Human IL-2 Research Articles

Abstract CT133: Trial in progress: a multicenter phase 1/1b dose escalation study of WTX-124 as a monotherapy and in combination with pembrolizumab in patients with selected advanced or metastatic solid tumors

Abstract Background: High-dose recombinant human IL-2 (HD IL-2) is an FDA-approved therapy that produces durable complete remissions in a subset of patients with advanced or metastatic cutaneous melanoma and renal cell carcinoma. However, the clinical benefit of HD IL-2 is counterbalanced by life-threatening toxicities such as capillary leak syndrome. WTX-124 is an engineered cytokine prodrug composed of a wild-type IL-2 fused via proprietary cleavable linkers to an inactivation domain and to a half-life extension domain. In the circulation and in normal tissues, WTX-124 is designed to remain inactive and not bind to high- (αβγ) and intermediate-affinity (βγ) IL-2 receptors. In contrast, proteolytic activation of WTX-124 in the tumor microenvironment (TME) is expected to liberate the IL-2 cytokine to stimulate antitumor immune responses. Preclinical data show that WTX-124 is activated by a range of dissociated human tumors in vitro but not by normal human cells or by patient serum. Moreover, WTX-124 demonstrates potent antitumor activity in multiple murine syngeneic tumor models with substantial expansion and activation of tumor-infiltrating lymphocytes. In the MC38 model, WTX-124 has a large therapeutic window of >19-fold compared to ~3-fold for recombinant human IL-2. Methods: This first-in-human, open-label phase 1/1b trial is investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity, and antitumor activity of WTX-124 administered as a monotherapy or in combination with the anti-PD-1 antibody pembrolizumab to patients with advanced or metastatic solid tumors (NCT05479812). All patients must have ≥ 1 measurable lesion per RECIST 1.1 and a tumor type for which immune checkpoint inhibitor (ICI) therapy is indicated. Dose escalation uses a modified toxicity probability interval-2 study design with WTX-124 administered intravenously (IV) every two weeks in 28-day cycles. If the safety of WTX-124 as a monotherapy is established in the first three cohorts, the study will then initiate dose escalation of WTX-124 in combination with pembrolizumab 400mg IV every six weeks. Dose expansion is anticipated to include four arms that will enroll patients with either advanced or metastatic cutaneous melanoma or renal cell carcinoma to receive WTX-124 monotherapy or combination therapy (N=20 patients per arm). Patients with melanoma assigned to the WTX-124/pembrolizumab combination arm may be naïve to all prior systemic therapy for advanced disease, but all other patients in expansion must have previously received a standard of care ICI regimen. Pre- and on-treatment tumor biopsies, archival tumor tissue, and blood samples will be used to investigate multiple exploratory biomarkers. As of January 12, 2023, WTX-124 monotherapy dose escalation is proceeding. Citation Format: Ildefonso Ismael Rodriguez-Rivera, Justin C. Moser, Mateusz Opyrchal, Brendan Curti, Mehmet A. Bilen, Saero Park, Marissa Bruno, Paul Windt, Kulandayan K. Subramanian, Sameer S. Chopra, Randi Isaacs. Trial in progress: a multicenter phase 1/1b dose escalation study of WTX-124 as a monotherapy and in combination with pembrolizumab in patients with selected advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT133.

Abstract CT115: Treatment of skin tumors with intratumoral interleukin 12 gene electrotransfer in the head and neck region: A phase I clinical trial

Abstract Immunotherapies with monoclonal antibodies (checkpoint inhibitors) are currently being intensively researched and have led to excellent response in many cases in various tumors. Another potential approach to immunotherapy is the targeted intratumoral administration of interleukin 12 (IL -12), a cytokine with proven anti-tumor activity. Due to its immunomodulatory effect, it can be used as an immunostimulatory component for in situ vaccination of local ablative therapies. We have developed a phIL12 plasmid without antibiotic resistance markers using a transgene for the human IL -12 p70 protein. The plasmid can be introduced intratumorally by gene electrotransfer. In gene electrotransfer, electrical pulses are applied to the tumor to deliver the plasmid DNA into the cells. Based on nonclinical studies of safety, pharmacokinetics, pharmacodynamics, tolerability, and immunogenicity, a phase I clinical trial of phIL12 gene electrotransfer was conducted (ISRCTN15479959, ClinicalTrials NCT05077033). The primary objective of the study is to evaluate the safety and tolerability of phIL12 gene electrotransfer in the treatment of basal cell carcinoma in patients with operable tumors of the head and neck. The study, an exploratory, dose-escalating Phase I study, will enroll 9 patients in three dose-escalating cohorts. Treatment consists of plasmid injection followed by application of electrical pulses to the tumors. Patients are monitored for 30 days, with tumors removed if a complete response is not achieved. To date, two cohorts of patients have completed the study. No adverse effects or treatment-related toxicities have been observed, and treatment has been well tolerated by patients. The results of this study will provide the basis for the use of phIL12 gene electrotransfer as adjuvant therapy to local ablative therapies to enhance their local effect and elicit a systemic response. Citation Format: Gregor Sersa, Maja Cemazar, Crt Jamsek, Primoz Strojan, Tanja Jesenko, Masa Bosnjak, Ales Groselj. Treatment of skin tumors with intratumoral interleukin 12 gene electrotransfer in the head and neck region: A phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT115.

Abstract 3970: NCG-hIL15 humanized mice: an ideal model for human immune reconstitution of NK cells

Abstract Introduction: IL-15 is a four α-helix bundle cytokine which is produced by dendritic cells, monocytes, and epithelial cells. IL-15 is necessary for the development, survival, and activation of natural killer (NK) cells. NK cells are unable to persist in immunodeficient NCG mice reconsituted with human NK cells, but after knocking in human IL-15 (hIL-15), NCG-hIL15 mice can support the reconstitution and survival of human NK cells. Results: Compared with NCG mice, the level of hIL-15 was significantly increased in NCG-hIL15 mice. After transplanting human PBMC (huPBMC) or NK (huNK) cells into NCG-hIL15 mice, the peripheral blood level of NK cells in huNK-NCG-hIL15 mice was much higher than in huPBMC-NCG-hIL15 mice. The expression of perforin in peripheral blood of huNK-NCG-hIL15 mice was significantly higher than that in huPBMC-NCG-hIL15 mice. Considering the obvious ADCC of rituximab acting on Raji cells in vitro, we evaluated the efficacy of rituximab in huNK-NCG-hIL15 mice subcutaneously engrafted with Raji cells. Rituximab significantly inhibited the tumor growth in huNK-NCG-hIL15 mice. Conclusion: NCG-hIL15 is a great mouse model to evaluate the anti-tumor efficacy of drugs targeting human NK cells without the interference of T cells. Citation Format: Xing Liu, Meirong Wu, Huiyi Wang, Weiwei Yu, Jianming Xu, Hongyan Sun, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. NCG-hIL15 humanized mice: an ideal model for human immune reconstitution of NK cells. [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 3970.

Abstract 1839: CLN-617 is a first-in-class fusion protein that retains IL-2 and IL-12 in the injected tumor and potently triggers systemic anti-tumor immunity

Abstract Despite clinical evidence of anti-tumor activity, cytokine therapies have been hampered by a narrow therapeutic window and limited efficacy. Two cytokines of interest are IL-2 and IL-12, which synergize to proliferate and activate T cells and natural killer cells. However, the only approved human IL-2 product (Proleukin) is rarely used in the clinic due to systemic toxicities, and no IL-12 product has been approved to date due to severe dose-limiting toxicities. Here, we describe CLN-617, a first-in-class therapeutic designed for intratumoral (IT) injection and retention, that co-delivers IL-2 and IL-12 in a safe and effective manner. CLN-617 is a single-chain fusion protein comprised of human IL-2, leukocyte-associated immunoglobulin-like receptor 2 (LAIR2), human serum albumin (HSA), and human IL-12. LAIR2 and HSA functionally retain CLN-617 in the injected tumor by binding collagen and increasing molecular weight, respectively. A murine surrogate of CLN-617, mCLN-617, was delivered either IV or IT in B16F10 tumor-bearing mice. The concentration of mCLN-617 as measured in serum two hours after IT administration was <5% that of the concentration following IV administration, demonstrating robust retention of mCLN-617 in the tumor. Following mCLN-617 treatment, durable and complete responses with no significant body weight loss were observed in checkpoint-refractory tumor models, including B16F10, CT26, and MC38. mCLN-617 triggered a robust systemic anti-tumor immune response based on three lines of evidence. First, in mice implanted with two MC38 tumors, 70% of injected tumors and 40% of non-injected tumors were eradicated. When combined with IV-administered anti-PD1 antibody, >90% of both injected and non-injected tumors showed a complete response, while anti-PD1 alone exhibited no anti-tumor activity. Second, in mice implanted with an MC38 tumor in the flank and with luciferase-expressing MC38 cells delivered to the liver via intra-splenic injection, the injected flank tumor exhibited ~90% growth inhibition with either mCLN-617 monotherapy or anti-PD1 co-administration. Growth of established metastatic tumors, as measured by luciferase signal, was inhibited by mCLN-617 monotherapy, and metastatic tumor burden significantly regressed in combination with anti-PD1, while anti-PD1 alone exhibited no anti-tumor activity in the metastases. Third, in mice bearing two MC38 flank tumors, tumor-specific CD8+ T cells expanded in both the injected and non-injected tumors and in peripheral blood. We conclude that IT-administered mCLN-617 is efficiently retained in the injected tumor, thereby preventing systemic toxicity of the two cytokines, yet can activate and remodel systemic immunosurveillance for clearance of non-injected distal lesions. A phase I trial of CLN-617 in patients with advanced solid tumors is expected to commence in 2023. Citation Format: Naveen K. Mehta, Kavya Rakhra, Kristan Meetze, K. Dane Wittrup, Jennifer S. Michaelson, Patrick A. Baeuerle. CLN-617 is a first-in-class fusion protein that retains IL-2 and IL-12 in the injected tumor and potently triggers systemic anti-tumor immunity [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 1839.

Abstract 587: A half-life extended, tumor-activated IL-12 increased the infiltration of effector immune cells into the tumor microenvironment and demonstrated anti-tumor activity in syngeneic mouse models

Abstract Interleukin-12 (IL-12) is a proinflammatory cytokine, which bridges innate and adaptive immunity via induction of T helper 1 differentiation and promotes cytolytic activity of natural killer and T cells. IL-12 has demonstrated potent antitumor activity in syngeneic mouse models and promising anti-tumor efficacy in humans. However, development of IL-12-based treatments has been limited by severe systemic toxicities in the clinical setting. To overcome toxicity and potentially improve the therapeutic index of IL-12 in a clinical setting, XTX301 was engineered as a half-life extended and masked IL-12. The masking domain of XTX301 is designed to pharmacologically inactivate IL-12 in circulation and non-tumor tissue, while enabling generation of an active IL-12 moiety upon cleavage of a linker sequence by matrix metalloproteinases that are enriched in the tumor microenvironment. To confirm the ability of human tumor associated proteases to activate XTX301, cleavage was assessed in primary human tumor samples and in plasma from cancer patients. We observed cleavage of XTX301 in the majority of human tumors samples tested, but no activation was observed in plasma from cancer patients. Human IL-12 does not bind and signal through the mouse IL-12 receptors, hence three murine surrogates were created for in vivo studies: mXTX301, a non-cleavable control, and an unmasked control molecule. Tumor growth inhibition was observed after a single dose of mXTX301 as low as 0.039 mg/kg. The non-cleavable form of mXTX301 was less potent than mXTX301, demonstrating that the anti-tumor activity of mXTX301 is dependent on protease activation. The unmasked control was not well tolerated, with a > 20% body weight loss observed by Day 6, resulting in 75% of animals being euthanized by Day 11. Unlike the unmasked control, mXTX301 was well-tolerated at all tested doses, as evidenced by no loss in animals’ body weights, and demonstrated minimal pharmacodynamic activity in non-tumor tissues. In tumors, mXTX301 stimulated the infiltration of cytotoxic CD8+ T cells and induced the expression of several immune-related genes including those associated with IFN-γ cell signaling, antigen processing and presentation, and defense response as well as enrichment of gene signatures for T cells, natural killer (NK) cells, macrophages and dendritic cells as determined by RNA sequencing. In non-human primates, the highest non-severely toxic dose of XTX301 was 2mg/kg dosed weekly for a total of 4 doses. In summary, a half-life extended tumor-activated IL-12 molecule, mXTX301, demonstrated anti-tumor activity in preclinical mouse models with improved tolerability compared to a systemically active IL-12 molecule suggesting that XTX301 has potential for exerting potent anti-tumor activity while widening the therapeutic index of IL-12 treatment. Citation Format: Natalia Malkova, Ekta Patel, Sallyann Vu, Damiano Fantini, Rebekah O'Donnell, Manoussa Fanny, Justin Greene, Wilson Guzman, David Crowe, Stephanie Hsiao, Parker Johnson, Megan McLaughlin, Oleg Yerov, Kurt Jenkins, Katarina Halpin-Veszeleiova, Hanumantha Rao Madala, Caitlin O'Toole, Jake Taylor, Magali Pederzoli-Ribeil, Benjamin Nicholson, Carl Uli Bialucha, Jennifer E. O'Neil. A half-life extended, tumor-activated IL-12 increased the infiltration of effector immune cells into the tumor microenvironment and demonstrated anti-tumor activity in syngeneic mouse models [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 587.

Abstract 6335: Developing anti-IL-22 therapeutics for inflammation and cancer

Abstract Chronic inflammation facilitates the development of cancer, age-related, inflammatory autoimmune and other diseases. Proinflammatory cytokines play a central role in this pathogenic process by driving activation of NF-ƙB, STAT3 and other signaling cascades. IL-22 is an inflammatory cytokine involved in the pathology of autoimmune diseases such as psoriasis, atopic dermatitis, and ulcerative colitis. IL-22 has also been shown to promote epithelial cell proliferation, stemness and tumorigenesis in cancer. Recent studies demonstrated that neutralization of IL-22 reduced dysplasia and tumor development in preclinical models. To date, there has not been a clear application for anti-IL-22 therapy for autoimmune diseases and cancer, likely due to identifying the right disease indication and to their low potency and low efficacy. Thus, there remains an opportunity to explore the potential of more potent anti-IL-22 therapeutics. Using BioAtla’s proprietary antibody discovery and engineering platforms, we have generated humanized anti-IL-22 antibodies with high affinities to human, cynomolgus and mouse IL-22. In vitro data demonstrated that our anti-IL-22 antibodies inhibited IL-22-induced p-STAT3, CXCL-1 and IL-10 activities. In addition, our antibodies showed a 10-fold increased binding activity to human IL-22 compared to Fezakinumab, which was previously developed by Pfizer for the treatment of autoimmune diseases. BioAtla’s high affinity anti-IL-22 antibodies were shown to be more potent relative to Fezakinumab using multiple in vitro assays. To test the functions of our antibodies in vivo, we also established mouse models to determine anti-IL22 efficacy. More specifically, to study whether targeting IL-22 in an inflammation-induced tumor microenvironment reduces tumor occurrence, we developed an inflammation-driven sporadic colitis-associated colorectal cancer mouse model for ongoing cancer studies that will be discussed. In addition, we evaluated our anti-IL22 antibodies in an imiquimod-induced psoriasiform skin inflammation mouse model. The mice treated with potent anti-IL-22 antibodies had significantly reduced skin lesions, including erythema, scales, and skin thickness compared to isotype antibody-treated mice. Q-PCR analysis of the mouse skin demonstrated that treatment with our anti-IL-22 antibody led to significantly decreased RNA levels of the imiquimod-induced inflammatory marker CXCL3. Thus, the enhanced anti-IL-22 therapy is a promising strategy for targeting anti-inflammation in autoimmune diseases, such as psoriasis and atopic dermatitis, and inflammatory cancers, including colorectal cancer. In conclusion, the development of a potent, species cross reactive anti-IL-22 antibody using BioAtla’s antibody discovery and engineering platforms addresses the challenges of anti-IL-22 therapeutics and allows for translational studies in relevant animal efficacy and safety models. Citation Format: Christina Wheeler, Jian Chen, Cathy Chang, Gerhard Frey, Haizhen Liu, Jing Wang, Kathryn Woodard, Solmarie Joyner, Wei Zhou, William J. Boyle, Jay M. Short. Developing anti-IL-22 therapeutics for inflammation and cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6335.

Abstract 1153: IL-4 depletion leads to the improvement of CART cell therapy

Abstract While chimeric antigen receptor T-cell therapy targeting CD19 (CART19) has shown remarkable success in the treatment of hematological malignancies, the durable response rates remain approximately 40% and there are limited solutions for CART cell therapy in the treatment of solid tumors. To further understand mechanisms of resistance, including CART cell exhaustion, we employed three independent approaches: 1) RNA and ATAC sequencing on unstimulated vs. exhausted healthy donor CART19 cells by utilizing an in vitro model for exhaustion, 2) RNA and ATAC sequencing on pre-infusion CART19 cell products from responders and non-responders in the Zuma-1 clinical trial that led to the FDA approval of axi-cel CART19 therapy, and 3) a genome-wide CRISPR knockout screen in healthy donor CART19 cells using our in vitro model for exhaustion. In each of these approaches, IL-4 was identified as a regulator of CART cell dysfunction. In approach 1, ingenuity pathway analysis of genes that were both differentially accessible and expressed in exhausted compared with unstimulated CART19 cells revealed IL-4 as a top upstream regulator (p = 5E-6). In approach 2, IL-4 was one of two genes that were both upregulated and more accessible in CART19 cell products from non-responders (p < 5E-2). Finally, in approach 3, gene ontology enrichment analysis of genes that were positively selected during the genome-wide CRISPR knockout screen, revealed regulation of the IL-4 pathway as one of the top affected pathways (p = 1E-4). Together, our data indicates a role for IL-4 in CART cell dysfunction caused by exhaustion. Investigating this mechanism further, we saw an increase in the production of IL-4 as CART cells became exhausted (p = 4E-3). Treatment of CART19 cells with human recombinant IL-4 (hrIL-4) resulted in dysfunction as evident by a decrease in antigen specific cytotoxicity (p = 4E-3) and proliferative ability (p= 6.5E-2), as well as exhaustion-specific signs of dysfunction such as an increase in the expression of the inhibitory receptor, TIM-3 (p = 3E-3) and an increase in the transcription of the exhaustion-related transcription factor EOMES (p = 1E-2). Finally, we tested whether IL-4 neutralization enhances CART19 cell functions. Using a CD19+ JeKo-1 xenograft mouse model, we compared the combination treatment of CART19 cells and an IL-4 neutralizing monoclonal antibody (10 mg/Kg, clone # MP4-25D2) to CART19 cells and an IgG control. IL-4 neutralization in combination with CART19 cells resulted in reduced tumor burden (p = 4.6E-2), increased CART cell proliferation (p = 8E-3), and prolonged overall survival (p= 5E-2). In summary, our data indicates that 1) IL-4 induces CART cell dysfunction through a state of exhaustion and 2) IL-4 neutralization with a monoclonal antibody enhances CART cell therapy. As such, this novel combination therapy holds the potential to be translated to the clinic to improve durable responses from CART cell therapy. Citation Format: Carli Stewart, Michelle J. Cox, Reona Sakemura, Ekene J. Ogbodo, Ismail Can, Claudia Manriquez Roman, Kun Yun, Olivia Sirpilla, James H. Girsch, Truc Huynh, Elizabeth L. Siegler, Jenny J. Kim, Mike Mattie, Nathalie Scholler, Simone Filosto, Saad S. Kenderian. IL-4 depletion leads to the improvement of CART cell therapy [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 1153.

Abstract 2918: In vivo delivery of CD3-directed CD19-CAR lentivectors leads to the generation of CAR T and NK-like (CAR-TaNK) cells capable of complete ablation of B cells in the blood, bone marrow, and tissue of NSG-SGM3 CD34+ humanized mice

Abstract Introduction: Direct in vivo delivery of lentiviral vectors (LV) to generate CD19 CAR+ cells without the need for ex vivo preparation represents a promising approach to transform autologous CAR therapy into an off-the-shelf treatment. In these studies, direct administration of a new LV encoding a CD19 CAR into humanized NOD SCID gamma (NSG) mice expressing human IL-3, GM-CSF, and SCF (NSG-SGM3) resulted in a dose-dependent elimination of B cells in the peripheral blood, peritoneal fluid, bone marrow, and tissue of treated mice. Methods: CD3-directed LV encoding a CD19 CAR with a novel synthetic driver element was manufactured utilizing a 25L clinical scale suspension-based process. NSG-SGM3 mice transplanted with human CD34+ cells from cord blood were injected with LV doses (1E6 IU, 1E7 IU, or 5E7 IU) intraperitoneally (IP) or 1E7 IU intravenously (IV). Quantification of CD19 CAR+ cells and CD20+ B cells in peripheral blood, peritoneal fluid, and bone marrow was assessed by flow cytometry. Additionally, immunohistochemical analysis was performed to evaluate the tissue-resident human B cells and for any other histopathological observations following test article administration. Results: All CD34+ humanized NSG-SGM3 mice were confirmed to exhibit efficient human hematopoietic engraftment by flow cytometry prior to test article administration (peripheral blood hCD45: 68.9% ± 4.93%; hCD19+ B cells: 53.7% ± 5.11%). Direct LV administration at the 1E7 IU and 5E7 IU doses demonstrated a dose-dependent reduction in circulating human B cells compared to the control and 1E6 IU dose (p < 0.05). The synthetic driver elements co-expressed with the CAR led to the formation of unique CD3+ CD8+ CD56+ T and NK-like (TaNK) CD19 CAR+ cells in circulation. The 1E7 IU IP dose demonstrated ablation of B cells (total cells/uL) in peripheral blood (control: 9.67 ± 3.72 vs. LV treated: 0.157 ± 0.117), intraperitoneal fluid (control: 0.322 ± 0.244 vs. LV treated: 0.038 ± 0.029), bone marrow (control: 9.16± 1.83 vs. LV treated: 0.734 ± 0.864), and splenic tissue. Both IP and IV routes of administration showed significant B cell depletion at the 1E7 IU dose. However, complete B cell elimination in splenic tissue was only observed at the 5E7 IU dose. Non-treated CD34+ humanized NSG-SGM3 mice exhibited hepatic portal inflammation and moderate graft-versus-host disease (GVHD) in the colon. Interestingly, mice treated with LV encoding CD19 CAR exhibited decreased inflammatory pathology, suggesting potential B cell involvement in the inflammatory response in this model. Conclusion: In this study, direct in vivo delivery of LV encoding CD19 CAR resulted in the generation of functionally active CD19 CAR TaNK cells capable of eliminating target B cells in peripheral blood, peritoneal fluid, bone marrow, and tissue. Citation Format: Frederic Vigant, Ani Kundu, Ramya Yarlagadda, Cody Gowan, Michael Betts, Jonathan Kato, Renata Soares, Alan Ponce, Lintao Liu, Junyi Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Suraj Kachgal, Gregory Schreiber, Wei Zhang, Gregory Wade, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of CD3-directed CD19-CAR lentivectors leads to the generation of CAR T and NK-like (CAR-TaNK) cells capable of complete ablation of B cells in the blood, bone marrow, and tissue of NSG-SGM3 CD34+ humanized mice [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 2918.

Abstract 5189: Comparisons of Rituximab and CD19xCD3 BiTE antitumor response across multiple humanized NSG strains engrafted with human hematopoietic stem cells

Abstract Humanized NSG mice engrafted with hematopoietic stem cells (HSCs) is a well-known in vivo platform for reconstitution of human immune system in a mouse host. In this study we tried to dissect the role of various human cytokines in the generation of the human immune system, expansion of human B cell lymphoma cells, and drug response to two anti-cancer therapies - Rituximab and CD19XCD3 BiTE (Bispecific T-cell Engager). To exclude donor variabilities, we first humanized five NSG-based, human cytokine-transgenic mouse strains (NSG, NSG-IL15, NSG-SGM3, NSG-SGM3-IL15, and NSG-FLT3L) with HSCs from one donor (0084). We found that tumor growth was enhanced in NSG-SGM3 mice when compared to NSG mice. In contrast, IL15 expression suppressed tumor growth in NSG-IL15 and NSG-SGM3-IL15 mice. Furthermore, NK cell frequencies in blood are higher in NSG-IL15 and NSG-SGM3-IL15 than in other strains. suggesting that NK cell-mediated cytotoxicity is involved in suppression of tumor cell growth in the strains expressing human IL-15. Rituximab treatment resulted in tumor growth inhibition and significant B cell depletions across NSG-IL15, NSG-SGM3, and NSG-SGM3-IL15 mice engrafted with 0084 PBMC. We also observed a donor variability of tumor growth rates in NSG-SGM3-IL15 mice engrafted with donors 0084, 0662A, and 0733. The tumor growth in NSG-SGM3-IL15 mice engrafted with 0084 PBMC was significantly slower than the one in mice engrafted with the other two donors (0084 vs. 0662A, P=0.002; 0084 vs. 0733, P=0.004 on day 21); interestingly, this strain of mice exhibited higher frequencies of CD8+ T cells and lower frequencies of NK cells. Next, we tested CD19XCD3 BiTE treatment in multiple NSG strains engrafted with HSCs from different donors. The BiTE administration inhibited Raji tumor growth and significantly depleted B cells in all tested strains as NSG-SGM3-IL15 showed highest frequencies of NK cells and CD8+ T cells. The combination of Rituximab and BiTE showed even better tumor inhibition efficacy. In conclusion, regardless of the donor and strain variations in tumor growth and human immune cell subset development, the administrations of rituximab or BiTE significantly and consistently inhibited tumor growth and depleted peripheral B cells across all mouse strains. These results indicate that utilizing a comprehensive humanized mouse platform could drive more impactful conclusions in terms of evaluating the anti-tumor efficacy of immune therapies. Citation Format: Won Yeong Kang, Guoxiang Yang, Ilian Radichev, Li-Chin Yao, Mingshan Cheng, James G. Keck. Comparisons of Rituximab and CD19xCD3 BiTE antitumor response across multiple humanized NSG strains engrafted with human hematopoietic stem cells. [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 5189.

Abstract 2972: Preclinical characterization of SAR445877, an anti-PD-1 antibody-IL-15 mutein fusion protein with robust anti-tumor efficacy as monotherapy and in combination with PD-L1 blockade

Abstract Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. A major unmet medical need in immuno-oncology (IO) is extending the benefits of these therapies to patients who never experience tumor regression (primary resistance) or who derive initial benefit followed by tumor progression (acquired resistance). To improve clinical benefits of anti-PD-1 antibody, we generated SAR445877 (formerly KD050), which is an antibody cytokine fusion molecule that combines a human anti-PD-1 antibody with a mutated fusion of human IL15 and human IL15Rα sushi domain. SAR445877 binds to human PD-1 and inhibits immunosuppressive signaling through the PD-1/PD-L1 pathway. The IL15-IL15Rα fusion contains a single amino acid mutation that confers reduced potency of IL2/15Rβγ stimulation relative to wild type IL-15, reduces binding to IL-2Rβ expressing HEK 293 cells, (EC50: >108 nM vs 4.05nM) and decreases pSTAT5 activation in HEK blue IL-2 reporter assay by more than 5-fold. Through its IL15-IL15Rα fusion moiety, SAR445877 stimulates cells expressing IL2/15Rβγ with much weaker activity than wild type IL-15 when measured by CTLL-2 cell proliferation in vitro (EC50: 398.8nM vs 1.68nM). Fusion of IL-15 to anti-PD-1 antibody mediates cis-activation of human PD-1+ cells, including both PD-1+ HEK blue reporter cell line and human primary T cells, with higher potency compared to non-targeted IL-15. This cis-activation could result in expansion and activation of antigen-specific CD8+ T cells with improved activity in the tumor microenvironment compared to non-targeted IL-15. Antitumor efficacy of SAR445877 was explored in the CT26 colorectal cancer model using the clinical candidate. Transgenic mice expressing human PD-1 and PD-L1 were implanted with CT26 tumors expressing human PD-L1 and treated with SAR445877. SAR445877 displayed a more potent antitumor efficacy than pembrolizumab. Further, treatment with SAR445877 increased the CD8/CD4 T cell ratio and significantly increased the percentage of effector memory CD8 T cells in tumors. In anti-PD-L1 antibody resistant Pan02 syngeneic tumor model, a murine surrogate molecule produced a remarkable antitumor activity and was well tolerated. Combination treatment of murine surrogate with anti-mPD-L1 displayed greater antitumor activity than single agent treatment (TGI = 99.30% vs 78.78%). In summary, SAR445877 can target potency-reduced IL-15 to PD-1+ T cells and induce cis-activation of T cells. SAR445877 exhibited potent anti-tumor efficacy in PD-1 resistant preclinical model with good tolerability. These results support further evaluation of SAR445877 in a phase I clinical trial (ClinicalTrials.gov #NCT05584670). Citation Format: Marie Bernardo, Yu-an Zhang, Dan Lu, Stella Martomo, Fatima Menas, Chen Zhu, Raymond Perez, Jeegar Patel, Donald Shaffer, Xiangming Li. Preclinical characterization of SAR445877, an anti-PD-1 antibody-IL-15 mutein fusion protein with robust anti-tumor efficacy as monotherapy and in combination with PD-L1 blockade [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 2972.

Abstract 5200: Novel Immuno-Oncology drugs evaluation by humanised immune cells and cancer co-inoculated models

Abstract Currently more and more immunotherapeutic drugs and engineered cells have been developed to use human immune system against cancer. While humanized peripheral blood mononuclear cell reconstitution in immune deficient mice is a forward-straight model for evaluating therapeutic antibodies, this conventional model has several drawbacks to hinder its widespread use, including the graft-versus-host-disease happened after one month of reconstitution, insufficient immune cell infiltration from reconstituted circulatory system and hardly retention of functional innate immune cells, such as macrophages and nature killer cells. To overcome this, LIDE has developed a specific human immune cell and cancer cell co-inoculation model. Cancer-priming PBMC and/or its derivates were well mixed with the fresh cancer cells in MatriGel, co-transferring into NCG mice to form a relatively “hot tumor” tissue for immunotherapy, including drugs targeting T cells, dendritic cells and macrophages. Additionally, we also take advantage of human IL15 transgenic mice to study the function of ex vivo expanded NK cells. This novel method has successfully helped evaluate biological function of immune checkpoint blockers and immune agonists in multiple cancers, such as melanoma, breast cancer, lung cancer, hepatocellular carcinoma and ovarian cancer. Citation Format: Bin Xie, Xin Hou, Hanglu Chen, Pengfei Yang, Yanbin Zhou, Danyi Wen. Novel Immuno-Oncology drugs evaluation by humanised immune cells and cancer co-inoculated models. [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 5200.

Abstract 6786: NT-I7 as an adjuvant to DNA neoantigen vaccination enhances and prolongs neoantigen-specific anti-tumor immunity

Abstract Background: IL-7 is an essential cytokine in the development and maintenance of antigen-specific T cells. NT-I7 (efineptakin alfa; NeoImmuneTech, Inc, Rockville, MD), is a long acting IL-7 composed of recombinant human IL-7 (rhIL-7) fused to a hybrid Fc antibody platform that has significantly improved in vivo stability and half-life compared to rhIL-7. DNA cancer neoantigen vaccines are a promising personalized cancer immunotherapy capable of generating strong anti-tumor immunity, but limited by a short-lived neoantigen-specific T cell response. We hypothesize NT-I7 as an adjuvant to a DNA neoantigen vaccine will enhance the magnitude and duration of neoantigen-specific anti-tumor immunity. Methods: C57BL/6 mice were vaccinated with DNA vaccine encoding neoantigens present in E0771 murine breast cancer model on days 0, 3, and 6. NT-I7 (5 mg/kg) was administered intravenously on day 4 or 13 during T cell expansion or contraction phase respectively. Primary T cell responses to neoantigens were assessed by IFN-γ ELISPOT. Tumor protection was evaluated by inoculating mice with 5×105 E0771 cells subcutaneously after receiving DNA vaccine with or without NT-I7. Duration of neoantigen-specific CD8 T cell immunity was determined by in vivo CFSE killing assay. Results: NT-I7 administered during the T cell contraction phase following the DNA vaccine increases the magnitude of neoantigen-specific T cell responses on day 20 compared to DNA vaccine alone. DNA vaccination and NT-I7 alone prior to tumor inoculation confers partial protection against tumor growth compared to control while DNA + NT-I7 during contraction phase rejects 100% of tumors. Compared to DNA vaccine alone, DNA vaccine + NT-I7 during contraction phase induces greater neoantigen-specific killing on day 41 (34.6% vs. 11.97%, p=0.03). Conclusion: NT-I7 as an adjuvant to a DNA neoantigen vaccine increases the magnitude and duration of neoantigen-specific anti-tumor immunity in a murine model. Clinical application of NT-I7 may help overcome immunologic shortcomings of current neoantigen vaccines. Citation Format: Michael Y. Chen, Ina Chen, Suangson Supabphol, Alexandra A. Wolfarth, Sara F. Martinez, Byung Ha Lee, Lijin Li, Xiuli Zhang, Peter S. Goedgebuure, William E. Gillanders. NT-I7 as an adjuvant to DNA neoantigen vaccination enhances and prolongs neoantigen-specific anti-tumor immunity [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 6786.

Abstract 2903: Epigenetic modulation of NK cells to improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma

Abstract Osteosarcoma is the most common primary malignant bone tumor. It metastasizes almost exclusively to the lung, and this constitutes the main cause of death in these patients. Immune therapy has shown limited benefit. We investigated whether epigenetic modulation of allogeneic NK cells by the HDAC inhibitor MS-275 will improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma (OS) lung metastases. Human NK cells were purified and expanded from donor buffy coats using recombinant human IL-2 and genetically engineered K562 feeder cells and treated with MS-275 before injection into mice. RNA seq was performed in untreated and MS-275 pre-treated NK cells to determine differentially expressed genes. We also used ChiPseq to determine the histone post-translational modifications associated with the differentially expressed genes induced by the treatment. Culture media from untreated and treated NK cells was collected for cytokine analysis using the Isolight/Isoplexis system. We used our OS-17 human OS experimental lung metastases mouse model and injected untreated or pre-treated NK cells (0.5uM MS-275) IV labeled with near-infrared DiR dye twice a week. Tumor burden was assessed, and NK cells were tracked by Bioluminescence imaging (BLI). Mice were either euthanized 24 hours after the final NK cell treatment for histologic analysis of the lungs or, in a second experiment, analyzed for survival across treatment groups. Some mice from each group were selected for 3D imaging using the Xerra cryofluorescence tomography (CFT) imaging system, allowing for greater resolution of NK cell distribution as compared to the IVIS imaging system. We identified 547 differentially expressed genes between the untreated and MS-275 treated NK cells. Four immune related genes (L1CAM, IFNG, C3 and CD28) were upregulated in the MS-275 treated NK cells and histone H3 and H4 acetylation was found at the identified immunomodulatory genes. Culture media from MS-275 treated NK cells showed significantly increased MIP-1b, IL-6 and IFN-g. Furthermore, there was a significant decrease in lung micro-metastases in the MS-275 pre-treated NK cell group as compared to the untreated control group (199 vs 97 p=0.05). Lastly, Xerra CFT imaging demonstrated greater DiR signal in the lungs of the mice that received MS-275 treated NK cells compared to those that received untreated NK cells, suggesting that MS-275 might lead to greater persistence of NK cells at tumor sites. We conclude that MS-275 has an immunomodulatory effect on NK cells (increase L1CAM, IFNG, C3 and CD28) and increases H3 and H4 acetylation which leads to transcriptional activation of the immune-related genes that contribute to NK cell increased cytolytic function. Pre-treatment of NK cells with MS-275 enhances NK cell therapeutic efficacy and localization and persistence in the lungs. Citation Format: Ariana Rupp Anjier, Nancy Gordon, Shinji Maegawa, Vidya Gopalakrishnan, Juan Bournat, Donghang Cheng, Yanwen Yang, Charles Kingsley, Adam Kulp. Epigenetic modulation of NK cells to improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma [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 2903.

Abstract 3580: Noninvasive immuno-PET imaging of CD8+T cell behavior in tumor bearing mice models treated with SAR444245

Abstract SAR444245 (formerly THOR-707) is a site-specific pegylated recombinant human IL-2 molecule which blocks IL-2R alpha-binding while retains near-native affinity for beta/gamma IL-2 receptor subunits. SAR444245 reduces B16-F10 tumor proliferation in C57BL/6 mice and induces a short transient migration of CD8+ T and NK cells from peripheral blood to lymphoid organs before a strong proliferation (Ptacin et al, Nat Commun 2021). Here we report CD8+ T cells imaging post SAR444245 as monotherapy in naive and tumor bearing mice using nuclear imaging. An anti-murine CD8+ PET probe was injected in mice 24 hours post SAR444245 administration and nuclear imaging was performed at different times over 6 days. In C57BL/6 naive and in poorly immunogenic B16-F10 tumor bearing animals, images reveal a decreased Standardized Uptake Value (SUV) within blood in treated animals at 3mg/kg compared to control ones. In parallel, blood SUV Ratio (SUVR) increases within spleen, lymph nodes and thymus after SAR444245 injection compared to control mice. On tumor site, a stronger SUVR is measured at 144 hours post injection in SAR444245 treated group than in the control group. A dose escalation ranging from 1 to 6 mg/kg of SAR444245 was performed in highly immunogenic CT26 tumor bearing animal. Blood SUV decreases in the treated groups compared to the control group. In lymphoid organs, all SUVR increase at 144 hours post injection in the treated groups compared to the control one. In CT26 tumors, a slight increase of the SUVR is observed for groups treated at 3 and 6 mg/kg. In parallel, a pronounced reduction of tumor growth is observed in the treated group at 6mg/kg. These studies non-invasively confirm the mode of action of SAR444245 with a rapid relocation of CD8+ T cells from peripheral blood to lymphoid organs and tumor site. Citation Format: Sebastion d'Heilly, Fabrice Tirode, Maxime Gaulin, Dudzicki Anne, Guillaume Bluet, Stéphane Guerif, Francis Descamps, Peter Casteels, Xiangming Li, Rui Wang, Robin Meng, Erwan Jouannot. Noninvasive immuno-PET imaging of CD8+T cell behavior in tumor bearing mice models treated with SAR444245. [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 3580.

Abstract 2155: Joint modeling of safety and peripheral mode-of-action (MoA) biomarkers to support RP2D identification in Phase 1 study of SAR444245 (SAR’245) as monotherapy (mono) or combined with pembrolizumab (pembro) in patients with advanced solid tumors

Abstract Background: SAR’245 is a clinical-stage site-specific pegylated human IL-2 that blocks IL-2 alpha receptor binding but retains near-native binding affinity for beta/gamma IL-2 receptor subunits. When administered in preclinical models, a unique ‘T-cell remodeling’ MoA characterized by robust increase of CD8+Teff/CD4+Treg coupled with potent NK-cell activation/expansion was observed. Circulating tumor DNA (ctDNA) can be used as a non-invasive biomarker of early clinical response whilst overcoming challenges of obtaining repeat tumor biopsies from patients. Previously, we reported results of the Phase 1 HAMMER study (NCT04009681); herein, we describe an innovative integrative approach that considers peripheral key MoA biomarkers with objective response and dose-limiting toxicity (DLT) rate to help identify the recommended Phase 2 dose (RP2D) for SAR’245. Methods: SAR’245 was given IV as mono Q3W [Cohort B] or Q3W + IV pembro 200 mg Q3W/400 mg Q6W [Cohort C]. Joint modeling was carried out to account for the relationship between dose and 1) MoA biomarkers, and NK cells in blood measured by flow cytometry; 2) response surrogate biomarker: ctDNA measured by Guardant Omni 500 panel; and 3) DLTs. In the model, a latent variable was used to model correlation between DLT and the MoA or response surrogate biomarkers, Bayesian approach was used to derive posterior probabilities (PP) at each dose level of the target region (defined by >20% probability of fold change of biomarker values post-treatment above a predefined threshold and DLT rate <33%) and the PP of the overdose region (defined by DLT rate ≥33%). RP2D dose level was determined by maximizing the PP of target region among doses with PP of the overdose region probability <40%. Results: Samples from 35 subjects (Cohort B) and 34 subjects (Cohort C) were available. The results from SAR’245 mono suggests that the CD8/CD4 ratio and the concentrations of NK, CD8, and CD4 achieve maximum probability of reaching meaningful modulation (based on pre-defined threshold) around 32 ug/kg. When SAR’245 was combined with pembro, the results with PoM biomarkers showed the best performance at 24-32 µg/kg, while results with ctDNA showed the best results at 16-24 µg/kg. When all parameters were considered, either 24 or 32 µg/kg could serve as an adequate dose at Q3W scheduling. Conclusions: In early oncology studies, joint modeling using non-invasive biomarkers, including MoA and response biomarkers, and a safety profile can inform dose-response relationships and support RP2D selection. This innovative integrative modeling will guide clinical study design. Studies of SAR’245 that further explore the dosing and scheduling are on-going. Disclosures: This study was sponsored by Synthorx, a Sanofi company. Citation Format: Siqing Fu, Gerald S. Falchook, Minal Barve, Meredith McKean, Tira J. Tan, Charlotte Lemech, Cheng E. Chee, Neyssa Marina, Giovanni Abbadessa, Robin Meng, Federico Rotolo, Hong Wang, Jason Deng, Wenting Wang, Rui Wang, Tarek Meniawy. Joint modeling of safety and peripheral mode-of-action (MoA) biomarkers to support RP2D identification in Phase 1 study of SAR444245 (SAR’245) as monotherapy (mono) or combined with pembrolizumab (pembro) in patients with advanced 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 2155.

Abstract 1824: Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity

Abstract Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. Although recombinant mIL18 has demonstrated efficacy in mouse tumor models, recombinant human IL18 has not been efficacious in clinical studies, likely due to upregulation of IL18 binding protein (IL18BP), a negative regulator of the IL18 signaling axis. IL18BP is induced 10 - 100 fold after IL18 administration, binds to IL18 with high affinity, and prevents IL18 binding to the IL18 receptor. Analysis of expression data from the TCGA database as well as archived patient samples from select indications revealed that both IL18 and IL18BP are co-expressed in a number of tumor types, suggesting there may be a preexisting pool of IL18/IL18BP complex at the site of the tumor in some patients. Inhibition of IL18/IL18BP binding using a monoclonal antibody may therefore allow release of IL18 in situ, leading to IL18-mediated proinflammatory effects and enhanced anti-tumor immunity. Although the interaction of IL18 with IL18BP is a high affinity interaction, we have been able to identify very high affinity antibodies which can both inhibit complex formation and disrupt existing mouse IL18/IL18BP complexes. These antibodies are active in a number of cell-based functional assays, resulting in potent stimulation of interferon gamma production from cell lines or splenocytes. In vivo, we observed efficacy in a mouse syngeneic tumor model using an anti-mIL18BP antibody. RNA and protein analysis demonstrated expression of both IL18BP and IL18 within the tumors. Combined, these data suggest that inhibition of IL18/IL18BP binding may prove efficacious in the treatment of cancers where existing pools of IL18/IL18BP complexes prevent an IL18-mediated proinflammatory response. Experiments exploring anti-IL18BP therapy in combination with other checkpoint inhibitors such as anti-PD-1 are under current investigation. Citation Format: H. Toni Jun, Deborah A. Witherden, Eric K. Elliot, Christine M. Chidester, Maggie Willen, Bryan R. Peguero, Merrick Chai, Robert A. Horlick, David J. King. Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity [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 1824.

Abstract 1850: A first-in-class PD1-IL18 immunocytokine (BPT567) targets PD-1+ IL18R+ CD8+ T effector cells enriched in the tumor microenvironment and exhibits potent antitumor efficacy with excellent tolerability

Abstract Immunocytokines (IC) leverage orthogonal mechanisms of action in one molecule to induce potent antitumor immune responses. PD-1-targeting ICs are of particular interest since they harbor the multifunctional ability to selectively target antigen-experienced PD-1+ CD8+ T cells enriched in the tumor microenvironment (TME), release them from PD-(L)1 pathway inhibition, be retained within the TME, and simultaneously deliver potent cytokine receptor stimulation to the same T cell in cis (cis-signaling). Interleukin (IL-18) is a proinflammatory cytokine that stimulates both innate and adaptive immunity and generates potent antitumor activity mediated by both T effector and NK cells. Recent evidence indicates that a subset of tumor-infiltrated PD-1+ CD8+ T effector cells hallmarked by high expression of IL-18 receptor exhibits a superior cytotoxic and proliferative phenotype. Hence, we developed a PD1-IL18 IC to specifically target and activate intratumoral IL-18R-expressing PD-1+ CD8+ T cells. We engineered a conjugatable variant of human IL-18 with enhanced potency and significant resistance to IL-18 binding protein (IL-18BP) and utilized this enhanced IL-18 payload to create a PD1-IL18 IC (BPT567) via site-specific chemical conjugation to a defined lysine residue within the heavy chain of an anti-human PD-1 antibody (Ab). Conjugation did not affect the basic properties of the Ab as neither binding to PD-1 nor the interaction with the neonatal Fc receptor (FcRn) or Fcγ receptors were significantly impacted. Of note, the conjugation handle in our enhanced IL-18 variant was inserted at a site distinct from its N- or C-terminus to preserve the full potency and selectivity of the IL-18 payload. As a result, BPT567 exhibits increased potency and marked resistance to IL-18BP inhibition compared to wild-type IL-18. In vivo, BPT567 shows striking antitumor efficacy in multiple syngeneic mouse tumor models that is superior to responses induced by an anti-PD-1 Ab alone, a non-targeted IL-18 IC or the combination of both agents. Our evidence suggests that the strong in vivo efficacy observed is attributed to a preferential activation of PD-1+ tumor-infiltrated immune cells. The excellent tolerability and large therapeutic window observed in mice may be driven by less potent immune cell activation in the periphery. Citation Format: Jean-Philippe Carralot, Kea Martin, Rubén Alvarez Sanchez, Roy Meoded, Caoimhe Herr, Philipp Moosman, Arnaud Goepfert, Andrew Chi, Vijaya J. Pattabiraman, Bertolt Kreft. A first-in-class PD1-IL18 immunocytokine (BPT567) targets PD-1+ IL18R+ CD8+ T effector cells enriched in the tumor microenvironment and exhibits potent antitumor efficacy with excellent tolerability [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 1850.

Abstract 5133: Oxazolone induced atopic dermatitis mouse model using hIL-4/hIL-4Ra double knock-in mice, a preclinical mouse model for evaluating Dupilumab biosimilars

Abstract Background. Th2 cytokines (e.g. IL-4, IL-5, IL13) drive defined allergic reactions and various autoimmune and inflammatory diseases including atopic dermatitis (AD), asthma and rhinitis. IL-4Ra is a receptor shared by both IL-4 and IL-13, thus blocking of IL-4Ra could prevent both IL-4 or IL-13 mediated Th2 inflammatory response. Dupilumab (Dupixent) is a humanized anti-IL-4Ra monoclonal antibody which binds to human IL-4Ra and attenuates IL-4/IL-13 mediated Th2 inflammation. It has been approved for treating moderate to severe AD and asthma. However, since Dupilumab isn’t cross-reacting to mouse IL-4Ra, there is an unmet need for a robust mouse model to evaluate Dupilumab biosimilars at preclinical stages. In this study, we described an oxazolone induced AD model in hIL-4/hIL-4Ra double knock-in mice and its application to evaluate Dupilumab and its biosimilars. Methods. The oxazolone induce AD mouse model was established in hIL-4/hIL-4Ra double knock-in mice by repeated topical applications of Oxazolone on ears. Oxazolone-induced mice received subcutaneous injection of Dupilumab at 25 mg/kg or 50 mg/kg. Dermatitis activity was assessed by measuring ear thickness, ear skin erosion, redness and scaling every other day. Serum were collected at the study end and IgE levels were measured by ELISA. In addition, spleens were also collected and their weights were compared. Ear were collected at study end and proceeded with cytokine analysis and pathology evaluation. Results. Repeated topical application of oxazolone on mouse ears induced AD-like phenotypes including ear skin swelling, erythema and scaling, drastic elevation of IgE in serum, excessive production of human IL-4 cytokine as well as excessive inflammatory cell infiltration in ear skin, which suggests oxazolone is able to induce AD in this hIL-4/hIL-4Ra double knock-in mice. Subcutaneous injection of Dupilumab at two different doses levels, 25 mg/kg and 50 mg/kg successfully ameliorated ear skin swelling, erythema and scaling at the study end. In addition, Dupilumab at both dose levels significantly reduced the IgE level in serum and mitigated the inflammatory cell infiltration, e.g. eosinophil infiltration, in mouse ear. Furthermore, Dupilumab treatment significantly reduced mouse IL-6 and mouse KC/GRO levels in mouse ear. However, Dupilumab at both dose levels did not change the serum levels of human IL-4 as well as some other Th1 cytokines such as IFN-γ, IL-1β, TNF-α, etc. in mouse ear. Conclusion. In summary, repeated challenge with Oxazolone in hIL-4/hIL-4Ra double knock-in mice resulted in the development of AD with a Th2-like hypersensitivity, mimicking the key features and pathology of the human disease, which responded to standard of care treatment Dupilumab. This provides a valuable translational model for the evaluation of future biosimilars of Dupilumab. Citation Format: Tao Yang, Rongfei Lu, Xiaofei Xu, Likun Zhang, Dawei Wang, Xinhe Feng, Xiaoyu An, Jessie (Jingjing) Wang, Xianfei He, Carl K. Edwards. Oxazolone induced atopic dermatitis mouse model using hIL-4/hIL-4Ra double knock-in mice, a preclinical mouse model for evaluating Dupilumab biosimilars. [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 5133.

Abstract 5654: A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells

Abstract Background: NEO-201 is a humanized IgG1 mAb reactive against multiple human cancers but not against most normal epithelial tissues. NEO-201 binds to core 1 or extended core 1 O-glycans expressed by its target cells, including neutrophils, various carcinomas, and some human hematological malignancies. NEO-201 can mediate antitumor activity through antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and blockade of the CEACAM5/CEACAM1 ICI pathway. A previous study using flow cytometry demonstrated that NEO-201+/CD4+ T cells were also CD25+/CD127−/Foxp3+/CD15s+ using PBMCs from healthy donors (HD). NEO-201 can kill these Treg cells through CDC in vitro. NEO-201 does not bind to the majority of CD4+ T cells and to other immune subsets. Human gMDSCs are increased in cancer patients and are a population of immature MDSCs deriving from immature neutrophils and alternative activation of mature neutrophils. gMDSC are characterized by HLA-DR−, CD11b+, CD33+, CD15+phenotype.We have shown that NEO-201 recognizes and kill human neutrophils through ADCC. This current investigation was designed to evaluate whether NEO-201 can target and mediate ADCC activity against human gMDSCs. Methods: gMDSCs were generated from human neutrophils from 5 HD isolated using EasySepTM direct human neutrophil isolation kit. Isolated neutrophils were cultured in complete RPMI1640 medium supplemented with human GM-CSF and human IL-6 for 7 days. Phenotypic analysis by flow cytometry was performed on the generated gMDSCs using NEO-201 and mAbs against human CD33, HLA-DR, CD15, CD14, CD66b. Flow cytometry based ADCC assay was performed using gMDSCs stained with both CD33 and HLA-DR as target. PBMCs from a separate HD were used as effectors at different E:T ratios. The ADCC activity of NEO-201 was evaluated comparing the percentage of CD33+/HLA-DR− viable cells in gMDSCs incubated with medium alone to the percentage of CD33+/HLA-DR− viable cells incubated with PBMCs alone and with PBMCs plus NEO-201. Results: Flow cytometry analysis revealed that gMDSCs can be generated from human neutrophils after 7 days of culture with GM-CSF and IL-6 and that they express the following phenotype: HLA-DR−/CD33+/CD15+/CD14−/CD66b+. NEO-201 bound to the majority of these gMDSCs. NEO-201 was functional in mediating ADCC to kill these gMDSCs. Conclusion: This study demonstrated that NEO-201 can be used to identify and kill suppressive gMDSCs in addition to Treg cells. Depletion of suppressive Tregs and gMDSCs in the TME could be an effective strategy to prevent hyperprogressive disease when anti-PD-1 is used in cancer immunotherapy. These data support the rationale for the ongoing phase II clinical trial using NEO-201 in combination with pembrolizumab in checkpoint refractory patients with metastatic solid tumors. Citation Format: Massimo Fantini, Christina M. Annunziata, Philip M. Arlen, Kwong Y. Tsang. A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells. [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 5654.

Recombinant Human Interleukin-2 Corrects NK Cell Phenotype and Functional Activity in Patients with Post-COVID Syndrome.

Post-COVID syndrome develops in 10-20% of people who have recovered from COVID-19 and it is characterized by impaired function of the nervous, cardiovascular, and immune systems. Previously, it was found that patients who recovered from infection with the SARS-CoV-2 virus had a decrease in the number and functional activity of NK cells. The aim of the study was to assess the effectiveness of recombinant human IL-2 (rhIL-2) administered to correct NK cell phenotype and functional activity in patients with post-COVID syndrome. Patients were examined after 3 months for acute COVID-19 of varying severity. The phenotype of the peripheral blood NK cells was studied by flow cytometry. It was found that disturbances in the cell subset composition in patients with post-COVID syndrome were characterized by low levels of mature (p = 0.001) and cytotoxic NK cells (p = 0.013), with increased release of immature NK cells (p = 0.023). Functional deficiency of NK cells in post-COVID syndrome was characterized by lowered cytotoxic activity due to the decreased count of CD57+ (p = 0.001) and CD8+ (p < 0.001) NK cells. In the treatment of patients with post-COVID syndrome with recombinant IL-2, peripheral blood NK cell count and functional potential were restored. In general, the effectiveness of using rhIL-2 in treatment of post-COVID syndrome has been proven in patients with low levels of NK cells.