The prognosis for children with recurrent and/or refractory neuroblastoma (NB) is dismal. The receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is highly expressed on the surface of NB cells, provides a potential target for novel immunotherapeutics. Anti-ROR1 chimeric antigen receptor engineered ex vivo expanded peripheral blood natural killer (anti-ROR1 CAR exPBNK) cells represent this approach. N-803 is an IL-15 superagonist with enhanced biological activity. In this study, we investigated the in vitro and in vivo anti-tumor effects of anti-ROR1 CAR exPBNK cells with or without N-803 against ROR1(+) NB models. Compared to mock exPBNK cells, anti-ROR1 CAR exPBNK cells had significantly enhanced cytotoxicity against ROR1(+) NB cells, and N-803 further increased cytotoxicity. High-dimensional analysis revealed that N-803 enhanced Stat5 phosphorylation and Ki67 levels in both exPBNK and anti-ROR1 CAR exPBNK cells with or without NB cells. In vivo, anti-ROR1 CAR exPBNK plus N-803 significantly (p < 0.05) enhanced survival in human ROR1(+) NB xenografted NSG mice compared to anti-ROR1 CAR exPBNK alone. Our results provide the rationale for further development of anti-ROR1 CAR exPBNK cells plus N-803 as a novel combination immunotherapeutic for patients with recurrent and/or refractory ROR1(+) NB.

Hemophagocytic lymphohistiocytosis (HLH), both primary/familial HLH and secondary HLH, is associated with multiorgan dysfunction caused by excessive immune activation and cytokine release. The high morbidity and mortality rates are in part due to diagnostic challenges leading to a delay in treatment initiation. The diagnosis, which uses the Histiocyte Society clinical criteria from 2004 and the HScore, remains challenging, with limited improvement in outcomes. No grading system is available for HLH. Although etoposide with dexamethasone remains the most frequently used first-line regimen, various new therapies are now being employed in the management of HLH. The interferon gamma inhibitor emapalumab, the Janus kinase signal transducer and activator of transcription pathway inhibitor ruxolitinib, and the interleukin 6 (IL-6) inhibitor tocilizumab have been trialed in HLH management, with additional treatment options being inhibition of IL-18 and tumor necrosis factor alpha. Here, we summarize current management options for HLH; we also propose a new grading system for HLH based on Common Terminology Criteria for Adverse Events version 5.0 as well as on known prognostic factors (eg, abnormal bilirubin and transaminase levels, elevated creatinine level, respiratory failure, neutropenia, hypertriglyceridemia, hypoalbuminemia, and coagulopathy), which could standardize the diagnosis and guide prompt and appropriate management.

Outcomes for pediatric patients with refractory or relapsed T-cell acute lymphoblastic leukemia (T-ALL) are poor, underscoring the need for improved therapeutic strategies. CD38, a type II transmembrane glycoprotein, is a promising target in T-ALL, with clinical trials evaluating CD38-targeting immunotherapies in frontline and relapsed settings. However, the biological role of CD38 in T-ALL has not been systematically defined. We interrogated CD38 biology through multimodal profiling of pediatric T-ALL samples. Bulk RNA sequencing of 1,335 primary tumors revealed that CD38 expression varies across genomic and immunophenotypic subtypes in T-ALL. Flow cytometry of 150 primary samples and CITE-sequencing of 40 cases demonstrated broad surface expression of CD38. A transcription factor CRISPR-screen identified RUNX1, RUNX3, and TP53 as candidate positive regulators of CD38. Metabolomic profiling of cell lines further revealed disruption of the polyamine pathway following CD38 perturbation. Supporting this finding, co-targeting CD38 with difluoromethylornithine (DFMO), a polyamine metabolism disruptor, improved survival in preclinical models. Across transcriptomic datasets, including primary tumors, cell lines, and patient-derived xenograft models, IL32 expression consistently decreased following CD38 loss or negativity, supporting an association between CD38 and inflammatory signaling pathways. Additionally, CD38 and LCK expression were positively correlated across majority of genomic subtypes, implicating SRC kinase signaling. Consistent with this, daratumumab in cell lines increased LCK phosphorylation, and combination therapy with dasatinib improved survival compared to monotherapy. Collectively, these findings define previously unrecognized interactions between CD38 and targetable pathways and genes in T-ALL and identify rational combinatorial strategies to enhance CD38-directed therapies and reduce relapse risk.

Background Patients following Allogeneic Hematopoietic Stem Cell Transplantation (AlloHSCT), Solid Organ Transplantation (SOT) and with Primary Immunodeficiency (PID) are at high risk of developing refractory ADV and CMV infections. Viral-specific (VS) T-cell immune reconstitution is essential to control ADV and CMV infections in these immunocompromised hosts. We developed a methodology of related donor-derived ADV and CMV CTLs by utilizing the IFN-γ cytokine capture system (CCS®) on the CliniMACS Prodigy® device by direct selection of VS-CTLs. Objective To evaluate genomic and immunomic characteristics, safety and efficacy of related donor-derived VS-CTLs in immunocompromised hosts with refractory viral infection. (IND 17449, NCT03266627, NCT03266640). Methods Patients following HSCT, SOT or with PID with refractory CMV or ADV infection and/or intolerant to anti-viral therapy were eligible. Related donors (HLA ≥ 3/6 matches) who screened positive for memory VS-CTLs underwent non mobilized apheresis collection and donor-derived VS-CTLs were enriched as we previously described by direct selection using the ADV or CMV PepTivator® generously provided by Miltenyi. 0.5 × 104 CD3 cells/kg were infused into recipients every two weeks with a maximum of 5 doses until CR or DLT. CTL characterization was performed utilizing scRNAseq, mass cytometry, NanoString® Immunoprofiling and multidimensional flow cytometry. Results Among the ADV and CMV CTLs recipients (N=20, N=19), median age and gender were (10.1 [0.11-19.1] and 8.0 [0.7-19.0] yrs), M/F (8/12, 13/6), haploidentical vs HLA-matched original AlloHSCT donor (68% vs 32%, and 74% vs 26%), AlloHSCT/SOT/PID (100%, 0, 0) (90%, 5%, 5%), respectively. ADV and CMV CTL post enrichment CD4+/IFN-γ+ and CD8+/ IFN-γ+ (mean ± SEM) percentages are 45 ± 10.1% and 39 ± 8.7%, 42 ± 9.6% and 67.1 ± 15.4%, respectively. Viral CMV CTL demonstrated significant increase in CD8+ TEM, TEMRA and CD4TH, CD4TFH1 subtypes (p < 0.05, 0.05, 0.001, 0.01, Fig 1), distinct gene expression profile highly enriched for IFN-γ and IL-2 (Fig 2A), CD4+ and CD8+ memory T cells CD27-CD45RO+ (Fig 2B) and significantly increased central memory and effector memory T-cells (p<0.01, 0.001), respectively. Both ADV and CMV CTLs were well tolerated. One Grade I and 3 Grade II transient aGVHD, no CRS, IRR, ICANS, GF nor neurotoxicity were reported and resulted in 85% and 84% CR median of 34 (6-112) and 30 (6-82) days, 1 yr OS of 70% and 74%, and 1yr viral associated mortality of 5% and 0%, respectively (Fig 3). Conclusions Related ADV and CMV CTLs enrichment by direct selection using the CliniMACS Prodigy® CCS (IFN-γ) is feasible, well tolerated and are characterized by central & effector memory T-cells secreting IFN-γ and IL-2 and associated with a high CR rate and low 1 yr viral associated mortality.

Immune checkpoint therapy (ICT) is designed to unleash the anti-tumor activity of Tlymphocytes. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibition and programmed death 1 (PD-1) inhibition are the most commonly utilized ICTs in clinical cancer therapy, and they enhance anti-tumor immunity by interrupting the inhibitory signals CTLA-4 and programmed death ligand 1 (PD-L1) respectively. In pediatric Hodgkin lymphoma, ICT has demonstrated remarkable efficacy in both high-risk and relapsed disease, with investigation into the efficacy in low-risk disease ongoing. Pediatric mature B-cell lymphomas have variable expression of PD-L1 with very limited experience incorporating ICT. Primary mediastinal B-cell lymphoma (PMBCL), anaplastic large cell lymphoma (ALCL), aggressive natural killer-cell lymphoma (ANKL), and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) all consistently express PD-L1, providing strong biologic rationale for the use of ICT in these diseases. In PMBCL, the Children’s Oncology Group (COG) and the National Cancer Institute (NCI) National Clinical Trials Network (NCTN) recently completed a randomized phase III trial of nivolumab in combination with chemo-immunotherapy in children and adults with newly diagnosed PMBCL. Results of this trial are expected in 2027. In ALCL and ANKL, ongoing clinical trials are evaluating the efficacy of ICT. Given the transformational role of ICT for pediatric Hodgkin lymphoma, there is significant promise for the use of ICT in multiple subtypes of pediatric non-Hodgkin lymphoma with increased expression of PD-L1.

Introduction Polatuzumab vedotin (Pv) is an antibody-drug conjugate consisting of a humanized monoclonal antibody targeting CD79b on human B cells conjugated to the antimitotic agent monomethyl auristatin E (MMAE).1 CD79b is expressed in Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), primary CNS lymphoma (PCNSL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and others. Pv was FDA approved in 2019 for relapsed or refractory (R/R) DLBCL, and later approved for upfront treatment of DLBCL in combination with R-CHP.2,3 DLBCL is the most common non-Hodgkin lymphoma (NHL) in adults. Forty to 50 percent of patients with DLBCL will relapse or have primary refractory disease. Standard of care for R/R DLBCL includes high dose chemotherapy followed by autologous stem cell transplantation (ASCT), however about 50 percent of patients will relapse following ASCT.4,5 There is an urgent need for effective maintenance therapies to decrease post-ASCT relapses. Objectives To evaluate the safety and tolerability of Pv following myeloablative conditioning (MAC) and ASCT in B-NHL. Secondary objectives include measurement of event-free, progression-free and overall survival; and measurement of overall response rate to Pv in patients with partial response (PR) or stable disease (SD) following MAC and ASCT. Methods Patients age 12 to 75 with BL, DLBCL, PCNSL, FL, MCL, marginal zone lymphoma, transformed FL, Richter syndrome, or Hodgkin lymphoma with adequate organ function and the following disease status are eligible: primary induction failure, or first through third relapse or progression having attained a complete response (CR), PR, or SD following reinduction therapy. Double or triple hit B-NHL or B-NHL with an IPI of 3 or greater having attained PR or CR are also eligible. Patients may receive MAC with BEAM (carmustine/etoposide/cytarabine/melphalan), CBV (cyclophosphamide/carmustine/etoposide), or for PCNSL, TT/BCNU (thiotepa/carmustine) at the discretion of the treating physician. Patients may begin Pv maintenance therapy between 30 and 60 days post-ASCT. Patients must not have progressive disease (PD) and must meet hematologic parameters to begin Pv. Pv is given at 1.8mg/kg/dose every 21 days for eight doses. Results Four patients have received at least one dose of maintenance therapy with Pv following MAC and ASCT from 2021 to present. There have been no dose-limiting toxicities or serious adverse events attributable to Pv. Diagnoses included DLBCL in three patients and primary CNS lymphoma in one patient. The patients have ranged in age from 13 to 49 years at enrollment. Two patients have completed the planned two-year follow-up period post-ASCT, and two have ongoing follow-up. All four patients remain in CR. Accrual is ongoing. Conclusion Maintenance therapy with Pv is a promising approach to prevent relapse in patients with CD79b positive lymphoma following MAC and ASCT.

Introduction Allogeneic HCT (AlloHCT) offers curative treatment for high-risk and relapsed T-cell acute lymphoblastic leukemia (T-ALL) or lymphoma (T-LLy), but post-HCT relapses remain an obstacle for long-term survival. CD38 is highly expressed in T-ALL/T-LLy, and significant activity of the anti-CD38 antibody, daratumumab (DARA), was seen in a recent clinical trial for relapsed/refractory disease (Bhatla et al., Blood 2024). We developed a phase 1 trial (NCT04972942) to evaluate targeted immunotherapy with DARA after AlloHCT for relapsed T-ALL/T-LLy. Objectives The primary objective is to determine the safety of targeted immunotherapy with DARA after total body irradiation (TBI)-based myeloablative conditioning and AlloHCT for children, adolescents, and young adults (CAYA) with high-risk T-ALL or T-LLy. Additional exploratory objectives will evaluate outcomes of receiving AlloHCT and post-HCT DARA on study and correlate those outcomes with novel biological and immunological studies, including measurable residual or minimal detectable disease (MRD/MDD). Methods Patients 1 (and ≥ 10 kg) to ≤ 39-years, with relapsed T-ALL in second or subsequent remission or relapsed T-LLy with complete response after reinduction therapy are eligible. Participants will undergo myeloablative TBI-based conditioning and AlloHCT with best available donor. Safety of post-HCT DARA will be determined through a phase 1, time-to-event Bayesian optimal interval study design, evaluating 3 dose levels of DARA IV (IND 159396) in 15 participants. If no dose-limiting toxicity (DLT) is observed, an additional 15 participants will be enrolled in a dose expansion cohort (DEC) for additional correlative studies and to guide selection of a recommended phase 2 dose (RP2D) (Figure 1). Participants will have pre- and post-HCT correlative blood, bone marrow, and cerebrospinal fluid studies collected, including NGS-based MRD/MDD. Results Three participants in CR2 enrolled to date, each at dose level 1 (8mg/kg). Participant 1 completed protocol therapy and is alive in CR2 at 27 months post-HCT. Participant 2 received 4 doses of DARA, came off protocol therapy due to moderate chronic GVHD, and is alive in CR2 at 17 months post-HCT. Early tacrolimus taper for EBV pneumonitis and mixed T-cell chimerism likely contributed to their chronic GVHD. Participant 3 completed protocol therapy and is alive in CR2 at 11 months post-HCT. No DLT related to DARA has been observed to date. Conclusions Novel strategies are needed to reduce relapse and improve survival after AlloHCT for relapsed T-ALL/T-LLy. ALLO-T-DART will (1) determine the safety and RP2D of post-HCT DARA and (2) collect data on novel biomarkers of response, MRD/MDD kinetics, and immune reconstitution after DARA treatment for relapsed T-ALL/T-LLy. Supported by DOD USAMRAA #HT94252310686, Johnson & Johnson Innovative Medicine, and Cures Within Reach.

Introduction Patients diagnosed with metastatic Ewing sarcoma (ES) have a dismal outcome. Lines of evidence have shown the importance of NK cell immunity against ES [1]. However, NK cell number and function are low in ES patients, in large part due to the immunosuppressive tumor microenvironment (TME) [2]. Objectives We aim to improve NK therapy against metastatic ES by strategic combinations that simultaneously enhance tumor specific targeting of NK cells via chimeric antigen receptor (CAR), increase NK migration and tumor infiltration by engineering CAR-NK cells to express tumor attracting chemokine receptor CXCR2, enhance NK cell persistence and the immune synapse between cancer and NK cells via a tri-specific killer engager camB7-H3 [3], and improve NK cell ADCC by a GD2 antibody dinutuximab. Methods NK cells were expanded using K562-mbIL-21-41BBL feeder cells and IL-2 [4,5,6]. Expanded NK cells were engineered to co-express ROR1-CAR and CXCR2 using mRNA electroporation. NK cell cytotoxicity was evaluated by luciferase-based cytotoxicity assay. In vitro transwell assays were performed to determine the migratory ability of CAR-NK and CAR-CXCR2-NK cells. An orthotopic mouse model generated by implanting ES cells into the tibia of NSG mice was utilized to evaluate in vivo tumor growth and animal survival. Results We found that compared to mock NK cells, ROR1-CAR-NK cells had significantly enhanced cytotoxicity against ES (A673) cells at various T:E ratios (p<0.05) (Fig 1A). While the in vitro cytotoxicity of CAR-NK and CAR-CXCR2-NK cells was very similar (Fig 1A), CXCR2-CAR-NK cells showed significantly enhanced migration towards the conditioned media of ES cells (p<0.05 and p<0.01) (Fig 1B). Both dinutuximab (Fig 1C) and camB7-H3 (Fig 1D) significantly enhanced in vitro cytotoxicity of CAR-CXCR2 NK cells against ES A673 cells (p<0.05 and p<0.01). Furthermore, CAR-CXCR2-NK cells combined with camB7-H3 and dinutuximab had significantly enhanced anti-tumor effects against ES in the orthotopic xenograft mouse model, as evidenced by significantly reduced tumor growth (p<0.05 and p<0.01) (Fig 1E) and prolonged animal survival (p<0.05 and p<0.01) compared to controls (Fig 1F). Conclusions Our data demonstrated enhanced anti-tumor efficacy of ROR1-CAR-CXCR2-NK cells combined with TriKE and anti-GD2 against ES in vitro and in vivo, suggesting a potential novel effective immunotherapy for patients with high-risk ES. Supported by U54 CA232561-01A1 and ALSF reach grant. Dinutuximab is generously provided by United Therapeutics.

Background: Children, adolescents, and young adults (CAYA) with advanced mature B non-Hodgkin lymphoma (MB-NHL) have achieved >= 90% overall survival with intensive chemoimmunotherapy and intrathecal chemotherapy (IT). However, patients receive multiple IT doses requiring sedation with general anesthesia which may be associated with long-term late effects in cognitive development. Liposomal cytarabine (LC) has a half-life of 100-263 h, potentially decreasing the number of IT doses required. LC has been associated with toxicities when used in combination with high-dose methotrexate (HD-MTX) and ARA-C in adults with MB-NHL. Methods: This phase II study incorporated 2 doses of LC to the French-American-British backbone in central nervous system (CNS) negative patients and 4 doses in CNS positive patients. Results: Of 41 patients, 33 (19 FAB Group B, LDH <= 2X ULN; 6 FAB Group B, LDH >= 2X ULN; 8 FAB Group C) received LC and only 1 (3%) had a transient Grade 3 adverse event, likely secondary to disease progression. We successfully reduced the number of IT in Group B patients from 9 to 5 and in Group C CNS negative patients from 10 to 7. CNS positive patients received 9 IT instead of 13. The 2-year event-free survival and overall survival were 94.5% and 97.2%, respectively. We demonstrated that IT LC in CAYA with MB-NHL was feasible and safe in the setting of HD- MTX and ARA-C when decadron was used as a premedication and the LC was given after MTX clearance. Conclusion: We maintained event free survival at numbers consistent with previous reports using the standard increased number of IT injections. We safely decreased the total number of IT without compromising CNS control.

Natural killer (NK) cells may be engineered with chimeric antigen receptors (CARs) to recognize tumor-associated antigens which bolsters their antitumor activity. More so than CAR-T cells, CAR-NK cell responses result from an integration of signals from a wider range of innate activating cytotoxic receptors, inhibitory receptors, and adhesion receptors in addition to the engineered CAR, making computational modeling of CAR-NK cell cytotoxicity more difficult than CAR-T cells. Uncovering mechanisms and predicting tumor cell responses to CAR-NK cytotoxicity is essential for improving therapeutic efficacy. The complexity of these effector-target interactions and the donor-to-donor variations in NK cell receptor (NKR) repertoire preclude the use of predictive models based on a single receptor, requiring function to be determined experimentally for each donor, CAR, and target combination. Computational modeling generates frameworks that allow the relationships of these factors to biologic outcomes to be explored without resource-consuming experiments. Here, we developed a computational mechanistic multiscale model which considers heterogenous expression of CARs, NKRs, adhesion receptors, and their cognate ligands, signal transduction, and NK cell-target cell population kinetics. The model is trained with quantitative flow cytometry and in-vitro cytotoxicity data and accurately predicts the short-term, long-term, and in-vivo cytotoxicity of CAR-NK cells. Furthermore, using Pareto optimization we explored the effect of CAR proportion and NK cell signaling on the differential cytotoxicity of CD33CAR-NK cells to cancer and healthy cells. This model can be extended to predict CAR-NK cytotoxicity across many antigens and tumor targets and serves as a tool to mechanistically explore CAR-NK signaling and biology.

BACKGROUND:The prognosis of patients with metastatic/relapsed/refractory Ewing sarcoma (ES) is dismal. Natural killer (NK) cells are highly cytotoxic to ES but limited by resistance within the ES tumor microenvironment (TME). Here we sought to overcome ES resistance to NK cells by a combinatorial immunotherapy approach simultaneously enabling NK tumor-specific-targeting via chimeric antigen receptor (CAR) against a novel ES target interleukin-1 receptor accessory protein (IL1RAP), circumventing transforming growth factor beta (TGFβ)-mediated NK immunosuppression by TGFβ1-imprinting, increasing NK cell antibody-dependent cellular cytotoxicity (ADCC) via an anti-GD2 antibody dinutuximab, and improving NK cell persistence and ADCC by an IL-15 agonist, NKTR-255. METHODS:Peripheral blood mononuclear cells were expanded into NK and TGFβ1-imprinted-NK (imNK) cells using antigen-presenting feeder cells co-expressing IL-21 and 4-1 BBL. Anti-IL1RAP-CAR messenger RNA was electroporated into NK or imNK cells. In vitro cytotoxicity assays were performed to investigate the efficacy of anti-IL1RAP-CAR-NK/imNK cell alone or combined with NKTR-255 and/or dinutuximab against ES cells. Xenograft mouse models of ES were used to investigate the antitumor efficacy of the combinatorial CAR-NK/imNK cell therapy against ES in vivo. Single-cell RNA sequencing and mass cytometry analyses of cells from xenograft tumors were performed to identify mechanisms of response/resistance to this combinatorial immunotherapy. RESULTS:We found that anti-IL1RAP-CAR-NK cells significantly and specifically enhanced NK cytotoxicity in vitro and decreased tumor growth and lung metastasis in vivo against IL1RAP+ES. TGFβ1-imprinting significantly enhanced in vitro cytotoxicity and tumor infiltration of CAR-NK cells, leading to significantly reduced tumor growth and improved animal survival in the ES orthotopic mouse model. Compared with single agent or double combinations, the triple combination of imprinted-CAR-NK (CAR-imNK) cells and NKTR-255 with dinutuximab had superior antitumor efficacy against IL1RAP+GD2+ ES. Mechanistic studies on single cells from the xenograft tumors revealed increased apoptosis of ES cells, upregulated expression of ligands to NK inhibitory receptors on ES cells, and enhanced mouse macrophage migration in the ES TME in response to the CAR-imNK+NKTR-255+dinutuximab therapy. CONCLUSIONS:Our preclinical data demonstrate that combinatorial innate immunotherapy leveraging tumor-targeting TGFβ1-imprinted IL1RAP-CAR-NK cells combined with an IL-15 agonist and an anti-GD2 antibody is a promising novel therapeutic strategy for targeting metastatic/relapsed/refractory ES.

Background Patients with relapsed/refractory AML have a dismal outcome (<20%) in large part secondary to chemotherapy resistance. Immune effector cell therapy is one approach to chemotherapy resistance. Different myeloid targets have been tested including CD33, expressed on over 90% of AML cells. IL-1RAP (Interleukin-1 receptor associated protein) is a novel target; it activates the IL-1RAP receptor as well as c-kit, FLT3, and other pathways associated with poor AML prognosis (Kelly/Steidl J Exp Med 2018). Immunotherapy with CAR-T cells is under investigation, but its clinical use is limited by the need for subsequent allo-HSCT. CAR NK cell persistence and function can be enhanced by IL-15 cytokines such as NKTR-255 (polymer-conjugated IL-15 agonist) or IL-15–containing TriKEs. Objectives To evaluate the cytotoxic effects of IL-1RAP CAR NK cells against AML cells. To assess whether the addition of NKTR-255 (a polymer-conjugated interleukin-15) or cam161533 TriKE, designed to bring together CD33 on AML cells and CD16 on NK cells, & includes IL-15—further enhances this effect. Methods NK cells were obtained from donor peripheral blood mononuclear cells and isolated using RosetteSep™ human NK cell enrichment kit via Ficoll separation. Cells were cultured and expanded in vitro with irradiated K562-mbIL21-41BBL feeder cells. AML cell lines (Molm13, MV4-11, HEL, THP-1) were used to evaluate NK-mediated cytotoxicity with NKTR-255 (generously provided by Nektar) or cam161533 TriKE. Cytotoxicity of unmodified NK cells was examined against IL-1RAP.CAR NK cells in AML cell lines after 24 hours of incubation at a 1:1 effector:target ratio. Results were analyzed using britelite plus luminescence assay & SoftMax Pro 6.3. Cytokine release was measured by ELISA. Results IL-1RAP.CAR NK cells exhibited significant cytotoxicity against different AML cell lines. The results were consistent even with low IL-1RAP expression (p < 0.001) Figures 1A & 1B. The cytotoxicity of IL-1RAP.CAR NK against AML cells was significantly higher than the cytotoxicity of unmodified NK cells (p < 0.001). Furthermore, the addition of NKTR-255 or cam161533 TriKE to IL-1RAP.CAR NK cells enhanced their cytotoxicity (p < 0.001). Compared to unmodified NK cells with NKTR-255 or TriKE, IL-1RAP.CAR NK cells with NKTR-255 or TriKE had significant cytotoxicity (p<0.001) Figure 1C. IL-1RAP.CAR NK cells showed significantly increased levels of cytokine release compared to unmodified NK cells (p <0.0001). The addition of NKTR-255 or TriKE further augmented the cytokine release from IL-1RAP.CAR NK cells, results were statistically significant Figures 2 A, B & C. Conclusions IL-1RAP is a novel therapeutic target for CAR NK cell therapy in AML. The addition of NKTR-255 or cam161533 TriKE significantly enhanced the cytotoxicity and cytokine release of IL-1RAP.CAR NK cells.Preclinical evaluation in AML xenograft models is ongoing

Introduction Outcomes for pediatric and AYA patients with advanced solid tumors remain dismal, with little improvement over decades. We developed a reduced-intensity haploidentical BMT (haploBMT) platform incorporating post-transplant cyclophosphamide (PTCy) to replace an incompetent, host, immune system with one from a healthy donor and limit GVHD followed by pre-emptive checkpoint inhibition to augment graft-versus-tumor (GVT) effects. Objectives To evaluate the safety and efficacy of haploBMT plus anti PD-1 therapy (aPD-1) as a consolidative intervention in high-risk solid tumors after achieving no evidence of disease or stable disease, and to define the immunologic mechanisms by which checkpoint blockade augments GVT. Methods Twelve patients (Ewing sarcoma n=5, rhabdomyosarcoma n=3, neuroblastoma n=3, medulloblastoma n=1) received haploBMT followed by aPD-1; 3 were treated at relapse and 9 received aPD-1 pre-emptively post-BMT (NCT03465592). Outcomes were compared with 25 haploBMT-only patients (Ewing n=5, rhabdomyosarcoma n=10, neuroblastoma n=3, desmoplastic small round cell n=3, other n=4). All patients were fully engrafted at the time of aPD-1 initiation and received 1-48 cycles. Peripheral blood and tumor specimens were analyzed with high-dimensional methods. Results (interim analysis) HaploBMT + aPD-1 significantly improved survival compared with haploBMT alone. Two-year OS rose from 25% with haploBMT alone to 65% with haploBMT + aPD-1 (p=0.03). Median OS was 1.0 vs 2.6 years, respectively (p=0.057). Survival curves diverged over time, with a subset of patients in the aPD-1 arm achieving a durable survival plateau beyond 3 years, consistent with sustained GVT activity. Treatment was well tolerated; one patient developed grade 3 GVHD (resolved) and two had serious immune-related events, managed with immunosuppression. Correlative analyses showed donor-derived lymphocytes remained checkpoint responsive, expanded in blood, and infiltrated tumors. Cytokine profiling revealed robust immune activation with increases in IFNγ, TNFα, and IL-12, consistent with Th1-skewed responses, together with modulation of PD-1, ICOS, and CCL5. Longitudinal profiling demonstrated expansion of cytotoxic CD8⁺ and effector CD4⁺ T cells with contraction of suppressive myeloid cells and Tregs, shifting the systemic milieu toward a pro-inflammatory state. Spatial multiplex immunofluorescence of tumor tissue revealed activated effector-like CD8⁺ and CD4⁺ T cells clustering with PD-L1⁺ myeloid subsets, reflecting a remodeled immune microenvironment conducive to antitumor responses. Conclusions HaploBMT plus aPD-1 is feasible, safe, and improves OS. Correlative studies show PD-1 blockade drives immune activation, effector T cell differentiation, and recruitment of tumor-reactive subsets, providing a mechanistic basis for durable GVT.

Objectives The prognosis of children, adolescents, and young adults (CAYA) with relapsed/refractory (R/R) Osteosarcoma (OSA) is extremely poor with an estimated 6 month overall survival of ≤ 5%, largely secondary to therapy resistance within the tumor microenvironment (TME) [1]. Our objective is to overcome the OSA TME resistance to NK cells by a combinatorial NK cell therapy that enhances NK tumor specific targeting via chimeric antigen receptor (CAR), and increases NK cell ADCC via anti-GD2 antibody dinutuximab. Methods PBMCs were expanded into NK cells using feeder cells and IL-2 [2]. By non-viral electroporation, expanded NK cells were modified to express CAR against MCAM, a novel target highly expressed on pediatric cancers including OSA [3]. Flow cytometry was performed to assess the expression levels of MCAM and GD2 on OSA cells. NK cell cytotoxicity was evaluated by luciferase-based cytotoxicity assay. An orthotopic mouse model of OSA was utilized to evaluate in-vivo anti-tumor efficacy of the CAR NK combinatorial therapy. Results We confirmed that MCAM and GD2 are both highly expressed on OSA U2OS cells (Fig 1A). MCAM CAR NK cells had a significantly enhanced in vitro cytotoxicity compared to mock NK cells against U2OS cells at various effector-to-target (E:T) ratios (p<0.01 and p<0.05) (Fig 1B). Dinutuximab significantly enhanced NK/CAR NK cell cytotoxicity in a dose-dependent manner (p<0.01 and p<0.05) (Fig 1C) at various E:T ratios (p<0.01 and p<0.05) (Fig 1D). Furthermore, compared to control, MCAM CAR NK cells alone and combined with dinutuximab significantly reduced tumor growth (p<0.05) in an orthotopic xenograft (U2OS) mouse model of OSA (Fig 1E). Conclusion Our data demonstrated enhanced anti-tumor efficacy of MCAM CAR NK cells alone and combined with dinutuximab against OSA in vitro and in vivo, suggesting a novel effective immunotherapy for CAYA with R/R OSA. This study was funded by St. Baldrick’s Foundation.