Potent Antitumor Efficacy Research Articles

Context-dependent tumor-suppressive BMP signaling in diffuse intrinsic pontine glioma regulates stemness through epigenetic regulation of CXXC5.

The most lethal subtype of diffuse intrinsic pontine glioma (DIPG) is H3K27M. Although ACVR1 mutations have been implicated in the pathogenesis of this currently incurable disease, the impacts of bone morphogenetic protein (BMP) signaling on more than 60% of H3K27M DIPG carrying ACVR1 wild-type remain unknown. Here we show that BMP ligands exert potent tumor-suppressive effects against H3.3K27M and ACVR1 WT DIPG in a SMAD-dependent manner. Specifically, clinical data revealed that many DIPG tumors have exploited the capacity of CHRDL1 to hijack BMP ligands. We discovered that activation of BMP signaling promotes the exit of DIPG tumor cells from 'prolonged stem-cell-like' state to differentiation by epigenetically regulating CXXC5, which acts as a tumor suppressor and positive regulator of BMP signaling. Beyond showing how BMP signaling impacts DIPG, our study also identified the potent antitumor efficacy of Dacinostat for DIPG. Thus, our study delineates context-dependent features of the BMP signaling pathway in a DIPG subtype.

Novel protein kinase inhibitor TT-00420 inhibits gallbladder cancer by inhibiting JNK/JUN-mediated signaling pathway.

This study aimed to investigate the efficiency of our chemically synthesized TT-00420, a novel spectrum-selective multiple protein kinase inhibitor, in cultured cells and animal models of gallbladder cancer (GBC) and explore its potential mechanism. Multiple GBC models were established to assess the anti-tumor efficiency, toxicity, and pharmacokinetics of TT-00420. Integrated transcriptomic, proteomic and phosphoproteomic analysis was conducted to identify potential downstream effectors of TT-00420. Western blotting, qRT-PCR, nuclear-cytoplasm separation, and immunofluorescence were performed to confirm the multi-omic results and explore the molecular mechanism of TT-00420. Immunohistochemistry was used to detect FGFR1 and p-FGFR1 expression levels in GBC samples. Autodock software was utilized to investigate the potential binding mode between the TT-00420 and the human FGFR1. We found that TT-00420 exerted potent growth inhibition of GBC cell lines and multiple xenograft models. Treatment of mice with 15mg/kg TT-00420 via gavage displayed a half-life of 1.8h in the blood and rapid distribution to the liver, kidneys, lungs, spleen, and tumors at 0.25h, but no toxicity to these organs over 2weeks. Multi-omic analysis revealed c-Jun as a potential downstream effector after TT-00420 treatment. Mechanistically, TT-00420 showed rigorous ability to block FGFR1 and its downstream JNK-JUN (S63/S73) signaling pathway, and induce c-Jun S243-dependent MEK/ERK reactivation, leading to FASLG-dependent tumor cell death. Finally, we found that FGFR1 and p-FGFR1 expression was elevated in GBC patients and these levels correlated with decreased patient survival. TT-00420 shows potent antitumor efficacy and may serve as a novel agent to improve GBC prognosis.

Targeted therapy of pyrrolo[2,3-d]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment

Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies that target unique vulnerabilities in the tumor microenvironment (TME) of EOC have largely been unrealized. One strategy to achieve selective drug delivery for EOC therapy involves use of targeted antifolates via their uptake by folate receptor (FR) proteins, resulting in inhibition of essential one-carbon (C1) metabolic pathways. FRα is highly expressed in EOCs, along with the proton-coupled folate transporter (PCFT); FRβ is expressed on activated macrophages, a major infiltrating immune population in EOC. Thus, there is great potential for targeting both the tumor and the TME with agents delivered via selective transport by FRs and PCFT. In this report, we investigated the therapeutic potential of a novel cytosolic C1 6-substituted pyrrolo[2,3-d]pyrimidine inhibitor AGF94, with selectivity for uptake by FRs and PCFT and inhibition of de novo purine nucleotide biosynthesis, against a syngeneic model of ovarian cancer (BR-Luc) which recapitulates high-grade serous ovarian cancer in patients. In vitro activity of AGF94 was extended in vivo against orthotopic BR-Luc tumors. With late-stage subcutaneous BR-Luc xenografts, AGF94 treatment resulted in substantial anti-tumor efficacy, accompanied by significantly decreased M2-like FRβ-expressing macrophages and increased CD3+ T cells, whereas CD4+ and CD8+ T cells were unaffected. Our studies demonstrate potent anti-tumor efficacy of AGF94 in the therapy of EOC in the context of an intact immune system, and provide a framework for targeting the immunosuppressive TME as an essential component of therapy.

In Vivo Evaluation of (-)-Zampanolide Demonstrates Potent and Persistent Antitumor Efficacy When Targeted to the Tumor Site.

Microtubule-stabilizing agents (MSAs) are a class of compounds used in the treatment of triple-negative breast cancer (TNBC), a subtype of breast cancer where chemotherapy remains the standard-of-care for patients. Taxanes like paclitaxel and docetaxel have demonstrated efficacy against TNBC in the clinic, however new classes of MSAs need to be identified due to the rise of taxane resistance in patients. (−)-Zampanolide is a covalent microtubule stabilizer that can circumvent taxane resistance in vitro but has not been evaluated for in vivo antitumor efficacy. Here, we determine that (−)-zampanolide has similar potency and efficacy to paclitaxel in TNBC cell lines, but is significantly more persistent due to its covalent binding. We also provide the first reported in vivo antitumor evaluation of (−)-zampanolide where we determine that it has potent and persistent antitumor efficacy when delivered intratumorally. Future work on zampanolide to further evaluate its pharmacophore and determine ways to improve its systemic therapeutic window would make this compound a potential candidate for clinical development through its ability to circumvent taxane-resistance mechanisms.

Apo-Form Selective Inhibition of IDO for Tumor Immunotherapy.

The pharmacological inhibition of IDO1 is considered an effective therapeutic approach for cancer treatment. However, the inadequate response of existing holo-IDO1 inhibitors and unclear biomarkers available in clinical practice limit the possibility of developing efficacious IDO1 inhibitors. In the current study, we aimed to elucidate the activity and mechanism of a potent 1H-pyrrole-2-carboxylic acid derivative (B37) targeting apo-IDO1 and to determine its role in tumor therapy. By competing with heme for binding to apo-IDO1, B37 potently inhibited IDO1 activity, with an IC50 of 22 pM assessed using a HeLa cell-based assay. The x-ray cocrystal structure of the inhibitor-enzyme complex showed that the B37-human IDO1 complex has strong hydrophobic interactions, which enhances its binding affinity, determined using isothermal titration calorimetry. Stronger noncovalent interactions, including π stacking and hydrogen bonds formed between B37 and apo-human IDO1, underlay the enthalpy-driven force for B37 for binding to the enzyme. These binding properties endowed B37 with potent antitumor efficacy, which was confirmed in a mouse colon cancer CT26 syngeneic model in BALB/c mice and in an azoxymethane/dextran sulfate sodium-induced colon carcinogenesis model in C57BL/6 mice by activating the host immune system. Moreover, the combination of B37 and anti-PD1 Ab synergistically inhibited tumor growth. These results suggested that B37 may serve as a unique candidate for apo-IDO1 inhibition-mediated tumor immunotherapy.

Glioma-associated mesenchymal stem cells-mediated PD-L1 expression is attenuated by Ad5-Ki67/IL-15 in GBM treatment

BackgroundGlioblastoma (GBM) is a highly immunosuppressive and vascular malignant brain tumor. Current therapeutic strategies targeting tumor cells have limited efficacy because of the immunosuppressive microenvironment and vascularization. Glioma-associated mesenchymal stem cells (GA-MSCs) have been identified as important stromal components of the tumor microenvironment, owing to their contribution to tumor angiogenesis and their potential to drive glioma stem cells. However, there are no reports on the effect of oncolytic Ad5-Ki67/IL-15 on programmed death ligand 1 (PD-L1) expression and angiogenesis induced by GA-MSCs.MethodsFlow cytometry was respectively performed to detect the PD-L1 of glioma cells and programmed death protein 1 (PD-1), CD3, CD4 and CD8 in lymphocytes, as well as distribution of the cell cycle. CCK-8 assay investigated the proliferation of glioma cells and GA-MSCs in vitro. Tumor-bearing nude mice were established with U87-Luc cells and treated with the viruses, and further the IVIS spectrum was utilized to obtain luciferase images. Finally, the expression of PD-L1 in tumor tissues was also investigated using western blotting.ResultsWe found that GA-MSCs had potential to induce PD-L1 upregulation and involved in vascular mimicry in vitro. Importantly, Ad5-Ki67/IL-15 reduced PD-L1 expression of glioma cells and neovascularization by targeting GA-MSCs. Furthermore, despite the presence of GA-MSCs, the virus has the ability to generate potent antitumor efficacy in vitro and vivo.ConclusionsThese findings suggest the use of oncolytic Ad5-Ki67/IL-15 targeting GA-MSCs to treat GBM, indicating potential clinical applications.

Synthesis and Evaluation of Small Molecule Drug Conjugates Harnessing Thioester-Linked Maytansinoids.

Ligand-targeting drug conjugates are a class of clinically validated biopharmaceutical drugs constructed by conjugating cytotoxic drugs with specific disease antigen targeting ligands through appropriate linkers. The integrated linker-drug motif embedded within such a system can prevent the premature release during systemic circulation, thereby allowing the targeting ligand to engage with the disease antigen and selective accumulation. We have designed and synthesized new thioester-linked maytansinoid conjugates. By performing in vitro cytotoxicity, targeting ligand binding assay, and in vivo pharmacokinetic studies, we investigated the utility of this new linker-drug moiety in the small molecule drug conjugate (SMDC) system. In particular, we conjugated the thioester-linked maytansinoids to the phosphatidylserine-targeting small molecule zinc dipicolylamine and showed that Zn8_DM1 induced tumor regression in the HCC1806 triple-negative breast cancer xenograft model. Moreover, in a spontaneous sorafenib-resistant liver cancer model, Zn8_DM1 exhibited potent antitumor growth efficacy. From quantitative mRNA analysis of Zn8_DM1 treated-tumor tissues, we observed the elevation of gene expressions associated with a “hot inflamed tumor” state. With the identification and validation of a plethora of cancer-associated antigens in the “omics” era, this work provided the insight that antibody- or small molecule-based targeting ligands can be conjugated similarly to generate new ligand-targeting drug conjugates.

Immunosuppressive Effects and Potent Anti-tumor Efficacy of mTOR Inhibitor Everolimus in Breast Tumor-bearing Mice.

To investigate the effects of everolimus, a mechanistic/mammalian target of rapamycin (mTOR) inhibitor, on tumor growth and immune response in a mouse model of breast cancer. Human hormone receptor-positive (HR+)/human epidermal growth receptor 2-negative (HER2-) MC4-L2 cell line was used to establish a mouse model of breast cancer. The inhibitory effects of high (10 mg/kg) and low (5 mg/kg) doses of everolimus were investigated on tumor growth. Additionally, the frequency of CD4+Foxp3+ regulatory T cells (Tregs), CD8+Foxp3+ Tregs, and CD4+ and CD8+ T cells expressing cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) was explored by flow cytometry in bone marrow, lymph nodes, and spleen. Our results showed that both 10 mg/kg and 5 mg/kg doses of everolimus efficiently inhibited tumor growth, resulting in reduced breast tumor volume. In addition, it was revealed that everolimus-treated mice induced a higher frequency of CD4+Foxp3+ Tregs, CD8+Foxp3+ Tregs, and CD4+Foxp3+CTLA-4+ Tregs as well as CD4+ and CD8+ T cells expressing CTLA-4 in their bone marrow, lymph nodes, and spleen compared with standard control (vehicle-treated) in a dose-dependent manner. Furthermore, we found that everolimus treatment with 10 mg/kg and 5 mg/kg increased the frequency of Helios+Foxp3+ Tregs in the bone marrow of treated mice compared with the control group. Our results indicate that treatment with everolimus not only inhibits tumor growth but also exerts an immunomodulatory effect by inducing Tregs in the lymphoid organs of breast cancer-bearing mice. The combination of therapy with other anti-cancer agents may negate immune suppression and improve the efficacy of mTOR-targeted breast cancer therapy.

Cowpea Mosaic Virus and Natural Killer Cell Agonism for In Situ Cancer Vaccination.

We have previously shown the plant virus Cowpea mosaic virus (CPMV) to be an efficacious in situ cancer vaccine, providing elimination of tumors and tumor-specific immune memory. Additionally, we have shown that CPMV recruits Natural Killer (NK) cells within the tumor microenvironment. Here we aimed to determine whether a combination of CPMV and anti-4-1BB monoclonal antibody agonist to stimulate tumor-resident and CPMV-recruited NK cells is an effective dual therapy approach to improve NK cell function and in situ cancer vaccination efficacy. Using murine models of metastatic colon carcinomatosis and intradermal melanoma, intratumorally administered CPMV + anti-4-1BB dual therapy provided a robust antitumor response, improved elimination of primary tumors, and reduced mortality compared to CPMV and anti-4-1BB monotherapies. Additionally, on tumor rechallenge there was significant delay/prevention of tumor development and improved survival, highlighting that the CPMV + anti-4-1BB dual therapy enables potent and durable antitumor efficacy.

Abstract 5525: An Fc-competent bispecific antibody targeting PD-L1 and TIGIT induces strong immune responses and potent anti-tumor efficacy

Abstract Background: TIGIT (T-cell immunoglobulin and ITIM domain), which is primarily expressed on activated and 'exhausted' T and NK cells, is one of the most promising 'next generation' immune checkpoint target. Engagement of TIGIT to its ligands (i.e., PVR and PVRL2) leads to inhibitory signaling in T cells and promotes the functional exhaustion of tumor-infiltrating T lymphocytes. Anti-TIGIT monoclonal antibodies have shown clinical benefit when combined with anti-PD-L1 agents in NSCLC. Here, we describe our novel anti-PD-L1 × TIGIT bispecific antibody (PD-L1 × TIGIT biAb) that blocks both the PD-L1/PD-1 and TIGIT/PVR/PVRL2 pathways and has the potential to exhibit equal clinical benefit compared to current combination therapies. Methods: PD-L1 × TIGIT biAb was engineered with a fully-human IgG targeting TIGIT in a g1-Fc backbone, fused to a VHH at the C-terminus targeting PD-L1. Binding affinities and specificity testing were studied by flow cytometry and biolayer interferometry. The co-binding of the PD-L1 × TIGIT biAb to TIGIT and PD-L1 was detected by ELISA. The immunomodulatory functions of the PD-L1 × TIGIT biAb were evaluated using luciferase reporter cell assays and mixed lymphocyte reaction (MLR) assays in vitro, and human PBMC models in vivo. Results: The PD-L1 × TIGIT biAb binds with high affinity to the extracellular domain of human TIGIT and PD-L1 and can bind to TIGIT and PD-L1 simultaneously. In a competition assay, the PD-L1 × TIGIT biAb efficiently blocked the interaction between TIGIT and PVR/PVRRL2, and likewise PD-L1 to PD-1. The PD-L1 × TIGIT biAb induced higher luciferase signals than the anti-TIGIT or anti-PD-L1 mAbs alone in a luciferase reporter-based cell system and enhanced IFN-γ production in an MLR assay. In vivo, the PD-L1 × TIGIT biAb demonstrates similar anti-tumor efficacy to the combination of anti-TIGIT and anti-PD-L1 mAbs, which is stronger than the single-agents alone. We have also completed GLP-toxicity studies that have shown excellent safety. Conclusion: We have discovered a novel PD-L1 × TIGIT biAb, which induces strong immune responses in vitro and in vivo, supporting its clinical development for the treatment of human cancers. Clinical trials shall be initiating in early 2022. Citation Format: Shuang Dai, Weifeng Huang, Zhijun Yuan, Shaogang Peng, Jiayi Si, Chao Wang, Yao Yan, Xiaoniu Miao, Yingda Xu, Joanne Sun, Xiaolin Liu, Andy Tsun, Tianhang Zhai. An Fc-competent bispecific antibody targeting PD-L1 and TIGIT induces strong immune responses and potent anti-tumor efficacy [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 5525.

Abstract 5555: A bispecific antibody targeting CLDN18.2 and 4-1BB induces potent anti-tumor efficacy with an extraordinary safety profile

Abstract Background: Gastric cancer is one of the most common cancers worldwide, which is estimated to have 32.1 and 13.2 cases per 100,000 individuals in males and females, respectively. Claudin-18 isoform 2 (CLDN18.2), a membrane-bound protein involved in the formation of tight junctions, has been identified to be highly expressed in various types of cancers such as gastric and pancreatic cancer. Targeting CLDN18.2 with the monoclonal antibody (mAb) Zolbetuximab has achieved moderate clinical benefit with controllable toxicity. 4-1BB is a potent stimulator of T cells and NK cells, and when activated, can improve effector and/or memory responses. However, inherent on-target related toxic effects in the liver was observed during the clinical development of the monoclonal agonist antibody Urelumab. Here, we report the discovery of a humanized bispecific antibody (biAb) anti-CLDN18.2 x 4-1BB, which activates immune cells such as T cells via CLDN18.2-mediated crosslinking of 4-1BB. This bispecific antibody induces potent anti-tumor efficacy with limited toxicity. Methods: The anti-CLDN18.2 x 4-1BB biAb was generated by the fusion of an anti-4-1BB VHH targeting the CRD4 domain for 4-1BB, to the C-terminus of an anti-CLDN18.2 mAb via a G4S linker. Binding affinity and specificity testing of each arm were studied by flow cytometry and on the Retrogenix Cell Microarray Technology platform, a human membrane/secreted protein binding array. The immunomodulatory functions were evaluated using luciferase reporter cell assays in vitro and human 4-1BB KI mouse models in vivo. Results: The CLDN18.2 antibody interacted specifically to CLDN18.2 on the Retrogenix Cell Microarray platform among over 5,500 proteins. Next, in vitro studies showed how the molecule specifically induced T cell activation and cytokine release against CLDN18.2-positive cells. More importantly, such activation was correlated to the expression level of CLDN18.2, which indicates that the molecule may preferentially recognize CLDN18.2-high-expressing cancer cells. In mouse models, the anti-CLDN18.2 x 4-1BB biAb induced superior anti-tumor efficacy compared to anti-CLDN18.2 and anti-4-1BB mAbs alone with acceptable PK attributes. Furthermore, the anti-CLDN18.2 x 4-1BB biAb induced strong immunological memory where tumors could not grow in re-challenged tumor-free mice. Finally, the CLDN18.2 x 4-1BB biAb showed a safe profile in a GLP NHP study of which no obvious signs of toxicity were observed at 50 mg/kg, 100 mg/kg or 150 mg/kg doses. Conclusion: In summary, a bispecific antibody targeting CLDN18.2 and 4-1BB was developed, which displayed potent anti-tumor efficacy with strong immunological memory and has shown good safety in NHPs. Moreover, the molecule has high selectivity to CLDN18.2. The molecule shall enter clinical trials by early 2022. Citation Format: Junying Chen, Weifeng Huang, Zhiquan Liang, Xiaoniu Miao, Shaogang Peng, Chao Wang, Tiantian Dong, Andy Tsun, Yi Luo. A bispecific antibody targeting CLDN18.2 and 4-1BB induces potent anti-tumor efficacy with an extraordinary safety profile [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 5555.

Abstract 383: ONCR-021 as a systemic intravenous synthetic RNA virus immunotherapy for the repeat treatment of cancer

Abstract Oncolytic viruses (OV) have shown great potential to improve clinical outcomes when dosed intratumorally, however, their therapeutic efficacy when intravenously administered is likely limited by the rapid emergence of neutralizing antibodies. To overcome this limitation, we developed Synthetic RNA viruses consisting of a replication competent viral genomic RNA (vRNA) encapsulated within a lipid nanoparticle (LNP) for IV administration. Upon dosing and delivery of this infectious RNA payload, the vRNA initiates viral replication and virus production in neoplastic cells leading to oncolysis and tumor destruction. This formulation enables repeat intravenous dosing of a replication competent oncolytic virotherapy even in presence of circulating neutralizing antibody to the virus. Here we present ONCR-021, an LNP formulation of Coxsackievirus A21 (CVA21) vRNA. ONCR-021 vRNA encodes a novel ICAM1-dependent strain of CVA21 that results in greater in vitro and in vivo oncolysis compared to the previously described CVA21 Kuykendall strain. ONCR-021 is broadly oncolytic in cancer cell lines in vitro and is intended for clinical development in NSCLC, RCC, and HCC based upon the viral tropism. IV-administration of ONCR-021 vRNA results in rapid initiation of viral replication, oncolysis, and potent anti-tumor efficacy, even in the presence of circulating CVA21 neutralizing antibodies. This efficacy is principally driven by CVA21 amplification in situ after delivery to tumor cells and we demonstrate viral replication, virion production and spread within the tumor after dosing. We also observe only modest and transient production of CVA21 in healthy tissues of transgenic mice expressing the CVA21 entry receptor human ICAM1. Consistent with these findings, high doses levels of ONCR-021 were well-tolerated in this model. Altogether, these preclinical data support the development of ONCR-021, a novel synthetic oncolytic virus designed to overcome the challenges of repeat intravenous administration of viral immunotherapy for the treatment of disseminated cancers. Citation Format: Jeffrey Bryant, Agnieszka Denslow, Jacqueline Hewett, Lingxin Cong, Ana De Almeida, Jennifer Lee, Judy Jacques, Sonia Feau, Daniel. Wambua, Adrienne Yanez, Pam Shou-Ping Wang, Jessica Deterling, Matthew Scott, Jason Auer, Brian B. Haines, Christophe Quéva, Lorena Lerner, Edward M. Kennedy. ONCR-021 as a systemic intravenous synthetic RNA virus immunotherapy for the repeat treatment of cancer [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 383.

Abstract 5528: A highly potent anti-CD39 biparatopic antibody and bispecific for cancer therapy

Abstract Background: There is an increasing demand for effective combinatory agents to improve upon PD-1/PD-L1-based therapeutics. One combinatory target axis is the adenosine metabolism pathway that consists of three major players, including CD39, CD73 and A2AR. Inhibition of any of these targets have shown enhanced preclinical efficacy in combination with PD-1/PD-L1 inhibitors. CD39 is an ectonucleotidase which degrades extracellular ATP to adenosine monophosphate (AMP). This is considered a rate-limiting step for the further degradation to adenosine by CD73. Adenosine is an immunosuppressive metabolite that can suppress NK and T cells. Blockade of CD39-mediated degradation of ATP to AMP may therefore recover anti-tumor immunity through preventing the enrichment of adenosine in the tumor microenvironment. Method: Two anti-CD39 VHH molecules were generated, named Ye-37 and Ye-46, which bind to two different epitopes on CD39. Binding experiments were carried out by bio-layer interferometry. Cell binding experiments were tested on CD39-overexpression cell lines by flow cytometry. Cellular CD39 enzymatic inhibition experiments were tested using an MOLP-8 cell line and PBMC via a luminescence-based assay. Soluble CD39 enzymatic tests were carried out on recombinant CD39 protein using a similar method. T cell proliferation assays were performed and observed on CD4+ or CD8+ T cell populations. In vivo efficacy studies were tested in B-NDG B2M-KO mice that were injected subcutaneously with A375-CD39+ tumor cells and human PBMC. An anti-PD1 x CD39 bispecific antibody was then generated by fusing the anti-CD39 biparatopic antibody to the C-terminus of an anti-PD1 IgG and tested using similar methods. Results: Two candidates, Ye-37 and Ye-46, were selected for their functional activity that recognize non-overlapping epitopes on CD39. The combination of Ye-37 and Ye-46 shows high potency in cell-based and soluble CD39 assays in blocking CD39 activity. Two biparatopic molecules were generated by fusing the biparatopic unit to the N- or C-termini of Fc (46-37-Fc and Fc-37-46) and showed similar activity to the combination. In vivo, we showed single-agent control of tumor growth and potentiation of tumor-growth inhibition when combined with anti-CD73 antibodies. An anti-PD1 x CD39 bispecific was generated and showed potent inhibition of PD-1/PD-L1 interactions by cell-based assays. Potent anti-tumor efficacy was shown, which was as effective as the combination of anti-PD1 plus anti-CD39 antibodies. Conclusion: Potent anti-CD39 and anti-PD1 x CD39 therapeutic candidates have been generated with promising activity as a combinatory or single agent, respectively. As such, we plan to file for clinical trial authorization of these programs by 2022. Citation Format: Zhenqing Zhang, Yunli Jia, Xiaoniu Miao, Weifeng Huang, Chao Wang, Zhijun Yuan, Wenchao Jiang, Liandi Chen, Zhiyuan Li, Andy Tsun. A highly potent anti-CD39 biparatopic antibody and bispecific for cancer therapy [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 5528.

Abstract 1826: Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers

Abstract Background: Disulfiram (DSF) in combination with copper pills has emerged as a safe and exciting anticancer strategy and is undergoing clinical trials in multiple human cancers. However, DSF breaks down in the gut or blood immediately and chances of cupric diethyldithiocarbamate (Cu-DDC), the active cytotoxic principle being formed at tumor sites is very low. Therefore, we have found that synthetic Cu-DDC is directly and potently cytotoxic against p53 mutant cancers. We report here for the first time the differential effects of Cu-DDC in robustly eliminating the mutant p53 proteins from tumor cells in contrast to its augmentation of the wild-type p53 protein and activation. Destabilization of Gain Of Function (GOF) p53 mutant proteins for overcoming oncogenic addiction and improving the therapeutic efficacy has been a long-sought, but unmet anticancer avenue. Methods: A panel of 7 P53 mutant cell lines (R248W, R273H,R173H), and p53 wt tumor and normal cells (HPBLs, dental pulp stem cells, IMR90 and HUVEC) were used. Immunoprecipitation, Western blotting, Annexin-V apoptotic assays, flow cytometry, immunocytochemistry were used to quantitate p53 protein. Mia-Paca2 xenografts developed in nude mice were administered Cu-DDC/BSA. Results: Cu-DDC generated peroxides but not superoxides in tumor cells. The GOF mutant p53 proteins, R248W and R273H in three different tumor cell lines were eliminated after Cu-DDC treatment in a time- and concentration-dependent manner. The loss of mutant p53 proteins was confirmed by three different procedures. The degradation occurred rapidly in 8-10 h at 100 nM Cu-DDC and was inhibited by proteasome blockers. The expression of the GRO-α oncogene, positively regulated by GOF R248W was decreased in Cu-DDC treated Mia-Paca2 cells. Cellular proteasome activity was not inhibited by Cu-DDC. Ubiquitination of mutant p53 proteins was not altered by Cu-DDC. In contrast, the wild-type p53 present in HCT116 and MCF-7 cells, and normal cell counterparts (HPBL, IMR-90 lung cells) was significantly upregulated and activated with enhanced p21cip1 expression. Cu-DDC triggered strong apoptosis irrespective of the p53 status, although the mutant p53 containing cells were 2 to 3-fold more sensitive in cytotoxicity assays (IC50 range 0.25-0.66 µM). A significant tumor growth delay accompanied by loss of mutant p53 protein was observed in MiaPaca2 xenografts. Conclusions: In moving the DSF anticancer repurposing forward, we describe the unique ability of Cu-DDC to rapidly eliminate oncogenic p53 mutant proteins and confer potent antitumor efficacy. The degradation was unrelated to the proteostasis involving the inactivation of NPL4 by Cu-DDC described previously (supported by a Cancer Prevention and Research Institute of Texas [CPRIT] grant RP170207 to KSS). Citation Format: Viswanath Arutla, Kalkunte S. Srivenugopal. Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers [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 1826.

Abstract 5518: Single cell RNA sequencing revealed that the mechanism of the synergistic efficacy of STA551 and immune checkpoint inhibitor results from the induction of highly activated T cells in tumor

Abstract Background: CD137 (4-1BB) is a co-stimulatory molecule which exerts activation signals in multiple immune cell subsets. However, agonist antibodies targeting CD137 have not been clinically successful due to severe systemic toxicity. Therefore, we generated STA551, a tumor selective CD137 agonist antibody. STA551 binds to CD137 only in the presence of ATP, which is known to be present at high levels in tumor tissue, and activates immune cells such as T cells. Previous studies have shown that STA551 exhibits antitumor efficacy without inducing a systemic immune response in a murine model. Combination studies with anti-PD-L1 antibody have also shown synergistic efficacy without inducing a systemic immune response. However, it was unclear why they exhibit strong combination effects. In this study, we elucidated the mechanism of STA551 and anti-PD-L1 antibody efficacy through comprehensive analysis of gene expression using single cell RNA sequencing. Method & Result: In a colon 38-bearing human CD137 knock-in mouse model, combination therapy with STA551 and anti-PD-L1 antibody showed potent anti-tumor efficacy. From immunohistochemistry of tumor tissue, increased numbers of CD8+ T cells and elevated expression of PD-L1 molecules were observed. To perform a detailed mechanistic analysis, tumor tissue was collected after antibody administration and CD3 positive and CD3 negative fractions were separated and gene expression was comprehensively analyzed using single cell RNA-seq. Next, the fraction expressing Cd8a gene was extracted from CD3 positive fraction and clustered. One cluster was found to be markedly increased in STA551 and anti-PD-L1 antibody combination compared to vehicle or monotherapy treatment. Differential gene expression analysis revealed that the cluster was enriched for genes related to cytokines and chemokines. This result revealed that the combination of STA551 and anti-PD-L1 antibody induced highly activated CD8+T cells. Conclusion: The combination of STA551 and PD-L1 showed synergistic anti-tumor efficacy in a colon38-bearing human CD137 knock-in mouse model. Detailed mechanistic analysis showed that the combination of STA551 and PD-L1 induced highly activated CD8+T cells expressing chemokines and cytokines compared to monotherapy. This cluster also express chemokines, suggesting that other immune cells have been recruited. These results suggest that even the activation of only tumor tissue, which is a characteristic of STA551, can induce a strong immune response. Citation Format: Ryo Uchikawa, Kazuya Takakuwa, Hirofumi Mikami, Shoichi Metsugi, Kenji Taniguchi, Yoshinori Narita, Mika Kamata-Sakurai. Single cell RNA sequencing revealed that the mechanism of the synergistic efficacy of STA551 and immune checkpoint inhibitor results from the induction of highly activated T cells in tumor [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 5518.

Abstract 5527: A novel fully human anti-TIGIT and PVRIG bispecific antibody that elicits potent anti-tumor efficacy in pre-clinical studies

Abstract Background: TIGIT (T-cell immunoglobulin and ITIM domain), which is primarily expressed on activated and 'exhausted' T and NK cells, is one of the promising 'next generation' immune checkpoint molecules. Engagement of TIGIT to its ligands (i.e., PVR and PVRL2) leads to inhibitory signaling in T cells, promoting functional exhaustion of tumor-infiltrating T lymphocytes. Anti-TIGIT monoclonal antibodies have shown clinical benefit when combined with anti-PD-L1 agents in NSCLC. However, the single-agent efficacy of anti-TIGIT therapies have been limited. PVRIG (PVR-related immunoglobulin domain containing), which is another coinhibitory receptor of the DNAM/TIGIT/CD96 nectin family, binds with high affinity to PVRL2 and suppresses T-cell function, and shows nonredundant inhibitory effects alongside the TIGIT/PVR/PVRL2 axis. Here, we report a fully-human anti-TIGIT × PVRIG bispecific antibody (anti-TIGIT × PVRIG biAb), which blocks both the PVRIG/PVRL2 and TIGIT/PVR/PVRL2 pathways, that maintains the efficacy of the combination of the two mono-agents. The anti-TIGIT × PVRIG biAb is also highly efficacious when combined with PD1/PD-L1 inhibitors in mouse tumor models. Methods: An anti-TIGIT × PVRIG biAb was generated through the fusing of a fully-human IgG targeting TIGIT with a wild type G1-Fc to a fully-human scFv at the c-terminus targeting PVRIG. Binding affinity and specificity analyses were studied by flow cytometry and biolayer interferometry. The co-binding of the anti-TIGIT × PVRIG biAb to TIGIT and PVRIG was detected by ELISA. The immunomodulatory functions of the anti-TIGIT × PVRIG biAb were evaluated using a luciferase reporter cell assay in vitro and human PBMC-based tumor models in vivo. Results: The anti-TIGIT × PVRIG biAb binds with high affinity to the extracellular domain of human TIGIT/PVRIG and can bind to TIGIT and PVRIG simultaneously. In a competition assay, the anti-TIGIT × PVRIG biAb efficiently blocked the interaction between TIGIT and PVR/PVRRL2, and PVRIG with PVRL2. In a luciferase reporter cell system, the anti-TIGIT × PVRIG biAb induced high levels of luciferase activity compared with the anti-TIGIT or anti-PVRIG mAbs alone. In vivo, the anti-TIGIT × PVRIG biAb demonstrated stronger anti-tumor efficacy than the anti-TIGIT and anti-PVRIG mAbs as monotherapies or combined with anti-PD-1 mAb. Conclusion: Our anti-TIGIT × PVRIG biAb, a fully human bispecific antibody, either alone or in combination with anti-PD-1 mAb promotes immune cell activation both in vitro and in vivo, supporting its clinical development for the treatment of human cancers. The molecule is currently under GLP-toxicity evaluation in NHP, and a first-in-human study is expected to begin in 2022. Citation Format: Shuang Dai, Weifeng Huang, Zhijun Yuan, Shaogang Peng, Jiayi Si, Chao Wang, Xiaoniu Miao, Yingda Xu, Joanne Sun, Xiaolin Liu, Andy Tsun, Tianhang Zhai. A novel fully human anti-TIGIT and PVRIG bispecific antibody that elicits potent anti-tumor efficacy in pre-clinical studies [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 5527.

Abstract 5523: LBL-019, a novel anti-TNFR2 antibody, shows a potent anti-tumor efficacy in a mouse MC38 model

Abstract Objective: Although immunotherapies, e.g., anti-PD-(L)1 has achieved great success in treating cancers, a large portion of patients still cannot benefit from it, due largely to primary refractory or acquired resistance leading to unsatisfied ORR or relapse. TNFR2 is a receptor for TNF-α, expressed highly selective in immune cells, and some tumor cells, transducing survival and immunostimulatory signal to cells expressing this receptor. Targeting TNFR2 may modulate this signaling, therefore a humanized anti-TNFR2 antibody, LBL-019, was developed for monotherapy or in combination with other anti-tumor therapies. Methods: LBL-019 is a monoclonal antibody of IgG1 subtype. The binding affinity to TNFR2 was determined by Fortebio. The epitope of TNFR2 was identified by differential binding to a serial human/mouse chimeric TNFR2 proteins. The binding of TNF-α to either TNFR2 protein or TNFR2 expressed in cells was examined by using ELISA and flow cytometry. The in vitro activity of LBL-019 was measured using several in vitro assays, e.g., cell proliferation and cytokine release in T cells, ADCC and CDC assays. The anti-tumor activity of LBL-019 was investigated in a model of human TNFR2 transgenic mice (Shanghai Model Organisms) implanted with MC38-OVA cells. Results: The binding affinity of LBL-019 was measured as 5.32 nM to human and 3.62 nM to cynomolgus TNFR2. It was found that TNFR2 was expressed in immune cells including T cells and monocytes examined in human PBMC, which was upregulated when T cells were activated. TNFR2 was also found highly expressed in some tumor cell lines and tumor TILs (from in vivo studies described below). The epitope of TNFR2 that LBL-019 binds was identified and mapped out CRD1 domain. The binding of TNF-α with either TNFR2 protein or TNFR2 expressed in immune cells was blocked by LBL-019. The growth of CD8+ T cells and CD4+ T cells was significantly enhanced, when incubated with LBL-019, along with elevated IFN-γ release. In a model of human TNFR2 transgenic mice implanted with MC38-OVA cells, LBL-019 was shown significant tumor growth inhibition (TGI) in a dose dependent manner as compared with vehicle treated mice (p<0.01). It appeared that the antibody with wild type Fc showed a better anti-tumor activity than that with silenced Fc, suggesting that Fc mediated NK killing of tumor cells or Treg may have contributed in part to the observed anti-tumor efficacy in this model. In addition, LBL-019 was also exhibited a synergy of anti-tumor activity, when dosed with anti-PD-1 (TGI reaching 79.51%). Conclusions: LBL-019, a humanized anti-TNFR2 antibody, shows great anti-tumor efficacy in a mouse tumor model, likely due to the mechanisms involving enhanced proliferation of Teff, elevated IFN-γ release, and possibly killing of tumor cells and Treg. The data support further development of LBL-019 as a novel therapeutic antibody for cancer, and clinical trial is expected to launch early 2022. Citation Format: Huan Lin, Xiao Huang, Yurong Qin, Yujia Dang, Peng Zhang, Jianming Sun, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-019, a novel anti-TNFR2 antibody, shows a potent anti-tumor efficacy in a mouse MC38 model [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 5523.

Abstract 5507: Generating optimal-affinity T cell receptors targeting the shared neoantigen KRAS G12V using the humanized TCR transgenic mouse platform HuTCR

Abstract Background T cell receptor (TCR)-engineered T cell therapy (TCR-T) is a promising therapeutic modality to address key limitations of targeting solid tumors. Neoantigens are excellent therapeutic targets for TCR-T cell therapy, because they are highly specific and often homogeneously expressed in the tumor. While most neoantigens are patient-specific, there are rare exceptions of widely shared neoantigens, such as the driver oncogene KRAS containing mutations at position 12. The KRAS mutations G12C, G12D, and G12V are among the most common mutations in solid tumors, including indications with high unmet medical need, such as pancreatic, colorectal, and non-small cell lung cancer. Previous TCR-T approaches targeting KRAS G12V have been primarily focused on HLA-A*02:01, which appeared to be unable to present a mutant KRAS epitope. Here, we aim to generate TCRs of optimal affinity and high specificity to a KRAS G12V epitope in an HLA-agnostic manner using our HuTCR mouse platform. Experimental procedures HuTCR mice represent a fully humanized TCR-MHC system and bear a diverse TCR repertoire, being transgenic for the entire human TCR alpha and beta gene loci and multiple human HLA class I molecules, while lacking murine TCRs and murine MHC class I molecules. The multi-HLA HuTCR mice were immunized with KRAS G12V peptides or the neoantigen-encoding adenovirus, leading to robust immune responses. Results Single-cell sequencing of responding T cells revealed HLA-A*11:01-restricted immune responses, indicating that the epitope presented on HLA-A*11:01 was more immunogenic than epitopes presented on other HLA alleles. HLA-A*11:01 is expressed in approximately 11% of the United States and the European Union population and in around 30% of Asian populations. We were able to generate 30 KRAS G12V-specific TCRs, which we characterized for efficacy and safety. We selected a lead candidate, which demonstrated potent anti-tumor efficacy and no detectable off-target toxicity, to be pursued further into the clinic. Conclusion Using the HuTCR mouse platform, we identified TCRs of optimal affinity and high specificity for the shared oncogenic KRAS G12V mutation. Importantly, the HuTCR platform was a powerful tool enabling us to determine the HLA allele responsible for generating the strongest immune response, and consequently, to define the patient population that may benefit most from targeting KRAS G12V. Citation Format: Alexei Leliavski, Lorenz Knackstedt, Jennifer Oduro, Claudia Selck, Arunraj Dhamodaran, Thomas Blankenstein, Elisa Kieback, Lucia Poncette. Generating optimal-affinity T cell receptors targeting the shared neoantigen KRAS G12V using the humanized TCR transgenic mouse platform HuTCR [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 5507.

Abstract 5554: A bispecific T cell engager targeting CEACAM5/6 exhibits strong anti-tumor efficacy in pre-clinical studies

Abstract Background: The carcinoembryonic antigen-related adhesion molecules (CEACAM), containing seven members, are involved in many biological processes including cell adhesion, proliferation, differentiation, and tumor suppression. Some variants of CEACAMs such as CEACAM5 and CEACAM6 have long been recognized to be overexpressed in many cancers and highly associated with cancer development. Immunotherapies targeting CEACAM have achieved some clinical benefit, e.g. Tusamitamab Ravtansine (anti-CEACAM5 ADC), that has shown decent disease control in NSCLC. Recently, with the success of CAR-T and T cell engager (TCE) therapies against blood cancers, TCEs against solid tumors have also been developed. For instance, Cibisatamab, targeting CEACAM5 and CD3, is currently under clinical investigation in CRC patients. Here, we report the discovery of a bispecific antibody (biAb) anti-CEACAM5/6 x CD3 that mediates direct T cell killing against CEACAM5- and/or CEACAM6-expressing cancer cells. Methods: Anti-CEACAM5/6 x CD3 TCE consists of two CEACAM5/6 binding Fabs for the preferential recognition of CEACAM5/6-high-expressing cancer cells, and one CD3 binding domain that permits CEACAM5/6-mediated CD3 crosslinking and T cell activation. To reduce off-target T cell stimulation there is no CH2 in this format to eliminate Fc receptor binding, while an anti-HSA VHH was included into this construct for half-life extension. Binding affinity and specificity were studied by flow cytometry, IHC and on the Retrogenix Cell Microarray Technology platform, a human membrane/secreted protein binding array. The immunomodulatory functions were evaluated using luciferase reporter cell assays, cell killing assays in vitro and via PBMC-based Hu-NSG mouse models in vivo. Results: Our CEACAM5/6 binding antibody only interacted with CEACAM5 and CEACAM6 on the Retrogenix Cell Microarray against over 5,500 proteins. Importantly, it showed a similar sensitivity to recognize CRC tumor tissues in an IHC array as the benchmark (BMK), but significantly better selectivity at recognition of tumor tissues vs. normal tissues. Next, in vitro testing illustrated CEACAM5/6-mediated T cell activation by the TCE, and such stimulation also correlated with the binding affinity of the anti-CD3 domain. The lead candidate with the optimized CD3 binding affinity, showed better anti-tumor efficacy than the BMK in mice. Furthermore, this molecule displayed a typical PK as a conventional antibody with a mean T1/2 of around 115hrs. Finally, we show high tolerance in NHP of which no obvious signs of toxicity were observed at 2.5 mg/kg. Conclusion: In summary, a bispecific TCE with high selectivity to CEACAM5/6 overexpressing cancer cells was engineered. It displays potent anti-tumor efficacy, and support from target selectivity testing shows promising safety traits. Citation Format: Liandi Chen, Weifeng Huang, Xiaoniu Miao, Jiayi Si, Chao Wang, Tiantian Dong, Andy Tsun, Yi Luo. A bispecific T cell engager targeting CEACAM5/6 exhibits strong anti-tumor efficacy in pre-clinical studies [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 5554.

Abstract 5532: An ‘anti-PD1-IL2 beta-only super-agonist’ displays potent anti-tumor efficacy

Abstract Introduction: Combination studies with engineered IL-2 variants and immune-checkpoint blockade have demonstrated synergy in both pre-clinical models and the clinic. Co-administration of those 2 agents can potentially face challenges related to overlapping toxicities in obtaining optimal doses for synergistic effects. We propose to potentiate the synergy by enabling cis activation of IL-2 agonism and blockade of PD-1/PDL-2 binding with a single anti-PD1-IL2 to generate Bifunctional SuperKines for ImmunoTherapy (BiSKIT). Both human (h) and mouse (m) versions of anti-PD1 are fused with MDNA109FEAA, an IL-2 superkine with enhanced affinity for IL-2Rβ (CD122) and no binding to IL-2Rα (CD25). Experimental Procedure: Studies included binding analyses with Biacore/SPR, signaling analyses in an IL-2 cell reporter assay, cell proliferation assay, in vitro pSTAT5 signaling in human PBMCs, PD1/PDL-1 cell reporter assay and efficacy in syngeneic tumor models. Summary of Data: Anti-hPD1-MDNA109FEAA and surrogate anti-mPD1-MDNA109FEAA were tested in both in vitro and in vivo assays. Both constructs demonstrated selective binding to either hPD1 or mPD1 as expected, and the MDNA109FEAA motif retained its receptor selectivity (enhanced affinity to CD122 and no binding to CD25). In both HEK-Blue IL-2 reporter and CTLL-2 proliferation bioassays, anti-PD1-MDNA109FEAA exhibited similar agonistic effect as MDNA109FEAA-Fc, demonstrating that potency towards the IL-2 axis was not diminished. This was further confirmed with pSTAT5 signaling in primary human PBMCs where anti-PD1-MDNA109FEAA showed enhanced activation of CD8 T-cells and NK cells with reduced activity on Treg when compared to rhIL-2. Both constructs were also effective at inhibiting PD1/PDL-1 pathway in respective human and mouse specific cell-based reporter assays. In a CT26 syngeneic colon tumor model, anti-mPD1-MDNA109FEAA exhibited superior efficacy over monotherapy and combination with anti-mPD1 and MDNA109FEAA-Fc when administered at equal molar dosage and same schedule. Conclusion: We have engineered anti-PD1-MDNA109FEAA to enable cis engagement with IL-2 receptor and PD1 as an approach to activate immune cells while reducing their exhaustion. Anti-mPD1-MDNA109FEAA achieved potent efficacy in a CT26 colon tumor model and is currently being evaluated in additional syngeneic tumor models with varying responsiveness to immune checkpoint blockade. Citation Format: Minh D. To, Manjunatha Ankathatti Munegowda, Rosemina Merchant, Aanchal Sharma. An ‘anti-PD1-IL2 beta-only super-agonist’ displays potent anti-tumor efficacy [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 5532.