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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

Supplementary methods. Supplementary Table S1. Antibodies used in immune cell characterization by flow cytometry. Supplementary Table S2. PK parameters of ALT-803 administered to cynomolgus monkeys. Supplementary Table S3. Representative clinical characteristics of cynomolgus monkeys following 4 weekly doses of ALT-803 administration (study day 26). Supplementary Table S4. Incidence of major histopathologic findings in cynomolgus monkeys following 4 weekly doses of ALT-803 administration (study day 26). Supplementary Figure 1. Comparative binding of ALT-803 to IL-15 receptors on mouse, cynomolgus monkey, and human immune cells.

Movie S3 from Dual Targeting of Tissue Factor and CD105 for Preclinical PET Imaging of Pancreatic Cancer

Effector CD8+ T cells conditioned with IL12 during activation mediate enhanced antitumor efficacy after adoptive transfer into lymphodepleted hosts; this is due in part to improved IL7 responsiveness. Therefore, we hypothesized that increasing the intensity or type of lymphodepletion would deplete more IL7-consuming host cells and improve the persistence and antitumor activity of IL12-conditioned CD8+ T cells. Using cyclophosphamide, fludarabine, and total body irradiation (TBI, 6 Gy) either individually or in combination, we found that combined lymphodepletion best enhanced T-cell engraftment in mice. This improvement was strongly related to the extent of leukopenia, as posttransfer levels of donor T cells inversely correlated to host cell counts after lymphodepletion. Despite the improvement in engraftment seen with combination lymphodepletion, dual-agent lymphodepletion did not augment the antitumor efficacy of donor T cells compared with TBI alone. Similarly, IL7 supplementation after TBI and transfer of tumor-reactive T cells failed to improve persistence or antitumor immunity. However, IL15 or IL2 supplementation greatly augmented the persistence and antitumor efficacy of donor tumor-reactive T cells. Our results indicate that the amount of host IL7 induced after single agent lymphodepletion is sufficient to potentiate the expansion and antitumor activity of donor T cells, and that the efficacy of future regimens may be improved by providing posttransfer support with IL2 or IL15.Significance: The relationship between lymphodepletion and cytokine support plays a critical role in determining donor T-cell engraftment and antitumor efficacy. Cancer Res; 78(11); 3067–74. ©2018 AACR.

This file contains the following figures: Supplemental Figure 1. The return of host lymphocytes is affected by the type of donor CD8+ T cells. Supplemental Figure 2. Return of myeloid cells after CTX leads to more rapid host cell reconstitution than TBI-containing regimens. Supplemental Figure 3. CTX and TBI differentially affect host T cells and NK cells. Supplemental Figure 4. Donor CD8+ T cells transferred into a TBI-conditioned host maintain functional efficacy. Supplemental Figure 5. ALT-803 (IL-15/IL-15Ra complexes) augment the persistence of Tc1 cells in irradiated hosts.

Determination of NOTA to Fab Fragment Ratio

Characteristics of primary iNHL patient samples used in this study.

Supplementary Figure 1-3 Legends

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

Supplementary Figure 1. Purification of heterodimer after click chemistry conjugation of ALT-836-Fab and TRC105-Fab Supplementary Figure 2. MALDI-TOF mass spectra of ALT-836-Fab: [M+H]+ = 47266.0, TRC105-Fab: [M+H]+ = 47454.7, and heterodimer: exact [M+H]+ = 103504.0. Supplementary Figure 3. Flow cytometry analysis in BXPC-3 after 1 h incubation of FITC-Fab conjugates for the comparison of their in vitro targeting Supplementary Figure 4. Radiolabeling efficiency at different copper: immunoconjugate ratios were used to determine the number of NOTA per antibody fragment molecule Supplementary Figure 5. PET imaging and biodistribution of 64CuCl2 in BXPC-3 tumor-bearing mice. Supplementary Figure 6. PET imaging and quantitative ROI analysis of 64Cu-NOTA-heterodimer with TF and CD105 dual-blocking in BXPC-3 tumor-bearing mice. Supplementary Figure 7. Coronal images and transverse PET/CT images of normal nude mice at 3, 15, 24 and 30 h following injection of 64Cu-NOTA-heterodimer Supplementary Figure 8. PET ROI-derived quantification of 64Cu-NOTA-heterodimer uptake in the tumor, normal pancreas, blood pool, liver, kidney and muscle at 3 h, 15 h, 24 h and 30 h p.i. of the tracer

Purpose: IL15 induces the activation and proliferation of natural killer (NK) and memory CD8+ T cells and has preclinical antitumor activity. Given the superior activity and favorable kinetics of ALT-803 (IL15N72D:IL15RαSu/IgG1 Fc complex) over recombinant human IL15 (rhIL15) in animal models, we performed this first-in-human phase I trial of ALT-803 in patients with advanced solid tumors.Patients and Methods: Patients with incurable advanced melanoma, renal cell, non–small cell lung, and head and neck cancer were treated with ALT-803 0.3 to 6 μg/kg weekly intravenously or 6 to 20 μg/kg weekly subcutaneously for 4 consecutive weeks, every 6 weeks. Immune correlates included pharmacokinetics, immunogenicity, and lymphocyte expansion and function. Clinical endpoints were toxicity and antitumor activity.Results: Twenty-four patients were enrolled; 11 received intravenous and 13 received subcutaneous ALT-803. Of these patients, nine had melanoma, six renal, three head and neck, and six lung cancer. Although total lymphocyte and CD8+ T-cell expansion were modest, NK cell numbers rose significantly. Neither anti–ALT-803 antibodies nor clinical activity were observed. Overall, ALT-803 was well tolerated, with adverse effects including fatigue and nausea most commonly with intravenous administration, whereas painful injection site wheal was reported most commonly with subcutaneous ALT-803.Conclusions: Subcutaneous ALT-803 produced the expected NK cell expansion and was well tolerated with minimal cytokine toxicities and a strong local inflammatory reaction at injection sites in patients with advanced cancer. These data, together with compelling evidence of synergy in preclinical and clinical studies, provide the rationale for combining ALT-803 with other anticancer agents. Clin Cancer Res; 24(22); 5552–61. ©2018 AACR.

Supplemental Figure 1. Effect of ALT-803 treatment on white blood cell and lymphocyte counts in i.v. dose cohorts Supplemental Figure 2. Circulating NK cell expansion during ALT-803 treatment Supplemental Figure 3. Effect of subcutaneous ALT-803 on circulating T cell expression of HLA-DR

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

Purpose: Pancreatic adenocarcinoma is a highly aggressive cancer, currently treated with limited success and dismal outcomes. New diagnostic and treatment strategies offer the potential to reduce cancer mortality. Developing highly specific noninvasive imaging probes for pancreatic cancer is essential to improving diagnostic accuracy and monitoring therapeutic intervention.Experimental Design: A bispecific heterodimer was synthesized by conjugating an anti-tissue factor (TF) Fab with an anti-CD105 Fab, via the bio-orthogonal “click” reaction between tetrazine (Tz) and trans-cyclooctene (TCO). The heterodimer was labeled with 64Cu for PET imaging of nude mice bearing BXPC-3 xenograft and orthotopic pancreatic tumors.Results: PET imaging of BXPC-3 (TF/CD105+/+) xenograft tumors with 64Cu-labeled heterodimer displayed significantly enhanced tumor uptake (28.8 ± 3.2 %ID/g; n = 4; SD) at 30 hours postinjection, as compared with each of their monospecific Fab tracers (12.5 ± 1.4 and 7.1 ± 2.6 %ID/g; n = 3; SD). In addition, the activity–concentration ratio allowed for effective tumor visualization (tumor/muscle ratio 75.2 ± 9.4 at 30 hours postinjection.; n = 4; SD). Furthermore, 64Cu-NOTA-heterodimer enabled sensitive detection of orthotopic pancreatic tumor lesions with an uptake of 17.1 ± 4.9 %ID/g at 30 hours postinjection and tumor/muscle ratio of 72.3 ± 46.7.Conclusions: This study demonstrates that dual targeting of TF and CD105 provided synergistic improvements in binding affinity and tumor localization of the heterodimer. Dual-targeted imaging agents of pancreatic and other cancers may assist in diagnosing pancreatic malignancies as well as reliable monitoring of therapeutic response. Clin Cancer Res; 22(15); 3821–30. ©2016 AACR.

PDF file - 304K, Growth pattern of 5T33P murine myeloma cells in BM of C57BL/6NHsd mice (S1); ALT-803 activity in murine myeloma models (S2); 5T33P cell apoptosis is not induce culturing with ALT-803 or IFNy (S3); Gating strategy and analysis of donors (S4).

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.

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.

Immunotherapeutics that aid in boosting natural immune defenses against cancers have revolutionized cancer treatment. Previously, we reported a novel heterodimeric bifunctional fusion molecule, HCW9218, designed using soluble tissue factor (TF)-based scaffold technology comprising extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin (IL)-15/IL-15 receptor α complex which exhibited both immune cell stimulatory and TGF-β neutralizing properties. Herein, we showed in two different syngeneic murine tumor models (B16F10, 4T1) that subcutaneous treatment with HCW9218 induces a proliferative burst of CD8+ T cells and NK cells in blood and a subsequent infiltration of these cells into established tumors. In vivo imaging of 4T1 tumor-bearing mice after treatment showed that HCW9218 was present both in lymph nodes and established tumors up to 24hr following treatment. Comprehensive analysis of tumor infiltrating lymphocytes (TILs) showed that HCW9218 mediated antitumor activity by expanding TCF+TIM3− ‘progenitor exhausted’ (Tpex) CD8+ T cells in tumors. Sphingosine-1-phosphate receptor blockade resulted in decreased tumor infiltration of CD8+ Tpex in B16F10 and 4T1 tumor-bearing mice indicating that these cells originate from tumor draining lymph nodes (TdLN). Increased ‘terminally exhausted‘ TCF-1−TIM3+ (Tex) CD8+ TILs were also observed in tumors of HCW9218-treated mice indicating increased antitumor activity. Tumor transplantation experiments further confirmed the mechanism of HCW9218 antitumor activity by increasing influx of CD45.1+ CD8+ T cells into transplanted tumors from CD45.2+ mice. Additionally, HCW9218 enhanced the therapeutic efficacy of PD-L1 treatment by increasing the infiltration of activated/memory CD8+ T cells into B16F10 tumors in mice leading to significant reduction in tumor volume. Collectively, the results of this study demonstrated that HCW9218 treatment of mice bearing solid tumors resulted in modulating the TdLN immune landscape and invigorating T cells for enhanced checkpoint blockade therapy. HCW9218 are currently in two clinical trials (clinicaltrials.org: NCT05322408, NCT05304936) against chemo-resistant/refractory solid tumors. Citation Format: Varghese George, Pallavi Chaturvedi, Niraj Shrestha, Leah Kanakraj, Crystal Gilkes, Nicole Encalada, Meng Wang, Xiaoyun Zhu, Bai Liu, Peter Rhode, Hing C. Wong. Bifunctional immunotherapeutic HCW9218 facilitates recruitment of immune cells from tumor draining lymph nodes to promote antitumor activity and enhance checkpoint blockade efficacy in solid tumors. [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 4441.

Therapy induced senescence (TIS) in tumors and TIS cancer cells secrete proinflammatory senescence-associated secretory phenotype (SASP) factors. SASP factors promote TIS cancer cells to re-enter the growth cycle with stemness characteristics, resulting in chemo-resistance and disease relapse. Herein, we show that the immunotherapeutic HCW9218, comprising transforming growth factor-β (TGF-β) receptor II and interleukin (IL)-15/IL-15 receptor α domains, enhances metabolic and cytotoxic activities of immune cells and reduces TIS tumor cells in vivo to improve the efficacy of docetaxel and gemcitabine plus nab-paclitaxel against B16F10 melanoma and SW1990 pancreatic tumors, respectively. Mechanistically, HCW9218 treatment reduces the immunosuppressive tumor microenvironment and enhances immune cell infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immuno-depletion analysis suggests that HCW9218-activated natural killer cells play a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhances efficacy of tumor antigen-specific and anti-programmed death-ligand 1 (PD-L1) antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment decreases TIS cells and lowers SASP factors in off-target tissues caused by chemotherapy of tumor-bearing mice. Collectively, HCW9218 has the potential to significantly enhance anti-tumor efficacy of chemotherapy, therapeutic antibodies, and checkpoint blockade by eliminating TIS cancer cells while reducing TIS-mediated proinflammatory side effects in normal tissues.

Background: A major mechanism of action for therapeutic antibodies is antibody-dependent cell-mediated cytotoxicity (ADCC). ALT-803 is an interleukin-15 superagonist complex that enhances ADCC against human carcinoma cells in vitro and exerts an antitumor activity in murine, rat, and human carcinomas in vivo. The authors investigated the ability of ALT-803 to modulate ADCC mediated by the humanized IgG1 monoclonal antibody (mAb) NEO-201 against human carcinoma cells. Materials and Methods: ALT-803 modulating activity on ADCC mediated by NEO-201 was evaluated on several NEO-201 ligand-expressing human carcinoma cells. Purified human natural killer (NK) cells from multiple healthy donors were treated with ALT-803 before their use as effectors in ADCC assay. Modulation of NK cell phenotype and cytotoxic function by exposure to ALT-803 was evaluated by flow cytometry and gene expression analysis. Results: ALT-803 significantly enhanced ADCC mediated by NEO-201. ALT-803 also upregulated NK activating receptors, antiapoptotic factors, and factors involved in the NK cytotoxicity, as well as downregulated gene expression of NK inhibiting receptors. Conclusions: These findings indicate that ALT-803 can enhance ADCC activity mediated by NEO-201, by modulating NK activation and cytotoxicity, suggesting a possible clinical use of ALT-803 in combination with NEO-201 for the treatment of human carcinomas.

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.

Purpose: IL15 induces the activation and proliferation of natural killer (NK) and memory CD8+ T cells and has preclinical antitumor activity. Given the superior activity and favorable kinetics of ALT-803 (IL15N72D:IL15RαSu/IgG1 Fc complex) over recombinant human IL15 (rhIL15) in animal models, we performed this first-in-human phase I trial of ALT-803 in patients with advanced solid tumors. Patients and Methods: Patients with incurable advanced melanoma, renal cell, non–small cell lung, and head and neck cancer were treated with ALT-803 0.3 to 6 μg/kg weekly intravenously or 6 to 20 μg/kg weekly subcutaneously for 4 consecutive weeks, every 6 weeks. Immune correlates included pharmacokinetics, immunogenicity, and lymphocyte expansion and function. Clinical endpoints were toxicity and antitumor activity. Results: Twenty-four patients were enrolled; 11 received intravenous and 13 received subcutaneous ALT-803. Of these patients, nine had melanoma, six renal, three head and neck, and six lung cancer. Although total lymphocyte and CD8+ T-cell expansion were modest, NK cell numbers rose significantly. Neither anti–ALT-803 antibodies nor clinical activity were observed. Overall, ALT-803 was well tolerated, with adverse effects including fatigue and nausea most commonly with intravenous administration, whereas painful injection site wheal was reported most commonly with subcutaneous ALT-803. Conclusions: Subcutaneous ALT-803 produced the expected NK cell expansion and was well tolerated with minimal cytokine toxicities and a strong local inflammatory reaction at injection sites in patients with advanced cancer. These data, together with compelling evidence of synergy in preclinical and clinical studies, provide the rationale for combining ALT-803 with other anticancer agents. Clin Cancer Res; 24(22); 5552–61. ©2018 AACR.

ALT-803 is a fusion protein complex consisting of an interleukin (IL)-15 superagonist and a dimeric IL-15 receptor alpha sushi domain IgG1 Fc fusion protein. When administered to mice, ALT-803 is capable of inducing natural killer (NK) and CD8(+) T cell proliferation and activation, and effectively promoting potent anti-tumor responses. Currently, ALT-803 is in clinical trials for treatment of various solid tumors and hematological malignancies. In the initial phase of these clinical studies, intravenous (iv) injection was used according to the route used in pre-clinical efficacy studies. In order to evaluate the possible advantage of subcutaneous (sc) injection versus iv injection, this study compared the biological activity of the two treatment regimens of ALT-803 in pre-clinical in vivo models. The pharmacokinetics, immune stimulation, and anti-tumor efficacy of iv and sc injection routes of ALT-803 in C57BL/6 mice were compared. The half-life of ALT-803 was 7.5 h for iv versus 7.7 h for sc with the maximal detected serum concentration of ALT-803 to be 3926 ng/ml at 0.5 h time-point following iv injection versus 495 ng/ml at 16 h post sc injection. Biodistribution studies indicated that sc ALT-803, similarly to iv ALT-803 as previously reported, has a greater tissue distribution and longer residence time in lymphoid tissues compared to recombinant IL-15. Notably, ALT-803 when administered either iv or sc induced comparable proliferation and activation of CD8(+) T and NK cells and resulted in similar reductions of tumor burden. A toxicity study of mice receiving multiple injections of ALT-803 for 4 weeks by iv or sc routes revealed equivalent immune-related changes. The gradual absorbance into the blood stream and lower maximal blood levels of ALT-803 in sc-injected mice, along with similar anti-tumor efficacy support the administration of ALT-803 by sc injection in patients with various malignancies and infectious diseases.

Summary Background Immunotherapy with PD-1 or PD-L1 blockade fails to induce a response in about 80% of patients with unselected non-small cell lung cancer (NSCLC), and many of those who do initially respond then develop resistance to treatment. Agonists that target the shared interleukin-2 (IL-2) and IL-15Rβγ pathway have induced complete and durable responses in some cancers, but no studies have been done to assess the safety or efficacy of these agonists in combination with anti-PD-1 immunotherapy. We aimed to define the safety, tolerability, and activity of this drug combination in patients with NSCLC. Methods In this non-randomised, open-label, phase 1b trial, we enrolled patients (aged ≥18 years) with previously treated histologically or cytologically confirmed stage IIIB or IV NSCLC from three academic hospitals in the USA. Key eligibility criteria included measurable disease, eligibility to receive anti-PD-1 immunotherapy, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received the anti-PD-1 monoclonal antibody nivolumab intravenously at 3 mg/kg (then 240 mg when US Food and Drug Administration [FDA]-approved dosing changed) every 14 days (either as new treatment or continued treatment at the time of disease progression) and the IL-15 superagonist ALT-803 subcutaneously once per week on weeks 1–5 of four 6-week cycles for 6 months. ALT-803 was administered at one of four escalating dose concentrations: 6, 10, 15, or 20 μg/kg. The primary endpoint was to define safety and tolerability and to establish a recommended phase 2 dose of ALT-803 in combination with nivolumab. Analyses were per-protocol and included any patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02523469; phase 2 enrolment of patients is ongoing. Findings Between Jan 18, 2016, and June 28, 2017, 23 patients were enrolled and 21 were treated at four dose levels of ALT-803 in combination with nivolumab. Two patients did not receive treatment because of the development of inter-current illness during enrolment, one patient due to leucopenia and one patient due to pulmonary dysfunction. No dose-limiting toxicities were recorded and the maximum tolerated dose was not reached. The most common adverse events were injection-site reactions (in 19 [90%] of 21 patients) and flu-like symptoms (15 [71%]). The most common grade 3 adverse events, occurring in two patients each, were lymphocytopenia and fatigue. A grade 3 myocardial infarction occurred in one patient. No grade 4 or 5 adverse events were recorded. The recommended phase 2 dose of ALT-803 is 20 μg/kg given once per week subcutaneously in combination with 240 mg intravenous nivolumab every 2 weeks. Interpretation ALT-803 in combination with nivolumab can be safely administered in an outpatient setting. The promising clinical activity observed with the addition of ALT-803 to the regimen of patients with PD-1 monoclonal antibody relapsed and refractory disease shows evidence of anti-tumour activity for a new class of agents in NSCLC. Funding Altor BioScience (a NantWorks company), National Institutes of Health, and Medical University of South Carolina Hollings Cancer Center.

Natural killer (NK) cells are innate cytotoxic lymphocytes that play a fundamental role in the immunosurveillance of cancers. NK cells of cancer patients exhibit impaired function mediated by immunosuppressive factors released from the tumor microenvironment (TME), such as transforming growth factor (TGF)-β1. An interleukin (IL)-15 superagonist/IL-15 receptor α fusion complex (IL-15SA/IL-15RA; ALT-803) activates the IL-15 receptor on CD8 T cells and NK cells, and has shown significant anti-tumor activity in several in vivo studies. This in vitro study investigated the efficacy of IL-15SA/IL-15RA on TGF-β1-induced suppression of NK cell-cytotoxic function. IL-15SA/IL-15RA inhibited TGF-β1 from decreasing NK cell lysis of four of four tumor cell lines (H460, LNCap, MCF7, MDA-MB-231). IL-15SA/IL-15RA rescued healthy donor and cancer patient NK cell-cytotoxicity, which had previously been suppressed by culture with TGF-β1. TGF-β1 downregulated expression of NK cell-activating markers and cytotoxic granules, such as CD226, NKG2D, NKp30, granzyme B, and perforin. Smad2/3 signaling was responsible for this TGF-β1-induced downregulation of NK cell-activating markers and cytotoxic granules. IL-15SA/IL-15RA blocked Smad2/3-induced transcription, resulting in the rescue of NK cell-cytotoxic function from TGF-β1-induced suppression. These findings suggest that in addition to increasing NK cell function via promoting the IL-15 signaling pathway, IL-15SA/IL-15RA can function as an inhibitor of TGF-β1 signaling, providing a potential remedy for NK cell dysfunction in the immunosuppressive tumor microenvironment.

National Institutes of Health (NIH) from the National Institute of Allergy and Infectious Diseases (NIAID) [R21 All 28970-01A1]; National Institutes of Health (NIH) from the NIH Office, and Martin Delaney BELIEVE collaborator NIAID award [P51 OD011092, UM1A1126617]; National Institute on Drug Abuse, National Institute of Mental Health, and National Institute of Neurological Disorders and Stroke

This mini review provides an overview and rationale for creating IL-15-based fusion protein complexes to be used as targeted immunotherapeutic agents. IL-15 stimulates proliferation and activation of CD8+ T and natural killer cells which result in augmentation of their anti-tumor activities. We have created ALT-803, an IL-15 superagonist complex which exhibits longer serum half-life, longer retention in lymphoid tissues, and better immunostimulatory and anti-tumor activities compared to native IL-15. When used alone or in combination with other immunotherapeutic molecules in various mouse tumor models, ALT-803 effectively reduces tumor burden and prolongs survival by stimulating the innate and adaptive arms of the immune system. To evaluate whether ALT-803 could be used as a protein scaffold to create IL-15-based tumor cell-specific molecules, we genetically fused it with a single chain anti-CD20 antibody derived from the variable regions of rituximab. This novel fusion protein exhibits enhanced anti-tumor activity compared to rituximab while maintaining IL-15 immunostimulating properties. Thus, ALT-803 may be exploited as a versatile scaffold to produce multivalent targeted fusion proteins to improve the anti-tumor efficacy of other therapeutic agents in the clinic.

BackgroundAllogeneic Natural Killer (NK) cells are emerging as a safe and effective modality for the treatment of cancer, overcoming several limitations associated with adoptive T cell therapies. Cytokine induced memory-NK cells offer several advantages over conventional NK cells, including enhanced functional persistence, efficacy, and metabolic fitness. Additionally, unlike iPSC and cord blood derived NK cells, they do not require engineering to enable functionality. Here we describe the use of WU-PRIME, a GMP-grade fusion protein complex to generate memory NK cells, and WU-EXPAND, a feeder cell free expansion system to expand memory-NK cells and create WU-NK-101. Further cryopreservation enables the large-scale, off-the-shelf manufacture of memory NK for cancer immunotherapy, with high anti-tumor activity.MethodsNK cells derived from healthy donor leukopheresate were either activated with WU-PRIME and then expanded with WU-EXPAND to form WU-NK-101 or immediately expanded with WU- EXPAND as controls and then cryopreserved. We compared NK cell expansion as well as post- thaw NK cell functionality as assessed by cytokine secretion and short-term and long-term anti- tumor functionality, long-term persistence in NSG mice, as well as anti-tumor activity in vivo.ResultsNK cells activated with WU-PRIME followed by WU-EXPAND (WU-NK-101), expand robustly in large-scale reactions, over 250-fold in 14 days. The cells maintain durable expression of CD25 after expansion, as well as several other hallmarks of the memory-NK phenotype as assessed by mass cytometry. As compared to cells expanded with WU-EXPAND only, WU-NK-101 cells have improved in vitro activity against K562 cells, as well as AML cell lines (TF-1, THP-1, and HL-60). Notably, this functionality is maintained long-term upon repeated challenge. In vivo, WU-NK-101 cells, compared to expanded NK cells have improved in vivo persistence (figure 1; 50,290 v. 9,623, pAbstract 188 Figure 1NK cell persistence in tumor-bearing mice. 10e6 cryopreserved NK cells were injected into K562 tumor-bearing mice, and supported with 50,000IU human IL-2 every other day. After 9 days, blood was harvested by cheek bleed and assessed for NK cells (hCD45+, CD56+, CD3) in the blood by flow cytometry.ConclusionsThe data demonstrate that WU-NK-101 generated using a feeder cell-free expansion system has a memory phenotype and improved in vitro and in vivo anti-tumor activity compared to conventional NK cells. This activation and expansion platform will enable the development and clinical translation of multiple allogeneic NK cell therapies.

Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies, including pancreatic cancer. Additionally, high TF expression was shown to strongly correlate with poor prognoses and decreased survival in pancreatic cancer patients. Herein, we exploited the potential targeting of TF for positron emission tomography (PET) imaging of pancreatic cancer. The TF-targeted tracer was developed through radiolabeling of the anti-human TF monoclonal antibody (ALT-836) with 89Zr. The tracer was characterized by fluorescence microscopy and flow cytometry assays in BXPC-3 and PANC-1 cells, two pancreatic cancer cell lines with high and low TF expression levels, respectively. Non-invasive PET scans were acquired in tumor-bearing mice injected with 89Zr-Df-ALT-836. Additionally, ex vivo biodistribution, blocking, and histological studies were performed to establish the affinity and specificity of 89Zr-Df-ALT-836 for TF in vivo. 89Zr-labeling of Df-ALT-836 was achieved in high yield and good specific activity. FACS and microscopy studies revealed no detectable difference in TF-binding affinity between ALT-836 and Df-ALT-836 in vitro. Longitudinal PET scans unveiled a lasting and prominent 89Zr-Df-ALT-836 uptake in BXPC-3 tumors (peak at 31.5 ± 6.0 %ID/g at 48 h post-injection; n=3), which was significantly abrogated (2.3 ± 0.5 %ID/g at 48 h post-injection; n=3) when mice were pre-injected with a blocking dose (50 mg/kg) of unlabeled ALT-836. Ex vivo biodistribution data confirmed the accuracy of the PET results, and histological analysis correlated high tumor uptake with in situ TF expression. Taken together, these results attest to the excellent affinity and TF-specificity of 89Zr-Df-ALT-836 in vivo. With elevated, persistent, and specific accumulation in TF-positive BXPC-3 tumors, PET imaging using 89Zr-Df-ALT-836 promises to open new avenues for improving future diagnosis, stratification, and treatment response assessment in pancreatic cancer patients.

// Cavan P. Bailey 1 , Tulin Budak-Alpdogan 3 , Christopher T. Sauter 1 , Michelle M. Panis 1 , Cihangir Buyukgoz 1 , Emily K. Jeng 2 , Hing C. Wong 2 , Neal Flomenberg 1 and Onder Alpdogan 1 1 Department of Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 2 Altor BioScience Corporation, Miramar, FL, USA 3 Department of Hematology and Oncology, MD Anderson Cancer Center at Cooper, Camden, NJ, USA Correspondence to: Onder Alpdogan, email: onder.alpdogan@jefferson.edu Keywords: stem cell transplantation, interleukin-15, cytokine therapy, graft-versus-tumor activity, animal models Received: August 15, 2016 Accepted: April 28, 2017 Published: May 15, 2017 ABSTRACT Interleukin-15 (IL-15) is a potent cytokine that increases CD8 + T and NK cell numbers and function in experimental models. However, obstacles remain in using IL-15 therapeutically, specifically its low potency and short in vivo half-life. To help overcome this, a new IL-15 superagonist complex comprised of an IL-15N72D mutation and IL-15RαSu/Fc fusion (IL-15SA, also known as ALT-803) was developed. IL-15SA exhibits a significantly longer serum half-life and increased in vivo activity against various tumors. Herein, we evaluated the effects of IL-15SA in recipients of allogeneic hematopoietic stem cell transplantation. Weekly administration of IL-15SA to transplant recipients significantly increased the number of CD8 + T cells (specifically CD44 + memory/activated phenotype) and NK cells. Intracellular IFN-γ and TNF-α secretion by CD8 + T cells increased in the IL-15SA-treated group. IL-15SA also upregulated NKG2D expression on CD8 + T cells. Moreover, IL-15SA enhanced proliferation and cytokine secretion of adoptively transferred CFSE-labeled T cells in syngeneic and allogeneic models by specifically stimulating the slowly proliferative and nonproliferative cells into actively proliferating cells. We then evaluated IL-15SA’s effects on anti-tumor activity against murine mastocytoma (P815) and murine B cell lymphoma (A20). IL-15SA enhanced graft-versus-tumor (GVT) activity in these tumors following T cell infusion. Interestingly, IL-15 SA administration provided GVT activity against A20 lymphoma cells in the murine donor leukocyte infusion (DLI) model without increasing graft versus host disease. In conclusion, IL-15SA could be a highly potent T- cell lymphoid growth factor and novel immunotherapeutic agent to complement stem cell transplantation and adoptive immunotherapy.

Here we demonstrate a novel application of ‘surface enhanced resonance Raman scattering nanoparticles’ (SERRS NPs) for imaging breast cancer (BC) lung metastases with much higher precision than currently feasible. The BC lung metastasis mouse model was established by intravenous injection of LM2 cells. SERRS NPs conjugated with ALT-836, an anti-tissue factor (TF) monoclonal antibody, were administered to these mice intravenously and subjected to Raman imaging to visualize the expression of TF both in vivo and ex vivo. Raman imaging indicated marked uptake of αTF-SERRS NPs by the lung metastases (n=4) compared to isotype (n=4) and blocking controls (n=3). Conversely, little uptake of αTF-SERRS NPs was observed in the lungs of healthy mice. Successful detection and delineation of pulmonary micrometastatic lesions as small as 200 μm, corroborated by histology, immunohistochemistry, and bioluminescence imaging (BLI) confirmed the suitability of both TF as a target and αTF-SERRS NPs as an effective contrast agent for imaging breast cancer lung metastases. Further advancements of this technique in the form of Raman endoscopes coupled with ultrabright SERRS NPs developed in this work could lead to minimally invasive detection and resection of lung metastases.

It has been reported that the Natural killer (NK) cell with FCGR3A (CD16a) V genotype is associated with enhanced clinical response to IgG1 monoclonal Ab (mAb) therapy such as trastuzumab, rituximab and cetuximab (1,2), suggesting a role of antibody-dependent cell-mediated cytotoxicity (ADCC) induced by NK cells. NK cells express three types of polymorphism of CD16; FcγRIIIa-158 VV, VF, FF, which are derived from the genotype of FCGR3A. It is a clinical challenge to improve the outcome in patients with FCGR3A 158FF genotype whose NK cells have lower affinity to mAb and mediate poor ADCC activity. The IL-15 superagonist/IL-15Rα-Fc fusion complex (ALT-803) activates the IL-15 receptor on CD8 T cells and NK cells, inducing their expansion, cytotoxity, and ADCC against B cell lymphoma (3, 4, 5). Here, we examined the effect of ALT803 on NK cell-mediated ADCC activity by the the anti-HER2 IgG1 mAb trastuzumab in HER2+ cell lines (SKBR3, BT474, MDA-MB-453). In addition, we used the anti-epidermal growth factor receptor(EGFR) IgG1 mAb cetuximab in EGFR positive TNBC cell lines (MDA-MB-231, SUM149, BT549). Finally, we examined the anti-PD-L1 IgG1 mAb avelumab was used for PD-L1 positive breast cancer cell lines (MDA-MB-231, BT549). Trastuzumab, cetuximab, and avelumab all significantly increased NK cell-induced lysis via ADCC. ALT803 significantly further increased both NK induced lysis and ADCC activity in all the cell lines. There was a significant positive correlation for the mean of ADCC lysis induced by NK cells from three FF (21%), three VF (33%), three VV (45%) donors. ALT803 significantly increased the mean of ADCC lysis by NK cells from all donors of each genotype to the same extent. ALT803 increased the expression of NK cell-activating receptors and cytotoxic granules regardless of the genotype of NK cell FCGR3A in terms VV, VF, or FF. We further examined the potential of ALT803 for NK cell-cytotoxicity suppressed by TGF-β1 which is one of the main barriers to immunity in the tumor microenvionment (TME). NK cells treated with TGF-β1 showed lower expression of activating receptors and cytotoxic granules, culminating in decreased lysis of MDA-MB231. ALT803 inhibited TGF-β1 from down-regulating the expression of NK cell-activating receptors and cytotoxic granules, and from suppressing the cytotoxicity of NK cells to MDA-MB231. In conclusion, the IL-15 superagonist ALT803 can potentially increase the clinical benefit of ADCC-based mAb therapy for breast cancer patients, regardless of the genotype of FCGR3A. Moreover, ALT803 prevented NK cell-cytotoxity from TGF-β1-induced suppression, providing a rationale for ALT803 therapy to overcome TME-mediated immunosuppression. References (1) Gavin et. al. JAMA Oncol.2017;3(3) (2) Musolino et. al. J Clin Oncol.2008;26(33) (3) Xu et. al. Cancer Res.2013;73(10) (4) Kim et. al. Oncotarget.2016;7(13) (5) Rosario et. al. Clin. Cancer Res. 2016; 22(3) Citation Format: Fujii R, Wong HC, Schlom J, Hodge JW. An IL-15 superagonist enhances antibody-dependent cell-mediated cytotoxicity against breast cancer cells regardless of FCGR3A (CD16) genotype and rescues NK cell from TGF-β1-induced immunosuppression [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-04.

Purpose: Pancreatic adenocarcinoma is a highly aggressive cancer, currently treated with limited success and dismal outcomes. New diagnostic and treatment strategies offer the potential to reduce cancer mortality. Developing highly specific noninvasive imaging probes for pancreatic cancer is essential to improving diagnostic accuracy and monitoring therapeutic intervention. Experimental Design: A bispecific heterodimer was synthesized by conjugating an anti-tissue factor (TF) Fab with an anti-CD105 Fab, via the bio-orthogonal “click” reaction between tetrazine (Tz) and trans-cyclooctene (TCO). The heterodimer was labeled with 64Cu for PET imaging of nude mice bearing BXPC-3 xenograft and orthotopic pancreatic tumors. Results: PET imaging of BXPC-3 (TF/CD105+/+) xenograft tumors with 64Cu-labeled heterodimer displayed significantly enhanced tumor uptake (28.8 ± 3.2 %ID/g; n = 4; SD) at 30 hours postinjection, as compared with each of their monospecific Fab tracers (12.5 ± 1.4 and 7.1 ± 2.6 %ID/g; n = 3; SD). In addition, the activity–concentration ratio allowed for effective tumor visualization (tumor/muscle ratio 75.2 ± 9.4 at 30 hours postinjection.; n = 4; SD). Furthermore, 64Cu-NOTA-heterodimer enabled sensitive detection of orthotopic pancreatic tumor lesions with an uptake of 17.1 ± 4.9 %ID/g at 30 hours postinjection and tumor/muscle ratio of 72.3 ± 46.7. Conclusions: This study demonstrates that dual targeting of TF and CD105 provided synergistic improvements in binding affinity and tumor localization of the heterodimer. Dual-targeted imaging agents of pancreatic and other cancers may assist in diagnosing pancreatic malignancies as well as reliable monitoring of therapeutic response. Clin Cancer Res; 22(15); 3821–30. ©2016 AACR.

Many signal transduction inhibitors are being developed for cancer therapy target pathways that are also important for the proper function of antitumor lymphocytes, possibly weakening their therapeutic effects. Here we show that most inhibitors targeting multiple signaling pathways have especially strong negative effects on T-cell activation at their active doses on cancer cells. In particular, we found that recently approved MEK inhibitors displayed potent suppressive effects on T cells in vitro. However, these effects could be attenuated by certain cytokines that can be administered to cancer patients. Among them, clinically available IL15 superagonists, which can activate PI3K selectively in T lymphocytes, synergized with MEK inhibitors in vivo to elicit potent and durable antitumor responses, including by a vaccine-like effect that generated resistance to tumor rechallenge. Our work identifies a clinically actionable approach to overcome the T-cell–suppressive effects of MEK inhibitors and illustrates how to reconcile the deficiencies of signal transduction inhibitors, which impede desired immunologic effects in vivo. Cancer Res; 76(9); 2561–72. ©2016 AACR.

Purpose: Anti-CD20 monoclonal antibodies (mAb) are an important immunotherapy for B-cell lymphoma, and provide evidence that the immune system may be harnessed as an effective lymphoma treatment approach. ALT-803 is a superagonist IL-15 mutant and IL-15Rα–Fc fusion complex that activates the IL-15 receptor constitutively expressed on natural killer (NK) cells. We hypothesized that ALT-803 would enhance anti–CD20 mAb-directed NK-cell responses and antibody-dependent cellular cytotoxicity (ADCC). Experimental Design: We tested this hypothesis by adding ALT-803 immunostimulation to anti-CD20 mAb triggering of NK cells in vitro and in vivo. Cell lines and primary human lymphoma cells were utilized as targets for primary human NK cells. Two complementary in vivo mouse models were used, which included human NK-cell xenografts in NOD/SCID-γc−/− mice. Results: We demonstrate that short-term ALT-803 stimulation significantly increased degranulation, IFNγ production, and ADCC by human NK cells against B-cell lymphoma cell lines or primary follicular lymphoma cells. ALT-803 augmented cytotoxicity and the expression of granzyme B and perforin, providing one potential mechanism for this enhanced functionality. Moreover, in two distinct in vivo B-cell lymphoma models, the addition of ALT-803 to anti-CD20 mAb therapy resulted in significantly reduced tumor cell burden and increased survival. Long-term ALT-803 stimulation of human NK cells induced proliferation and NK-cell subset changes with preserved ADCC. Conclusions: ALT-803 represents a novel immunostimulatory drug that enhances NK-cell antilymphoma responses in vitro and in vivo, thereby supporting the clinical investigation of ALT-803 plus anti-CD20 mAbs in patients with indolent B-cell lymphoma. Clin Cancer Res; 22(3); 596–608. ©2015 AACR.

IL15, a potent stimulant of CD8+ T cells and natural killer (NK) cells, is a promising cancer immunotherapeutic. ALT-803 is a complex of an IL15 superagonist mutant and a dimeric IL15 receptor αSu/Fc fusion protein that was found to exhibit enhanced biologic activity in vivo, with a substantially longer serum half-life than recombinant IL15. A single intravenous dose of ALT-803, but not IL15, eliminated well-established tumors and prolonged survival of mice bearing multiple myeloma. In this study, we extended these findings to demonstrate the superior antitumor activity of ALT-803 over IL15 in mice bearing subcutaneous B16F10 melanoma tumors and CT26 colon carcinoma metastases. Tissue biodistribution studies in mice also showed much greater retention of ALT-803 in the lymphoid organs compared with IL15, consistent with its highly potent immunostimulatory and antitumor activities in vivo. Weekly dosing with 1 mg/kg ALT-803 in C57BL/6 mice was well tolerated, yet capable of increasing peripheral blood lymphocyte, neutrophil, and monocyte counts by >8-fold. ALT-803 dose-dependent stimulation of immune cell infiltration into the lymphoid organs was also observed. Similarly, cynomolgus monkeys treated weekly with ALT-803 showed dose-dependent increases of peripheral blood lymphocyte counts, including NK, CD4+, and CD8+ memory T-cell subsets. In vitro studies demonstrated ALT-803–mediated stimulation of mouse and human immune cell proliferation and IFNγ production without inducing a broad-based release of other proinflammatory cytokines (i.e., cytokine storm). Based on these results, a weekly dosing regimen of ALT-803 has been implemented in multiple clinical studies to evaluate the dose required for effective immune cell stimulation in humans. Cancer Immunol Res; 4(1); 49–60. ©2015 AACR.

Chemotherapeutic agents not only cause apoptosis and necrosis of cancer cells but also induce cellular senescence (TIS), resulting in a persistent cell population that re-enters the cell cycle with stemness characteristics via the senescence-associated secretory phenotype. These senescent cancer cells, therefore, seed disease relapses if they are not effectively eliminated. TIS cancer cells lack HLA expression and cannot be targeted by T cells. Herein, we show that chemotherapy-induced senescent B16F10 cells express the NK ligands Rae1e and Ulbp1, and are effectively killed by mouse NK cells in vitro. Docetaxel treatment also induced senescent B16F10 cells in vivo. We have previously shown that the TGF-β trap/IL-15 fusion protein, aka HCW9218, effectively activates NK cells in vivo. Therefore, we treated B16F10 tumor bearing mice with docetaxel and then a single dose of HCW9218 in combination with the ADCC antibody TA99. This treatment regimen exhibited potent antitumor activity compared to docetaxel alone. End point analysis showed that HCW9218-treated tumors have lower mitotic activity, reduced expression of senescence markers, and increased frequencies of infiltrating CD8 T cells. We further showed in this model that HCW9218 treatment followed by anti-PD-L1 therapy was more effective than anti-PD-L1 therapy followed by HCW9218. These results suggest that the optimal sequence of cancer treatment is: (1) standard-of-care chemotherapy to control tumor burden and induce cellular senescence, (2) immunotherapy for removal of senescent cells and enhanced tumor infiltration of CD8+ T cells and (3) checkpoint blockade to maintain infiltrated T cell effector function to enhance therapeutic responses and treatment durability.

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.

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.

Background Neuroblastoma (NB) is the most common malignant extracranial solid tumor in children. Despite significant survival improvements in multiple pediatric malignancies, children with relapsed neuroblastoma (NB) have dismal outcomes. ROR1 is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al, Can Imm Res 2015). N-803 (ALT-803) is a novel IL15 superagonist complex where mutant IL-15N72D is bound to an IL-15RαSu-Fc fusion protein with higher potency and longer half-life compared to IL-15 (Han/Wong, Cytokine, 2011). Objectives To determine if anti-ROR1 CAR engineered NK cells can efficiently target ROR1+ NB and if the cytotoxicity can be further enhanced in combination with N-803. Methods ExPBNK cells were expanded with lethally irradiated K562-mbIL21cells (Denman/Lee Plos One, 2012) and isolated as previously described (Chu/Cairo, Can Imm Res, 2015). ExPBNK cells were electroporated with anti-ROR1-CAR mRNA (ROR1-CAR was generously provided by Riddell S) using maxcyte electroporator. ALT-803 was generously provided by Altor BioScience. CHLA-255 and CHLA-136 were generously provided by Seeger R. In vitro cytotoxicity of anti-ROR1 CAR NK against NB cell lines were examined at different E:T ratios. Granzyme B, Perforin and IFN-γ levels were evaluated by ELISA assays. High dimentionsal CyTOF analysis was performed to character the immunophenotying of anti-ROR1-CAR-NK. NB xenografted NSG mice were generated and tumor burden was monitored as we previously described (Chu/Cairo, Oncoimmunology 2017). Results Anti-ROR1-CAR was efficiently expressed on exPBNK after CAR mRNA-electroporation at 24 hrs. Anti-ROR1-CAR-NK were effective in killing NB cells even at very low E:T ratio compared to mock NK with enhanced granzyme B, perforin and interferon- release (p Conclusion anti-ROR1-CAR NK had significantly enhanced cytotoxicity against ROR1+ NB cells in vitro and in vivo using human NB xenografted NSG mice compared to controls. ALT-803 further enhanced the anti-tumor effect of anti-ROR1 CAR NK (supported by W81XWH-17-1-0420).

Here we describe a novel bifunctional fusion protein, designated N-809. This molecule comprises the IL-15/IL15Rα superagonist complex containing the Fc-domain of IgG1 (N-803, formerly designated as ALT-803) fused to two single chain anti-PD-L1 domains. The fully human IgG1 portion of the N-809 molecule was designed to potentially mediate antibody dependent cellular cytotoxicity (ADCC). The studies reported here show that N-809 has the same ability to bind PD-L1 as an anti-PD-L1 monoclonal antibody. RNAseq studies show the ability of N-809 to alter the expression of an array of genes of both CD4+ and CD8+ human T cells, and to enhance their proliferation; CD8+ T cells exposed to N-809 also have enhanced ability to lyse human tumor cells. An array of genes was differentially expressed in human natural killer (NK) cells following N-809 treatment, and there was increased expression of several surface activating receptors; there was, however, no increase in the expression of inhibitory receptors known to be upregulated in exhausted NK cells. N-809 also increased the cytotoxic potential of NK cells, as shown by increased expression of granzyme B and perforin. The lysis of several tumor cell types was increased when either NK cells or tumor cells were exposed to N-809. Similarly, the highest level of ADCC was seen when both NK cells (from donors or cancer patients) and tumor cells were exposed to N-809. These studies thus demonstrate the multi-functionality of this novel agent.