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Table 4 Excel Document

Supplemental Figures 1-8, Supplemental Tables 1-3

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)–grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Legends for Supplementary Figures 1-8 and Supplementary Tables 1-5

Table 5 Excel Document

Supplemental figures S1-S11

Supplementary patient data, supplementary materials and methods

Supplemental patient tables (1-3) and mass cytometry reagent table (4)

Additional Methods

CITE-seq reveals N-803 and rituximab mediated activation of PB NK cells

Supplementary Table S1

Supplementary Fig. S3. Differentiation of ML NK cells and Preactivated-Expanded CB-NK cells.

Supplementary Materials and Methods// Supplementary Figure 1. Supporting Figure 1. MIR142 mutations attenuate miR-142-3p and miR-142-5p functions.// Supplementary Figure 2. Supporting Figure 2. Mir142 loss alters differentiation in mature hematopoietic populations.// Supplementary Figure 3. Supporting Figure 3. Mir142 loss alters differentiation in MPPs and HSCs.// Supplementary Figure 4. Supporting Figure 4. Mir142-/- bone marrow exhibits reduced repopulating activity with a loss of HSC lymphoid potential.// Supplementary Figure 5. Supporting Figure 7. Mir142 loss enhances IDH2R172K disease initiating capacity.

Characteristics of primary iNHL patient samples used in this study.

Summary of AntiDrug Antibody Data Incidence by Route of Administration

Spleen NK cell numbers and percentages in SCID mice analyzed in Figures 4E/F.

Supplementary Materials and Methods

Point mutations in the seed sequence of miR-142-3p are present in a subset of acute myelogenous leukemia (AML) and in several subtypes of B-cell lymphoma. Here, we show that mutations associated with AML result both in loss of miR-142-3p function and in decreased miR-142-5p expression. Mir142 loss altered the hematopoietic differentiation of multipotent hematopoietic progenitors, enhancing their myeloid potential while suppressing their lymphoid potential. During hematopoietic maturation, loss of Mir142 increased ASH1L protein expression and consequently resulted in the aberrant maintenance of Hoxa gene expression in myeloid-committed hematopoietic progenitors. Mir142 loss also enhanced the disease-initiating activity of IDH2-mutant hematopoietic cells in mice. Together these data suggest a novel model in which miR-142, through repression of ASH1L activity, plays a key role in suppressing HOXA9/A10 expression during normal myeloid differentiation. AML-associated loss-of-function mutations of MIR142 disrupt this negative signaling pathway, resulting in sustained HOXA9/A10 expression in myeloid progenitors/myeloblasts and ultimately contributing to leukemic transformation.Significance: These findings provide mechanistic insights into the role of miRNAs in leukemogenesis and hematopoietic stem cell function. Cancer Res; 78(13); 3510–21. ©2018 AACR.

Purpose:N-803 is an IL15 receptor superagonist complex, designed to optimize in vivo persistence and trans-presentation, thereby activating and expanding natural killer (NK) cells and CD8+ T cells. Monoclonal antibodies (mAbs) direct Fc receptor–bearing immune cells, including NK cells, to recognize and eliminate cancer targets. The ability of IL15R agonists to enhance tumor-targeting mAbs in patients has not been reported previously.Patients and Methods:Relapsed/refractory patients with indolent non-Hodgkin lymphoma were treated with rituximab and intravenous or subcutaneous N-803 on an open-label, dose-escalation phase I study using a 3+3 design (NCT02384954). Primary endpoint was maximum tolerated dose. Immune correlates were performed using multidimensional analysis via mass cytometry and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) which simultaneously measures protein and single-cell RNA expression.Results:This immunotherapy combination was safe and well tolerated and resulted in durable clinical responses including in rituximab-refractory patients. Subcutaneous N-803 plus rituximab induced sustained proliferation, expansion, and activation of peripheral blood NK cells and CD8 T cells, with increased NK cell and T cells present 8 weeks following last N-803 treatment. CITE-seq revealed a therapy-altered NK cell molecular program, including enhancement of AP-1 transcription factor. Furthermore, the monocyte transcriptional program was remodeled with enhanced MHC expression and antigen-presentation genes.Conclusions:N-803 combines with mAbs to enhance tumor targeting in patients, and warrants further investigation in combination with immunotherapies.

Human NK cell engraftment and proliferation in NSG mice with ALT-803 administration.

Purpose:Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation.Experimental Design:We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood–derived NK cells was investigated in vitro and in vivo.Results:We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13–NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo.Conclusions:We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

New therapies are needed for patients with relapsed/refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) who do not benefit from or are ineligible for stem cell transplant and chimeric antigen receptor therapy. The CD30-targeted, antibody-drug conjugate brentuximab vedotin (BV) and the immunomodulator lenalidomide (Len) have demonstrated promising activity as single agents in this population. We report the results of a phase 1/dose expansion trial evaluating the combination of BV/Len in rel/ref DLBCL. Thirty-seven patients received BV every 21 days, with Len administered continuously for a maximum of 16 cycles. The maximum tolerated dose of the combination was 1.2 mg/kg BV with 20 mg/d Len. BV/Len was well tolerated with a toxicity profile consistent with their use as single agents. Most patients required granulocyte colony-stimulating factor support because of neutropenia. The overall response rate was 57% (95% CI, 39.6-72.5), complete response rate, 35% (95% CI, 20.7-52.6); median duration of response, 13.1 months; median progression-free survival, 10.2 months (95% CI, 5.5-13.7); and median overall survival, 14.3 months (95% CI, 10.2-35.6). Response rates were highest in patients with CD30+ DLBCL (73%), but they did not differ according to cell of origin (P = .96). NK cell expansion and phenotypic changes in CD8+ T-cell subsets in nonresponders were identified by mass cytometry. BV/Len represents a potential treatment option for patients with rel/ref DLBCL. This combination is being further explored in a phase 3 study (registered on https://clinicaltrials.org as NCT04404283). This trial was registered on https://clinicaltrials.gov as NCT02086604.

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B cell cancers yet fail to induce durable remissions for nearly half of patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven anti-tumor T cell function. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation of CAR T cells from a patient with progressive lymphoma confirmed activation of this novel program in a failing clinical product. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Interest in adoptive cell therapy for treating cancer is exploding owing to early clinical successes of autologous chimeric antigen receptor (CAR) T lymphocyte therapy. However, limitations using T cells and autologous cell products are apparent as they (1) take weeks to generate, (2) utilize a 1:1 donor-to-patient model, (3) are expensive, and (4) are prone to heterogeneity and manufacturing failures. CAR T cells are also associated with significant toxicities, including cytokine release syndrome, immune effector cell–associated neurotoxicity syndrome, and prolonged cytopenias. To overcome these issues, natural killer (NK) cells are being explored as an alternative cell source for allogeneic cell therapies. NK cells have an inherent ability to recognize cancers, mediate immune functions of killing and communication, and do not induce graft-versus-host disease, cytokine release syndrome, or immune effector cell–associated neurotoxicity syndrome. NK cells can be obtained from blood or cord blood or be derived from hematopoietic stem and progenitor cells or induced pluripotent stem cells, and can be expanded and cryopreserved for off-the-shelf availability. The first wave of point-of-care NK cell therapies led to the current allogeneic NK cell products being investigated in clinical trials with promising preliminary results. Basic advances in NK cell biology and cellular engineering have led to new translational strategies to block inhibition, enhance and broaden target cell recognition, optimize functional persistence, and provide stealth from patients’ immunity. This review details NK cell biology, as well as NK cell product manufacturing, engineering, and combination therapies explored in the clinic leading to the next generation of potent, off-the-shelf cellular therapies for blood cancers.

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)–grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

In many cancers, including lymphoma, males have higher incidence and mortality than females. Emerging evidence demonstrates that one mechanism underlying this phenomenon is sex differences in metabolism, both with respect to tumor nutrient consumption and systemic alterations in metabolism, i.e., obesity. We wanted to determine if visceral fat and tumor glucose uptake with fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) could predict sex-dependent outcomes in patients with diffuse large B-cell lymphoma (DLBCL). We conducted a retrospective analysis of 160 patients (84 males; 76 females) with DLBCL who had imaging at initial staging and after completion of therapy. CT-based relative visceral fat area (rVFA), PET-based SUVmax normalized to lean body mass (SULmax), and end-of-treatment FDG-PET 5PS score were calculated. Increased rVFA at initial staging was an independent predictor of poor OS only in females. At the end of therapy, increase in visceral fat was a significant predictor of poor survival only in females. Combining the change in rVFA and 5PS scores identified a subgroup of females with visceral fat gain and high 5PS with exceptionally poor outcomes. These data suggest that visceral fat and tumor FDG uptake can predict outcomes in DLBCL patients in a sex-specific fashion.

Introduction: Offering the optimal frontline treatment to a patient with acute myeloid leukemia (AML) requires trading off expected benefit and risk. Typical standard of care intensive induction chemotherapy (e.g., cytarabine plus idarubicin (7+3)) results in high clinical response rates. However, many patients receive a less intensive regimen (e.g., venetoclax plus decitabine (VenDec)) because their individual toxicity risk is high based on lack of medical fitness. Predicting an individual patient’s clinical response prior to treatment has the potential to increase the benefit/risk ratio (therapeutic index) for some patients and optimize their treatment selection. Here, we demonstrate the ability of an automated high-throughput, multi-color flow cytometry predictive precision medicine platform (PPMP) to predict response to 7+3 or VenDec. Methods: To assess correlation between PPMP-predicted and actual clinical response to 7+3 or VenDec in clinical trial NCT04263181, pre-induction blood samples were collected from 31 patients of which 18 received 7+3 (all newly diagnosed (ND) AML) and 13 VenDec (7 ND AML, 5 secondary AML, 1 MDS). We measured drug effects on leukemic blasts by applying a cutoff at the total blast count that optimizes separation between predicted responders and non-responders (“conventional approach”) or by a machine learning (ML) approach considering multiple cell populations. For the former approach, training sets represented 13 patients for 7+3 and 8 for VenDec. Both 7+3 and VenDec models were validated with 5 patients. For the ML approach, the model was trained on all 13 VenDec patients and monitored using leave-one-out cross-validation. Results: For the conventional approach, predicted and true clinical responses were highly correlated for 7+3 (AUC = 0.91) and VenDec (AUC = 0.81), with 100% precision (positive predictive values (PPV)) for both, i.e., all predicted responders were true clinical responders. Some true clinical responders were not identified (negative predictive value (NPV) = 67% for 7+3 and 57% for VenDec), resulting in an accuracy of 94% (7+3) and 77% (VenDec). To maximize NPV and accuracy for predicting VenDec clinical outcomes, we applied a novel ML-based algorithm to integrate the behavior of malignant and non-malignant cell populations, yielding a model with 100% accuracy (100% PPV and NPV). Additional outcome data, including overall survival, are under evaluation. Summary: Total blast-based predictions yielded accuracies of 94% for 7+3 and 77% for VenDec. An ML algorithm for VenDec considering additional cell populations increased the accuracy to 100%. Further studies will expand patient numbers. We plan to use this platform to inform our frontline decision making with the goal to maximize the therapeutic benefit/risk ratio and ensure that the most appropriate frontline therapy is used for each individual patient. Citation Format: Meagan A. Jacoby, John S. Welch, Peter Westervelt, Matthew Christopher, Geoffrey L. Uy, Ravi Vij, Keith E. Stockerl-Goldstein, Brad S. Kahl, Iskra Pusic, John F. DiPersio, Mark A. Schroeder, Miriam Y. Kim, Todd A. Fehniger, Armin Ghobadi, Christine J. Gu, Wade Anderson, Kathryn Vanderlaag, Kamran Ali, Camille Pataki, Markus D. Lacher. Predictive precision medicine platform accurately predicts individual patient response to AML treatments to maximize outcomes. [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 4342.

Metastatic (m) colorectal cancer (CRC) is an incurable, frequently lethal disease present in approximately 25% of newly diagnosed CRC patients, and almost 50% of the patients with CRC will develop metastatic disease. For mCRC, while systemic therapies (cytotoxic therapy, targeted therapy, immunotherapy or their combinations) have extended patient’s life expectancy, not all patients are candidates for these treatments. Immunotherapy has achieved significant curative effects in patients with solid tumors; however, a considerable proportion of mCRC patients (~90%) are not responsive to immune checkpoint blockade (ICB) leaving a gap in the CRC immunotherapy options. Natural killer (NK) cells are cytotoxic innate lymphoid cells that display potent effector responses against a wide variety of tumor cells; however, they are frequently dysfunctional in cancer patients. NK cells from CRC patients exhibit decreased expression of activating receptors and reduced cytokine production after stimulation with CRC cells with a more accentuated phenotype in advanced stages of the disease. Memory-like (ML) NK cells differentiated after IL-12, IL-15, and IL-18 activation have been shown to overcome limitations associated with deficient tumor recognition and poor anti-tumor activity. In clinical trials, ML NK cells were safe and active against acute myeloid leukemia (Romee R et al, Sci Transl Med, 2016). Preclinically, ML NK cells exhibited improved responses against melanoma (Marin ND et al, Clin Cancer Res, 2021) and ovarian cancers, compared to conventional NK cells. Here, we hypothesized that memory-like differentiation will enhance multiple aspects of the NK cell response against CRC cells. Allogeneic ML NK cells displayed enhanced IFN-γ production against four CRC cell lines DLD-1 (p=0.015), SW480 (p=0.0005), HT-29 (p=0.0005) and HCT116 (p=0.001), as well as primary patient derived CRC tumoroids (p=0.0156), compared to conventional (c) NK cells. ML NK cells also exhibited superior and sustained killing of CRC cell lines over time compared to cNK cells, as measured by Incucyte assays and using CRC tumoroids. Furthermore, IFN-γ production was significantly reduced after blockade of NKG2D, DNAM-1 and NKp46 (p Collectively, these findings demonstrate that ML NK cells exhibit enhanced responses against CRC cells, and thus warrants further investigation in clinical trials for CRC patients, especially those who are not candidates for standard of care therapy or failed ICB. Citation Format: Nancy D. Marin, Michelle Becker-Hapak, Quazim A. Alayo, Melissa Berrien-Elliot, Lynne Marsala, Naomi Sonnek, Miriam T. Jacobs, Jennifer A. Foltz, Alice Zhou, Jennifer Tran, Pamela Wong, Celia Cubit, Kimberly Hwang, Timothy Schappe, Ryan C. Fields, Matthew A. Ciorba, Todd A. Fehniger. Memory like differentiation enhances in vitro and in vivo NK cell responses against colorectal cancer [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 893.

Natural killer (NK) cells are an emerging cancer cellular therapy and potent mediators of antitumor immunity. Cytokine-induced memory-like (ML) NK cellular therapy is safe and induces remissions in patients with acute myeloid leukemia (AML). However, the dynamic changes in phenotype that occur after NK-cell transfer that affect patient outcomes remain unclear. Here, we report comprehensive multidimensional correlates from ML NK cell–treated patients with AML using mass cytometry. These data identify a unique in vivo differentiated ML NK–cell phenotype distinct from conventional NK cells. Moreover, the inhibitory receptor NKG2A is a dominant, transcriptionally induced checkpoint important for ML, but not conventional NK-cell responses to cancer. The frequency of CD8α+ donor NK cells is negatively associated with AML patient outcomes after ML NK therapy. Thus, elucidating the multidimensional dynamics of donor ML NK cells in vivo revealed critical factors important for clinical response, and new avenues to enhance NK-cell therapeutics. Significance: Mass cytometry reveals an in vivo memory-like NK-cell phenotype, where NKG2A is a dominant checkpoint, and CD8α is associated with treatment failure after ML NK–cell therapy. These findings identify multiple avenues for optimizing ML NK–cell immunotherapy for cancer and define mechanisms important for ML NK–cell function. This article is highlighted in the In This Issue feature, p. 1775