be achieved with rationally designed combination strategies of immunetherapy, moleculartherapy, and chemotherapy. A number of such combinations are enrolling patients with AML in variousclinical settings. Biomarkers to select the optimal combination regimen for individual patients are critical.

Abstract B111: Characterization of APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecule for redirected T-cell cytotoxicity, in preclinical models of AML and nonhuman primates

Abstract Introduction: CD123 has emerged as a promising target for T-cell directed immunotherapy in acute myeloid leukemia due to its high level of expression on leukemic blasts and stem cells and low frequency of expression on normal cell types. CD123 is expressed by rare normal leukocyte populations, including basophils and plasmacytoid dendritic cells in circulation and hematopoietic progenitor cells in the bone marrow. We have developed APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR molecule for redirecting T-cell cytotoxicity to CD123-expressing tumor cells. Results are presented that examine the in vitro and in vivo activity of APVO436 in preclinical models of AML, as well as pharmacokinetics and tolerability in nonhuman primates. Methods: CHO-produced APVO436 protein was used for in vitro functional studies with CD123+ AML tumor cell lines and primary human T-cell populations. Cytotoxic activity was determined using chromium release assays. On-cell binding, T-cell activation and proliferation were assessed using multicolor flow cytometry. Cytokine levels were measured using multiplex assay kits. In vivo studies to examine tumor growth inhibition activity were performed using NSG mice with established AML tumors followed by treatment with APVO436 coadministered with T cells. Tumor growth was assessed by bioluminescent imaging. In vivo studies to determine pharmacokinetics of APVO436 following single intravenous injections were performed in healthy BALB/c mice and cynomolgus monkeys. Results: APVO436 bound to CD123 and CD3 expressing cells with high affinity. APVO436 induced concentration-dependent lysis of CD123+ AML cell lines with primary human T cells at low effector-to-target ratios, accompanied by T-cell activation and proliferation. These activities were dependent on the expression of CD123 by the tumor target cells. Treatment of established disseminated tumors in mice with APVO436 resulted in a significant reduction in tumor burden. A single 10 mg/kg IV injection of APVO436 in normal BALB/c mice demonstrated an extended elimination serum half-life of approximately 300 hours. Single IV injections of APVO436 ranging from 0.25 mg/kg to 1 mg/kg were well tolerated in cynomolgus monkeys. The observed elimination half-life in cynomolgus serum was up to 84 hours with normal clearance and volume distribution. Conclusions: These data demonstrate APVO436 has potent in vitro activity against a number of CD123-expressing tumor cell lines and in vivo significantly reduces established tumor burden in xenograft models. APVO436 demonstrates an extended serum half-life in normal mice and cynomolgus monkeys. These data are supportive of further investigation of APVO436 as a potential treatment option for AML and other hematologic malignancies. Citation Format: Michael R. Comeau, Robert E. Miller, Jeannette Bannink, Starrla Johnson, Robert Bader, Rebecca Gottschalk, Lynda Misher, Danielle Mitchell, Lara Parr, Melissa DeFrancesco, David Bienvenue, Catherine J. McMahan, Gabriela H. Hoyos, Jane A. Gross. Characterization of APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecule for redirected T-cell cytotoxicity, in preclinical models of AML and nonhuman primates [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B111.