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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.

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Table 5 Excel Document

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.

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.

Natural killer (NK) cells are innate lymphoid cells that eliminate cancer cells, produce cytokines, and are being investigated as a nascent cellular immunotherapy. Impaired NK cell function, expansion, and persistence remain key challenges for optimal clinical translation. One promising strategy to overcome these challenges is cytokine-induced memory-like (ML) differentiation, whereby NK cells acquire enhanced antitumor function after stimulation with interleukin-12 (IL-12), IL-15, and IL-18. Here, reduced-intensity conditioning (RIC) for HLA -haploidentical hematopoietic cell transplantation (HCT) was augmented with same-donor ML NK cells on day +7 and 3 weeks of N-803 (IL-15 superagonist) to treat patients with relapsed/refractory acute myeloid leukemia (AML) in a clinical trial (NCT02782546). In 15 patients, donor ML NK cells were well tolerated, and 87% of patients achieved a composite complete response at day +28, which corresponded with clearing high-risk mutations, including TP53 variants. NK cells were the major blood lymphocytes for 2 months after HCT with 1104-fold expansion (over 1 to 2 weeks). Phenotypic and transcriptional analyses identified donor ML NK cells as distinct from conventional NK cells and showed that ML NK cells persisted for over 2 months. ML NK cells expressed CD16, CD57, and high granzyme B and perforin, along with a unique transcription factor profile. ML NK cells differentiated in patients had enhanced ex vivo function compared to conventional NK cells from both patients and healthy donors. Overall, same-donor ML NK cell therapy with 3 weeks of N-803 support safely augmented RIC haplo-HCT for AML.

Both tumor cell-intrinsic signals and tumor cell-extrinsic signals from cells within the tumor microenvironment influence tumor cell dissemination and metastasis. The fibrillar collagen receptor tyrosine kinase (RTK) discoidin domain receptor 2 (DDR2) is essential for breast cancer metastasis in mouse models, and high expression of DDR2 in tumor and tumor stromal cells is strongly associated with poorer clinical outcomes. DDR2 tyrosine kinase activity has been hypothesized to be required for the metastatic activity of DDR2; however, inhibition of DDR2 tyrosine kinase activity, along with that of other RTKs, has failed to provide clinically relevant responses in metastatic patients. Here, we show that tyrosine kinase activity-independent action of DDR2 in tumor cells can support Matrigel invasion and in vivo metastasis. Paracrine actions of DDR2 in tumor cells and cancer-associated fibroblasts (CAFs) also support tumor invasion, migration and lung colonization in vivo. These data suggest that tyrosine kinase-independent functions of DDR2 could explain failures of tyrosine kinase inhibitor treatment in metastatic breast cancer patients and highlight the need for alternative therapeutic strategies that inhibit both tyrosine kinase-dependent and -independent actions of RTKs in the treatment of breast cancer. This article has an associated First Person interview with the first author of the paper., Summary: The collagen receptor DDR2, a receptor tyrosine kinase, exhibits tyrosine kinase-independent functions that influence paracrine regulation of breast tumor cell invasion and breast cancer metastasis.

Neoantigens are novel peptide sequences resulting from somatic mutations in tumors that upon loading onto major histocompatibility complex (MHC) molecules allow recognition by T cells. Accurate neoantigen identification is thus critical for designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly inferring whether the presenting peptide sequence can successfully induce an immune response. As the majority of somatic mutations are SNVs, changes between wildtype and mutant peptide are subtle and require cautious interpretation. An important, yet underappreciated, variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient’s specific HLA alleles. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. However, a systematic method for determining anchor locations for the wide range of HLA alleles present in the population and application of these to evaluate MT/WT peptide pairs arising in tumors has not been reported. As a result, many neoantigen studies have either failed to adequately consider this crucial factor or have used conventional assumptions to guide their neoantigen identification process. Here, we provide a computational workflow for predicting anchor locations for a wide range of HLA alleles, using a reference dataset generated from clinical and The Cancer Genome Atlas (TCGA) patient samples. We calculated high probability anchor positions for different peptide lengths for over 300 common HLA alleles. Analysis of these results showed clusters of different anchor trends among the HLA alleles analyzed. A subset of these HLA anchor results were orthogonally validated using protein crystallography structures. Analysis of 923 tumor samples showed that 7-41% of neoantigen candidates were potentially misclassified in the neoantigen selection process and can be rescued using allele-specific knowledge of anchor positions. These anchor predictions are currently undergoing experimental validation using both peptide-MHC stability assays as well as fluorescence-based competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, such as pVACtools, we hope to formalize and streamline the identification process for relevant clinical studies. Citation Format: Huiming Xia, Joshua McMichael, Michelle Becker-Hapak, Onyinyechi C. Onyeador, Rico Buchli, Ethan McClain, Patrick Pence, Suangson Supabphol, Megan M. Richters, Anamika Basu, Cody A. Ramirez, Cristina Puig-Saus, Kelsy C. Cotto, Jasreet Hundal, Susanna Kiwala, S. Peter Goedegebuure, Tanner M. Johanns, Gavin P. Dunn, Antoni Ribas, Christopher A. Miller, William Gillanders, Todd A. Fehniger, Obi L. Griffith, Malachi Griffith. Computational prediction of MHC anchor locations guide neoantigen prediction and prioritization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5639.

Neoantigens are novel peptide sequences resulting from sources such as somatic mutations in tumors. Upon loading onto major histocompatibility complex (MHC) molecules, they can trigger recognition by T cells. Accurate neoantigen identification is thus critical for both designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly predicting whether the presenting peptide sequence can successfully induce an immune response. As the majority of somatic mutations are single nucleotide variants, changes between wildtype and mutated peptides are typically subtle and require cautious interpretation. A potentially underappreciated variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient’s specific MHC molecules. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. We computationally predicted high probability anchor positions for different peptide lengths for 328 common HLA alleles and identified unique anchoring patterns among them. Analysis of 923 tumor samples shows that 6-38% of neoantigen candidates are potentially misclassified and can be rescued using allelespecific knowledge of anchor positions. A subset of anchor results were orthogonally validated using protein crystallography structures. Representative anchor trends were experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize, streamline and improve the identification process for relevant clinical studies.One Sentence SummaryNeoantigen prediction accuracy is significantly influenced by the mutation position within the neoantigen and its relative position to the patient’s allele-specific MHC anchor locations.

Adoptive cell therapy (ACT) using NK cells is a promising armament in the fight against cancer. Cytokine induced memory like (CIML) NK cells have been shown in clinical studies to have potent antitumor activity with superior in vivo persistence. Currently, the expansion of NK cells for clinical development is mainly based on feeder cells, which imposes significant regulatory hurdles and increases the costs for manufacturing. We have developed fusion proteins, HCW9201 and HCW9206 comprising of IL-15/IL-18/IL-12 and IL15/IL-7/IL-21, respectively, capable of priming memory-like differentiation and expanding CIML NK cell products without using feeder cells. This “Kick and Expand” strategy allows greater than 100x expansion of CIML NK cells from donor PBMCs in as little as 14 days without the use of exogenous feeder cells. Continued expansion can yield sufficient CIML NK cells for cryopreservation and multiple ACT infusions. The NK cells generated have bona fide memory-like properties: enhanced antitumor activity across multiple cancer cell lines, higher metabolic capacity, stable epigenetic demethylation of the IFN-γ promoter and increased persistence in NSG mice, when compared to conventional NK cells. In conclusion, this “Kick and Expand” process supports generation of abundant CIML NK cells for multiple ACT infusions and provides simpler, more regulatory friendly, off-the-shelf platform for generating NK cell products, including those with chimeric antigen receptor (CAR) constructs.

Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses and exhibit innate memory following stimulation with IL-12, IL-15, and IL-18, thereby differentiating into cytokine-induced memory-like (ML) NK cells. ML NK cells have well-described enhanced anti-tumor properties; however, the molecular mechanisms underlying their enhanced functionality are not well-understood. Initial reports of allogeneic donor ML NK cellular therapy for relapsed/refractory (rel/ref) acute myeloid leukemia (AML) demonstrated safety and a 47% CR/CRi rate (PMID32826231). In this setting, allogeneic ML NK cells are rejected after 3 weeks by recipient T cells, which precludes long-term evaluation of their biology. To address this limitation, we conducted a clinical trial for rel/ref AML patients that added adoptive transfer of same-donor ML NK cells on day +7 of a reduced-intensity conditioning (RIC) MHC-haploidentical HCT, followed by 4 doses of IL-15 (N-803) over 2 weeks (NCT02782546). Since the ML NK cells are from the HCT donor, they are not rejected, but remain MHC-haploidentical to the patient leukemia. Using samples from these patients, we profiled the single cell transcriptomes of NK cells using multidimensional CITE-seq, combining scRNAseq with a custom NK panel of antibodies. To identify donor ML NK cells in an unbiased fashion, we developed a CITE-seq ML NK classifier from in vitro differentiated paired conventional NK (cNK) and ML NK cells. This classifier was applied via transfer learning to CITE-seq analyzed samples from the donor (cNK cells) and patients at days +28 and +60. This approach identified 28-40% of NK cells as ML at Day +28 post-HCT. Only 1-6% of donor peripheral blood NK cells and 4-7% of NK cells in comparator leukemia patients at day +28 after conventional haplo-HCT alone were identified as ML NK cells (Fig 1A). These ML NK cells had a cell surface receptor profile analogous to a previously reported mass cytometry phenotype. Within the CITE-seq data, ML NK cells expressed a transcriptional profile consistent with enhanced functionality (GZMK, GZMA, GNLY), secreted proteins (LTB, CKLF), a distinct adhesome, and evidence of prior activation (MHC Class II and interferon-inducible genes). ML NK cells had a unique NK receptor repertoire including increased KIR2DL4, KLRC1(NKG2A), CD300A, NCAM1(CD56) , and CD2 with decreased expression of the inhibitory receptor KLRB1(CD161). Furthermore, ML NK cells upregulated HOPX, a transcription factor implicated in memory T cells and murine CMV adaptive NK cells. Additionally, ML NK cells downregulated transcription factors related to terminal maturation (ZEB2) and exhaustion (NR4A2). We next sought to identify changes during ML differentiation in patients post-HCT from day +28 to +60 post-HCT. Trajectory analysis identified a ML NK cell state distinct from cNK cells that was present at least 60 days post-HCT (Fig 1B). The ML transcriptional phenotype continued to modulate during late differentiation, including downregulation of GZMK and NCAM1, and upregulation of maturation related transcription factors, while maintaining high expression of HOPX. ML NK cells retained their enhanced functionality during in vivo differentiation, as patient ML NK cells had significantly increased IFNγ production compared to cNK cells after restimulation with leukemia targets or cytokines using mass cytometry (Fig. 2). Subsequently, we confirmed the ML CITE-seq profile in an independent clinical trial treating pediatric AML relapsed after allogenic HCT with same-donor ML NK cells (NCT03068819). In this setting, ML NK cells expressed a similar transcriptional signature and persisted for at least 2 months in the absence of exogenous cytokine support. Thus, ML NK cells possess a distinct transcriptional and surface proteomic profile and undergo in vivo differentiation while persisting within patients for at least 2 months. These findings reveal novel and unique aspects of the ML NK cell molecular program, as well as their prolonged functional persistence in vivo in patients, assisting in future clinical trial design. Figure 1 Figure 1. Disclosures Foltz: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Bednarski: Horizon Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; ImmunityBio: Research Funding; Kiadis: Other; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; OrcaBio: Other; Indapta: Other.

Biomechanical changes in the tumor microenvironment influence tumor progression and metastases. Collagen content and fiber organization within the tumor stroma are major contributors to biomechanical changes (e., tumor stiffness) and correlated with tumor aggressiveness and outcome. What signals and in what cells control collagen organization within the tumors, and how, is not fully understood. We show in mouse breast tumors that the action of the collagen receptor DDR2 in CAFs controls tumor stiffness by reorganizing collagen fibers specifically at the tumor-stromal boundary. These changes were associated with lung metastases. The action of DDR2 in mouse and human CAFs, and tumors in vivo, was found to influence mechanotransduction by controlling full collagen-binding integrin activation via Rap1-mediated Talin1 and Kindlin2 recruitment. The action of DDR2 in tumor CAFs is thus critical for remodeling collagen fibers at the tumor-stromal boundary to generate a physically permissive tumor microenvironment for tumor cell invasion and metastases.

Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final committed step in the biosynthesis of triglycerides. DGAT-1 knockout mice have been shown to be resistant to diet-induced obesity and have increased insulin sensitivity. Thus, inhibition of DGAT-1 may represent an attractive target for the treatment of obesity or type II diabetes. Herein, we report the discovery and characterization of a potent and selective DGAT-1 inhibitor PF-04620110 (3). Compound 3 inhibits DGAT-1 with an IC50 of 19 nM and shows high selectivity versus a broad panel of off-target pharmacologic end points. In vivo DGAT-1 inhibition has been demonstrated through reduction of plasma triglyceride levels in rodents at doses of ≥0.1 mg/kg following a lipid challenge. On the basis of this pharmacologic and pharmacokinetic profile, compound 3 has been advanced to human clinical studies.