Direct Killing Research Articles

One cisplatin dose provides durable stimulation of anti-tumor immunity and alleviates anti-PD-1 resistance in an intraductal model for triple-negative breast cancer

ABSTRACT Aggressive triple-negative breast cancer (TNBC) is classically treated with chemotherapy. Besides direct tumor cell killing, some chemotherapeutics such as cisplatin provide additional disease reduction through stimulation of anti-tumor immunity. The cisplatin-induced immunomodulation in TNBC was here investigated in-depth using immunocompetent intraductal mouse models. Upon primary tumor transition to invasive carcinoma, cisplatin was injected systemically and significantly reduced tumor progression. Flow cytometric immunophenotyping was corroborated by immunohistochemical analyses and revealed both differential immune cell compositions and positivity for their programmed death (PD)-1 and PD-ligand (L)1 markers across body compartments, including the primary tumor, axillary lymph nodes and spleen. As key findings, a significant decrease in immunosuppressive and a concomitant increase in anti-tumor lymphocytic cell numbers were observed in the axillary lymph nodes and spleen, highlighting their importance in cisplatin-stimulated anti-tumor immunity. These immunomodulatory effects were already established following the first cisplatin dose, indicating that early cisplatin-mediated events may determine (immuno)therapeutic outcome. Furthermore, a single cisplatin dose sufficed to alleviate anti-PD-1 resistance in a 4T1-based model, providing add-on disease reduction without toxic side effects as seen upon multiple cisplatin dosing. Overall, these results highlight cisplatin as immunotherapeutic ally in TNBC, providing durable immunostimulation, even after a single dose.

PD-L1 Amino Acid Position 88 Represents a Hotspot for PD-L1 Stability With Relevance for PD-L1 Inhibition.

The two most common antibody targeting principles in oncology are the induction of direct antitumor effects and the release of antitumor T cell immunity by immune checkpoint blockade. These two principles, however, may be overlapping if the targeted checkpoint molecule is not located on the immune cell but on the tumor cell itself. Secondary resistance by epitope escape may therefore remain a challenge in both settings. We previously reported epitope escape through L88S and truncating programmed cell death ligand 1 (PD-L1) gene mutations in colorectal cancer patients on selective pressure with avelumab, a PD-L1-directed checkpoint blocker that—in addition to T cell disinhibition—allows direct tumor cell killing via its unmodified Fc portion. Here, we confirmed this principle by liquid biopsy monitoring in a colorectal cancer patient from an independent clinical trial. In this patient, both PD-L1 L88E and L88fs mutations emerged under selective pressure with avelumab. By ectopically expressing PD-L1 L88E, we show that this mutation leads to a reduction of full-length glycosylated PD-L1 and greatly reduced avelumab surface binding. Further experiments indicated that PD-L1 L88E represents a phosphomimetic variant of PD-L1 L88S leading to loss of protein stability and increased proteasomal degradation. The association of this PD-L1 mutation with the high-affinity FCGR3A single nucleotide polymorphism rs396991 confirms prior evidence that patients harboring this polymorphism experience the strongest selective pressure by avelumab. Together, position 88 of PD-L1 is a hotspot residue critically regulating PD-L1 cell surface expression with clinical significance in the context of immune checkpoint blockade.

Intersexual differences in the number of genes differentially expressed in wild mammals in response to predation risk

Predation is a psychological stressor in prey animals. Besides direct killing and consumption by predators, the perception of predation risk indirectly influence prey population behavior, dynamics and physiology. Few studies identified the transcriptomic response associated with predator presence/abundance in natural populations and uncontrolled settings. However, to our knowledge, intersexual differences in the number of genes whose expression change in response to high predation risk have not been previously reported in wild mammals. Here, by using publicly available gene expression data in wild yellow-bellied marmots (Marmota flaviventer), we found that the number of differentially expressed genes in response to predator stress is higher in female marmots (n = 516) than males (n = 387). Only a small percentage of these differentially expressed genes (n = 36) are shared between the sexes, and that the most of the differentially expressed genes are expressed in a sex-specific manner in response to predation stress. Overall, our results provide new insight into sex-specific variation in gene expression changes in wild mammals under high predation risk.

Regulation of juvenile hormone and ecdysteroid analogues on the development of the predatory spider, Pardosa pseudoannulata, and its regulatory mechanisms

Insecticides harm the beneficial organisms, such as predatory spiders, through direct killing or regulation of the development and reproduction. In this study, the bioassay showed that the treatment of juvenile hormone (JH) analogue fenoxycarb delayed the moulting of Pardosa pseudoannulata, a dominant predatory spider in paddy fields. In order to figure out the regulatory mechanism of fenoxycarb on the spider development, we systematically analyzed JH biosynthesis in P. pseudoannulata. All genes involved in JH biosynthesis pathway were retrieved from the genome of P. pseudoannulata, except for CYP15A1. The absence of CYP15A1 was in agreement with the identification of methyl farnesoate (MF) rather than JH III in the spider. The delayed moulting and decreased expression of JH biosynthesis-related genes in the MF-applied spiderlings supported that MF was an active JH. Fenoxycarb treatment significantly upregulated the transcriptional level of JH biosynthesis-related genes and consequently delayed the spiderling moulting. In the spider development, ecdysteroid played the opposite role, in contrast to MF, to accelerate the development, as our previous study. Here we found that the treatment of ecdysteroid analogue tebufenozide accelerated P. pseudoannulata spiderling moulting, which resulted from the expressional suppression of ecdysteroid biosynthesis-related genes. In total, the JH and ecdysteroid analogues affected the development of P. pseudoannulata by the expressional regulation of biosynthesis-related genes, which would be helpful for the evaluation of hormone analogue insecticides in environmental safety, and useful for the protection and application of P. pseudoannulate and related spider species.

Non-consumptive effects of intraguild predator Blattisocius dentriticus (Berlese) on the development and prey consumption of Neoseiulus cucumeris (Oudemans)

Predator-prey interactions are fundamental to the understanding of ecological communities. The non-consumptive effects of predators on the food web can be more prominent than direct killing. However, the non-consumptive effects in intraguild interaction have rarely been studied. This study determined the non-consumptive effects of an intraguild predator Blattisocius dentriticus on Neoseiulus cucumeris. The responses of N. cucumeris to the predation risk of B. dentriticus adult females were examined in a laboratory test. The developmental response of N. cucumeris to predation risk was sex-specific: the presence of B. dentriticus cues prolonged the immature development of N. cucumeris males and increased their consumption rates of Tyrophagus putrescentiae eggs, but did not affect the development and prey consumption of female N. cucumeris. The size at maturity of N. cucumeris was not affected by predation risk from B. dentriticus. This study demonstrated that intraguild predators can have substantial non-consumptive influence on intraguild prey.

Natural Killer Cells in SARS-CoV-2 Infection: Pathophysiology and Therapeutic Implications.

Natural Killer (NK) cells are lymphocytes of the innate immunity that play a crucial role in the control of viral infections in the absence of a prior antigen sensitization. Indeed, they display rapid effector functions against target cells with the capability of direct cell killing and antibody-dependent cell-mediated cytotoxicity. Furthermore, NK cells are endowed with immune-modulatory functions innate and adaptive immune responses via the secretion of chemokines/cytokines and by undertaking synergic crosstalks with other innate immune cells, including monocyte/macrophages, dendritic cells and neutrophils. Recently, the Coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally. Although the specific role of NK cells in COVID-19 pathophysiology still need to be explored, mounting evidence indicates that NK cell tissue distribution and effector functions could be affected by SARS-CoV-2 infection and that a prompt NK cell response could determine a good clinical outcome in COVID-19 patients. In this review, we give a comprehensive overview of how SARS-CoV-2 infection interferes with NK cell antiviral effectiveness and their crosstalk with other innate immune cells. We also provide a detailed characterization of the specific NK cell subsets in relation to COVID-19 patient severity generated from publicly available single cell RNA sequencing datasets. Finally, we summarize the possible NK cell-based therapeutic approaches against SARS-CoV-2 infection and the ongoing clinical trials updated at the time of submission of this review. We will also discuss how a deep understanding of NK cell responses could open new possibilities for the treatment and prevention of SARS-CoV-2 infection.

Local tumour nanoparticle thermal therapy: A promising immunomodulatory treatment for canine cancer

Distinct thermal therapies have been used for cancer therapy. For hyperthermia (HT) treatment the tumour tissue is heated to temperatures between 39 and 45°C, while during ablation (AB) temperatures above 50°C are achieved. HT is commonly used in combination with different treatment modalities, such as radiotherapy and chemotherapy, for better clinical outcomes. In contrast, AB is usually used as a single modality for direct tumour cell killing. Both thermal therapies have been shown to result in cytotoxicity as well as immune response stimulation. Immunogenic responses encompass the innate and adaptive immune systems and involve the activation of macrophages, dendritic cells, natural killer cells and T cells. Several heat technologies are used, but great interest arises from nanotechnology-based thermal therapies. Spontaneous tumours in dogs can be a model for cancer immunotherapies with several advantages. In addition, veterinary oncology represents a growing market with an important demand for new therapies. In this review, we will focus on nanoparticle-mediated thermal-induced immunogenic effects, the beneficial potential of integrating thermal nanomedicine with immunotherapies and the results of published works with thermotherapies for cancer using dogs with spontaneous tumours, highlighting the works that evaluated the effect on the immune system in order to show dogs with spontaneous cancer as a good model for evaluated the immunomodulatory effect of nanoparticle-mediated thermal therapies.

S206: OBINUTUZUMAB PLUS CHEMOTHERAPY DEMONSTRATES LONG-TERM BENEFIT OVER RITUXIMAB PLUS CHEMOTHERAPY IN PATIENTS WITH PREVIOUSLY UNTREATED FOLLICULAR LYMPHOMA: FINAL ANALYSIS OF THE GALLIUM STUDY

Background: Immunochemotherapy with rituximab plus chemotherapy (R-chemo) has significantly improved outcomes for patients (pts) with previously untreated follicular lymphoma (FL). However, most pts still experience disease relapse, progression, or death. Obinutuzumab (G) is a type II anti-CD20 monoclonal antibody with enhanced direct cell killing, antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis versus R. The safety and efficacy of G-chemo versus R-chemo was assessed in pts with previously untreated, advanced stage FL in the randomized, Phase III GALLIUM (NCT01332968) study. In the primary analysis, G-chemo demonstrated a significant improvement in progression-free survival (PFS) versus R-chemo, with a manageable safety profile (Marcus, et al. 2017); this efficacy benefit was maintained after 5 years of observation (Townsend, et al. 2020). Aims: To report the final analysis of the GALLIUM study after a median observation time of 8 years. Methods: Pts aged ≥18 years with previously untreated histologic Grade 1–3a FL requiring treatment were enrolled. Pts were randomized 1:1 to receive G 1000mg intravenously (IV; Days [D]1, 8 and 15 of Cycle 1 and D1 of subsequent cycles) or R 375mg/m2 IV (D1 of each cycle) plus chemo for 6 or 8 cycles depending on the chemo backbone selected at each institution (cyclophosphamide, doxorubicin, vincristine, and prednisolone [CHOP]; cyclophosphamide, vincristine, and prednisolone [CVP]; or bendamustine). Pts attaining a complete or partial response received maintenance with the same antibody every 2 months for 2 years or until disease progression (PD). The primary endpoint was investigator-assessed PFS; secondary endpoints included time-to-next anti-lymphoma treatment (TTNLT), overall survival (OS) and incidence of adverse events (AEs). All pts provided written informed consent. Results: 1202 pts with FL were enrolled (G-chemo, n=601; R-chemo, n=601). As of July 30, 2021, median observation time was 8 years. Seven-year PFS was improved with G-chemo (63.4%) versus R-chemo (55.7%; hazard ratio [HR], 0.77; 95% confidence interval [CI]: 0.64–0.93; p=0.006; Figure). TTNLT was also improved with G-chemo versus R-chemo (HR, 0.71; 95% CI: 0.58–0.87; p=0.001); the proportion of pts who had not started their next treatment at 7 years was 74.1% and 65.4%, respectively. Disease transformation was observed in 4.2% of pts with G-chemo and 5.0% of pts with R-chemo. Seven-year OS was similar in both arms, 88.5% with G-chemo versus 87.2% with R-chemo (HR, 0.86; 95% CI: 0.63–1.18; p=0.36). Seventy-five pts in the G-chemo arm and 86 pts in the R-chemo arm had died, most commonly due to PD (4.2% and 6.0%, respectively). The incidence of serious AEs (SAEs) was 48.9% with G-chemo (28.2% and 24.4% during induction and maintenance, respectively) and 43.4% with R-chemo (24.6% and 21.7%, respectively). Rates of fatal AEs were similar with G-chemo (4.4%) and R-chemo (4.5%); pneumonia was the most common fatal AE (0.8% [n=5] and 0.2% [n=1], respectively) and SAE (5.9% [n=35] and 6.2% [n=37], respectively). Second malignancies occurred in 13.1% of pts in the G-chemo arm and 9.9% of pts in the R-chemo arm. After adjusting for observation time, rates were similar between arms. These safety findings are consistent with previous analyses. Image:Summary/Conclusion: After a median observation time of 8 years, a meaningful improvement in PFS was maintained with G-chemo versus R-chemo in pts with previously untreated FL, confirming the role of G-chemo as a standard of care for the first-line treatment of pts with FL.

Abstract 5504: Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to restrain acquired resistance from antigen-loss-variant tumor cells

Abstract Adoptive cell therapy (ACT) with tumor-specific T cells expressing T-cell-receptors (TCR) or chimeric antigen receptors (CAR) has not mediated sustained responses in solid tumors. Several factors limit the efficacy of ACT in solid tumors and lead to primary resistance and acquired resistance (refers to recurrence after the initial response). These include but not limited to the insufficient proliferation of T cells, the low capacity of T cells to penetrate stroma-rich solid tumors, and heterogeneity of antigen expression in solid tumors, which allows the escape of T cell attack by tumor cells when becoming antigen-loss-variant cancer cell (ALV). Hence, it is urgent to develop novel ACT strategies to eradicate solid tumors and prevent acquired resistance. We have previously reported (Yong Lu, Cancer Cell, 2018) that tumor-specific IL-9-producing CD4+ Th9 cells represent a novel T cell paradigm for ACT - they are less exhausted, fully cytolytic, and hyperproliferative. This hyperproliferation of Th9 cells is driven by a unique Th9 master transcriptional factor Pu.1 mediated Pu.1-Traf6-NFκB signaling to ensure Th9 cells persist as long as “stem cell-like” T cells, which is not presented in other known CD4+ T cells subsets. In our most recent study, we demonstrate that murine and human Th9 cells, but not Th1/Tc1 or Th17 cells, expressing tumor-specific T-cell-receptors (TCR) or chimeric antigen receptors (CAR), eradicate advanced tumors that contain ALVs. This unprecedented antitumor capacity of Th9 cells is attributed to both enhanced direct tumor cell killing and bystander antitumor effects promoted by intratumor release of IFNα/β. Mechanistically, tumor-specific Th9 cells increase the intratumor accumulation of extracellular ATP (eATP, released from dying tumor cells), because of a unique feature of Th9 cells that lack the expression of ATP degrading ectoenzyme CD39. Intratumor enrichment of eATP promotes the monocyte infiltration and stimulates their production of IFNα/β by inducing eATP-endogenous retrovirus-TLR3/MAVS pathway activation. These results identify tumor-specific Th9 cells as a unique T cell subset endowed with the unprecedented capacity to eliminate ALVs for curative responses. Citation Format: Yong Lu. Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to restrain acquired resistance from antigen-loss-variant tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5504.

Abstract 2902: Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies

Abstract CD20 and CD37 are co-expressed on most B-NHL and CLL tumors. Systemic depletion of normal and malignant B-cells by anti-CD20 antibodies (e.g. rituximab and obinutuzumab) and anti-CD37 antibodies (e.g. otlertuzumab and BI 836826) has been well tolerated in clinical studies. Depletion of B-cells is reversible, with the ability to repopulate B-cells from hematopoietic stem cells. Dual targeting CD20 and CD37 may improve the clinical efficacy and decrease drug resistance. PSB202 is a novel biological entity composed of two engineered monoclonal antibodies which are produced from a single stable CHO cell line - a humanized anti-CD20 antibody (PSB102) and a humanized anti-CD37 antibody (PSB107) at a 1:1 molecular ratio. In vitro binding studies demonstrated that PSB202 specifically binds to human CD20 and CD37. The antibodies’ binding to FcγRIIIa was enhanced by reducing the fucose level on each antibody component. PSB202 directly induced potent autonomous killing via target engagement. PSB202 mediated depletion of human and monkey blood B-cells and malignant human B-cell lines through ADCC activity at EC50 of double digit pM and CDC activity at EC50 of low single digit nM. Importantly, PSB202 effectively depleted B-cells in ex vivo cultures of blood cells from CLL and NHL patients who were refractive to rituximab treatment. The antitumor activity of PSB202 was demonstrated in human xenograft models in mice. PSB202 was able to reduce different human lymphoma tumors by up to ~97.9% in a dose-dependent manner, suggesting that PSB202 might be potentially efficacious against multiple types of B-cell malignancies. Synergy was demonstrated for PSB202 in combination with lenalidomide. Toxicokinetic assessment in cynomolgus monkeys showed a dose proportional exposure for both antibody components, with mean terminal elimination half-life (T1/2) of ~60 and ~68 hours for the anti-CD20 and anti-CD37 component, respectively. PBS202 was well tolerated, with observed toxicities mostly related to its activity as a B-cell depletion agent. As expected, PSB202 when administered at ≥ 10 mg/kg resulted in nearly complete depletion of B lymphocytes 24 hours following the 1st dose. B-cells returned to near baseline within 2 months after the last dose at 10 mg/kg. PSB202 can deplete normal and malignant B-cells by targeting two clinically validated targets through multiple distinct mechanisms including direct B-cell killing, enhanced ADCC activity, and CDC activity. PSB202 offers the potential to improve clinical efficacy, decrease drug resistance, provide administration convenience especially when combining with additional therapeutics, lower the cost for treatment, and reduce the unwanted side effects from chemotherapy. PSB202 is currently being evaluated in patients with previously treated, relapsed, indolent B-cell malignancies (NCT05003141). Citation Format: Paul Algate, Zhi Liu, Saying Yuan, Hua Liu, Cristina Domeier, Haiming Jiang, Yuanzhi Lao, Yanling Xu, William C. Fanslow, Ron Schoner, Wei Yan. Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies [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 2902.

Abstract 1281: SGN-B7H4V shows immunomodulatory activity through induction of immunogenic cell death

Abstract SGN-B7H4V is a novel investigational antibody-drug conjugate composed of a B7-H4-directed human monoclonal antibody conjugated to the validated vedotin drug linker, which incorporates the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linkage. This vedotin drug linker system has been clinically validated by multiple ADC programs, including brentuximab vedotin, enfortumab vedotin, tisotumab vedotin, and polatuzumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumor types, including breast, ovarian, and endometrial tumors. SGN-B7H4V is designed to bind and internalize the B7-H4/ADC complex from the surface of malignant cells and release the cytotoxic payload MMAE. The antitumor activity of SGN-B7H4V may be multimodal as SGN-B7H4V can induce tumor cell death through several mechanisms, including MMAE-mediated direct cytotoxicity and bystander killing as well as antibody-mediated functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Previously, vedotin ADCs have been described to elicit antitumor immune responses in part through induction of immunogenic cell death (ICD) mediated by the MMAE payload. These immunomodulatory effects potentially position vedotin ADCs to uniquely synergize with checkpoint inhibitors, supported by recent clinical activity observed when vedotin ADCs are paired with anti-PD1 agents. Here, we characterize SGN-B7H4V-mediated ICD and subsequent immunomodulatory activity. We also evaluate the contribution of SGN-B7H4V-induced immune activation to antitumor activity in combination with an anti-PD1 agent in an immunocompetent mouse model. In vitro, tumor cells treated with SGN-B7H4V showed several hallmarks of immunogenic cell death, including calreticulin exposure and release of ATP. In vivo, treatment of B7-H4-expressing tumors with SGN-B7H4V led to immune changes in the tumor microenvironment, including recruitment of macrophages and T cells. Finally, SGN-B7H4V drove robust, curative activity in an immunocompetent tumor model as a monotherapy and paired well with an anti-PD1 agent. Altogether, these data support the evaluation of SGN-B7H4V as a monotherapy in a first-in-human phase 1 clinical study and potential future clinical combinations with immunotherapies. Citation Format: Elizabeth E. Gray, Michelle Ulrich, Angela Epp, Patrick Younan, Kelly Hensley, Sean Allred, Julie Hahn, Kristen Gahnberg, Piper M. Treuting, John J. Gosink, Robert Thurman, Alyson J. Smith, Jason Schrum, Natalya Nazarenko, Shyra J. Gardai. SGN-B7H4V shows immunomodulatory activity through induction of immunogenic cell death [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 1281.

Abstract 2652: Efficacy of a small molecule MCT1 and MCT4 transporter inhibitor in cancer

Abstract Targeting metabolic pathways has been a major clinical interest to effectively treat cancer patients. FDA approval of TIVSOVO(R) (IDH1 inhibitor) has validated the clinical importance of targeting tumor metabolism in cancer. Although MCT1-targeting small molecule inhibitor is currently under investigation for lymphoid cancers, the therapeutic efficacy of this compound is compromised by compensatory overexpression of MCT4 by tumor cells. Therefore, there is a need for a dual MCT1/4 inhibitor to target cancer. We have developed a first-in-class orally bioavailable small molecule inhibitor that targets lactate transport through MCT1 and MCT4 transporters. In 4T1 syngeneic model, we observed a significant dose-dependent reduction of tumor growth by NGY-B treatment that also correlated with serum lactate levels. Immunoprofiling of tumors revealed that NGY-B promoted anti-tumor immune response in 4T1 tumor bearing mice indicating simultaneous immune activation. Also, the combination of NGY-B with a checkpoint inhibitor led to greater efficacy and overall survival in this model suggesting a potential combination therapy in the clinic. Furthermore, we demonstrated the direct tumor cell killing effect of NGY-B in multiple human CDX and PDX models. Therefore, NGY-B intervenes two key hallmarks of cancer - tumor metabolism and anti-tumor immunity, and provides a novel modality to therapeutically target cancer. Citation Format: Sambad Sharma, Gregory Goreczny, Nicole Bowman, Jennifer Duffy, Daliya Banerjee, Sanath Wijerathna, John Dzuris, Kerui Wu, Shih-Ying Wu, Abhishek Tyagi, Kounosuke Watabe, Jaime Escobedo, Vincent Sandanayaka. Efficacy of a small molecule MCT1 and MCT4 transporter inhibitor in 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 2652.

Abstract 1286: Oral administration of poly gamma glutamic acid significantly enhances the antitumor immune response of doxorubicin in a murine cancer model through regulating tumor microenvironment

Abstract Immunogenic cell death (ICD) is a type of cancer cell death triggered by certain chemotherapeutic drugs, oncolytic viruses, and other therapies. In particular, doxorubicin (DOX), one of the chemo-drugs, is well known to induce ICD, which involves the activation of the immune system against cancer in immunocompetent hosts. ICD comprises the release of damage-associated molecular patterns (DAMPs) from dying tumor cells that result in the activation of tumor-specific immune responses, thus eliciting long-term efficacy of anticancer drugs by combining direct cancer cell killing and antitumor immunity. However, the antitumor immunity can be achieved by overcoming the immunosuppressive tumor microenvironment (TME). Most current treatment strategies harnessing the tumor microenvironment focus on T cell-immune response, either by promoting activating signals or suppressing inhibitory ones.We here present a novel approach to cope with the inhibitory TME, combinatory treatment of DOX with poly gamma glutamic acid (γ-PGA). PGA, a safe edible biomaterial secreted by B. subtillis, has been evaluated in the clinical trials with the patients diagnosed as cervical intraepithelial neoplasia 1 (CIN 1). From the study using murine cancer models, including MC38, we found the combinatory treatment with γ-PGA restored the downregulation of NK cell activity caused by DOX, enhanced ICD and T cell responses, and reduced proinflammatory cytokines in the tumor tissues. Taken together, co-administration of γ-PGA exhibited synergistic anti-cancer effects by favoring the immunosuppressive TME in the combinatory treatment with conventional chemo-drugs. Citation Format: Joungwoo Choi, Solmin Jung, Yena Oh, Jaepyeong Jang, Yeondong Cho, Do young Lee, Youngcheol Park, Kwangil Jeong. Oral administration of poly gamma glutamic acid significantly enhances the antitumor immune response of doxorubicin in a murine cancer model through regulating tumor microenvironment [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 1286.

Abstract 5567: hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing cancers

Abstract Background and Hypothesis: Mesothelin is an attractive target for CAR-T cell therapy since it is highly expressed in many solid tumors but has limited expression on normal human tissues. However, anti-mesothelin CAR-T cells have shown limited efficacy in patients. Since most antibodies to mesothelin bind the membrane distal epitopes we generated antibodies that bind mesothelin in a membrane-proximal region. We hypothesize that CAR-T cells recognizing a membrane proximal epitope on mesothelin will be more efficacious. Study Design: We developed CAR-T cells targeting a membrane-distal (SS1CAR-T) and a membrane-proximal epitope (hYP218 CAR-T) of mesothelin. Using mesothelin-expressing cancer cell lines, anti-tumor efficacy of CAR-T cells was assessed in vitro by measuring direct killing of tumor cells by the CAR-T cells and quantifying cytokine-release by the CAR-T cells. In vivo anti-tumor efficacy of CAR-T was determined in NSG mice using three different solid tumor models of high-mesothelin expression: ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived xenograft (NCI-Meso63). Blood and tumor infiltration of CAR-T cells in vivo was determined via flow cytometry analysis. Results: Upon co-culture with tumor cells with high mesothelin expression (A431-H9, OVCAR-8, KLM-1, H226, NCI-Meso63, NCI-Meso29), hYP218 CAR-T cells killed cancer cells more efficiently than SS1 CAR-T cells, with a 2-4-fold lower ET50 value (Effector to Target ratio for 50% killing of tumor cells). Additionally, compared to SS1 CAR-T cells, hYP218 CAR-T cells secreted significantly higher level of cytokines (IL-2, TNFα, IFNγ) in a co-culture with tumor cells. In three solid tumor models, single treatment of tumor-bearing mice with hYP218 CAR-T cells lead to improved tumor response and survival compared to SS1 CAR-T, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared to SS1 CAR-T cells, higher hYP218 CAR-T cell infiltration was detected both in the blood and tumors isolated from hYP218 CAR-T treated mice seven days post-treatment. Conclusion: We demonstrate that hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are more potent than similar CAR-T cells targeting a membrane distal epitope. Increased accumulation of hYP218 CAR-T cells in the tumor may explain their increased efficacy. These results support its clinical development to treat patients with mesothelin expressing cancers. Citation Format: Sakshi Tomar, Jingli Zhang, Nan Li, Ira Pastan, Mitchell Ho, Raffit Hassan. hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing 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 5567.

Phytochemical natural killer cells reprogram tumor microenvironment for potent immunotherapy of solid tumors

Natural killer cells (NKs) hold great promise in cancer treatment, but their application in solid tumors remains a great challenge because current solutions hardly can overcome various difficulties that faced. Herein, we endow NKs with the phytochemical feature for effective immunotherapy of solid tumors. NKs are decorated with natural thylakoid (Tk) membranes through an efficient and convenient membrane fusion strategy. Tk engineering effectively activates NKs, because the antioxidase on Tk induce glycogen synthase kinase-3β inhibition, and subsequently increase the expression of activating receptor and cytotoxic effector molecules in NKs. After systemic administration, the phytochemical NKs (PC-NKs) can target tumor tissues, and then profoundly reprogram tumor microenvironment (TME) with the help of catalase on Tk, resulting in significantly enhanced direct killing of PC-NKs and immune activated TME. Therefore, potent therapeutic effects with few abnormalities are achieved, providing a novel idea for the development of highly efficient NKs for solid tumors.

Abstract CT152: Phase I study of MT-6402, a novel engineered toxin body (ETB) targeting PD-L1, in patients with PD-L1 expressing advanced solid tumors

Abstract This is the first-in-human study of MT-6402, a unique, first-in-class potent PD-L1-targeted engineered toxin body (ETB) capable of direct killing of PD-L1 expressing cells via rapid PD-L1-mediated internalization of a fused Shiga-like toxin A subunit (SLTA) resulting in permanent ribosomal inactivation. MT-6402 also delivers an HLA-A*02 restricted pp65 cytomegalovirus (CMV) antigen into PD-L1 expressing tumor cells leading to MHC-I presentation to existing CMV-specific cytotoxic T cells (antigen seeding). MT-6402 functions by targeting tumor and inhibitory immune cells directly and altering tumor immunophenotype to re-direct antiviral cytotoxic T cells into the tumor microenvironment. MT-6402 shows picomolar cytotoxic activity across several PD-L1 expressing cancer cell lines and treatment of human PBMCs results in selective depletion of PD-L1 positive cells. MT-6402 was well tolerated in non-human primates at doses above those which induce pharmacodynamic (PD) effect (reduction of CD14+ monocytes) in humans. This first-in-human study in patients with PD-L1-expressing advanced solid tumors will employ a modified toxicity probability interval design to determine MTD and will then enroll additional subjects at the MTD, to further explore safety and efficacy and determine RP2D. Results of the first dose cohort (16 micrograms/kg) are presented. Six patients received MT-6402. A significant and sustained reduction in CD14+ monocytes starting in cycle 2 was observed in patients on therapy beyond one cycle and was maintained with each MT-6402 administration, indicating HLA-independent PD effect consistent with preclinical observations. One HLA-A*02 and CMV+ patient with osseous metastases from NSCLC showed marked CMV-specific T-cell extravasation at day 8 and serum cytokine signatures consistent with antigen dependent responses and T cell mobilizations, suggesting engagement of MT-6402 antigen seeding. This patient has reduced intensity of metastatic bone lesions with 3/4 lesions resolving; the remaining lesion showing reduced uptake. Two patients had cytokine elevations at day 15, manifested by transient (1-12h), grade 2 infusion-related reactions and grade 2 cytokine release syndrome, which were subsequently prevented by steroid premedication. These results describe a novel approach to checkpoint modulation, leveraging direct PD-L1 cell kill and antigen seeding technology by the ETB. The results support further dose escalation and hold promise for development of MT-6402 for solid tumors, including in the R/R setting. Citation Format: David R. Spigel, Eugene Ahn, Herbert L. Duvivier, Drew Rasco, Agnes Rethy, Chris Moore, Amy Yuet, Sandra Hankins, Swati Khanna, Joseph Dekker, Brian Van Tine. Phase I study of MT-6402, a novel engineered toxin body (ETB) targeting PD-L1, in patients with PD-L1 expressing advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT152.

Abstract 2836: Genome engineered natural killer cell immunotherapy against osteosarcoma

Abstract Over the last decade Chimeric Antigen Receptor based T cell therapy (CAR-T) has developed into an effective immunotherapy for some cancers. Unfortunately, CAR-T cell therapies have several shortcomings and clinical success has primarily been limited to hematological cancers. Challenges of CAR-T cell therapy include tumor immune evasion through loss of target antigen expression by tumor cells and inhibition of CAR-T cell function by tumor expressed inhibitory molecules. Natural killer (NK) cells present an alternative to T cells that could be more effective due to their ability to perform both antigen dependent and independent killing. NK cells have demonstrated antigen specific killing when engineered to express CARs and NK cells also mediate the direct killing of transformed cells with reduced or absent MHC expression. Moreover, NK cells carry out antibody dependent cell mediated cytotoxicity (ACDD) of cells bound by antibodies via the NK cell CD16A receptor. Due to the multiple modalities for cancer cell killing, there is an increased interest in NK cells for cancer immunotherapy. As NK cells are not associated with graft versus host disease, neurotoxicity, long-term autoimmunity, nor cytokine release syndrome, they are more suited for use in allogeneic settings than T cells and have significant clinical potential for use as off-the-shelf products. However, previous publications and clinical trials have demonstrated that the use of unmanipulated NK cells to treat cancer is minimally effective, likely due to limited engraftment, little in vivo expansion or persistence, and suppression by the tumor microenvironment. NK cells activated and expanded with engineered feeder cells expressing membrane bound interleukin-21 (mbIL-21) and 4-1BBL have shown promising results clinically with high-risk myeloid malignancies and preclinically in several solid tumor models. Therefore, we hypothesize that activated/expanded CAR-NK cells that have been genetically edited can be used to successfully treat osteosarcoma, a disease for which patient outcome has not improved in over forty years. Our proposed objectives are to evaluate the baseline response of rested- and activated/expanded-NK cells against various osteosarcoma cell lines, knockout negative regulators of NK cell function (specifically, c-CBL, IL-1R8, and SMAD3), and implement several CARs alone or in combination that optimally activate NK cell antigen-specific killing. Genetically engineered CAR-NK cells will be evaluated for enhanced therapeutic efficacy and safety in osteosarcoma models. Our preliminary data strongly supports the hypothesis that CAR-NK cell-based cancer immunotherapy can be fully realized using activated, genome engineered CAR-NK cells. Citation Format: Gabrielle M. Robbins, Kenta Yamamoto, Joshua Krueger, Walker Lahr, Joseph Skeate, Branden Moriarity. Genome engineered natural killer cell immunotherapy against osteosarcoma [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 2836.

Promising Therapeutic Strategies for Colorectal Cancer Treatment Based on Nanomaterials.

Colorectal cancer (CRC) is a global health problem responsible for 10% of all cancer incidences and 9.4% of all cancer deaths worldwide. The number of new cases increases per annum, whereas the lack of effective therapies highlights the need for novel therapeutic approaches. Conventional treatment methods, such as surgery, chemotherapy and radiotherapy, are widely applied in oncology practice. Their therapeutic success is little, and therefore, the search for novel technologies is ongoing. Many efforts have focused recently on the development of safe and efficient cancer nanomedicines. Nanoparticles are among them. They are uniquewith their properties on a nanoscale and hold the potential to exploit intrinsic metabolic differences between cancer and healthy cells. This feature allows them to induce high levels of toxicity in cancer cells with little damage to the surrounding healthy tissues. Graphene oxide is a promising 2D material found to play an important role in cancer treatments through several strategies: direct killing and chemosensitization, drug and gene delivery, and phototherapy. Several new treatment approaches based on nanoparticles, particularly graphene oxide, are currently under research in clinical trials, and some have already been approved. Here, we provide an update on the recent advances in nanomaterials-based CRC-targeted therapy, with special attention to graphene oxide nanomaterials. We summarise the epidemiology, carcinogenesis, stages of the CRCs, and current nanomaterials-based therapeutic approaches for its treatment.

Identification of ADS024, a newly characterized strain of Bacillus velezensis with direct Clostridiodes difficile killing and toxin degradation bio-activities

Clostridioides difficile infection (CDI) remains a significant health threat worldwide. C. difficile is an opportunistic, toxigenic pathogen that takes advantage of a disrupted gut microbiome to grow and produce signs and symptoms ranging from diarrhea to pseudomembranous colitis. Antibiotics used to treat C. difficile infection are usually broad spectrum and can further disrupt the commensal gut microbiota, leaving patients susceptible to recurrent C. difficile infection. There is a growing need for therapeutic options that can continue to inhibit the outgrowth of C. difficile after antibiotic treatment is completed. Treatments that degrade C. difficile toxins while having minimal collateral impact on gut bacteria are also needed to prevent recurrence. Therapeutic bacteria capable of producing a range of antimicrobial compounds, proteases, and other bioactive metabolites represent a potentially powerful tool for preventing CDI recurrence following resolution of symptoms. Here, we describe the identification and initial characterization of ADS024 (formerly ART24), a novel therapeutic bacterium that can kill C. difficile in vitro with limited impact on other commensal bacteria. In addition to directly killing C. difficile, ADS024 also produces proteases capable of degrading C. difficile toxins, the drivers of symptoms associated with most cases of CDI. ADS024 is in clinical development for the prevention of CDI recurrence as a single-strain live biotherapeutic product, and this initial data set supports further studies aimed at evaluating ADS024 in future human clinical trials.

A phase 1 trial of D2C7-it in combination with an Fc-engineered anti-CD40 monoclonal antibody (2141-V11) administered intratumorally via convection-enhanced delivery for adult patients with recurrent malignant glioma (MG).

e14015 Background: D2C7 immunotoxin (D2C7-IT) is a dual-specific recombinant immunotoxin comprising an EGFR wild-type and mutant-specific (EGFRvIII) monoclonal antibody (Ab) fragment and a genetically engineered form of the Pseudomonas exotoxin. When injected directly into the tumor by convection enhanced delivery (CED), immunotoxins induce both direct tumor killing and secondary immune responses by activation of CD4+ and CD8+ T-cells. Tumor-associated macrophages (TAMs) are the most prominent glioma-infiltrating immune cells and constitute up to 40% of the tumor mass. Upon binding of D2C7-IT to EGFR and cellular internalization, the Pseudomonas exotoxin moiety of the D2C7-IT kills residual GBM cells, upregulates proinflammatory CD40, and induces pattern recognition receptor pathway transcriptome expression. This potentially creates a proinflammatory glioma microenvironment where TAM activation may be further stimulated by sequential CED of 2141-V11, an Fc engineered anti-human CD40 agonist antibody developed at Rockefeller University. We are conducting a first in human trial of the combination of D2C7-IT + 2141-V11 administered via CED in recurrent MG patients. Methods: Eligibility includes adult patients with recurrence of a solitary supratentorial WHO grade 3 or 4 MG; ≥ 4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; and KPS ≥ 70%. Cohorts of 3 patients are treated with increasing levels of 2141-V11 to determine the maximum tolerated dose (MTD) of the compound administered intratumorally in conjunction with D2C7-IT. Dose escalation and de-escalation are managed using a modified Bayesian optimal interval (BOIN) design to identify the MTD. Intratumoral administration of D2C7-IT via CED (4612 ng/mL over 72 hours) is followed by a 7-hour infusion of 2141-V11, both infused at 0.5 mL/hr. 2141-V11 is dose-escalated to determine the MTD when combined with D2C7-IT. Four dose levels (DL) are planned: #1: 0.70 mg; #2: 2.0 mg; #3: 7.0 mg; #4: 21.0 mg. Results: As of February 7, 2022, three patients were treated at DL1 and DL2, and two patients at DL3. No DLTs have been observed, and all eight patients remain alive and in observation on study after 7.0, 6.5, 6.0, 4.4, 2.8, 2.4, 0.9 and 0.5 months. Early signs of tumor response have been observed, with one patient at DL1 and 2 patients at DL2 without radiographic evidence of active tumor. Grade 2 or higher AEs due to D2C7-IT and/or 2141-V11 include: headache (grade 3, n = 1; grade 2, n = 2); paresthesia (grade 3, n = 1; grade 2, n = 1); dysphasia (grade 3, n = 1); pyramidal tract disorder (grade 3, n = 1; grade 2, n = 1); and depressed level of consciousness (grade 2, n = 1). Enrollment is ongoing. Conclusions: Intratumoral administration of D2C7-IT + 2141-V11 via CED is safe, and encouraging efficacy results have been observed. Clinical trial information: NCT04547777.