Immune-related adverse events (irAE) may affect almost any organ system and occur at any point during treatment with immune checkpoint inhibitors (ICI). We present a patient with advanced lung cancer receiving antiprogrammed death 1 checkpoint inhibitor who developed a delayed-onset visual irAE treated with corticosteroids. Through assessment of longitudinal biospecimens, we analyzed serial autoantibodies, cytokines, and cellular populations. Months after ICI initiation and preceding clinical toxicity, the patient developed broad increases in cytokines (most notably interleukin-6 (IL-6), interferon-γ (IFNγ), C-X-C motif chemokine ligand 2 (CXCL2), and C–C motif chemokine ligand 17 (CCL17)), autoantibodies (including anti-angiotensin receptor, α-actin, and amyloid), CD8 T cells, and plasmablasts. Such changes were not observed in healthy controls and ICI-treated patients without irAE. Administration of corticosteroids resulted in immediate and profound decreases in cytokines, autoantibodies, and inflammatory cells. This case highlights the potential for late-onset changes in humoral and cellular immunity in patients receiving ICI. It also demonstrates the biologic effects of corticosteroids on these parameters. Application of humoral and cellular immune biomarkers across ICI populations may inform toxicity monitoring and management.

Immunotherapy with T-cells expressing bispecific T-cell engagers (ENG T-cells) is a promising approach to improve the outcomes for patients with recurrent/refractory acute myeloid leukemia (AML). However, similar to T-cells expressing chimeric antigen receptors (CARs), their antitumor activity is limited in the setting of chronic antigen stimulation. We therefore set out to explore whether transgenic expression of IL15 improves the effector function of ENG T-cells targeting CD123-positive AML. T-cells expressing CD123-specific ENG (CD123-ENG) ± IL15 were generated by retroviral transduction from peripheral blood T cells from healthy donors or patients with AML. In this study, we characterized in detail the phenotype and effector functions of ENG T-cell populations in vitro and in vivo. IL15-expressing CD123-ENG (CD123-ENG.IL15) T-cells retained their antigen-specificity and effector function in the setting of chronic antigen exposure for more 30 days of coculture with AML blasts in contrast to CD123-ENG T-cells, whose effector function rapidly eroded. Furthermore, CD123-ENG.IL15 T-cells remained in a less differentiated state as judged by a high frequency of naïve/memory stem T-cell-like cells (CD45RA+CCR7+/CD45RO−CD62L+ cells) without evidence of T-cell exhaustion. Single cell cytokine profiling using IsoPlexis revealed enhanced T-cell polyfunctionality of CD123-ENG.IL15 T-cells as judged by effector cytokine production, including, granzyme B, IFN-γ, MIP-1α, perforin, TNF-α, and TNF-β. In vivo, CD123-ENG.IL15 T-cells exhibited superior antigen-specific anti-AML activity and T-cell persistence in both peripheral blood and tissues (BM, spleens, and livers), resulting in a significant survival advantage in one AML xenograft model and two autologous AML PDX models. In conclusion, we demonstrate here that the expansion, persistence, and anti-AML activity of CD123-ENG T-cells can be significantly improved by transgenic expression of IL15, which promotes a naïve/TSCM-like phenotype. However, we also highlight that targeting a single tumor antigen (CD123) can lead to immune escape, reinforcing the need to develop approaches to target multiple antigens. Likewise, our study demonstrates that it is feasible to evaluate autologous T cells in AML PDX models, which will be critical for future preclinical evaluations of next generation AML-redirected T-cell therapies.

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p<0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p<0.01 Ven+ or CD123-ENG T-cells versus control; p<0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

Background: CD123 is frequently expressed on hematologic malignancies including 96-98% of AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) showed a promising approach anti-AML activity, however T-cell persistence was limited. Interleukin-15 (IL15) has emerged as a candidate immunomodulator as it enhances T-cell expansion and persistence, and induces long-lasting memory T-cells. To improve the efficacy and persistence of CD123-ENG T-cells we developed IL15 expressing CD123-ENG T-cells. Here, we report on the characterization and efficacy of IL15-secreting CD123-ENG T cells in vitro and in vivo models of adult AML. Methods/Results: A cDNA encoding IL15 was cloned into retroviral vectors encoding CD123-ENG or CD19-ENG (CD123-ENG.IL15; CD19-ENG.IL15). ENG T-cells were generated from human peripheral blood mononuclear cells (PBMCs) from normal donors or T-cells from AML patients by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells and T-cells expressing CD123 (CD123-ENG T-cells) served as controls. IL15 production of CD19-ENG.IL15 and CD123-ENG.IL15 T cells was confirmed by ELISA (144-159 pg/ml vs 38 and 46 pg/ml of NT and CD123-ENG T cells, p<0.01, n=6). Both CD123-ENG and CD123-ENG.IL15 T-cells recognized CD123+ AML cells as judged by IL2 and interferon γ (IFNγ) production (p<0.01, n=5). In contrast, NT and CD19-ENG.IL15 T-cells did not, confirming specificity. In addition, CD123-ENG.IL15 and CD123-ENG T-cells induced killing of only CD123-positive target cells as well as of primary adult patients' AML blasts in luciferase- or 7AAD-based cytotoxicity assays (p<0.001, n=10). CD123-ENG.IL15 T-cells showed greater cytolytic activity than CD123-ENG T-cells as determined by luciferase activity (p=0.0002, n=3). In a Molm13 AML xenograft model, CD123-ENG.IL15 and CD123-ENG T-cells exhibited potent anti-leukemic activity as judged by bioluminescence imaging. Moreover, CD123-ENG.IL15 T cells had enhanced anti-leukemic activity and greater persistence in BMs, spleens, and livers in comparison to CD123-ENG T cells, resulting in improved anti-AML activity (Figure 1, p<0.01 vs CD123-ENG T-cell group, n=12 per group) and extended survival (Figure 2, p=0.0097 vs CD123-ENG T-cell group). Finally, AML PDX models and ENG T-cells were generated from AML blasts and T-cells from 3 patients with active AML. Infusion of autologous ENG T-cells (1.5x106 cells/mouse, n=7) in AML PDX#6697688 mouse model revealed significant reduction of leukemia burden in the CD123-ENG.IL15 or CD123-ENG T-cells mouse groups but not in the mouse group with NT or CD19-ENG.IL15 T-cells or PBS (p=0.004, n=6-8). We are currently monitoring survival of these PDX models. Conclusion: We here demonstrated that transgenic expression of IL15 in CD123-ENG T-cells results in improved expansion and persistence, and anti-AML activity. These results warrant further exploration of IL15-modified CD123-targeted T-cells as immunotherapy for AML. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:EMD Serono: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; ASSISI fundation of Memphis: Research Funding; TESSA Therapeutics: Other: Research Collaboration; ViraCyte: Consultancy; NHLBI: Research Funding; America Lebanese Syrian Associated Charities: Research Funding; California Institute for Regenerative Medicine: Research Funding; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Sanofi: Honoraria; Tidal: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy. Velasquez:St. Jude: Patents & Royalties: Patent Applications in the Fields of Cell and Gene Therapy ; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees. Andreeff:Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstaZeneca: Consultancy; Amgen: Consultancy; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; CPRIT: Research Funding; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding.

CXC chemokine receptor 4 (CXCR4)/CXC motif ligand 12 (CXCL12) and E-(endothelial)-selectin/E-selectin ligands (E-selectin-L) axes play critical roles in leukemia cell homing to the bone marrow niche and are closely associated with resistance to FLT3-targeted therapy in FLT3-mutant acute myeloid leukemia (AML) patients. Hence, it is imperative to co-target CXCR4/E-selectin/FLT3 in FLT3 mutant AML. Herein, we determined whether FLT3 inhibition modulates CXCR4/E-selectin-L levels and whether co-targeting CXCR4/E-selectin enhances the anti-leukemia effects and reduces bone marrow niche-mediated resistance in FLT3-targeted therapy. Our results demonstrate that CXCR4/E-selectin-L are transcriptionally upregulated by FLT3 inhibition. Concomitant blockage of CXCR4/E-selectin with the dual inhibitor GMI-1359 disrupts leukemia cell homing and migration to bone marrow niches. Combination treatment with GMI-1359 and quizartinib significantly reduced leukemia cell burden and extended mouse survival in a patient derived xenograft AML mouse model. These findings provide pre-clinical rationale for combined CXCR4/E-selectin/FLT3 targeting in FLT3-mutant AML.