4-1BB agonists for cancer immunotherapy have shown good preliminary efficacy in clinical trials, but several of the first-generation 4-1BB agonistic antibodies entering the clinic have failed due to safety issues. Selenium nanoparticles (SeNPs) exhibit anti-inflammatory, anti-tumor, antioxidant, and immune-modulating properties. In addition, they have been shown to have detoxifying effects and prevent oxidative liver damage. In this study, we used an anti-4-1BB antibody in combination with SeNPs to evaluate the anti-lung cancer effects in in vitro and in vivo experiments and explore the underlying mechanisms by pathological analyses, quantitative PCR, and enzyme-linked immunoassay. We found that 5 μmol·L–1 anti-4-1BB antibody combined with 1 μmol·L–1 SeNPs increased the expression of IFN-γ and promoted the killing effects of peripheral blood mononuclear cells on Lewis lung carcinoma cells, with a lethality rate up to 56.88 %. Experiments in tumor-bearing mice showed that the tumor inhibition rate was 58.61 % after treatment with 3.5 mg/kg anti-4-1BB antibody combined with 0.25 mg/kg SeNPs, and the liver function index returned to normal. When the combined treatment was compared with the antibody treatment alone, detection of immune relevant factors demonstrated that the expression of FOXP3, IL-2, IL-12, and TNF-α in the spleen was downregulated, whereas the expression of IFN-γ in the spleen, serum, and tumor was upregulated, accompanied by increased Fas ligand expression in the tumor tissues. Based on these findings, we get the conclusion that anti-4-1BB antibody combined with SeNPs may alleviate the immunosuppression of regulatory T cells, promote the immune cell proliferation and metastasis to synergistically kill tumor cells. This combination also reduces the inflammatory damage to normal tissues and slows overstimulation of the splenic immune response.

Background Resistance to immune checkpoint inhibitor (ICI) therapy narrows the efficacy of cancer immunotherapy. Although 4-1BB is a promising drug target as a costimulatory molecule of immune cells, no 4-1BB agonist has been given clinical approval because of severe liver toxicity or limited efficacy. Therefore, a safe and efficient immunostimulatory molecule is urgently needed for cancer immunotherapy. Methods HK010 was generated by antibody engineering, and the Fab/antigen complex structure was analyzed using crystallography. The affinity and activity of HK010 were detected by multiple in vitro bioassays, including enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), flow cytometry, and luciferase-reporter assays. Humanized mice bearing human PD-L1-expressing MC38 (MC38/hPDL1) or CT26 (CT26/hPDL1) tumor transplants were established to assess the in vivo antitumor activity of HK010. The pharmacokinetics (PK) and toxicity of HK010 were evaluated in cynomolgus monkeys. Results HK010 was generated as an Fc-muted immunoglobulin (Ig)G4 PD-L1x4-1BB bispecific antibody (BsAb) with a distinguished Fab/antigen complex structure, and maintained a high affinity for human PD-L1 (KD: 2.27 nM) and low affinity for human 4-1BB (KD: 493 nM) to achieve potent PD-1/PD-L1 blockade and appropriate 4-1BB agonism. HK010 exhibited synergistic antitumor activity by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously, and being strictly dependent on the PD-L1 receptor in vitro and in vivo. In particular, when the dose was decreased to 0.3 mg/kg, HK010 still showed a strong antitumor effect in a humanized mouse model bearing MC38/hPDL1 tumors. Strikingly, HK010 treatment enhanced antitumor immunity and induced durable antigen-specific immune memory to prevent rechallenged tumor growth by recruiting CD8+ T cells and other lymphocytes into tumor tissue and activating tumor-infiltrating lymphocytes. Moreover, HK010 not only did not induce nonspecific production of proinflammatory cytokines but was also observed to be well tolerated in cynomolgus monkeys in 5 week repeated-dose (5, 15, or 50 mg/kg) and single-dose (75 or 150 mg/kg) toxicity studies. Conclusion We generated an Fc-muted anti-PD-L1x4-1BB BsAb, HK010, with a distinguished structural interaction with PD-L1 and 4-1BB that exhibits a synergistic antitumor effect by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously. It is strictly dependent on the PD-L1 receptor with no systemic toxicity, which may offer a new option for cancer immunotherapy.

Background: CD137 (TNFRSF9, 4-1BB) is a member of the tumor necrosis factor receptor superfamily that functions as a costimulatory molecule of immunocytes and is mainly expressed on the surface of immune cells including activated T and natural killer (NK) cells. Agonists against CD137 have shown promising therapeutic activity in mouse tumor models but have suffered setback in the clinic due to notably hepatotoxicity, or suboptimal agonistic potency. Here, we developed a fully human agonistic anti-CD137 monoclonal IgG4 antibody, ZG033, with an exclusive epitope, to achieve a better efficacy and safety profile for immunotherapy. Methods: The antigen binding specificity and affinity of ZG033 were determined by ELISA, surface plasmon resonance (SPR). X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms. The activities were determined using cell-based and reporter gene assays. In vivo antitumor activities were evaluated in human 4-1BB transgenic mice bearing with syngeneic tumors, and possible toxicity was evaluated in humanized mice and cynomolgus monkeys. Cytokine release assay was conducted by incubating ZG033 with PBMCs from healthy donors. The pharmacokinetic (PK) behavior of ZG033 were characterized in cynomolgus monkeys. A phase 1 study was initiated to evaluate the safety and preliminary clinical activities of ZG033, as well as the immunogenicity and pharmacokinetics. Results: ZG033 showed high specificity for human CD137 without cross-species reactivity across mouse, rat and exhibited potent CD137 agonist activity dependent on FcγR crosslinking. The binding affinity of ZG033 determined by surface plasmon resonance (SPR) was moderate (KD=10 nM). X-ray crystallography revealed that the Fab of ZG033 binds CD137 at an epitope overlap with the ligand binding interface. ZG033 increased CD137-driven NFκB reporter gene activation and the production of IFN-γ by human T cells in a FcγR-dependent manner. In human CD137 transgenic mice, ZG033 showed robust antitumor activity and induced durable antigen-specific immunological memory that prevented the tumor formation in the rechallenged mice. There were no signs of toxicity in human cytokine release assay which confirmed that ZG033 did not induce pro-inflammatory cytokines in the absence of TCR stimulation. Toxicology data in mice showed that ZG033 was well-tolerated with normal ALT and AST levels. ZG033 was further confirmed to well tolerated up to 180 mg/kg (MTD≥180mg/kg) dose without systemic toxicity in cynomolgus monkeys. Patients with advanced tumors, who are refractory after exhausting available therapies, were enrolled for ZG033 treatment. We will report the results from early dose escalation part of the study. Conclusion: These data demonstrate that ZG033 is an FcγR crosslinking-dependent CD137 agonist that displays a favorable safety profile and has potential in either mono- or combinational immunotherapies. Citation Format: Yang Huang, Liangwei Li, Wenting Liu, Dayan Zhang, Pengfei Zhou, Qing Fang, Weiming Zhou, Fengrong Wang, Guodong Shen, Liansheng Cheng. A novel CD137 agonist exhibits potent antitumor efficacy with a good safety profile [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 1880.

Background: Checkpoint inhibitors targeting the PD-1/PD-L1 have changed the treatment and prognosis for patients with advanced solid tumors. There remains a unmet need for additional therapies due to treatment resistance. Tumor-necrosis superfamily members, such as CD137 and CD40, have been shown to synergize with the checkpoint inhibitors in preclinical studies. However, agonists antibodies have exhibited limited clinical activity for severe liver toxicity. Here we have developed a Fc-silenced bispecific antibody targeting PD-L1 and CD137, HK010, which could overcome these limitations by tumor-directed T-cell activation and checkpoint blockade. Methods: The antigen binding specificity and affinity of HK010 were determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) and flow cytometry. The functional activity of HK010 was determined using in vitro cell-based assays and reporter gene assay. Transgenic mice expressing human PD-L1 and 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumor activity. Cytokine release assay was conducted by incubating HK010 with peripheral blood mononuclear cells (PBMCs) from healthy donors (n=6). The pharmacokinetic (PK) behavior and safety profiles of HK010 were characterized in cynomolgus monkeys. Results: HK010 is a IgG4 PD-L1 × 4-1BB bsAb, which was designed to maintain a high affinity for human PD-L1 and a low affinity for human 4-1BB (KD PD-L1: 2.27 nM; KD 4-1BB: 493 nM) to achieve strong blocking of PD-1/PD-L1 and appropriate agonism of 4-1BB. HK010 retained full blockade activity on PD-1/PD-L1 signaling and enhances T-cell proliferation and IFN-γ production in vitro primary cell assays and Mixed lymphoid reaction (MLR). In addition, it led to a dose-dependent increase in the CD137-driven NFκB reporter gene activation through the bridging of PD-L1 on target cells and CD137 on effector cells. In humanized PD-L1/4-1BB transgenic mice bearing with MC38/hPDL1 tumor, HK010 showed robust single agent anti-tumor activity and induced durable antigen-specific immunological memory that prevents the growth of the same tumor cells in the mice re-challenged. There were no signs of toxicity in human cytokine release assay for HK010. Toxicology data in cynomolgus monkeys showed that HK010 was well-tolerated up to 150 mg/kg (MTD≥150mg/kg) dose without systemic toxicity. Furthermore, HK010 was confirmed to be highly safe in the 5-week repeated-dose toxicity study with no observed treatment-related mortality, abnormality in hematological indexes, important organs. Conclusion: These data demonstrate that HK010, an anti-PD-L1 × CD137 bispecific antibody, may exert a strong localized anti-tumor therapeutic efficacy with a low risk of liver toxicity through tumor-directed T-cell activation and checkpoint blockade in tumors. Citation Format: Liangwei Li, Wenting Liu, Guodong Shen, Dayan Zhang, Weiming Zhou, Fengrong Wang, Xiaoli Zeng, Yang Huang, Liansheng Cheng. HK010, a novel anti-PDL1 × CD137 bispecific antibody, exhibits potent anti-tumor immunity and low toxicity. [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 5657.

Background: Interleukin-12 (IL12) is a heterodimeric pro-inflammatory cytokine (70 kDa) composed of two different polypeptide chains: an α-chain (p35 subunit) and a β-chain (p40 subunit). IL12 could induce the proliferation and cytotoxicity of T and NK cell and production of interferon-γ (IFN-γ), promote the differentiation of T helper 1 (TH1) cells and regulate innate resistance and adaptive immunity. However, the clinical efficacy of IL12 has been hindered for its high dose-related toxicities and short serum half-life. Here we have developed a heterodimeric Fc-fused IL12, HK054-V, with reduced potency to improve tolerability and prolong half-life. Methods: We assessed the binding, bioactivity, anti-tumor efficacy and safety of HK054-V. The binding ability of IL12-Fc lead variants to activated PBMCs was analyzed by flow cytometry. The bioactivity of HK054-V was determined by assessing the activated T-cell proliferation and IFN-γ production in human PBMCs, and STAT-4 phosphorylation in a reporter gene assay. A murine surrogate, mHK054-V, was engineered to mimic HK054-V to evaluate the antitumor efficacy in MC38 and CT26 syngeneic tumor mouse models. Cytokine release assay was conducted by incubating HK054-V with peripheral blood mononuclear cells (PBMCs) from healthy donors. The safety profiles and pharmacokinetic (PK) behavior of HK054-V were characterized in non-tumor-bearing Balb/c mice. Results: HK054-V is a heterodimeric Fc-fused IL12, which was designed with reduced potency. HK054-V was observed to stimulate the proliferation of activated T cells and NK cells and induce production of IFN-γ in a dose-dependent manner. HK054-V could activate pSTAT4 signal in luciferase reporter gene assay and showed up to 100-fold reduction of potency compared to wild-type IL12-Fc. Human IL12 is inactive in mice, so in vivo anti-tumor activity was assessed by a murine surrogate, mHK054-V. mHK054-V demonstrated anti-tumor efficacy in syngeneic mouse models and was better tolerated than WT mIL12-Fc. Furthermore, mHK054-V has improved PK and therapeutic index in mice. Conclusion: These data demonstrate that HK054-V, a heterodimeric Fc-fused IL12 with reduced potency, retain strong anti-tumor efficacy with a favorable tolerability profile, which may provide a practical alternative to the systemic administration of IL12 for antitumor therapy. Citation Format: Yang Huang, Wenting Liu, Dayan Zhang, Pengfei Zhou, Weiming Zhou, Xiaoli Zeng, Qun Zhao, Liangwei Li, Guodong Shen, Liansheng Cheng. Heterodimeric Fc-fused IL12 shows potent antitumor activity and low toxicity. [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 6336.

Background: 4-1BB (CD137) is not only expressed on the surface of activated T cells and NK cells, but also a marker for Treg cells. Mesothelin (MSLN) is a ~71 kDa cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in many types of tumors. The fastest progress of MSLN-targeted therapy is in clinical phase II currently, including antibodies, ADCs, immunotoxins, vaccines and CAR-T. Here, we developed a bispecific antibody (bsAb) targeting both MSLN and 4-1BB with an intact Fc fragment from human IgG1. It can simultaneously exert the cytotoxic effect of CD8+T cells and and NK cells on tumor cells expressing MSLN to achieve better antitumor efficacy. Methods: Firstly, two bsAbs with IgG-scFv format were constructed with IgG1 and IgG4, named as HK013-G1 and HK013-G4 respectively. Secondly, we evaluated the binding activities of two candidates to tumor cells with different MSLN expression levels and their effects on 4-1BB+ cell activation mediated by MSLN-crosslinking. Subsequently, the cell-killing abilities of NK and cytotoxic T cells induced by bsAbs were quantified. Finally, we compared the ability of bsAbs to inhibit tumor growth in vivo and their effects on intratumoral CD8+T cells and Tregs. The capacity of bsAb candidates to induce liver toxicity was investigated simultaneously in these immunocompetent mice. Moreover, to further confirm the safety of HK013-G1, the risk of cytokine release storm (CRS) and activation of 4-1BB signal mediated by Fc receptors (FcRs), which are responsible for the potential side effects of 4-1BB mAbs were also evaluated. Results: HK013-G1 is able to bind different MSLN-expressing cancer cells and bridge MSLN+ cells and 4-1BB+ cells. In luciferase reporter assay, the bsAb-induced 4-1BB activation is dependent on expression level of MSLN. While incubated with CD8+T cells, HK013-G1 increased IFN-γ production only in the presence of MSLN+ cells. Furthermore, HK013-G1 could exert its affects via both NK cell and activated PBMC. In MC38-hMSLN tumor models, HK013-G1 showed a more significant growth inhibition effect than HK013-G4, and did not show detectable liver damage. In addition, HK013-G1 could enhance the antitumor effect of anti-PD-1 in CT26-hMSLN tumor models. Also, HK013-G1 was shown no stronger ability to inducing CRS and activating 4-1BB signal in the presence of FcRs than HK013-G4 and parent anti-4-1BB mAb in vitro. Conclusions: HK013-G1, a MSLN × 4-1BB bsAb with human IgG1 Fc fragment prevents tumor development by killing tumor cells directly via effector functions mediated by NK and cytotoxic T cells. These results show that this bsAb has the potential to develop into a new clinical therapy for cancer types with high-level MSLN expression. Citation Format: Liangwei Li, Dayan Zhang, Guodong Shen, Wenting Liu, Xiaoli Zeng, Lingling Wu, Qun Zhao, Weiming Zhou, Yang Huang, Liansheng Cheng. Development of a novel MSLN × 4-1BB bispecific antibody. [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 6338.

Resistance to HER2-targeted therapy narrows the efficacy of cancer immunotherapy. Although 4-1BB/CD137 is a promising drug target as a costimulatory molecule of immune cells, no therapeutic drug has been ap-proved in the clinic because of systemic toxicity or limited efficacy. Previously, we developed a humanized anti-HER2 monoclonal antibody (mAb) HuA21 and anti-4-1BB mAb HuB6 with distinct antigen epitopes for cancer therapy. Here, we generated an Fc-muted IgG4 HER2/4-1BB bispecific antibody (BsAb) HK006 by the fusion of HuB6 scFv and HuA21 Fab. HK006 exhibited synergistic antitumor activity by blocking HER2 signal transduction and stimulating the 4-1BB signaling pathway simultaneously and strictly dependent on HER2 expression in vitro and in vivo. Strikingly, HK006 treatment enhanced antitumor immunity by increasing and activating tumor-infiltrating T cells. Moreover, HK006 did not induce nonspecific production of proinflammatory cytokines and had no obvious toxicity in mice. Overall, these data demonstrated that HK006 should be a promising candidate for HER2-positive cancer immunotherapy.

Abstract Background Mesothelin (MSLN) is a 70 KD glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in a variety of cancers, including mesothelioma, ovarian cancer, pancreatic cancer and breast cancer et.al. 4-1BB is a member of the tumor necrosis factor receptor superfamily that functions as a co-stimulatory molecule. Agonistic antibodies bind to 4-1BB, triggering a signaling cascade leading to T-cell activation and expansion of cytotoxic CD8+ T lymphocytes. Here, we developed two bispecific antibodies (bsAbs) targeting both MSLN and 4-1BB with an intact Fc fragment from human IgG1 or IgG4, named HK013-G1 and HK013-G4 respectively. We suspected that HK013-G1 can simultaneously exert the cytotoxic effect of CD8+T cells and NK cells on tumor cells expressing MSLN to achieve better antitumor efficacy. Methods Both HK013-G1 and HK013-G4 were constructed by fused a single-chain variable fragment (scFv) targeting hu4-1BB to the C terminus of an anti-MSLA nanobody. And their affinity was optimized to making it highly effective in tumor localization. Next, we tested the killing ability of bsAbs-mediated PBMC or NK92 against tumor cells with different expression levels of MSLN in vitro. And the IFN-γ secretion was detected when CD8+T cells co-cultured with MSLN+ or MSLN- cells in the presence of antibodies. Also, the 4-1BB agonist activity of bsAbs was measured in a luciferase report gene assay. To confirm the safety of HK013-G1, non-specific activation of 4-1BB signal mediated by Fc receptor and CRS was evaluated in vitro. Finally, we compared the antitumor activity of two bispecific antibodies in both MC38/hMSLN and CT26/hMSLN tumor model and hepatotoxicity as well as cardiotoxicity was evaluated. Results Affinity-optimized HK013-G1 has an order of magnitude greater affinity for MSLN(KD≈10−9M) than 4-1BB (KD≈10−8M). HK013-G1 induced stronger PBMC against tumor cells than MOARb009 while HK013-G4 does not. Also, HK013-G1 could only mediate the killing of NK92 on MSLN-positive tumor cells. In co-cultured assay, HK013-G1 had superior ability to stimulate CD8+T cell secretion of IFN-γ than urelumab in the presence of MSLN. In luciferase reporter assay, the bsAbs-induced 4-1BB activation is dependent on expression level of MSLN. In addition, HK013-G1 was shown no stronger ability to inducing non-specific activation of 4-1BB signal mediated by Fc receptor and CRS in vitro. Compared with HK013-G4, HK013-G1 showed a more significant anti-tumor effect in both MC38/hMSLN and CT26/hMSLN tumor model. And, HK013-G4 showed significant hepatotoxicity in mice while HK013-G1 not. Moreover, HK013-G1 can protect mice against tumor re-challenge. Conclusions HK013-G1, an MSLN×4-1BB bsAb with human IgG1 Fc fragment, prevents tumor development by killing tumor cells directly via effector functions mediated by NK and cytotoxic T cells. More importantly, HK013-G1 showed no stronger toxic side effects both in vitro and in vivo. These results show that HK013-G1 has the potential to develop into a new clinical therapy for cancer types with MSLN expression.

Abstract Mesothelin (MSLN) is a 70 KDa cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in a variety of cancers, including mesothelioma, ovarian cancer, pancreatic cancer and breast cancer. Binding of MSLN with CA125/MUC16 promotes cell proliferation, local infiltration and metastasis. CD137 is mainly expressed on the surface of activated T cells, NK cells and some other immune cells, mediating the costimulatory signal to enhance the function of T cells and improve the anti-tumor ability of immune cells. Here, we report the development of a bispecific antibody, HK013, targeting both MSLN and CD137 for the treatment of solid tumors. The bispecific antibody HK013 was generated by introducing an anti-MSLN nanobody to the C-terminus of the heavy chain of an anti-CD137 antibody. Two stages were involved: first, we compared the MSLN nanobodies with different binding epitopes to select the optimal candidate. Two nanobodies, R2G12 and R3C7, with the binding affinity of 10-9M to human MSLN were selected. R3C7 blocks the binding of MORAb-009 to MSLN, while R2G12 does not. However, the bi-specific antibody containing R2G12 exhibited stronger anti-tumor activity than that containing R3C7 in both N87 and H226 mouse xenograft models. We then determined the best isotype and format of the bispecific antibody. While IgG1 or IgG4 (FALA) were selected as the candidate isotypes, the anti-MSLN VHH were linked to the N-terminal or C-terminal of the heavy chain of anti-CD137 antibody. Results showed that in the presence of H226 cells, IFN-γ secretion of CD8+T cells was slightly higher as triggered by IgG1 isotype than that by IgG4. However, IgG1-based molecules were not as potent as those in IgG4 in N87 and H226 xenograft models. Moreover, IgG1-based molecules showed cytotoxicity on CD4+T and CD8+T cells while those in IgG4 did not. In addition, HK013 exhibited the minimal ability of non-specific activation of CD137 signaling pathway by Fc receptor in a NFκB-luciferase reporter assay.These data indicate that our bispecific antibodies containing MSLN nanobodies with different epitopes exhibit varied antitumor activity, and antibodies with similar epitopes to MORAb-009 show poor antitumor activity. Molecules in IgG1 may potentially reduce the antitumor activity due to the cytotoxicity to the effector T cells. The bispecific formats are related to the Fc receptor-mediated non-specific activation of CD137 signal, which may potentially cause safety issue. The strong antitumor activity and the lowest non-specific activation of CD137 signal of HK013 makes it the candidate worthwhile of further evaluation of its safety and efficacy. Citation Format: Xing Xu, Dayan Zhang, Wenting Liu, Guodong Shen, Liansheng Cheng. The development of HK013, a bispecific antibody targeting MSLN and CD137, for the treatment of MSLN+ solid tumors [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 LB508.

CD137 (TNFRSF9, 4-1BB) is a member of the tumor necrosis factor receptor superfamily that functions as a co-stimulatory molecule of immunocytes. Agonistic antibodies against CD137 have shown promising therapeutic activity in mouse tumor models. However, molecules in the clinical development have shown limitations due to either dose-dependent severe liver toxicity (e.g. Urelumab), or modest antitumor activity (e.g. Utomilumab). We developed ZG033, a fully human IgG4 agonist of CD137 that engages with a exclusive epitope, to achieve a better efficacy and safety profile for immunotherapy. The biophysical properties and anti-tumor activities were determined using in vitro assays and in vivo mice xenograft models, respectively. ZG033 is a safe and potent antibody targeting a ligand competitive epitope, that conserved in human, cynomolgus monkey, and is associated with a differentiated pharmacology and toxicology profile. The complex structure of ZG033/CD137 was determined by X-ray crystallography. The binding affinity to human 4-1BB of ZG033 determined by Surface plasmon resonance (SPR) was moderate (KD=10 nM). ZG033 increased the production of IFN-γ by human CD8+T,CD4+T, NK cells in a Fcγ receptor-dependent manner, displaying an intermediate activation level between that of Urelumab and Utomilumab. In addition, it led to a dose-dependent increase in the CD137-driven NFκB reporter gene activation. In syngeneic mouse tumor models, ZG033 showed robust single agent anti-tumor activity starting from 0.3 mg/kg and induced durable antigen-specific immunological memory that prevents the growth of the same tumor cells in the mice re-challenged as compared to Utomilumab. Furthermore, ZG033 combined with an anti-PD-L1 antibody (Atezolizumab) significantly inhibited the growth of colorectal tumor in a human PBMC-engrafted humanized mouse model. Data from toxicology test in humanized CD137 transgenic mice showed that ZG033 was well-tolerated with normal ALT and AST levels. Even high dose of ZG033 (MTD≥180mg/kg) was achieved in cynomolgus monkeys with no abnormality in hematological indexes, important organs. These data demonstrate that ZG033 is a novel CD137 agonist that displays an favorable safety profile and has a potential in either mono- or combinational immunotherapies. Citation Format: Liangwei Li, Wenting Liu, Guodong Shen, Dayan Zhang, Xiaoli Zheng, Liansheng Cheng. ZG033, a novel CD137 agonist, induces superior anti-tumor activity without hepatotoxicity rely on Fc-mediated crosslinking [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 LB509.

Background Colorectal cancer (CRC) is one of the most common malignancies and the patient survival rate remains unacceptably low. The anti-programmed cell death-1 (PD-1)/programmed cell death ligand 1 (PD-L1) antibody-based immune checkpoint inhibitors have been added to CRC treatment regimens, however, only a fraction of patients benefits. As an important co-stimulatory molecule, 4-1BB/CD137 is mainly expressed on the surface of immune cells including T and natural killer (NK) cells. Several agonistic molecules targeting 4-1BB have been clinically unsuccessful due to systemic toxicity or weak antitumor effects. We generated a humanized anti-4-1BB IgG4 antibody, HuB6, directed against a unique epitope and hypothesized that it would promote antitumor immunity with high safety. Methods The antigen binding specificity, affinity and activity of HuB6 were determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), biolayer interferometry (BLI) and flow cytometry. The antitumor effects were evaluated in humanized mice bearing syngeneic tumors, and possible toxicity was evaluated in humanized mice and cynomolgus monkeys. Results HuB6 showed high specificity and affinity for a binding epitope distinct from those of other known 4-1BB agonists, including utomilumab and urelumab, and induced CD8 + T, CD4 + T and NK cell stimulation dependent on Fcγ receptor (FcγR) crosslinking. HuB6 inhibited CRC tumor growth in a dose-dependent manner, and the antitumor effect was similar with urelumab and utomilumab in humanized mouse models of syngeneic CRC. Furthermore, HuB6 combined with an anti-PD-L1 antibody significantly inhibited CRC growth in vivo. Additionally, HuB6 induced antitumor immune memory in tumor model mice rechallenged with 4 × 10 6 tumor cells. Toxicology data for humanized 4-1BB mice and cynomolgus monkeys showed that HuB6 could be tolerated up to a 180 mg/kg dose without systemic toxicity. Conclusions This study demonstrated that HuB6 should be a suitable candidate for further clinical development and a potential agent for CRC immunotherapy.

Background Mesothelin (MSLN) is a ~71kDa cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in a variety of cancers.1 4-1BB is not only expressed on the surface of activated T cells and NK cells but also a marker for Treg.2 Moreover, 4-1BB shows high selectivity for human tumor-derived Tregs and is associated with worse survival outcomes in patients with multiple tumor types, such as bladder cancer, glioblastoma, prostate cancer, or renal clear cell cancer.3 Here, we developed a IgG1-based bi-specific antibody, HK013-1, targeting both MSLN and 4-1BB to achieve better antitumor therapeutic efficacy. Methods We tested the binding ability of HK013-1 to tumor cells with different expression levels of MSLN, and tested the killing ability of HK013-1-mediated NK cells against these tumor cells in vitro. Moreover, the 4-1BB agonist activity of HK013-1 was detected using CD8+T cells co-cultured with MSLN+ or MSLN- cells. To confirm the safety of HK013-1, non-specific activation of 4-1BB signal mediated by Fc receptor and killing potency to CD8+T cells and Tregs induced by HK013-1 was evaluated. In vivo, we verified the ability to inhibit tumor growth of HK013-1 and examined the effects of HK013-1 on spleen and tumor CD8+T cells and Tregs. Results HK013-1 could bind to various tumor cells that differentially expressed MSLN and induce NK cells to kill these cells. In co-cultured assay, HK013-1 increased IFN-γ production only in the presence of MSLN+ cells. Compared with anti-4-1BB parent antibody and urelumab, HK013 induced weaker FcγR-mediated 4-1BB activation. Furthermore, HK013-1 engaged NK cells to kill Treg but not CD8+T cells. In 4-1BB humanized transgenic mice, HK013-1 was revealed to reduce the proportion of Treg cells in tumor but had no effect on CD8+T cells, and CD8+T and Treg cells in the spleen. Compared with IgG4-based bi-specific antibody,IgG1-based HK013-1 showed a more significant anti-tumor effect in MC38/MSLN tumor model. Conclusions IgG1-based HK013-1 prevents tumor development by directly killing tumor cells and depleting Treg to relieve immunosuppression. Preclinical studies have shown that IgG1-based HK013-1 has good antitumor activity and safety, which may further develop its clinical potential. References Weidemann S , Gorbokon N , Hflmayer D, et al. Abstract 2833: Mesothelin expression in human tumor types: a tissue microarray study on more than 13,000 tumor samples. Proceedings: AACR Annual Meeting 2021; April 10-15. Luu K, Patwardhan MV, Zeng Q, et al. Regulatory T Cells Inhibit T Cell Activity by Downregulating CD137 Ligand via CD137 Trogocytosis. Cells. 2021;10(2):353. Freeman ZT, Nirschl TR, Hovelson DH, et al. A conserved intratumoral regulatory T cell signature identifies 4-1BB as a pan-cancer target. The Journal of Clinical Investigation. 2020;130(3).