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.
Abstract
Atherosclerosis is the leading cause of coronary heart disease and stroke. Sterile Inflammation, resulting from activation of the inflammasome NLRP3, plays a key role in the pathophysiology ...of atherosclerosis. Studies suggested that low-dose rIL-2 may be effective in treating autoimmune and inflammatory diseases by inducing Treg cells. Since rIL-2 has unfavorable pharmacokinetic profile in humans, we constructed a fusion protein, designated HCW9302, containing two IL-2 domains linked by an extracellular tissue factor domain, to extend the half-live of IL-2 for Treg expansion. In vitro treatment of human PBMCs with HCW9302 significantly expanded CD4+/CD25+/FoxP3+ Treg cells without activating CD4+ and CD8+ T cells over a broader concentration range than rIL2. Subcutaneous injection of HCW9302 (3 mg/kg every 2 – 3 weeks) into atherosclerosis-prone mice (ApoE−/− mice and LDLR−/− mice fed the Western Diet (WD) for 6 weeks) significantly increased the percentage of Treg cells, NK cells and CD8+ T cells in blood compared to the PBS-treated control group (p<0.001). Cytokines associated with atherosclerosis, including IL-1β and MCP-1, were also significantly decreased in the HCW9302-treated group, suggesting HCW9302 can moderate inflammasome NLRP3 activities. Remarkably, HCW9302 lowered the fasting blood glucose and insulin resistance. In aortas evaluated by the en face method at week 13 of WD, the ratio of plaque size to aortic surface was also significantly lower in HCW9302-treated mice compared to controls. HCW9302 treatment was well tolerated in both models. In summary, HCW9302 can effectively prevent the development of atherosclerosis in ApoE−/− mice and LDLR−/− mice, potentially through expansion of Treg cells.
Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. However, novel immunotherapeutics with these dual functions are not frequently reported. ...Here we describe the creation of a heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology. This complex comprises extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin-15 (IL-15)/IL-15 receptor α complex. HCW9218 can be readily expressed in CHO cells and purified using antibody-based affinity chromatography in a large-scale manufacturing setting. HCW9218 potently activates mouse natural killer (NK) cells and CD8+ T cells in vitro and in vivo to enhance cell proliferation, metabolism, and antitumor cytotoxic activities. Similarly, human immune cells become activated with increased cytotoxicity following incubation with HCW9218. This fusion complex also exhibits TGF-β neutralizing activity in vitro and sequesters plasma TGF-β in vivo. In a syngeneic B16F10 melanoma model, HCW9218 displayed strong antitumor activity mediated by NK cells and CD8+ T cells and increased their infiltration into tumors. Repeat-dose subcutaneous administration of HCW9218 was well tolerated by mice, with a half-life sufficient to provide long-lasting biological activity. Thus, HCW9218 may serve as a novel therapeutic to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors.
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A bifunctional fusion protein, HCW9218, was created using tissue factor-based scaffold technology. HCW9218 comprises the extracellular domains of human transforming growth factor-β receptor II and human interleukin-15 (IL-15)/IL-15 receptor α. This fusion protein represents a potent anti-cancer immunotherapeutic to simultaneously provide immune stimulation and lessen immunosuppressive activities associated with tumors.
Abstract
We have constructed two heterodimeric multi-cytokine fusions, HCW9201 and HCW9206, to support a “Kick and Expand” approach to activate and induce proliferation of purified NK cells for ...adoptive cell therapy (ACT). HCW9201 is a heterodimeric fusion protein complex comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-18 and IL-12. HCW9206 is heterodimeric fusion protein comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-7, and IL-21. When purified human NK cells from peripheral blood were activated with HCW9201 for three hours and then incubated with HCW9206 in combination with a capture antibody, the HCW9201-activated NK cells expanded approximately 100–300 folds within 14 days. The expanded NK cells exhibit a cytokine-induced memory-like (CIML) phenotype with a high metabolic rate and respiratory capacity, remarkable anti-tumor activity, and persistence when they were adoptively transferred into NSG mice. They also retained their heightened responsiveness when re-stimulated with tumor targets. Cytokine dependent epigenetic demethylation imprints of the Ifng promoter region were also observed for at least 10 days after the NK cells were adoptively transferred to NSG mice. In conclusion, a simple, scalable, non-feeder-cell-based “Kick and Expand” process was developed to support the generation of large numbers of CIML NK cells for multiple rounds of ACT using peripheral blood NK cell. We also provide data to show that these CIML NK cells are an excellent source for generation of CAR-NK cells.
Abstract
Interleukin-15 (IL-15) is a promising cytokine for cancer therapy. However, it has shown limited antitumor efficacy as monotherapy in clinical trials to date. Removal of immunosuppression of ...the tumor microenvironment is considered a promising approach to enhance IL-15-mediated immune responses. TGF-β is a key component for creating an immunosuppressive tumor microenvironment. Therefore, we constructed a novel bifunctional fusion protein complex, designated HCW9218, comprising a soluble fusion of two TGFβRII, human tissue factor, and human IL-15, and a second soluble fusion of two TGFβRII and a sushi domain of IL-15Rα. HCW9218 activates IL-15R signaling and the dimeric TGFβRII functions as “trap” for all the three TGF-β isoforms. In healthy and tumor-bearing C57/BL6 mice, subcutaneously administrated HCW9218 was well tolerated with a long serum half-life and was able to stimulate and induce proliferation of CD8+ T cells and NK cells. Splenocytes from HCW9218-treated mice showed enhanced metabolic activity and cytotoxicity against Yac-1 target cells compared to untreated mouse splenocytes. In the syngeneic B16F10 melanoma mouse model, a single-dose of HCW9218 exhibited strong antitumor activity in combination with chemotherapy and TA99 antibody. HCW9218 also significantly increased immune cell infiltration into tumors. Mutagenesis studies demonstrated that both the TGFβRII and IL-15/IL-15Rα domains are essential for antitumor efficacy of HCW9218. Collectively, our preclinical data demonstrate that HCW9218 elicits potent immune responses with a remarkable safety profile and serves as a novel immunotherapeutic against cancer alone or in combination with therapeutic antibodies.
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