Abstract T cell engagers (TCEs), mainly in the format of Bispecific T-cell Engagers (BiTE), are promising anti-cancer immunotherapies for hematological malignancies. However, solid tumors create immunosuppressive microenvironments and physical structures that hinder the effectiveness of T- cell engagers and preclude tumor infiltration by CD8+ T cells. Our hypothesis for overcoming these barriers is to add components to the BiTE format that alleviate immunosuppression and promote activation and tumor infiltration of CD8+ T cells. To test this hypothesis, we used our novel TRBC platform to construct HCW11-018b, a tetravalent heterodimeric TCE. The BiTE portion of HCW11-018b contains an antibody that targets human tissue factor (TF), which is overexpressed in a wide spectrum of solid tumors; an anti-CD3 single-chain antibody; and an IL-15Rα domain. The other chain of HCW11-018b comprises a dimeric soluble TGFβRII domain (i.e., TGFβ trap), TRβC1 and a soluble IL-15. The plasmids carrying the coding regions of the two fusion proteins were co-transfected into CHO cells and the fully functional TCE was purified from culture supernatant. In vitro, we found HCW11-018b induced robust, antigen-specific tumor cell killing, which was sustained up to five rounds. Increased phosphorylation of STAT5, expression of activation markers (CD69, CD25), chemokine receptor (CCR5), and anti-apoptosis marker (BCL2) were observed on T cells by HCW11-018b treatments. RNAseq analysis revealed that increased expression of BCL2 and other genes associated with T-cell effector function were specifically attributable to the IL-15 component of HCW11-018b. In SCID mice subcutaneously (s.c.) implanted human AsPC-1 pancreatic cancer cells with human PBMCs, we demonstrated that s.c. administered HCW11-018b could infiltrate into implants and activate bystander CD8+ T cells for potent anti-tumor activities. In AsPC-1 tumors (NSG mice models) and adoptively transferred human T cells, HCW11-018b stimulated expression of CCR5 and promoted tumor infiltration of human T cells, upregulated T cell expression of CD25, NKG2D, DNAM1, Granzyme B, and IFNγ, and enhanced their cytotoxicity against cancer cells. Treatment also reduced tumor cell metastasis from the primary site. We further demonstrated that the upregulation of NKG2D and DNAM1 on HCW11-018b-activated CD8+ T cells played a role in the cytotoxicity against AsPC-1 tumor cells. In a PDX model, we further demonstrated the potency of HCW11-018b which directed hPBMCs against TF+ patient-derived pancreatic cancer tissues. HCW11-018b was well tolerated in mice and non-human primates with s.c. administration. HCW11-018b is currently in IND-enabling studies for clinical development against solid tumors. In summary, we demonstrate that the addition of TGFβ-trap and IL-15 components onto BiTE using our novel TRBC platform can overcome the deficiencies of BiTE for solid tumor therapy. Citation Format: Hing C. Wong, Xiaoyun Zhu, Varghese George, Hamidreza Farzaneh, Crystal Gilkes, Natalia Valderrama, Alyssa Thompson, Lijing You, Lucas Gomez, Christian Echeverri, Niraj Shrestha, Peter R. Rhode, Jack Egan, . An innovative approach to improve bispecific T-cell engagers for solid tumor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1629.

BACKGROUND:Stem-like progenitor exhausted CD8+ T cells (TPEX), located within the tumor-draining lymph nodes (TDLNs), are responsible for maintaining tumor-specific responses in cancer. Although cytokines such as interleukin (IL)-15 are known to expand CD8+ T-cell subsets, transforming growth factor (TGF)-β in the TDLN is known to arrest the egress of these TPEX to the tumor microenvironment. We hypothesized that combining IL-15 stimulatory and TGF-β blocking activity would boost antitumor responses mediated by TPEX in the TDLN. METHODS:We developed a bifunctional TGF-βRII/IL-15 protein complex (HCW9218) and evaluated its antitumor activity in two murine models of melanoma and breast cancer. Peripheral blood, TDLN and tumor-infiltrating CD8+ T cells were characterized by flow cytometry following a single subcutaneous dose (s.c.) of HCW9218. Transcription profiling of CD8+ T cells in both murine models was performed. Synergistic activity of HCW9218 with immune-checkpoint inhibitors (ICIs) was evaluated. Finally, safety and immune profiling in patients with chemo-refractory/relapsed solid tumors was performed in a Phase 1 dose-escalating trial. RESULTS:HCW9218 was capable of localizing to the TDLNs and tumors after s.c. administration, neutralized TGF-β, expanded TPEX in TDLNs, increased chemokine-expressing effectors in peripheral circulation and promoted their infiltration into murine tumors. These data were corroborated in RNA sequencing analysis of TDLNs. ICIs significantly enhanced the effects of HCW9218 on TPEX and synergistically improved HCW9218 antitumor efficacy in melanoma and reduced spontaneous lung metastasis in breast cancer models. In a Phase 1 clinical trial, HCW9218 monotherapy was well-tolerated, reduced serum TGF-β levels, promoted and sustained CD8+ T-cell expansion in peripheral blood and CD8+ T-cell infiltration in tumor biopsies. Stable disease was reported for four of six subjects (67%) with advanced ovarian cancer treated with HCW9218. CONCLUSIONS:Our findings demonstrate that combination therapy targeting immune cells critical for antitumor responses and blocking immune-suppressive environment significantly improves antitumor therapeutic efficacy. These findings provide a strong basis for using HCW9218 to enhance the efficacy of ICIs against solid tumors in the clinical setting.

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming (“Prime” step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 (“Expand” step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting “Prime and Expand” ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the “Prime and Expand” strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

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.

Abstract 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.

Background HCW9218 is a bifunctional protein complex comprising dimeric extracellular domains of human transforming growth factor beta (TGF-β) receptor II and interleukin-15 (IL-15). HCW9218 1) stimulates immune effector cells and 2) sequesters soluble immunosuppressive TGF-β.1 2 The objectives of our pre-clinical and first-in-human studies are to explore the antitumor mechanism of action (MOA) and determine the RP2D. Methods Two syngeneic mouse tumor models were used to explore the MOA of HCW9218. A phase I dose escalation study (NCT05322408) of HCW9218 monotherapy (Q3W, subcutaneous) has been completed in adult patients (n=12) with refractory advanced/metastatic solid tumors and is expanding at the highest dose level (1.2mg/kg).3 Correlative analyses included pharmacokinetics, serum cytokines, blood lymphocytes, and cellular/molecular profiling of tumors. Results In B16F10 melanoma and 4T1 breast cancer murine models, HCW9218 promotes proliferation and activation of progenitor exhausted (Tpex) and exhausted transitory effector (Tem) CD8+ T cells in draining lymph nodes, and CD8+ memory T cells and NK cells in peripheral blood. HCW9218 increases tumor infiltrating Tpex, Tem, and memory CD8+ T cells and enhances antitumor potency of immune checkpoint blockade therapy, likely due to stimulation of Tpex and Tem cells. HCW9218 protein localizes in tumors and significantly lowers TGF-β levels and increases proinflammatory cytokines. In the first-in-human clinical trial, there were no dose limiting toxicities. The most common adverse events are grade 1–2 injection site reactions and transient lymphopenia. Patients receiving ≥0.25 mg/kg HCW9218 exhibit robust NK cell and CD8+ T cell proliferation through day 15 and recurred with each treatment cycle, a biologic effect beyond what has been previously observed with other IL-15 agonists. At ≥0.5 mg/kg HCW9218, serum TGF-β1 levels decrease to baseline through day 8. Immunofluorescent staining of patients' tumors show that HCW9218 increases CD8+ T cell infiltration with elevated tumor Tpex and/or fully differentiated memory CD8+ T cells correlating with disease stabilization. Single-cell RNA-seq tumor analysis demonstrates that HCW9218 reduces expression of genes associated with tumor invasion, immunosuppression, and inflammation and upregulates genes involved in differentiation of Tpex, TCR signaling, and inflammatory response, while downregulating proinflammatory response genes of tumor lymphocytes. HCW9218 has a serum half-life of ~25h in patients. Conclusions Repeated HCW9218 administration at ≥0.5 mg/kg in heavily pretreated advanced solid tumor patients resulted in immune cell activation, proliferation, and infiltration into the tumor microenvironment without causing unacceptable toxicity. HCW treatment presents a promising approach to enhancing the antitumor activity of immune checkpoint inhibitors in patients with solid tumors. Trial Registration NCT05322408 References Liu B, Zhu X, Kong L, et al. Bifunctional TGF-beta trap/IL-15 protein complex elicits potent NK cell and CD8(+) T cell immunity against solid tumors. Mol Ther 2021;29:2949–62. Chaturvedi P, George V, Shrestha N, et al. Immunotherapeutic HCW9218 augments anti-tumor activity of chemotherapy via NK cell-mediated reduction of therapy-induced senescent cells. Mol Ther 2022;30:1171–87. Geller M, Patel M, Wong H, et al. A phase I study of HCW9218, a bifunctional TGF-β antagonist/IL-15 protein complex, in advanced solid tumors. J Immunother Cancer 2022;10. doi: 10.1136/jitc-2022-SITC2022.0724 Ethics Approval This study was approved by the University of Minnesota's Institutional Review Board; approval number: 00015102. Consent Written informed consent was obtained from the patients included in the clinical trial for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Accumulation of senescent cells (SNCs) with a senescence-associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age-related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF-β and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet β cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro-inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single-nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell-cycle, and senescence-associated pathways in naturally aged mice. Long-term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.

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).

Atherosclerosis is a chronic inflammatory disease caused by deposition of oxidative low-density lipoprotein (LDL) in the arterial intima which triggers the innate immune response through myeloid cells such as macrophages. Regulatory T cells (Tregs) play an important role in controlling the progression or regression of atherosclerosis by resolving macrophage-mediated inflammatory functions. Interleukin-2 (IL-2) signaling is essential for homeostasis of Tregs. Since recombinant IL-2 has an unfavorable pharmacokinetic profile limiting its therapeutic use, we constructed a fusion protein, designated HCW9302, containing two IL-2 domains linked by an extracellular tissue factor domain. We found that HCW9302 exhibited a longer serum half-life with an approximately 1000-fold higher affinity for the IL-2Rα than IL-2. HCW9302 could be administered to mice at a dosing range that expanded and activated Tregs but not CD4+ effector T cells. In an ApoE-/- mouse model, HCW9302 treatment curtailed the progression of atherosclerosis through Treg activation and expansion, M2 macrophage polarization and myeloid-derived suppressor cell induction. HCW9302 treatment also lessened inflammatory responses in the aorta. Thus, HCW9302 is a potential therapeutic agent to expand and activate Tregs for treatment of inflammatory and autoimmune diseases.

Supplemental Figures 1-8, Supplemental Tables 1-3

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Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. Here, we report a novel heterodimeric bifunctional fusion molecule, HCW9218, constructed using our 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. This fusion complex exhibited TGF-β neutralizing activity in vitro and sequestered plasma TGF-β in vivo. Subcutaneous administration of HCW9218 was well tolerated in mice, with a half-life sufficient to provide long lasting biological activity. HCW9218 enhanced metabolic and cytotoxic activities of immune cells (CD8+ T cells, NK cells) and reduced tumor progression in models of chemotherapy induced senescence (TIS, docetaxel for B16F10 tumors and gemcitabine plus nab-paclitaxel for SW1990 tumor models) and non-TIS in vivo. Mechanistically, HCW9218 treatment not only affected the immunosuppressive tumor microenvironment but also enhanced CD8+ T cells and NK cells infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immune-depletion studies showed that HCW9218-activated NK cells played a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhanced efficacy of tumor antigen-specific and anti-PDL-1 antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment lowered senescence associated secretory phenotype (SASP) factors in off-target tissues in chemotherapy treated tumor-bearing mice. Thus, HCW9218 may serve as a novel therapeutic (monotherapy or in combination with chemotherapy) to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors. HCW9218 has been cleared by the U.S. Food and Drug Administration to proceed for evaluation in a first-inhuman Phase 1b clinical trial in patients with advanced pancreatic cancer. Citation Format: Pallavi Chaturvedi, Varghese George, Bai Liu, Niraj Shrestha, Meng Wang, Micheal Dee, Xiaoyun Zhu, Jack Egan, Jin-an Jiao, Catherine Spanoudis, Jilan Xing, Victor Gallo, Christian Echeverri, Lijing You, Lin Kong, Gabriela Muniz, Peter Rhode, Hing Wong. Immunotherapeutic hcw9218 enhances anti-tumor activity of chemotherapy by facilitating immune-mediated elimination of therapy induced senescent cells [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 1299.

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.

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-beta (TGF-beta) receptor II and interleukin (IL)-15/IL-15 receptor alpha 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.

Regulatory T cells (CD4+CD25+FoxP3+) (Tregs) are a subset of CD4 T cells that suppress the activities of other immune cells and have applications in the treatment of autoimmune and inflammatory diseases. Their use as an adoptive cell therapy has been limited by the practicality of expanding and purifying clinically sufficient numbers of cells. HCW9213 and HCW9302 are fusion proteins based on HCW Biologics’ TOBI™ technology platform, consisting of anti-CD3/anti-CD28 antibody domains and IL-2 domains, respectively. When used in combination, these fusion proteins were capable of expanding human Treg cells in vitro without the use of anti-CD3/CD28 magnetic beads and/or feeder cells, improving the overall yield, process efficiency and overcoming regulatory hurdles in manufacturing. Tregs generated with these molecules displayed similar phenotypes and suppressive cytokine production as Tregs expanded with recombinant human IL-2. Using a proprietary anti-CD39 antibody to isolate CD39+ Tregs, we have also been able to generate a Treg population with twice the suppressive activity against CD4+ T responder cells as traditional CD4+CD25+CD127lo Tregs. Thus, using its novel fusion proteins, HCW Biologics has been able to develop a superior Treg cell product ideal for the use in adoptive cell transfer. Additionally, this Treg platform can potentially be further optimized with addition of disease-targeted chimeric antigen receptors (CAR).