Acute Myeloid Leukemia Tumor Cells Research Articles

The Role and Mechanism of NDUFS2 in FLT3-Mutated Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by poor prognosis and high recurrence rates. Disease relapse and the lack of precise treatment tailored to each patient pose challenges in AML treatment. The mitochondria, crucial for cellular energy center and signaling, are essential for maintaining the metabolic flexibility of tumor cells. However, their complex and elusive functions in cancer pose challenges for targeted mitochondrial therapies. Therefore, there is an urgent need to discover new mitochondrial-targeted drugs and novel targeted therapeutic strategies for AML. In this study, we explored the expression of NDUFS2 in AML using real-time quantitative PCR and immunohistochemistry. Subsequently, we performed knockdown and overexpression experiments of NDUFS2 in MOLM-13 and MV4-11 cell lines and assessed its effects on AML cell proliferation, apoptosis, cell cycle, reactive oxygen species (ROS) levels, and mitochondrial membrane potential using flow cytometry. Additionally, we evaluated the impact of NDUFS2 on mitochondrial morphology using transmission electron microscopy and confocal microscopy. Seahorse energy metabolism assay kits were utilized to examine the influence of NDUFS2 on AML cell energy metabolism, while mitochondrial complex I and II assay kits were employed to assess its effect on mitochondrial enzyme activity. Furthermore, Western blot analysis was conducted to evaluate the effects of NDUFS2 knockdown on the AMPK pathway, endoplasmic reticulum stress pathway, and apoptosis pathway in AML cells, with reverse validation performed using AMPK and PERK inhibitors. Finally, we established xenograft models of FLT3-mutated AML tumor cells to investigate the impact of NDUFS2 on tumor growth and the synergistic effect of NDUFS2 with FLT3 inhibitor Gilteritinib against FLT3-mutated AML. Concurrently, we developed xenograft models of relapsed/refractory FLT3-mutated AML patients to explore the synergistic effect of mitochondrial complex I inhibitor IACS-010759 with Gilteritinib against relapsed/refractory FLT3-mutated AML. We validated that NDUFS2 exhibits the highest expression level in AML, especially in FLT3-mutated AML cells. Knockdown of NDUFS2 inhibited the proliferation of FLT3-mutated AML cell lines, caused cell cycle arrest, induced mitochondrial fission, decreased mitochondrial membrane potential, increased ROS levels, impaired the function of mitochondrial complex I, altered cellular energy metabolism by reducing oxidative phosphorylation (OXPHOS) levels, and activated the AMPK/PERK pathways, leading to apoptosis. Knockdown of NDUFS2 enhanced the cytotoxicity of Gilteritinib against FLT3-mutated AML cells, and the combination of NDUFS2 knockdown and Gilteritinib reduced mitochondrial membrane potential, increased ROS levels, and activated the AMPK/PERK pathways to induce cell apoptosis. In vivo experiments confirmed that knockdown of NDUFS2 enhanced the antitumor effect of Gilteritinib. Furthermore, IACS-010759 and Gilteritinib exhibited synergistic activity against FLT3-mutated AML by synergistically inducing cell apoptosis, inhibiting cell proliferation, affecting mitochondrial function, activating the AMPK/PERK pathways. In vivo experiments in mice also confirmed the synergistic killing effect on FLT3-mutated AML tumor cells without affecting normal hematopoiesis or the normal function of other organs. We found that NDUFS2 is associated with the prognosis of AML and is highly expressed in FLT3-mutated AML. Knockdown of NDUFS2 suppresses the proliferation of FLT3-mutated AML cells, induces excessive ROS production, disrupts mitochondrial membrane potential, inhibits the enzyme activity of mitochondrial complex I, thereby suppressing OXPHOS, decreasing ATP production, and activating the AMPK/PERK pathways, ultimately leading to cell apoptosis. Targeting NDUFS2 can exert synergistic anti-FLT3-mutated AML effects with FLT3 inhibitors by activating the AMPK/PERK pathways. Additionally, the inhibition of mitochondrial complex I by the inhibitor IACS-010759 can synergize with Gilteritinib to combat FLT3-mutated AML. This indicates that NDUFS2 may serve as a potential target for the treatment of FLT3-mutated AML and enhance the sensitivity of targeted drugs to FLT3-mutated AML.

A novel bispecific T-cell engager using the ligand-target csGRP78 against acute myeloid leukemia

Current medical therapies for treating acute myeloid leukemia (AML) remain unmet, and AML patients may benefit from targeted immunotherapy approaches that focus on specific tumor antigens. GRP78, which is upregulated in various malignant tumors such as AML, is partially expressed as cell surface GRP78 (csGRP78) on the cell membrane, making it an ideal target for redirecting T cells, including T-cell engagers. However, considering the conventional approach of using two scFv segments to construct a bispecific T-cell engager (BiTE), we have undertaken the development of a novel BiTE that utilizes a cyclic peptide ligand to specifically target csGRP78, which we refer to as GRP78-CD3/BiTE. We studied the effects of GRP78-CD3/BiTE on treatments for AML in vitro and in vivo and assessed the pharmacokinetics of this engager. Our findings demonstrated that GRP78-CD3/BiTE could not only effectively mediate the cytotoxicity of T cells against csGRP78-expressing AML cells but also specifically eliminate primary AML tumor cells in vitro. Furthermore, GRP78-CD3/BiTE exhibited a longer half-life despite having a lower molecular weight than CD19-CD3/BiTE. In a xenograft mouse model of AML, treatment with GRP78-CD3/BiTE prolonged the survival time of the mice. Our findings demonstrate that GRP78-CD3/BiTE is effective and selective for eliminating csGRP78-expressing AML cells and suggest that this approach to targeted immunotherapy could lead to effective new treatments for AML.

Abstract 6323: Preclinical evaluation of CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy, that exhibits specific and potent toxicity in acute myeloid leukemia (AML) xenograft models

Abstract Background: CLL-1 is a compelling therapeutic target for AML as it is highly expressed on AML tumor cells and leukemic stem cells but is not expressed on hematopoietic stem cells. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology and leverages both checkpoint disruption and immune cloaking armoring strategies to potentially improve antitumor activity. The CB-012 anti-CLL-1 CAR was developed with a fully human scFv and the CD28 costimulatory domain and is currently in development for the treatment of relapsed or refractory AML (r/r AML). Here we describe preclinical studies that supported the CB-012 IND clearance by the FDA in October 2023. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012 through prevention of GvHD, PD-1 checkpoint disruption, and suppression of allograft rejection. In vitro and in vivo studies evaluated the specificity of antigen binding, antigen-dependent activity, and toxicologic potential. Results: CB-012 demonstrated potent antigen-dependent expansion and cytotoxic activity against CLL-1+ human AML cell lines and patient-derived cells in co-cultures. In AML xenograft models, a single dose of CB-012 CAR-T cells resulted in robust tumor control, leading to significant prolongation of survival. CB-012 co-culture with multiple CLL-1-negative cell types representing vital tissues demonstrated that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated highly specific interaction with human CLL-1, with no detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised murine models. Conclusion: CB-012, the first allogeneic anti-CLL-1 CAR-T cell therapy using both checkpoint disruption and immune cloaking armoring, demonstrated specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was demonstrated in an unbiased protein-binding study and no adverse safety signals were observed from CB-012 in murine toxicology models. These preclinical studies supported the IND clearance of CB-012, which is being evaluated in the AMpLify trial, a Phase 1, first-in-human clinical trial for patients with r/r AML (NCT06128044). Citation Format: Brian Francica, Elizabeth Garner, Sai Namburi, Cian Colgan, Tristan Fowler, Devin Mutha, Art Aviles, Morena Stanaway, Raymond Guo, Zili An, Erin Kelly, Emilie Degagne, George Kwong, Leslie Edwards, Emma Jakes, McKay Shaw, Benjamin Schilling, Jeremy Huynh, Ricky Luu, Max Sidorov, Rhonda Mousali, Mikk Otsmaa, Peter Lauer, Justin Skoble, Steven Kanner. Preclinical evaluation of CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy, that exhibits specific and potent toxicity in acute myeloid leukemia (AML) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6323.

Leukemic mutation FLT3-ITD is retained in dendritic cells and disrupts their homeostasis leading to expanded Th17 frequency.

Dendritic cells (DC) are mediators between innate and adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for acute myeloid leukemia (AML) patients. Having a shared myeloid lineage it can be difficult to distinguish bone fide DCs from AML tumor cells. To date, there is little information on the effects of FLT3-ITD in DC biology. To further elucidate this relationship we utilized CITE-seq technology in combination with flow cytometry and multiplex immunoassays to measure changes to DCs in human and mouse tissues. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with AML to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet- phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4+ T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4+ T cells in the bone marrow and spleen compartments. Ex vivo stimulation of CD4+ T cells also showed increased Th17 phenotype in AML mice. Moreover, co-culture of AML mouse-derived DCs and naïve OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4+ T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes and their downstream effects on Th polarization.

Dual-targeting CD33/CD123 NANOBODY T-cell engager with potent anti-AML activity and good safety profile

AbstractNovel therapies are needed for effective treatment of acute myeloid leukemia (AML). Relapse is common and salvage treatment with cytotoxic chemotherapy is rarely curative. CD123 and CD33, 2 clinically validated targets in AML, are jointly expressed on blasts and leukemic stem cells in >95% of patients with AML. However, their expression is heterogenous between subclones and between patients, which may affect the efficacy of single-targeting agents in certain patient populations. We present here a dual-targeting CD33/CD123 NANOBODY T-cell engager (CD33/CD123-TCE) that was designed to decrease the risk of relapse from possible single antigen-negative clones and to increase coverage within and across patients. CD33/CD123-TCE killed AML tumor cells expressing 1 or both antigens in vitro. Compared with single-targeting control compounds, CD33/CD123-TCE conferred equal or better ex vivo killing of AML blasts in most primary AML samples tested, suggesting a broader effectiveness across patients. In a disseminated cell-line–derived xenograft mouse model of AML, CD33/CD123-TCE cleared cancer cells in long bones and in soft tissues. As cytokine release syndrome is a well-documented adverse effect of TCE, the compound was tested in a cytokine release assay and shown to induce less cytokines compared to a CD123 single-targeting control. In an exploratory single-dose nonhuman primate study, CD33/CD123-TCE revealed a favorable PK profile. Depletion of CD123 and CD33 expressing cells was observed, but there were neither signs of cytokine release syndrome nor clinical signs of toxicity. Taken together, the CD33/CD123 dual-targeting NANOBODY TCE exhibits potent and safe anti-AML activity and promises a broad patient coverage.

Engineered Natural Killer Cells Expressing Chimeric Ilt Receptors (CIR) Effectively Target HLA-G Positive AML Tumor Cells

Background Human leukocyte antigen-G (HLA-G), the immune checkpoint of pregnancy, directs immunosuppression by binding to Ig-like transcript (ILT) receptors (ILT2 in lymphoid cells and ILT4 in myeloid cells) via immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling domains. HLA-G is expressed in more than 50% of solid tumors and leukemias as a mechanism to evade immune attack. As a result, HLA-G is an excellent tumor-specific antigen since its expression in normal tissues is low except for cytotrophoblasts in the placenta where it protects the fetus from the mother's immune system. We present here a novel approach to target HLA-G-expressing Acute Myeloid Leukemia (AML) cells by utilizing the natural receptors of ILT2 and ILT4 coupled to positive intracellular signaling domains. Such chimeric ILT receptors, or CIRs drive activation of Natural Killer (NK) cells. Further, we have identified unique intracellular activation modules that function to robustly enhance cytotoxicity and expansion of NK cells upon engagement with tumor cells. Methods CD56 + NK cells were isolated from PBMCs, cultured with IL-15 and activated with a feeder-free cocktail of an immobilized cytokine and activating ligand. Activated NK cells were transduced with γ-retroviruses directing expression of CIR proteins (CIR.4-1BB.CD3ζ or CIR.X.Y where X and Y are candidate activation and coactivation moieties), soluble IL-15 and a ΔCD19 marker or CAR control constructs directed to HLA-G or CD33. After 8 days of expansion, CIR- or CAR-NK cells were cocultured with GFP ffluc-expressing AML target cell lines with or without transgenic HLA-G (G1, G2 and G5) isoforms. Cytotoxicity of CIR-NK cells was determined by reduction of GFP fluorescence in Incucyte or Celligo instruments or luciferase activity and supernatants were analyzed for IFN-γ and TNF-α release by ELISA. Transduced NK cells were labeled with nuclear-RFP for measurement of expansion in coculture. In comparative experiments, T cells from PBMCs were transduced to produce CIR-T cells by standard methods. Results CIR (ILT2 or ILT4.4-1BB.ζ) constructs were efficiently and stably expressed in T and NK cells (>80% ΔCD19 + 8- and 14-days post activation). Mock-transduced NK cells displayed innate killing activity against HLA-G negative AML cells (THP1 and KG1) that was not augmented by CIR constructs (mock = 6.16E6 ± 0.77E6 vs. CIR = 6.43E6 ± 0.37E6 in coculture against KG1 cells). In cocultures against THP1 and KG1 target cells expressing exogenous G1, G2 or G5 isoforms, CIR-NK cells or CIR-T cells displayed enhanced target cell killing in 2 and 7-day assays relative to mock-transduced effector cells (mock = 1.47E6 ± 0.49E6 vs. CIR = 0.57E6 ± 0.6 in coculture against KG1-HLA-G1). Further, while mock NK cells transduced with RFP alone could kill one-third of AML cells expressing endogenous HLA-G1 and G5 (Molm13 and Kasumi1), CIR-NK cells eliminated more than 90% of these AMLs and their activivation resulted in 4-fold higher IFN-γ secretion. Notably, robust cytotoxicity of CIR-NK cells was maintained even at 28 days post activation (mock = 36.95E6 ± 21.85E6 vs. CIR = 7.38E6 ± 1.92 in coculture against Kasumi1). To further enhance the functionality of the CIR constructs, a screen of novel costimulatory molecules to replace 4-1BB and/or CD3ζ resulted in several potent candidates ultilizing MyD88 signaling that greatly improved the cytotoxicity (> 80% tumor elimination), proliferation (> 2-fold expansion) and proinflammatory cytokine production (IFN-γ and TNF-α) of NK cells in cocultures. A subset is displayed in Figure 1. Conclusions The use of natural receptors ILT2 and ILT4 to target HLA-G takes advantage of the physiological pairing of the ILT receptors with the multiple immunosuppressive HLA-G isoforms thereby circumventing the possibility of antigen escape often observed in CAR-directed therapy. We demonstrated that engineered CIR-NK cells can convert HLA-G expression in AML from a potent inhibitor of leukocyte activity into a target for anti-tumor control.

CLL-1-Targeted Allogeneic CAR-T Cells Exhibit High on-Target Specificity and Potent Cytotoxicity in Preclinical Models of Acute Myeloid Leukemia

Background: CLL-1 is a compelling therapeutic target for acute myeloid leukemia (AML) as it is highly expressed on AML tumor cells and leukemic stem cells, but is not expressed on hematopoietic stem cells. An allogeneic anti-CLL-1 CAR-T cell therapy (CB-012) is in development for relapsed or refractory (r/r) AML. The CAR was generated with a fully human scFv targeting CLL-1 that was selected from a panel of scFvs. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology to leverage both checkpoint disruption and immune cloaking for potentially improved antitumor activity. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to reduce graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding to prolong antitumor activity. This multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012. In vitro and in vivo studies evaluated specificity for antigen binding, antigen-dependent activity, and preclinical safety assessments. Results: CB-012 CAR-T cells express a fully human anti-CLL-1 scFv-containing CAR construct and demonstrate potent antigen-dependent cytotoxic activity in human AML cell line co-cultures. In AML xenograft models, a single-dose administration of CB-012 CAR-T cells exerted robust tumor control, leading to significant prolongation of survival. Cell binding studies suggested that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity when examined in the context of a collection of cell types representing vital tissues. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated specific interaction with human CLL-1, without detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised mouse models. Conclusion: CB-012 demonstrated highly specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was further demonstrated by screening in an unbiased protein-binding study and no adverse safety signals were observed in murine models. These data support advancing the development of CB-012 into a first-in-human clinical trial for patients with r/r AML.

Abstract 2902: Off-the-Shelf CAR-NK cells engineered to express calibrated release IL-15 exhibit enhanced persistence and anti-tumor activities

Abstract Chimeric antigen receptor (CAR) natural killer (NK) cell therapy is an attractive immunotherapy strategy due to potential for increased anti-tumor activity and favorable safety. Allogeneic donor derived NK cells have shown promising clinical outcomes in both hematological and solid tumor malignancies but have a short lifespan in the absence of cytokine support. Interleukin (IL)-15 is a pleiotropic cytokine that promotes the survival, proliferation, and cytotoxicity of NK cells. In this study, we engineered human peripheral blood-derived NK cells to co-express a CAR and calibrated release (cr) IL-15 and studied how crIL-15 affected CAR-NK cell functions. Senti Bio has designed the calibrated release technology to simultaneously produce membrane bound and secreted cytokines, such as IL-15 and IL-12, to provide autocrine and paracrine activity. We further validated crIL-15 activity in vitro and demonstrated enhanced NK cell persistence, tumor killing and autocrine activity in CAR NK cells as well as paracrine stimulation of neighboring T cells and NK cells via pSTAT5 activation. Additionally, we examined in vivo persistence and biodistribution of NK cells engineered with crIL-15 and a nanoluc reporter. crIL-15 NK cells were intravenously (i.v.) injected into NSG mice. These NK cells were detected in the lung, femur, spleen, liver and stomach 1 day post injection by bioluminescence imaging and polymerase chain reaction (PCR) and lasted up to 20 days in the lung. In addition, crIL-15 increased CAR-NK cells persistence in a dose-dependent manner compared to unengineered NK cells. Injection of higher CAR-NK cell numbers (15× 106 vs 7.5× 106 CAR+ cells per mouse) or multiple NK dosages (1 vs 3 doses) both contributed to prolonged persistence. Finally, we studied how crIL-15 affected CAR-NK cell anti-tumor function in acute myeloid leukemia (AML) xenograft models. MV4-11-Fluc AML tumor cells were co-injected with engineered NK cells expressing a FLT3 and/or CD33 bivalent activating CAR, a EMCN inhibitory CAR and crIL-15 into NSG-Tg (Hu-IL15) mice via i.v. injection. Our data showed that these CAR-NK cells significantly reduced MV4-11 tumor burden and prolonged mouse survival compared to unengineered NK cells. The improved anti-tumor functions correlated with IL-15 levels produced by the CAR-NK cells. In conclusion, our results demonstrated that crIL-15 can improve persistence and anti-tumor activity of CAR-NK cells. Citation Format: Chen-Ting Lee, Michelle Hung, Andrew Banicki, Wenqi Song, Niran Almudhfar, Deepika Kaveri, Priscilla Wong, Lawrence Naitmazi, Marcela Guzman, Alice Lam, Gozde Yucel, Timothy Lu, Alba G. Junca, Brian Garrison, Philip Lee. Off-the-Shelf CAR-NK cells engineered to express calibrated release IL-15 exhibit enhanced persistence and anti-tumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2902.

Preclinical Evaluation of ISB 1442, a First-in-Class CD38 and CD47 Bispecific Antibody Innate Cell Modulator for the Treatment of AML and T-ALL

ISB 1442 is a bispecific antibody (BsAb) using Ichnos' proprietary Bispecific Engagement by Antibodies based on the TCR (BEAT®) platform. A fully human BsAb with anti-CD38 and CD47 binding arms, ISB 1442 was developed for the treatment of hematologic malignancies. The CD38 binding arm consists of two bi-paratopic Fabs that strongly bind to CD38 through avidity-induced interactions. The anti-CD47 arm comprises a single Fab designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes. With this design, the CD38 Fab arm preferentially drives binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. Hence, ISB 1442 is anticipated to induce minimal effects on red blood cells (RBC) compared to benchmark anti-CD47 monoclonal antibodies (mAb), such as magrolimab. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Preclinical activity of ISB 1442 for the treatment of relapsed/refractory multiple myeloma (rrMM), including increased killing potency relative to daratumumab and magrolimab benchmarks as well as more favorable off tumor/on target specificity, was reported previously (Sammicheli et al. ASH 2021). ISB 1442 is currently in a Phase 1 clinical trial for rrMM. Beyond MM, CD38 and CD47 are expressed in other hematologic cancers, including acute myeloid leukemia (AML) and T cell acute lymphoblastic leukemia (T-ALL). In AML, unleashing tumor cell phagocytosis using magrolimab shows clinical benefit in combination with demethylating agents and BCL-2 inhibitors. T-ALL is characterized with high CD38 expression, and daratumumab in combination with chemotherapy is under investigation (NCT03384654) for this indication. ISB 1442 has the potential to treat AML and T-ALL patients with its first-in-class molecular attributes of co-targeting CD38 and CD47 by a single antibody with enhanced Fc effector functions. ISB 1442 induced potent phagocytosis of both AML and T-ALL cell lines in vitro. The induction of phagocytosis was more prominent in cell lines expressing high level of CD38. ISB 1442 also induced higher phagocytosis of both AML and T-ALL tumor cells as compared to daratumumab and anti-CD47 (5F9) in cell lines expressing low level of CD38 and CD47. In addition to phagocytosis, ISB 1442 induced potent ADCC of both AML and T-ALL cell lines. To characterize the complex mechanisms of action of ISB 1442 in a single assay, a multiple mode of action of killing (MMoAK) in vitro assay was established where macrophages and PBMCs are incubated with AML or T-ALL tumor cells and human serum. With this approach, tumor cells can be targeted simultaneously by NK cells from PBMCs (ADCC Mechanism of Action (MOA)), macrophages (ADCP MOA), and complement from human serum (CDC MOA). ISB 1442 induced higher killing of AML and T-ALL cell lines in MMoAK assays as compared to daratumumab or anti-CD47 (5F9). ISB 1442 induced potent killing of primary tumor samples from both AML and T-ALL patients in a heterologous assay using macrophages from healthy donors as effector cells, suggesting that target cancer cells do not have intrinsic mechanisms protecting them from the immune cell-mediated MOA of ISB 1442 killing . In patients' samples having detectable levels of myeloid or NK cells in bone marrow aspirates, ISB 1442 induced killing of tumor cells in the autologous setting in a direct killing assay ex vivo. In summary, these data support the clinical development of 1442 in AML and T-ALL. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to have antitumor activity in AML and T-ALL patients in a single antibody relative to anti-CD38 or anti-CD47 monoclonal antibodies as well as their combination.

Defining Clinical and Molecular Biomarkers for Venetoclax-Based Drug Combinations to Augment AML Therapy

Background: The combination of the BCL2 inhibitor venetoclax with azacytidine (Ven+Aza) is efficacious for elderly patients with acute myeloid leukemia (AML) who are unfit for standard chemotherapy. However, the overall and progression-free survival of these patients after 1 year of therapy is approximately 30-40%, and resistance to Ven+Aza, either primary or adaptive through clonal evolution, limits its durability. Therefore, we evaluated new venetoclax combinations to elucidate mechanisms and biomarkers of response and resistance. Methods: We tested a set of 31 venetoclax (Ven) combinations using an ex vivo assay on primary AML patient specimens (n=433, though not all combinations were screened for all samples). Enhanced efficacy was determined using a combination ratio (CR), defined as the sensitivity of the combination (by AUC) divided by the sensitivity of the most potent single agent AUC. Drug combination synergy was evaluated using the highest single agent (HSA) method. Clinical, genetic, and disease status information were collated, and ex vivo combination sensitivities were compared to matched single-agent data by Friedman test, across groups by Wilcoxon rank sum or Kruskal-Wallis test, and with continuous variables by Spearman rank correlation. For patient specimens with RNAseq data collected (n=279 patients), transcriptomic gene signatures of AML tumor cell state (van Galen, Cell 2019) were correlated with combination sensitivity by Spearman rank. Uni- and multi-variate analyses were applied to the full dataset to identify biomarkers of differential sensitivity to each combination. For one combination, Ven plus the JAK1/2 inhibitor ruxolitinib (Rux), additional genome wide CRISPR screens and follow up in vivo patient xenograft and mechanistic validation experiments using AML cell line models were performed. Results: Using Ven+Aza as a benchmark, we found several venetoclax combinations that exceeded both the ex vivo activity and enhanced efficacy observed with Ven+Aza in primary AML patient samples (Fig. 1A), while showing limited to no effect on stromal cell controls. Evaluation of clinical and genetic data with combination sensitivity revealed many significant transcriptomic, genetic, and immunophenotypic associations. The strong bias in sensitivity of Ven with respect to transcriptomic signatures of primitive tumor cell states compared to more mature, monocytic tumor states was unchanged with Ven+Aza and select other combinations. Importantly, this differential sensitivity was overcome when pairing Ven with several new drug partners such as Rux or the p38 MAPK inhibitor doramapimod (Fig. 1B). Uni- and multi-variate analyses further revealed an array of different biomarkers involving clinical and genetic features that correlated with significant differential sensitivity to each combination. Many of these likewise involved markers of cell differentiation state, such as CD14. To elucidate the mechanisms of enhanced efficacy of the Ven+Rux combination, which is currently under phase 1 clinical investigation, in vivo murine patient xenograft models and genome-wide CRISPR screens in OCI-AML2 cells were performed. Ven+Rux demonstrated superior inhibition of tumor growth in an AML xenograft model compared to either single-agent. The Ven+Rux CRISPR screen identified many genes whose knockdown was enriched in combination-resistant cells, including three (MDM2, MED13L, MEIS2) that were confirmed to confer resistance in subsequent immunoblotting and drug sensitivity validation experiments. In comparison, analysis of a control CRISPR screen using Ven alone revealed numerous gene hits in the JAK/STAT signaling pathway as enhancing the sensitivity of Ven. Conclusion: To expand the utility of venetoclax-inclusive combinations, we prospectively profiled a set of drug combinations and identified several with activity that exceeds Ven+Aza in ex vivo assays. Associations of clinical features with sensitivities to these drug combinations provide insight into their effectiveness which may be used to align the most promising combinations with translation to treatment of discrete subsets of AML patients. Together, these findings support opportunities for expanding the impact of Ven-based drug combinations in AML by leveraging clinical and molecular biomarkers of response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lymphocyte Exhaustion in AML Patients and Impacts of HMA/Venetoclax or Intensive Chemotherapy on Their Biology.

Simple SummaryPatients with acute myeloid leukemia (AML) are routinely treated with either intensive chemotherapy or DNA hypomethylating agents (HMA) in combination with the Bcl-2 inhibitor, venetoclax. While both treatment regimens are highly cytotoxic to the aggressive AML tumor cells, they are also toxic to immune cells. Therefore, we sought to establish the detrimental impacts of these therapies on lymphocytes and their recovery over time in AML patients. Even prior to treatment initiation, the patients were found to have exhausted lymphocytes in peripheral blood, and additional signs of exhaustion were noted after treatment with HMA/venetoclax. In fact, the lymphocytes were still suppressed for two to three months after the initiation of induction therapy. Furthermore, T cells in a subset of patients subsequently found to be resistant to venetoclax therapy exhibited a higher expression of perforin and CD39 and more pronounced IFN-γ production.Acute myeloid leukemia (AML) is an aggressive malignancy that requires rapid treatment with chemotherapies to reduce tumor burden. However, these chemotherapies can compromise lymphocyte function, thereby hindering normal anti-tumor immune responses and likely limiting the efficacy of subsequent immunotherapy. To better understand these negative impacts, we assessed the immunological effects of standard-of-care AML therapies on lymphocyte phenotype and function over time. When compared to healthy donors, untreated AML patients showed evidence of lymphocyte activation and exhaustion and had more prevalent CD57+NKG2C+ adaptive NK cells, which was independent of human cytomegalovirus (HCMV) status. HMA/venetoclax treatment resulted in a greater fraction of T cells with effector memory phenotype, inhibited IFN-γ secretion by CD8+ T cells, upregulated perforin expression in NK cells, downregulated PD-1 and 2B4 expression on CD4+ T cells, and stimulated Treg proliferation and CTLA-4 expression. Additionally, we showed increased expression of perforin and CD39 and enhanced IFN-γ production by T cells from pre-treatment blood samples of venetoclax-resistant AML patients. Our results provide insight into the lymphocyte status in previously untreated AML patients and the effects of standard-of-care treatments on their biology and functions. We also found novel pre-treatment characteristics of T cells that could potentially predict venetoclax resistance.

P871: ISB 1442, A FIRST-IN-CLASS CD38 AND CD47 BISPECIFIC ANTIBODY INNATE CELL MODULATOR FOR THE TREATMENT OF CD38 POSITIVE HEMATOLOGICAL MALIGNANCIES

Background: ISB 1442 is a first in class 2 + 1 biparatopic bispecific antibody targeting CD38 x CD47 using the BEAT® 2.0 (Bispecific Engagement by Antibodies based on the TCR) platform. The CD38 binding arm consists of bi-paratopic Fabs that strongly bind to CD38 in two different epitopes on tumor cells and do not functionally compete with daratumumab. The anti-CD47 arm comprises a single Fab arm designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes (including macrophages, monocytes and dendritic cells). With this design, the high affinity anti-CD38 Fab arms preferentially drive binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Aims: ISB 1442 was developed for the treatment of hematologic malignancies that express CD38, including multiple myeloma (MM), acute myeloid leukemia (AML), and T-acute lymphoblastic leukemia (T-ALL), with the intention of overcoming mechanisms of resistance to CD38-targeted therapies such as daratumumab and isatuximab, and minimizing hemagglutination/hemolysis on red blood cell (RBC) observed with anti-CD47 monoclonal antibodies (mAb) such as magrolimab. Methods: ISB 1442 was tested for its capacity in vitro to induce ADCP, ADCC and CDC relative to daratumumab and magrolimab across a broad range of MM, AML and T-ALL cell lines expressing different levels of CD38 and CD47. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells (ADCC), autologous macrophages (ADCP), and complement from human serum (CDC). In vivo, ISB 1442 was assessed in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice compared to daratumumab and magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. Results: In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing MM, AML and T-ALL tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. In the CDC, ADCC and MMoAK assays, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab in MM cell lines. In vivo, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. In monkeys, ISB 1442 showed more pronounced binding on RBC than magrolimab due to higher expression of CD38, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Summary/Conclusion: In summary, we report a novel approach for the treatment for CD38 positive hematologic malignancies by co-targeting CD38 and CD47 using a first in class multispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance.

FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML

Background: While chimeric antigen receptor (CAR) cell therapies have provided extraordinary clinical responses in some hematological malignancies, developing effective CAR cell therapies for acute myeloid leukemia (AML) has been challenging due to: (a) the lack of a single target antigen robustly expressed across both AML leukemic stem cell (LSC) and immature leukemic blast cell subpopulations, and (b) the lack of truly AML-specific target antigens, since current targets are also expressed on healthy tissues and may result in off-tumor toxicity. Using logic gated gene circuits, we are engineering our SENTI-202 CAR-NK cell therapy to overcome these long-standing challenges to treating AML patients.Methods: To maximize clearance of AML tumor subpopulations and minimize off-tissue toxicities, we used a proprietary bioinformatics paired antigen discovery platform to identify the optimal combinations of AML tumor-associated and healthy tissue antigens to target using an OR and NOT logic gated CAR gene circuit approach. The SENTI-202 therapeutic candidate is a FLT3 OR CD33 NOT Endomucin (EMCN) gene circuit-enabled allogeneic CAR-NK cell, designed to broadly target FLT3 and/or CD33-expressing AML tumor cells (including both LSCs and blasts) but not healthy hematopoietic stem cells (HSCs).Results: First, for the OR GATE portion of the logic circuit we demonstrated that engineered primary human NK cells expressing activating CARs (aCARs) that recognize both FLT3 and CD33 outperformed more traditional single target CAR approaches with FLT3 (p<0.05) or CD33 (p<0.01), and exhibited >80% cytotoxicity and significant cytokine secretion (GrB, IFN-g, and TNF-a) against multiple leukemia cell lines in vitro, including MOLM13, THP1, and SEM. We successfully engineered FLT3 OR CD33 CAR-NK cells using both bicistronic and bivalent CAR configurations, where bicistronic CARs possess separate FLT3 and CD33 CARs linked via a 2A peptide, and bivalent CARs use a loop structure to connect FLT3 and CD33 scFvs within the same CAR. While both approaches demonstrated robust efficacy against AML cells, the bivalent approach enabled greater CAR expression and cytotoxicity (p<0.05). Importantly, our FLT3 OR CD33 CAR-NK cells demonstrated significant cytotoxicity against primary AML patient samples (p<0.01-0.001) and significantly reduced tumor burden and improved mouse survival in MOLM13 (p<0.05) and MV4-11 (p<0.01) xenograft AML models. We believe that our strategy of concurrently targeting FLT3 and CD33 will result in a more robust synergistic anti-tumor effect, leading to a more durable remission with decreased risk of relapse due to single antigen escape.Second, for the NOT GATE portion of the logic circuit to protect healthy HSCs, we developed NK and T cell inhibitory CARs (iCARs) consisting of an scFv against a healthy cell antigen, hinge and transmembrane domains, and functional intracellular domains derived from inhibitory co-receptors containing immunoreceptor tyrosine-based inhibitory motifs. In the case of SENTI-202, the iCAR scFv recognizes EMCN, a surface antigen expressed on up to 76% of healthy HSCs but not on AML cells. Using two different iCAR configurations, we demonstrated that FLT3 (CD28z) aCAR-NK cells engineered with an EMCN-specific iCAR protected up to 67% (iCAR#1, p<0.01) or 50% (iCAR#2, p<0.01) of FLT3+ EMCN+ cells from FLT3 aCAR-mediated cytotoxicity. Next, to replicate a clinical context more closely, we mixed FLT3+ EMCN- (AML-like) and FLT3+ EMCN+ (HSC-like) target cells and demonstrated that FLT3 NOT EMCN CAR-NK cells exhibit preferential killing of FLT3+ EMCN- target cells (p<0.0001), demonstrating that our NOT GATED gene circuit controls NK-mediated responses on a cell-by-cell basis.Conclusion: SENTI-202 is a novel NK cell product candidate to be engineered with both OR and NOT logic gated CAR gene circuits, wherein the OR gate is designed to increase AML LSC/blast tumor clearance (to prevent relapse), and the NOT gate is designed to protect healthy HSCs from off-tumor toxicity, enabling regeneration of a healthy hematopoietic system and mitigating the need for a bone marrow transplant. Beyond AML, OR and NOT logic gated CAR-NK cell therapy has applicability to other cancer-associated antigen targets that are potentially limited by antigen escape and/or off-tumor toxicity, increasing the potential for enhanced efficacy and reduced risk of undesirable side effects. DisclosuresNo relevant conflicts of interest to declare.

279 ILT3 blockade reduces tumor blast cells in acute myeloid leukemia PBMC and inhibits tumor cell growth in a humanized AML tumor model

BackgroundImmunoglobulin-like transcript 3 (ILT3), an immune-inhibitory receptor expressed on myeloid cells, is highly expressed in acute myeloid leukemia (AML) with monocytic differentiation (M4 and M5 subtypes) and clinically is negatively correlated with overall survival. We examined the effects of a highly selective and potent chimeric anti-ILT3 mAb (c52B8) on tumor cell survival/growth and human immune cell modulation in AML PBMC and AML tumor cell lines in pre-clinical in vitro and in vivo models.MethodsILT3 mRNA and cell surface expression levels were measured by RNAseq and FACS, respectively. PBMC from AML patients with different levels of ILT3 cell surface expression were treated with c52B8 in vitro.A systemic AML model was generated by IV inoculation of NSG mice with luciferase-transfected MV-4-11 human AML cells, with or without human PBMC engraftment. Tumor bearing mice were treated weekly with c52B8, modified to contain the human Fc of either the IgG1 or IgG4 subclass. MV-4-11 growth in vivo was measured by bioluminescence imaging (BLI).CyTOF was performed to phenotype AML PBMC in in vitro culture and bone marrow samples from the systemic MV-4-11 model after the treatment.THP-1 cells/human T cell co-cultures stimulated with anti-CD3/CD28 antibodies were treated with c52B8. Human T cell activity was assessed by levels of IFNγ production in the culture using MSD ELISA.ResultsIn ILT3 high AML patient PBMC, c52B8 treatment in vitro decreased the frequency of tumor blasts, modulated Tregs, and increased monocytic myeloid cells. In the MV-4-11 model, weekly treatment with c52B8 significantly reduced tumor burden in vivo, regardless of the IgG subclass. Importantly, tumor cell growth inhibition by c52B8 occurred in the absence of human PBMC. Additional post-mortem CyTOF analysis of bone marrow cells from MV-4-11/human PBMC inoculated mice confirmed a reduction in MV-4-11 cells, identified by CD3-CD19- human myeloid cells following c52B8 treatment. In THP-1/human T cell co-cultures, c52B8 treatment reversed THP-1 induced T cell suppression, as measured by enhanced IFNγ production.ConclusionsAnti-ILT3 mAb (c52B8) is efficacious in blocking progression of AML with monocytic differentiation by directly influencing tumor cell growth and enhancing human T cell activity in pre-clinical models. These studies support a potential role for this compound in humans.Ethics ApprovalAll animal work was reviewed and approved by Merck IACUC before experiments were conducted.

Abstract 525: Novel DARPin multi-specific T-cell engager with an improved therapeutic window to overcome dose limiting toxicities in AML therapies

Abstract Purpose: The medical need due to high mortality in acute myeloid leukemia (AML) remains high, and the treatment of relapsed or refractory AML continues to be therapeutically challenging. MYLOTARG, the only approved anti-CD33 antibody drug conjugate (ADC), has provided proof-of-concept for targeted immunotherapies in AML. Currently, a plethora of ADCs and T-cell engager (TCE) therapies have entered clinical development in AML, but those therapies are often accompanied by dose limiting toxicities, preventing dose escalation to desired anti-tumor efficacy. The biggest challenges seem to be limited target specificity and hyperstimulation of the immune system leading to e.g. myelotoxicities and cytokine release syndrome, respectively. Therefore, more selective therapies are needed to allow for robust anti-tumor activity with a more acceptable safety profile. Experimental design: To address the selectivity challenge, we have generated multi-specific T-cell engaging DARPin® molecules, targeting two different tumor associated antigens (TAAs) with optimized affinity for their targets. In order to find the right target combination, the optimal affinity to increase tumor specificity via avidity, as well as the best molecular architecture, we took advantage of our unique modular DARPin® platform and screened 1000s of combinations of multi-specific DARPin® molecules, binding simultaneously to multiple TAAs in conjunction with our CD3-binding DARPin® molecule. Results: We constructed multi-specific TCEs targeting two different AML antigens with optimized affinity leading to a substantial avidity gain when both targets are co-expressed on tumor cells. The avidity gain resulted in strongly enhanced in vitro potency as shown by activation of both CD8+ and CD4+ T cells and subsequent killing of AML tumor cells, with bioactivities in the range of established TCE benchmark formats (e.g. BiTE® and DART®). In contrast, in an ex vivo whole blood assay the multi-specific DARPin® constructs induced profoundly less cytokine release as compared to benchmark molecules indicating an improved therapeutic window. Finally, we also demonstrated tumor regression in PMBC humanized mouse models bearing MOLM-13 tumors, using both half-life extended (HLE) and non-HLE lead constructs. In conclusion, we have generated TCEs based on multi-specific DARPin® constructs with high potency, selectivity and ultimately with the potential for an improved therapeutic window for the treatment of AML. Citation Format: Nina Reschke, Thamar Looser, Jennifer Krieg, Matteo Bianchi, Patricia Schildknecht, Nicole Bassler, Yvonne Gruebler, Sebastian Grimm, Laura Jeanbart, Tanja Hospodarsch, Alexandra Neculcea, Daniel Steiner, Bernd Schlereth, Christian Reichen. Novel DARPin multi-specific T-cell engager with an improved therapeutic window to overcome dose limiting toxicities in AML therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 525.

Autologous T Cells Modified to Co-Express CD33-Specific Chimeric Antigen Receptor and a Kill Switch for Treatment of CD33+ Acute Myeloid Leukemia

Relapsed/refractory acute myeloid leukemia (AML) is an aggressive malignancy with poor outcomes underscoring the need to implement new therapies. Adoptive transfer of genetically modified T cells with specificity redirected through a chimeric antigen receptor (CAR) has resulted in clinical responses, particularly for B-cell malignancies. CD33 is an attractive target for immunotherapy of AML as this transmembrane protein is expressed on majority of AML blasts. On target, but off tumor effects are anticipated as this target is also expressed on normal myeloid cells and on a subset of activated T and NK cells. Therefore, we generated a lentiviral vector co-expressing a CD33-specific CAR with a kill switch, HER1t, for the generation of CD33-specific CAR+HER1t+ T (CD33-CAR-T) cells. HER1t improves the safety profile by providing a mechanism for selective in vivo depletion of these genetically modified T cells upon addition of the clinically-available monoclonal antibody, cetuximab. Co-expression of CAR and HER1t on transduced T cells was confirmed by flow cytometry and western blot analyses. The redirected specificity was demonstrated by the specific killing of CD33+ tumor cells as well as significant release of cytokines IFNγ, TNFα, IP-10, IL-13, IL-18, and LIF upon co-culture with AML tumor cells. The ability to eliminate AML in vivo was determined in immunocompromised (NSG) mice bearing MOLM-13 (CD33+CD19neg) tumor. A single administration of human CD33-CAR-T cells resulted in a significant reduction in tumor burden (Figure) and improvement in overall survival with an overall median survival time of 46 days compared to 15 days for control treated mice (saline only, untransduced (CARneg) T cells, or CD19-specific CAR+ T (CD19-CAR-T) cells). Assessment from the plasma of CD33-CAR-T treated mice revealed production of cytokines consistent with observations from in vitro activation of genetically modified T cells. CD33-CAR-T cells were effectively eliminated in the presence of cetuximab both in vitro in an antibody dependent cell cytotoxicity assay and in an in vivo adoptive T cell transfer model in immunocompromised NOD/SCID mice. In preparation for a clinical trial, we manufactured CD33-CAR-T cells from an individual with relapsed AML. As with T cells from healthy volunteers, transduced T cells from the patient co-expressed CAR and HER1t and were effective at specifically eliminating autologous CD33+ tumor as well as producing cytokines in response to CAR signaling. These pre-clinical data provide a strong rationale to evaluate CD33-CAR-T therapy in a clinical trial for treatment of AML. The FDA has approved an investigational new drug application to initiate phase 1 clinical trial at MD Anderson Cancer Center for treatment of CD33+relapsed/refractory AML with autologous CD33-CAR-T cells. [Display omitted] DisclosuresSong:Intrexon Corporation: Employment. Tian:Intrexon Corporation: Employment. Carvajal-Borda:Intrexon Corporation: Employment. Plummer:Intrexon Corporation: Employment. Shah:Intrexon Corporation: Employment. Wierda:Emergent: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria; Karyopharm: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; GSK/Novartis: Consultancy, Honoraria, Research Funding; Genentech/Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Kite: Research Funding; Genzyme: Consultancy, Honoraria; The University of Texas MD Anderson Cancer Center: Employment; Pharmacyclics: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Sanofi: Consultancy, Honoraria; Acerta: Research Funding; Juno: Research Funding. Cooper:Organovo Holdings: Equity Ownership; Sangamo Biosciences: Patents & Royalties; Miltenyi Biotec: Honoraria; Ampliphi Biosciences: Equity Ownership; Argos Therapeutics: Equity Ownership; Intrexon Corporation: Equity Ownership, Patents & Royalties; Procter & Gamble: Equity Ownership; Targazyme, Inc.: Equity Ownership; Ferring: Consultancy; Immatics: Equity Ownership, Patents & Royalties, Research Funding; Ziopharm: Employment, Equity Ownership, Research Funding. Chan:Intrexon Corporation: Employment.

Overcoming target epitope masking resistance that can occur on low-antigen-expresser AML blasts after IL-1RAP chimeric antigen receptor T cell therapy using the inducible caspase 9 suicide gene safety switch

Although chimeric antigen receptor CAR) T cell immunotherapies are an undeniable and unequivocal success, knowledge obtained from the monitoring of the first clinical trials targeting the CD19 antigen in B malignancies, either refractory/relapsed acute lymphoid leukemia (ALL) or lymphomas, contributed to the identification of tumor cell escape in about 30–50% of B-ALL. Resistance occurred due to loss of surface expression of the antigen (rCD19−) or to the early disappearance or inactivation of CAR T cells (rCD19+). In a recently reported clinical case, rCD19− relapse resulted from masking of the antigen by the CAR at the surface of B-ALL leukemia cells following the unexpected viral transduction of a leukemic cell present in the cytapheresis sample. The objective of this work was to reproduce this epitope-masking resistance model, in the context of acute myeloid leukemia (AML), based on our immunotherapeutic CAR T cell model targeting the accessory protein of the interleukin-1 receptor (IL-1RAP) expressed by leukemic stem cells. As AML primary blasts express different levels of IL-1RAP, we modeled transduction of different AML tumor cell lines screened for density of antigenic sites with our lentiviral vectors carrying a third-generation IL-1RAP CAR, an iCASP9 suicide gene, and a truncated CD19 surface gene. We demonstrated that primary AML blasts can be easily transduced (74.55 ± 21.29%, n = 4) and that CAR T cytotoxicity to IL-1RAP is inversely correlated with epitope masking in relation to the number of antigenic sites expressed on the surface of IL-1RAP+ lines. Importantly, we showed that, in vitro, a 24-h exposure of IL-1RAP+/CAR+ leukemia lines to Rimiducid eliminated >85% of the cells. We confirmed that the expression of IL-1RAP CAR by an IL-1RAP+ leukemic cell, by decreasing the membrane availability of the targeted antigen, can induce resistance while a high epitope density maintains sensitivity to CAR T cells. Moreover, the presence of the iCASP9/Rimiducid suicide system safety switch makes this immunotherapy approach safe for application in a future phase 1 clinical trial.

High PD‑L1 expression drives glycolysis via an Akt/mTOR/HIF‑1α axis in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematological malignancy derived from immature myeloid cells, which have the characteristics of abnormal proliferation and differentiation. Glycolysis has been a popular topic of research in recent years, with increasing uptake and consumption of glucose. The present study aimed to investigate the glycolysis of tumor cells in patients with AML; in particular, how programmed cell death 1 ligand 1 (PD‑L1) regulates tumor cells glycolysis using real time PCR (RT‑PCR), western blotting and flow cytometry. PD‑L1 high expression predicted poor outcome in patients with AML in the public database Gene Expression Profiling Interactive Analysis. PD‑L1 expression was decreased in the samples from patients with AML with complete remission compared to that in patients with relapsed or refractory AML. In AML cell lines, glycolysis‑associated genes ALDOA, PGK1, LDHA and HK2 were highly expressed in a PD‑L1 high‑expressed cell line. Overexpressed PD‑L1 enhanced glucose consumption and the extracellular acidification rate, accompanied by decreased apoptosis and accumulation of cells in the S phase. In contrast, the apoptosis rate of tumor cells and the percentage of cells in the S phase were significantly increased following PD‑L1 knockdown in the THP1 cell line. HK2 and LDHA expression decreased after AML tumor cells were treated with Akt inhibitor or rapamycin. In addition, the PD‑L1‑overexpressed cell line (PD‑L1‑OV) MOLM‑13 exhibited rapid tumor progression. Glycolysis‑associated genes were highly expressed in tumor tissues of PD‑L1‑OV MOLM‑13, with increased Ki67. Based on these findings, PD‑L1 may be considered as a suitable marker for prognosis and treatment in the clinical setting.

Preclinical Characterization of Prgn-3006 Ultracar-T™ for the Treatment of AML and MDS: Non-Viral, Multigenic Autologous CAR-T Cells Administered One Day after Gene Transfer

Relapsed/refractory (r/r) acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis and limited treatment options underscoring the need for new therapies. Traditional methods for generating CAR-T cells require extensive ex vivo expansion following viral vector transduction, adding time and high cost to manufacturing with a centralized manufacturing process and prolonging the waiting period between apheresis and administration of CAR-T therapy to a patient. Time is of the essence for advanced cancer patients including patients with r/r AML. The UltraCAR-T™ platform addresses shortcomings of current CAR-T technologies by streamlined rapid manufacturing process that can be performed in a local hospital's GMP facility following apheresis from the patient providing a potential solution for broader adoption of CAR-T therapies. The UltraCAR-T™ platform utilizes Precigen's advanced non-viral multi-gene delivery system and the rapid manufacturing process with high cell viability that allows for administration of autologous CAR-T cells one day after gene transfer. PRGN-3006 UltraCAR-T™ cells, utilizing Precigen's UltraVector® platform, co-express CD33 CAR, membrane bound IL-15 (mbIL15) and a kill switch. Expression of mbIL15 provides CAR-T cells with a source of cytokine without the need for exogenous cytokines to further enhance the expansion potential in the presence of tumor antigen and the overall persistence for PRGN-3006 in. The expression of mbIL15 on PRGN-3006 eliminates any need for ex vivo expansion of T cells prior to cell administration. Co-expression of kill switch provides a mechanism for selective depletion of PRGN-3006 post administration and improves therapeutic control. We manufactured PRGN-3006 UltraCAR-T™ cells using non-viral gene transfer and rapid streamlined manufacturing process from multiple donor T cells. Co-expression of CAR, mbIL15 and kill switch was confirmed by flow cytometry and western blot analyses. PRGN-3006 demonstrated robust expansion in presence of CD33 antigen, lack of autonomous expansion in absence of CD33, and prolonged persistence in absence of exogenous cytokines in in vitro tests. The redirected specificity was demonstrated by the specific killing of CD33+ tumor cells as well as significant release of inflammatory cytokines such as IFNγ upon co-culture with AML tumor cells. We also demonstrated specific elimination of PRGN-3006 cells by kill switch activator antibody treatment. In vivo, a single administration of PRGN-3006 UltraCAR-T™ cells only one day after gene transfer effectively eliminated tumor burden and significantly improved overall survival (p&lt;0.01) of tumor bearing mice compared to CAR-T cells lacking mbIL15 expression (conventional CAR-T) in an aggressive xenograft model of AML in immunocompromised (NSG) mice. PRGN-3006 demonstrated engraftment and significantly higher expansion and persistence in mice compared to CAR-T cells lacking mbIL15 (conventional CAR-T). These pre-clinical data provide a strong rationale to evaluate PRGN-3006 UltraCAR-T™ in a clinical trial for treatment of AML. The FDA has approved an investigational new drug application for PRGN-3006 UltraCAR-T™ and the first in human Phase 1 clinical trial for treatment of patients with relapse/refractory AML and high-risk myeloid dysplastic syndrome is currently ongoing (ClinicalTrials.gov Identifier: NCT03927261). Figure: Antitumor activity of PRGN-3006 UltraCAR-T™ cells in an established model of AML. MOLM-13 tumor cells were engrafted into NSG mice on Day 0 and PRGN-3006 were administered on Day 6 (arrow). Data shown is tumor burden (expressed as total flux in photons/sec) as the mean±SEM for each group of mice shown, with n=6-7 mice mice/group at the start of the study. Figure Disclosures Chan: Precigen: Employment. Ma:Precigen, Inc.: Employment. Carvajal-Borda:Precigen, Inc.: Employment. Velez:Precigen, Inc.: Employment. Plummer:Precigen, Inc.: Employment. Shepard:Precigen, Inc.: Employment. Shah:Precigen, Inc.: Employment; Intrexon Corp.: Equity Ownership. Sallman:Celyad: Membership on an entity's Board of Directors or advisory committees. Sabzevari:Precigen, Inc.: Employment.

CRISPR/Cas9-Mediated Protection of Normal Hematopoiesis Combined with the CD33/CD3 Bispecific T-Cell Engager (BiTE) Antibody AMG330 for Improved AML Therapy

While survival for patients with acute myeloid leukemia (AML) has increased in recent years, relapse and treatment-related mortality remain a significant problem, especially in older patients. Although the use of gemtuzumab ozogamicin (GO) has validated the concept of antibody directed therapy for AML targeting CD33, complications from myelosuppression and opportunistic infections due to on-target toxicity to normal CD33+ hematopoietic cells have limited this approach. In addition, only a subset of patients derives long-term benefit from GO. Thus, to improve the outcomes of CD33-targeted immunotherapy, more potent drugs are needed such as CD33/CD3 bispecific T-cell Engagers (BiTEs), as are strategies to protect normal blood cells from the cytotoxic effects of these agents. In this study, we assessed the in vivo activity of the clinically exploited CD33/CD3 BiTE AMG330 using our previously described MATCH mouse model (Haworth et al., MTM 2017), in which human CD34+ stem/progenitor cells (HSPCs) are transplanted into neonate mice. This model enables the in vivo maturation of human HSPCs to establish a fully reconstituted blood and immune system, and in particular functional T cells that permit the testing of BiTEs without risks of graft-versus-host disease. Since CD33 is expressed on normal myeloid cells, causing significant on-target/off-leukemia effects, we also investigated whether our previously validated CRISPR/Cas9-based strategy (Humbert et al., Leukemia 2019) protects hematopoiesis from AMG330 activity. Mice were engrafted with mock-(Cas9 only) or CRISPR/Cas9-edited human CD34+ HSPCs to ablate CD33 expression. Normal multilineage engraftment was observed in both experimental groups, resulting in hematopoiesis that either expressed or almost entirely lacked CD33. Mice were also infused with AML tumor cells to validate AMG330 activity, which was administered intravenously to the animals for 5 consecutive days at 0.2mg/kg. AMG330 rapidly eliminated CD33+ Molm14 tumor cells within 5 days (Figure 1A) and reduced the number of circulating CD14+ monocytes in the mock group (Figure 1B), thus confirming the functionality of in vivo-matured autologous T-lymphocytes generated. In contrast, CRISPR/Cas9-edited cells were fully protected from AMG330 cytotoxicity, as evidenced by steady CD14+ counts pre- and post-treatment (Figure 1B). Together, these results validate the MATCH model for testing the efficacy and toxicity of CD33-directed immunotherapies, in particular novel BiTEs. In addition, the combination of HSPC protection and improved CD33 targeting to more effectively kill AML should be of great value for the development of novel BiTEs and other bispecific antibodies for the treatment of AML. Figure 1: A) Leukemia burden measured by luciferase expression in mice from the two experimental groups treated or not with AMG330. B) Quantification of CD14+/CD33+ and CD14+/CD33− cells from peripheral blood of AMG330-treated animals in Cas9 only (n=3) and CRISPR/Cas9 (n = 3) groups. In A) and B), 'pre' is before AMG330 treatment and 'post' is 5 days after administration. Figure 1 Disclosures Walter: New Link Genetics: Consultancy; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Boehringer Ingelheim: Consultancy; Aptevo Therapeutics: Consultancy, Research Funding; Boston Biomedical: Consultancy; Covagen: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Kite Pharma: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy; Seattle Genetics: Research Funding; Agios: Consultancy; Amgen: Consultancy. Kiem:Magenta Therapeutics: Consultancy; Rocket Pharma: Consultancy, Equity Ownership; Homology Medicines: Consultancy, Equity Ownership; CSL Behring: Consultancy.