Bystander Killing Effect Research Articles

Abstract 1133: A B7-H4 targeting antibody-drug conjugate shows anti-tumor activity in PARPi and platinum resistant cancers with B7-H4 expression

Abstract Purpose: Platinum and PARP inhibitors (PARPi) demonstrate activity in breast and ovarian cancers, but drug resistance ultimately emerges. B7-H4 is over expressed in breast and ovarian cancer compared to normal tissues making it a promising target for therapies, however, to date it has not been evaluated if the expression is maintained in drug resistant cancer. Here we examine B7-H4 expression in high grade serous ovarian carcinoma (HGSOC) tumors at diagnosis and post-platinum or PARPi-resistance. We also evaluate the activity of a novel B7-H4-directed antibody-drug conjugate (ADC) bearing the pyrrolobenzodiazepine-dimer payload tesirine, in preclinical models of breast and ovarian cancer, including those resistant to standard of care therapies. Experimental Design: B7-H4 expression was measured by quantitative flow cytometry and immunohistochemistry. ADC efficacy was tested against multiple cell lines in vitro and patient-derived xenografts (PDX) in vivo. The effect of ADC treatment on cell cycle, DNA damage, and apoptosis was measured using flow cytometry. Results: B7-H4 was over-expressed in 92% of HGSOC tumors at diagnosis (n = 12), persisted in recurrent matched samples after platinum treatment, and was expressed at similar levels at multiple metastatic sites after acquired multi-drug resistance (n = 4 donors). Treatment with the ADC resulted in target-specific growth inhibition in a panel of ten B7-H4 expressing ovarian and breast cancer cell lines (IC50 6.18-273.9 pM, median 48.3 pM). Co-cultures of B7-H4 +/- lines support bystander killing effects leading to further tumor cell death in B7-H4- clones. In platinum- or PARPi-resistant ovarian cancer cells, ADC treatment significantly decreased viability and colony formation (P < 0.001) while increasing S or G2/M phase cell cycle arrest (P < 0.001) and DNA damage (P < 0.0001), ultimately leading to apoptosis (P < 0.01). A single 0.3 mg/kg dose of the ADC resulted in tumor regression in 61% of breast and ovarian PDX models tested (n = 23), where lower activity was identified in B7-H4 low or negative expressing models (P = 0.048). In PARPi- and platinum-resistant HGSOC models (n = 3), continuous B7-H4 ADC treatment (dosed once every 28 days) resulted in sustained anti-tumor activity, leading to complete (7 of 16) or partial responses (3 of 16) and increased survival (P < 0.004). Conclusions: These data support B7-H4 as an attractive ADC target for treatment of drug-resistant HGSOC. Citation Format: Sarah B. Gitto, Margaret Whicker, Gareth Davies, Sushil Kumar, Krista Kinneer, Arthur Lewis, Srinivas Mamidi, Sergey Medvedev, Judith Anderton, Jessica Tang, Benjamin Ferman, Steve Coats, Robert W. Wilkinson, Eric J. Brown, Daniel J. Powell, Fiona Simpkins. A B7-H4 targeting antibody-drug conjugate shows anti-tumor activity in PARPi and platinum resistant cancers with B7-H4 expression [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 1133.

Abstract P4-01-12: BAT8006, a novel FRα ADC with strong bystander effect, for the treatment of advanced solid tumor

Abstract FRα is overexpressed in a number of cancers, including ovarian, breast, lung cancer, and endometrial carcinoma, yet limited expression in normal tissue. Efficacy of anti-FRα ADC have been demonstrated in clinical trials for high FRα expression ovarian cancer. These results validate FRα as a viable target for cancer treatment. We have developed a novel FRα ADC with strong bystander killing effect, BAT8006, which consists of a high-affinity antibody, an enzymatically cleavable linker and a topoisomerase I inhibitor as the payload, with DAR of 8. BAT8006 shows high affinity for the FRα antigen (KD < 0.5 nM) and FRα-expressing tumor cells. Upon binding to FRα on cell surface, BAT8006 internalizes effectively and traffics to the lysosome where payload is proteolytically released. BAT8006 demonstrated potent in vitro cell growth inhibitory activity to FRα-positive cells with IC50 values of ~ 1 nM. In an in vitro bystander killing assay, proliferation of FRα-negative cells was potently inhibited by addition of culture medium of BAT8006-treated FRα-positive cells, but not that of BAT8006-treated FRα-negative cells, indicating the bystander killing effect of the released payload in the culture medium. Less than 0.03% of the payload was released from BAT8006 when incubated with human or monkey plasma for 4 days in 37oC, suggesting the stability of BAT8006 in blood circulation. BAT8006 led to potent and dose-dependent tumor regression in patient-derived xenograft (PDX) model of SCLC. Furthermore, single dose of 2.5 mg/kg of BAT8006 showed complete eradication of tumor in choriocarcinoma JeG-3 xenograft mice model. In PDX of ovarian cancer, 2.5 mg/kg of BAT8006 (DAR8) demonstrated superior tumor inhibition activity than 5 mg/kg of BAT8006 with DAR4, suggesting the high DAR approach of BAT8006 would be more effective. In addition, BAT8006 showed favorable pharmacokinetic and safety profiles in cynomolgus monkeys with the highest non-severely toxic dose (HNSTD) of 30 mg/kg when dosed once every three weeks for 3 times. Together these results indicate that BAT8006 could potentially provide a therapeutic benefit to treat FRα-positive tumors including breast cancer in clinical trial. Citation Format: siqi mai, xingxing Mei, weijia Tang, Xin Zhou, Xuekang qi, Zhi Zhong, Shuoxu Li, Jianjun fan, Jirong Gan, Binghua Tan, Yao Qi, Yanling Guo, Shengfeng li, Jin-Chen Yu. BAT8006, a novel FRα ADC with strong bystander effect, for the treatment of advanced solid tumor [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-12.

Abstract P4-01-32: BAT8008, a novel Trop-2 ADC with strong bystander effect, for the treatment of Trop-2 positive cancer

Abstract Trophoblast cell surface antigen 2 (Trop-2) is overexpressed on many epithelial carcinomas, yet is expressed at much lower level on normal tissue. Elevated expression of TROP-2 is often associated with tumor invasion/aggression, progression, and metastasis. Efficacy of anti-TROP2 ADC have been demonstrated in clinical trials for both TNBC (triple negative breast cancer) and HR+/HER2- breast cancer. We have developed a novel Trop-2 ADC with strong bystander killing effect, BAT8008, which consists of an enzymatically cleavable linker and a topoisomerase I inhibitor as the payload, with DAR of 6. BAT8008 demonstrated potent in vitro cell growth inhibitory activity to Trop2-positive cells with IC50 values of < 1 nM. In an in vitro bystander killing assay, proliferation of Trop-2-negative cells was potently inhibited by addition/transfer of culture medium of BAT8008-treated Trop-2-positive cells, but not that of BAT8008-treated Trop-2-negative cells, indicating the bystander killing effect of the released payload in the culture medium. Less than 0.1% of the payload was released from BAT8008 when incubated with human or monkey plasma for 7 days in 37°C, suggesting the stability of BAT8008 in blood circulation. Single dose of BAT8008 at 1 and 2.5 mg/kg could inhibit 73% and 81% tumor growth in the Trop-2-positive MDA-MB-468 and MX-1 xenograft mice model, respectively. BAT8008 also demonstrated superior tumor inhibition activity than a modified ADC using Daiichi’s ADC technology or Trodelvy in a pancreatic BxPC-3 xenograft mice model. In addition, BAT8008 showed favorable pharmacokinetic and safety profiles in cynomolgus monkeys with the highest non-severely toxic dose (HNSTD) of at least 25 mg/kg when dosed once every two weeks for 3 times. Together these results indicate that BAT8008 could potentially provide a therapeutic benefit to treat TROP2-positive breast cancers in clinical trial. Citation Format: Xingxing Mei, Weijia Tang, Xin Zhou, Xuekang Qi, Siqi Mai, Zhi Zhong, Shuoxu Li, Jianjun Fan, Jirong Gan, Binghua Tan, Yao Qi, Yanling Guo, Jin-Chen Yu, Shengfeng Li. BAT8008, a novel Trop-2 ADC with strong bystander effect, for the treatment of Trop-2 positive cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-32.

Trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody–drug conjugate with topoisomerase I inhibitor payload, shows antitumor activity in uterine and ovarian carcinosarcoma with HER2/neu expression

ObjectivesCarcinosarcomas are highly aggressive gynecologic malignancies containing both carcinomatous and sarcomatous elements with heterogeneous HER2/neu expression and limited therapeutic options. We compared the efficacy of trastuzumab deruxtecan (DS-8201a), a novel HER2/neu-targeting antibody-drug conjugate (ADC) to an ADC isotype control (MAAA-9199) against primary uterine and ovarian carcinosarcomas in vitro and in vivo. MethodsTwelve primary carcinosarcoma (CS) cell lines were evaluated for HER2/neu surface expression by immunohistochemistry (IHC) and by flow cytometry, and gene amplification by fluorescence in situ hybridization (FISH) assays. The in vitro experiments included cytotoxicity and bystander killing effect assays on three cell lines of variable HER2/neu expression. In vivo activity was studied in a mouse CS xenograft model of 3+ HER2/neu uterine CS. ResultsIn vitro studies showed that DS-8201a was highly effective against uterine and ovarian CS cell lines demonstrating 3+ HER2/neu expression compared to MAAA-9199 control; there was no significant improvement in the 0 HER2/neu CS cell line. However, DS-8201a induced efficient bystander killing of 0 HER2/neu tumor cells when admixed with 3+ HER2/neu cells. In vivo studies confirmed that DS-8201a was more effective than MAAA-9199 in 3+ HER2/neu-expressing CS xenografts. ConclusionDS-8201a may represent a novel and highly effective ADC against HER2/neu-expressing CS.

Targeted nano-sized drug delivery to heterogeneous solid tumor microvasculatures: Implications for immunoliposomes exhibiting bystander killing effect

Targeted drug delivery to cancer cells utilizing antibodies against oncogenic cell-surface receptors is an emerging therapeutical approach. Here, we developed a computational framework to evaluate the treatment efficacy of free Doxorubicin (Dox) and immunoliposome at different stages of vascular solid tumors. First, three different stages of vascularized tumor progression with various microvascular densities (MVDs) are generated using mathematical modeling of tumor-induced angiogenesis. Fluid flow in vascular and interstitial spaces is then calculated. Ultimately, convection-diffusion-reaction equations governing on classical chemotherapy (stand-alone Dox) and immunochemotherapy (drug-loaded nanoparticles) are separately solved to calculate the spatiotemporal concentrations of therapeutic agents. The present model considers the key processes in targeted drug delivery, including association/disassociation of payloads to cell receptors, cellular internalization, linker cleavage, intracellular drug release, and bystander-killing effect. Reducing MVD led to a decrease in the interstitial fluid pressure, allowing higher rates of the drug to enter the intratumoral environment. The current model also confirms the heterogeneous accumulation of Dox in the perivascular regions during classical chemotherapy. On the other hand, immunoliposomes exhibiting bystander-killing effect yield higher drug internalization during immunochemotherapy. The bystander-killing effect alongside intracellular Dox release and persistence of immunoliposomes within tumor over a longer period lead to more homogeneous drug distribution and a much greater fraction of killed cancer cells than the stand-alone chemotherapy. Present results can be used to improve the treatment efficacy of drug delivery at different stages of vascular tumors.

Computational model of CAR T-cell immunotherapy dissects and predicts leukemia patient responses at remission, resistance, and relapse

BackgroundAdaptive CD19-targeted chimeric antigen receptor (CAR) T-cell transfer has become a promising treatment for leukemia. Although patient responses vary across different clinical trials, reliable methods to dissect and predict patient...

253 (PB033) - The Trop-2-targeting antibody drug conjugate DB-1305 has higher antitumor activity and a potentially better safety profile compared with DS-1062

Background: Trop-2 is a well validated oncology therapeutic target. DB-1305 is an antibody drug conjugate designed by conjugating an anti-Trop-2 antibody with a novel topoisomerase I inhibitor P1021 via an enzymatically cleavable tetrapeptide linker. Material and Methods: To characterize DB-1305 and the payload P1021, thorough studies were performed including in vitro functional assays, in vivo efficacy and PK/PD studies with murine CDX models, as well as PK/TK and GLP toxicity studies in monkeys and rats. Results: The results show that in vitro, DB-1305 selectively bound to Trop-2-positive cells and was endocytosed into their lysosomes. DB-1305 led to Trop-2-dependent cytotoxicity in Trop-2-expressing cells but not Trop-2-negative cells in a concentration-dependent manner. DB-1305 demonstrated tumor cell proliferation inhibition activities comparable to or stronger than DS-1062 produced in-house, as well as intriguingly displayed a stronger bystander killing effect. in vivo, DB-1305 showed strong anti-tumor activity in mouse models of MDA-MB-468, COLO205 and DMS-53, with significantly higher efficacy compared with DS-1062. In Trop-2 negative SHP77 model, DB-1305 did not suppress tumor growth, indicating the dependence of DB-1305 activity on Trop-2 expression. The PK/PD study performed by treatment of DB-1305 in the MDA-MB-468 model demonstrated substantially increased ratios of P1021 exposure (AUC) to the exposure of DB-1305-total antibody or DB-1305-ADC in the tumor than in serum, suggesting that P1021 is effectively released in the tumor. The pharmacokinetics parameters of DB-1305 (total antibody, ADC, and released payload) were obtained from the PK/TK studies in monkeys. The PK/TK profile of P1021 was assessed in rats in addition to the PK/TK study of DB-1305 in monkeys. The exposure levels of DB-1305 and P1021 had no obvious gender difference and no apparent accumulation in monkeys and rats. The systemic exposure AUC0-last and Cmax of ADC showed similar values to those of total antibody, and both parameters were increased approximately dose proportionally from 1 mg/kg to 10 mg/kg. Particularly, in monkeys, the peak concentration of P1021 in the DB-1305 10 mg/kg group was reduced to 1/171 compared with the P1021 0.14 mg/kg (equivalent to the molar mass of P1021 in 10 mg/kg DB-1305), while the t1/2 was prolonged from 0.651 h to 194 h. DB-1305 was well tolerated in the GLP toxicity studies in Cynomolgus monkeys with the highest non-severely toxic dose (HNSTD) of 80 mg/kg, which is significantly higher than HNSTD of 30 mg/kg for DS-1062. Conclusions: Taken together, this study has demonstrated higher efficacy and a potentially better safety profile of DB-1305 compared with DS-1062, supporting further clinical development of DB-1305 and indicating the great potential of DB-1305 in the treatment of Trop-2-positive cancers. No conflict of interest.

Potent bystander effect and tumor tropism in suicide gene therapy using stem cells from human exfoliated deciduous teeth.

Herpes simplex virus thymidine kinase (HSVTK)/ganciclovir (GCV) suicide gene therapy has a long history of treating malignant gliomas. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are collected from deciduous teeth and have excellent harvestability, ethical aspects, and self-renewal, have been attracting attention mainly in the field of gene therapy. In the present study, we assessed SHED as a novel cellular vehicle for suicide gene therapy in malignant gliomas, as we have previously demonstrated with various cell types. SHED was transduced with the HSVTK gene (SHEDTK). In vitro experiments showed a significant bystander effect between SHEDTK and glioma cell lines in coculture. Furthermore, apoptotic changes caused by caspase 3/7 activation were simultaneously observed in SHEDTK and glioma cells. Mice implanted with a mixture of U87 and SHEDTK and treated with intraperitoneal GCV survived for longer than 100 days. Additionally, tumors in treatment model mice were significantly reduced in size during the treatment period. SHEDTK implanted at the contralateral hemisphere migrated toward the tumor crossing the corpus callosum. These results suggested that SHEDTK-based suicide gene therapy has potent tumor tropism and a bystander-killing effect, potentially offering a new promising therapeutic modality for malignant gliomas.

Sustained potentiation of bystander killing via PTEN-loss driven macropinocytosis targeted peptide-drug conjugate therapy in metastatic triple-negative breast cancer

While conventional approaches for PTEN-loss cancers mainly focus on turning off growth promoting process through modulation of PI3K/AKT pathways, no effective therapeutic treatments that target PTEN-loss cancer cells have yielded results. Moreover, conventional targeted therapies, which are potent against only a subset of cancer cells with limited specificity, bring on temporary response. Here, we report the development of albumin-binding caspase-3 cleavable peptide-drug conjugate (PDC), which utilizes the enhanced albumin metabolism pathway in PTEN-loss cancer cells to enhance the intracellular delivery of chemotherapeutic payload that could exert a bystander killing effect. Albumin metabolism-mediated apoptosis triggered expression of caspase-3 allows the continuous activation of the PDC, accumulation of payloads, sustained upregulation of tumoral caspase-3, and intensified in-situ apoptosis. Importantly, PDC strategy exerts potent therapeutic efficacy against PTEN-loss metastatic triple-negative breast cancer, the highly aggressive and heterogenous nature of which remains a challenge conventional targeted therapies need to overcome. This study thus presents a conceptually novel approach to treat PTEN-loss cancer and creates new translational perspectives of exploiting PTEN-loss for providing an avenue to advance current targeted therapy.

Trastuzumab deruxtecan (DS-8201a), a HER2-Targeting Antibody-Drug Conjugate with Topoisomerase I Inhibitor Payload, Shows Antitumor Activity in Uterine and Ovarian Carcinosarcoma with HER2/neu Expression (046)

<b>Objectives:</b> Carcinosarcomas (CSs) are highly aggressive gynecologic malignancies containing both carcinomatous and sarcomatous elements with heterogeneous HER2/neu expression. We evaluated the preclinical <i>in vitro</i> and <i>in vivo</i> activity of trastuzumab deruxtecan (DS-8201a), a novel HER2/neu-targeting antibody-drug conjugate (ADC) against primary uterine and ovarian CSs overexpressing HER2/neu<i>.</i> <b>Methods:</b> Twelve primary uterine and ovarian CS cell lines were evaluated for HER2/neu surface expression by immunohistochemistry (IHC) and by flow cytometry, and gene amplification by ¹³cytotoxicity, viability, and bystander killing effect assays. <i>In vivo</i> activity was studied in mouse CS xenograft models. <b>Results:</b> DS-8201a, as compared to MAAA-9199 control ADC, was found to be highly effective against uterine and ovarian CS cell lines demonstrating 3+ HER2/neu expression. Specifically, the mean half-maximal inhibitory concentrations (IC50's) were 0.02931 μg/mL and 9.75 μg/mL (p<0.0001) against 3+ HER2/neu uterine cell line, and 0.0285 μg/mL and 20.9 μg/mL (p<0.0001) against 3+ ovarian HER2/neu cell lines for DS-8201a versus MAAA-9199, respectively. There was no significant difference between DS-8201a and MAAA-9199 against CS cell lines with low/negligible HER2/neu expression. Importantly, DS-8201a induced an efficient bystander killing effect of HER2/neu negative tumor cells when admixed with 3+ HER2/neu cells. <i>In vivo</i> studies confirmed that DS-8201a was significantly more effective than MAAA-9199 in decreasing tumor volume and prolonging life in 3+ HER2/neu-expressing CS xenografts (attached Figure). There was no significant toxicity demonstrated between treatment and control groups. <b>Conclusions:</b> Trastuzumab deruxtecan (DS-8201a) is a novel ADC with remarkable activity against CS with strong (3+) HER2/neu expression. Clinical studies with DS-8201a in patients harboring chemotherapy-resistant CS with high HER2/neu expression are warranted. DS-8201a showing significant tumor growth inhibition in 3+ HER2/neu expression mice. Day 8 mean tumor volumes in treated mice with DS-8201a (0.271 cm3) versus ADC isotope control (0.859 cm3, p=0.0011) and vehicle control (0.804 cm3, p=0.0047). Day 11 mean tumor volumes in treated mice with DS-8201a (0.179 cm3) versus ADC isotope control (1.166 cm3, p <0.0001) and vehicle control (1.151 cm3, p <0.0001).Fig. 1

Efficacy of HSV-TK/GCV system suicide gene therapy using SHED expressing modified HSV-TK against lung cancer brain metastases

Lung cancer is one of the most common cancers, and the number of patients with intracranial metastases is increasing. Previously, we developed an enzyme prodrug suicide gene therapy based on the herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system using various mesenchymal stem cells to induce apoptosis in malignant gliomas through bystander killing effects. Here, we describe stem cells from human exfoliated deciduous teeth (SHED) as gene vehicles of the TK/GCV system against a brain metastasis model of non-small cell lung cancer (NSCLC). We introduced the A168H mutant TK (TKA168H) into SHED to establish the therapeutic cells because of the latent toxicity of wild type. SHED expressing TKA168H (SHED-TK) exhibited chemotaxis to the conditioned medium of NSCLC and migrated toward implanted NSCLC in vivo. SHED-TK demonstrated a strong bystander effect in vitro and in vivo and completely eradicated H1299 NSCLC in the brain. SHED-TK cells implanted intratumorally followed by GCV administration significantly suppressed the growth of H1299 and improved survival time. These results indicate that the TKA168H variant is suitable for establishing therapeutic cells and that intratumoral injection of SHED-TK followed by GCV administration may be a useful strategy for therapeutic approaches.

Abstract 5242: Rationally designing a radionuclide drug conjugate for heterogeneous genomically stable gastric cancer

Abstract Background: Gastric cancer (GC) is one of common malignant tumors with a 5-year survival rate less than 10% for patients in advanced stages. Genomically stable gastric cancer (GSGC), featuring the worst prognosis of four subtypes of GC based on its molecular characteristics, receives limited benefits from recently approved HER2-targeted antibody drug conjugates (ADCs) due to the lack and heterogeneity of tumoral HER2 expression. Currently, radionuclide drug conjugates (RDCs) have attracted great attention in targeted therapy, owing to its unique advantages of radiation-associated direct DNA damages and relatively long radiation distances. These unique advantages enable RDCs to efficiently and broadly ablate both cancer cells and cancer-associated stromal cells in heterogeneous tumor microenvironment without being internalized by target cells, featuring a novel and more efficient “bystander killing” effect than linker-cleavable ADCs. Here, a RDC was developed by rationally screening GSGC-targeting antibodies and radionuclides for targeted treatment of GSGCs. Meanwhile, the “bystander killing” effect of the RDC will be quantitatively determined and compared with conventional linker-cleavable ADCs. Results: First, ICAM1 was identified as a more preferable molecular target relatively to HER2 in human GSGCs via flow cytometric analyses. Next, a RDC was constructed by covalently conjugating anti-ICAM1 antibody with radionuclide 131I via indogen chemistry. 131I, the RDC warhead emitting β-particles with an emission range of approximately 0.8 mm, is a clinically-used radionuclide with FDA approval for treating several tumors. Then, the half maximum inhibitory concentrations (IC50s) of constructed 131I conjugated ICAM1 antibody (131I-ICAM1) was evaluated in human GSGC cell lines. Its inherent bystander killing effect was validated by measuring the viability of ICAM1 negative cells co-incubated with ICAM1 positive cells in the treatment of 131I-ICAM1, in comparison with ADC of the same target. Ultimately, the anti-tumor efficacy of 131I-ICAM1 was verified in heterogeneous GSGC models. Simultaneously, the histopathological damages to normal organs by 131I-ICAM1 was minimized by optimizing its dose. Conclusions: In this study, we constructed a rationally designed 131I-ICAM1 by conjugating ICAM-1 antibody and clinical available radionuclide 131I and investigated the intrinsic bystander killing effect of this RDC in ablating heterogeneous GSGCs. Our research explored the potential of 131I-ICAM1 as a promising targeted therapeutic candidate for treating heterogeneous GSGCs. Citation Format: Le Yang, Rui Xu, Peng Guo, Heqing Yi, Xiangdong Cheng. Rationally designing a radionuclide drug conjugate for heterogeneous genomically stable gastric 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 5242.

Abstract 6153: MyD88-mediated chimeric antigen receptor macrophage suppresses brain metastasis by target-specific phagocytosis and bystander effect through TNF-α

Abstract Treating patients with brain metastasis of lung cancer poses significant challenges because a surgical approach is limited due to the sensitive nature of the brain and also because most chemotherapeutic drugs are ineffective owing to the blood-brain barrier (BBB). Although chimeric antigen receptor (CAR)-T cell and immune checkpoint inhibitors have been used in clinical trials for patients with recurrent brain tumors, the efficacy of these treatments has been limited due to severe side effects and poor penetrance of these reagents through BBB. Published data as well as the results of our own study indicate that macrophage is the major component of infiltrated immune cells in the brain metastatic lesions, suggesting that engineering to reprogram macrophages is a viable therapeutic strategy for patients with brain metastasis. Given the unique ability of macrophages to cross BBB and exert their phagocytic function to tumor cells, we genetically engineered macrophage that expressed tumor-specific antibody (scFv) to Mesothelin (MSLN) and activated the anti-tumor immune signaling molecule, MyD88. We hypothesize that MyD-88-driven chimeric antigen receptor macrophage (MyD-CARMA) suppresses brain metastasis of lung cancer by phagocytosis and increasing anti-tumor immune response. To investigate the effect of MyD-CARMA on brain metastasis in vivo, mice were intracranially transplanted with lung cancer brain metastasis cell (H2030BrM) which expresses a high level of MSLN followed by administration of MyD-CARMA every 2 weeks for 40 days. This CARMA indeed penetrated BBB and drastically decreased brain metastasis growth in our humanized mouse model. The MyD-CARMA showed antigen-specific phagocytosis activity against MSLN+ tumor cells. We then utilized NCI-H522 cell which lacks MSLN expression and overexpressed MSLN in this cell line followed by co-culturing with CARMA. We found that CARMA suppressed tumor growth of only MSLN+ but not MSLN- cells. However, when CARMA was co-cultured with both MSLN+ and MSLN- cells, CARMA exhibited a bystander killing effect even on MSLN- cells. When TNF-α was knocked out in CARMA by CRISPR/Cas9, CARMA lost the ability to show the bystander effect on MSLN- cells, suggesting that CARMA exhibited the bystander killing effect via up-regulation of TNF-α upon engagement with MSLN+ cells. Importantly, CARMA showed much less liver and neuron toxicities compared to CAR-T. In conclusion, our results showed that these unique features of MyD-CARMA represent significant advantage over other immune therapies, and therefore, MyD-CARMA may serve as a promising therapeutic tool for the treatment of brain metastasis. Citation Format: Shih-Ying Wu, Kerui Wu, Abhishek Tyagi, Fei Xing, Dan Zhao, Ravindra Pramod Deshpande, Yin Liu Liu, Kounosuke Watabe. MyD88-mediated chimeric antigen receptor macrophage suppresses brain metastasis by target-specific phagocytosis and bystander effect through TNF-α [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 6153.

Caspase-3 mediated switch therapy of self-triggered and long-acting prodrugs for metastatic TNBC.

Triple-negative breast cancer (TNBC) is characterized by its highly heterogeneous microenvironment and propensity for aggressive behavior, both of which represent, along with poor prognosis and high incidence of relapse, the main challenges of curing the disease. Although recent progress in targeted chemotherapy combinations has shown promising outcomes, conventional targeted chemotherapeutic approaches have relied on exploiting the expression of certain molecules or proteins overexpressed on cancer cells as drug targets, which have demonstrated limited clinical benefit against metastatic cancers. Here, we describe a tumoral caspase-3 mediated peptide-doxorubicin conjugates (PDC) switch therapy that adopts two different caspase-3 cleavable PDCs, RGDEVD-DOX (TPD1) and EMC-KGDEVD-DOX (MPD1), for targeting metastatic triple-negative breast cancer (mTNBC). First, using TPD1, an integrin αVβ3 based targeted strategy was utilized to target tumor cells or tumor vasculature associated with the highly malignant progression of mTNBC. TPD1 triggered the tumor cell-specific initial apoptosis and the induction of caspase-3 expression in the target tumor site. Then MPD1 was administered sequentially, which is an albumin-binding prodrug, and activated by induced caspase-3 in order to maintain the tumoral caspase-3 level and release the cytotoxic payload. The PDC switch therapy markedly accumulated doxorubicin in the tumor site and augmented tumor-specific in situ amplification of apoptosis. Importantly, the PDC switch therapy exerted a bystander killing effect on the neighboring cancer cells thus demonstrating potent therapeutic efficacy against both local and metastatic cancers. Given the limited therapeutic outcomes with conventional targeted therapies, our strategy of regulating the expression of caspase-3 level as a drug target could provide as a more durable and effective alternative in the treatment of highly heterogeneous mTNBC.

Oncolytic peptide nanomachine circumvents chemo resistance of renal cell carcinoma

Due to intrinsic and acquired chemo/radiotherapy-resistance, renal cell carcinoma shows limited therapeutic response to clinically utilized targeting drugs. Here a tumor-activated oncolytic peptide nanomachine is devised to selectively lysing tumor cell membrane without causing drug resistance. Specifically, in the acidic tumor microenvironment, the oncolytic peptide nanomachine automatically activated through morphologically transformation from nanoparticles to nanofibrils with restoring α-helical conformation, which physically bind to tumor cell membrane with multiple (spatially correlated and time-resolved) interactions and subsequently lyse local cell membrane. The IC50 of the oncolytic peptide nanomachine is as low as 2.44 μM and it inhibit up to 90% of tumor cells within 2 h with unique bystander killing effect. In vivo, the tumor inhibition rate of the oncolytic peptide nanomachine is 71% without off-target activity and hemolytic activity. These results support that tumor-selective oncolytic peptide nanomachine represent a promising alternative approach for multidrug-resistant tumor treatments by inducing cell membrane lysis.

Bystander effect of antibody-drug conjugates: fact or fiction?

Summarizing the current preclinical and clinical evidence about bystander effect of antibody-drug conjugates (ADCs) in solid tumors. One of the main challenges of treating solid tumors with ADCs is the heterogeneous expression of the target antigen (Ag), which however may be overcome by the so-called bystander killing effect. This unique, but still debated, feature of certain ADCs is represented by the unintentional payload diffusion from Ag-positive tumor cells to adjacent Ag-negative tumor cells. Some pharmacological characteristics, such as a hydrophobic payload or a cleavable linker, seem to play a major role in this effect. Abundant preclinical evidence of the bystander effect has emerged, and the clinical activity of ADCs in tumors with a heterogeneous Ag expression suggests the relevance of this feature. Additional studies are required to investigate if the bystander effect is necessary for achieving a solid activity with ADCs.

Albumin metabolism targeted peptide-drug conjugate strategy for targeting pan-KRAS mutant cancer

Despite recent breakthroughs in the development of direct KRAS inhibitors and modulators, no drugs targeting pan-KRAS mutant cancers are clinically available. Here, we report a novel strategy to treat pan-KRAS cancers using a caspase-3 cleavable peptide–drug conjugate that exploits enhanced albumin metabolism in KRAS altered cancers to deliver a cytotoxic agent that can induce a widespread bystander killing effect in tumor cells. Increased albumin metabolism in KRAS mutant cancer cells induced apoptosis via the intracellular uptake of albumin-bound MPD1. This allowed caspase-3 upregulation activated MPD1 to release the payload and exert the non-selective killing of neighboring cancer cells. MPD1 exhibited potent and durable antitumor efficacy in mouse xenograft models with different KRAS genotypes. An augmentation of anti-cancer efficacy was achieved by the bystander killing effect derived from the caspase-3 mediated activation of MPD1. In summary, albumin metabolism-induced apoptosis, together with the bystander killing effect of MPD1 boosted by caspase-3 mediated activation, intensified the efficacy of MPD1 in KRAS mutant cancers. These findings suggest that this novel peptide-drug conjugate could be a promising breakthrough for the treatment in the targeting of pan-KRAS mutant cancers.

Generation and functional characterization of a multigene-modified NK101 cell line exerting diverse mechanisms of antitumor action

ABSTRACT Clonal cell line-based, multigene-modified, off-the-shelf NK cell therapeutics are emerging as the new frontier of adoptive cellular immunotherapy. Here, we utilized a newly established NK cell line, NK101, as a backbone to derive multifaceted killer cells armored with various antitumor modalities through repeated cycles of genetic modification and clonal selection. First, NK101 cells were transduced with a tricistronic lentiviral vector expressing CD7, CD28, and cytosine deaminase (CD). The resulting cell line demonstrated enhanced cytotoxicity against B7+ tumors and exerted bystander killing effects on neighboring tumor cells upon 5-FC treatment. Second, engineered NK101 cells were again transduced with a bicistronic vector expressing membrane-bound interleukin-15 (mbIL-15) and dominant negative TGFβ type II receptor (DNTβRII). Ectopic expression of mbIL-15 resulted in further augmentation of lytic activities against all tested target cells by inducing upregulation of multiple activating receptors, while that of DNTβRII allowed the cells to maintain heightened cytotoxicity in the presence of TGFβ. Finally, dual-transduced NK101 cells were modified to express chimeric antigen receptors (CARs) targeting either a solid tumor antigen (EpCAM) or a hematologic tumor antigen (FLT3). The final engineered products not only demonstrated antigen-specific killing activities in vitro but also exerted strong tumor-inhibitory effects in preclinical models of metastatic solid tumor and hematologic malignancy. Notably, combined treatment with 5-FC further enhanced antitumor efficacy of engineered NK101 in the solid tumor model. Our results demonstrate successful generation of multigene-modified NK101 cell therapeutics exerting diverse mechanisms of antitumor action – activation receptor-mediated innate killing, antigen-specific killing, and bystander effect-mediated killing.

Abstract 955: Preclinical characterization of a novel SSEA-4-targeting antibody drug conjugate, OBI-998

Abstract Stage-specific embryonic antigen-4 (SSEA-4) ceramide, a globo-series hexasaccharide glycosphingolipid, has been reported to be a tumor-associated antigen. Here, we present a novel antibody-drug conjugate (ADC) targeting SSEA-4 to evaluate its potential as a therapeutic agent for cancer treatment. OBI-998 is an ADC comprising the humanized anti-SSEA-4 antibody (OBI-898) that is conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE) through maleimide and PEGylated cleavable linkers. We demonstrated the specificity of the naked antibody to SSEA-4 by screening against a panel of 21 related carbohydrate antigens using ELISA. OBI-998 displayed potent cytotoxic activity against cells (SKOV3) with a high level of SSEA-4 expression at sub-nanomolar potency but no effect on the viability of cells (SKBR3) with negligible SSEA-4 expression. The bystander killing effect of OBI-998 was shown by the transferring of conditioned medium from SKOV3 cells treated with 1 or 5 nM of OBI-998 to SKBR3 cells. Furthermore, significant bystander effects were observed by co-culturing high (NCI-N87) and low (PANC-1/GFP) SSEA-4-expressing cell lines at different ratios in the presence of 5 or 10 nM of OBI-998. OBI-998 was found to be rapidly internalized into cancer cells within 5 minutes upon binding to its target on the cell surface by confocal microscopy. OBI-998 showed significant anti-tumor efficacy in multiple cancer cell–derived xenograft models at doses of 1, 3, and 10 mg/kg in a dose-dependent manner. More importantly, OBI-998 at a dose of 10 mg/kg showed complete tumor regression in an EGFR-triple mutation non–small cell lung cancer xenograft model, which is resistant to the current last-line tyrosine kinase inhibitor, osimertinib. Pharmacokinetic analysis of OBI-998 revealed that total antibody and the conjugated antibody exhibited similar pharmacokinetic profiles, suggesting OBI-998 is highly stable in vivo. The biodistribution study in HCC1428 tumor-bearing mice indicated that MMAE accumulated in the tumor site at a higher level compared with other blood-rich organs. In addition, the MMAE levels in tumors reached peak level at 24 hours post-treatment, which was much higher than the maximum levels in other organs, and the level was sustained for 168 hours with tumor-to-muscle ratio of 329. OBI-998 is a novel ADC targeting SSEA-4 that possesses desired properties such as high target specificity, rapid internalization, potent cytotoxicity, and significant bystander effects. Furthermore, OBI-998 showed a high level of deposition and a persistent presence of MMAE in tumors and significant anti-tumor efficacy in a variety of animal models. Taken together, these results support the further development of OBI-998 as a therapeutic agent for SSEA-4-targeting cancer therapy. Citation Format: I-Ju Chen, Chun-Chung Wang, Chi-Sheng Shia, Chung-Chen Su, Chi-Huan Lu, Hui-Wen Chang, Ping-Tzu Chiu, Yueh-Chin Wu, Ming-Tain Lai, Wei-Chien Tang, Hsin-Yi Tung, Ren-Yu Hsu. Preclinical characterization of a novel SSEA-4-targeting antibody drug conjugate, OBI-998 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 955.

Abstract 945: Trastuzumab deruxtecan (T-DXd) sensitivity in various levels of HER2 expressing gastric cancer cells

Abstract Trastuzumab, a humanized anti-HER2 monoclonal antibody, has shown clinically significant benefits for HER2-positive GC patients. Also, all anti-HER2 agents approved by the FDA are used for HER2-positive. However, since HER2-positive is only 10-20% of GC, targeted therapies for non-HER2-positive GC is needed. To overcome these limitations, antibody-drug conjugates (ADCs), which directly deliver cytotoxic drugs to cancer cells, have been developed. T-DXd (Daiichi Sankyo, Japan), an anti-HER2 ADC, can induce the death of neighboring cells because it has a bystander killing effect due to the high membrane permeability of the payload. However, biomarkers for predicting drug response have not yet been studied other than HER2 overexpression/amplification. In this study, we investigated the correlation between HER2 expression level and anti-tumor effect of T-DXd in a 49 GC cell line panel and the HER2-associated molecules related to T-DXd sensitivity. For HER2 status, we analyzed multiple genetic and molecular characteristics using WES, SISH, IHC, flow cytometry, and immunoblotting. GC cell lines (n=49) were divided into 3 groups according to the HER2 expression level through flow cytometry using 4 breast cancer cell lines (SK-BR-3, ZR-75-1, MCF-7, and HCC1937) for quantitative control. Groups with HER2 over- and moderate/low-expression were characterized with higher expression levels compared to ZR-75-1 (HER2 2+) and HCC1937 (HER2-negative), respectively. HER2 non-expression had lower expression levels compared to HCC1937. The sensitivity of T-DXd was evaluated in 49 GC cell lines by cell viability assay. The inhibition rate of T-DXd was calculated at 10 µg/ml. Also, expression levels of RTKs were determined by immunoblotting, and HER2 extracellular domain (ECD) and NRG1 expression were evaluated by ELISA. GC cell lines were divided into 6 HER2 over-, 26 HER2 moderate/low-, and 17 HER2 non-expression cell lines. As expected, HER2 over-expression cell lines were sensitive to T-DXd (5/6, 83.3%). T-DXd also had anti-tumor effects in the HER2 moderate/low-expression cell lines (12/26, 46.2%). There were correlations between HER2 expression level and anti-tumor effect of T-DXd in both HER2 over and moderate/low-expression cell lines. HER2 over and moderate/low-expression cell lines with high HER2 ECD expression were sensitive to T-DXd. NRG1 had relatively low expression in all GC cell lines and was not correlated with T-DXd. Additionally, even HER2 non-expression cell lines were sensitive to T-DXd (10/17, 58.8%). We found that HER2 non-expression cell lines had an RTK amplification (9/17, 52.9%) and among those cell lines, MET amplified cells showed sensitivity to T-DXd. Currently, the evaluation of the underlying mechanisms is ongoing. Our results indicate that T-DXd shows efficacy in HER2-amplified GC cell lines, HER2 moderate/low-expression, and some cell lines with HER2 non-expression. Citation Format: Seo Young Yu, Juin Park, Woo Sun Kwon, Inhye Jeong, Sun Kyoung Kang, Hyun Joo Bae, Tae Soo Kim, Hyun Cheol Chung, Sun Young Rha. Trastuzumab deruxtecan (T-DXd) sensitivity in various levels of HER2 expressing gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 945.