Superior Tumor Research Articles

Abstract 3308: Chemoradiation sensitization effect of PARPi in non-small cell lung cancer

Abstract Chemoradiation (CRT) is widely used to treat locally advanced non-small cell lung cancer (NSCLC) patients, but only around 30% of patients achieve pathological complete response. Here, we utilized an in vitro high-content clonogenic survival screening method to identify CRT sensitizers from the NCI Cancer Therapy Evaluation Program (CTEP) portfolio. Talazoparib (PARPi) was identified among the top candidates to show potent CRT sensitization effects in both KRAS mutant and TP53 mutant NCSLC cell lines. We validated the CRT sensitization effect of PARPi on lung tumors in vivo with cell line xenografts mouse models. To further investigate alterations in the tumor microenvironment (TME) induced by PARPi, we utilized syngeneic mouse models and showed that the combination of radiotherapy, PARPi, and PD-L1 blockade achieved superior tumor control effects. By applying single cell RNA sequencing technologies to the mouse tumors, we found that PARPi combined with CRT or immunotherapy significantly reprogrammed the tumor cells and TME, resulting in the increase of tumor-infiltrating T cells which elicit potent tumor control effects. Our data showed that PARPi sensitizes NSCLC tumors to CRT and immunotherapy through a combination of tumor-intrinsic and extrinsic phenomenon. Citation Format: Rui Ye, Kaile Wang, Yawei Qiao, Zhenna Xiao, Yun Yan, Jianzhong He, Nan Li, Yifan Wang, Min Hu, Shanshan Bai, Emi Sei, Nicholas Navin, Steven Lin. Chemoradiation sensitization effect of PARPi in non-small cell lung 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 3308.

Abstract 569: Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME)

Abstract Targeting solid tumors with CAR T cells has proven to be more difficult due to their heterogenous target antigen expression, antigen escape, and due to their hostile tumor microenvironment (TME). Mesothelin represents a promising surface tumor antigen, since it has been associated with tumor invasion and is highly expressed on various cancer types, including pancreatic adenocarcinoma. As clinical trials of mesothelin targeting CAR have yet to show efficacy, we hypothesized that tumor stromal cells such as cancer-associated fibroblasts (CAFs) may play a role in this resistance. To provide a more favorable TME for CAR T cells, we generated a bicistronic lentiviral vector encoding a mesothelin CAR along with a secreted T cell engaging molecule (TEAM) that targets fibroblast activation protein (FAP), which is expressed by CAFs. We termed our constructs CARTEAM, and in this case, mesoFAP. We have assessed the activation and proliferative capacity of these CARTEAM through in vitro assays. We showed that TEAMs secreted by CAR T cells bind their appropriate target antigen by adding supernatant from CARTEAM to target cells expressing FAP. In a co-culture assay using a transwell system we demonstrated the cytotoxic effect of secreted TEAM interacting and recruiting bystander T cells against CAFs. In a real-time cell analysis (RTCA) co-culture assay with a pancreatic cancer cell line (AsPC1) and FAP expressing CAFs, we showed cell death of AsPC1 upon CAR recognition and cell death of CAFs through TEAM-mediated recruitment of bystander T cells and CART cells. We show in these co-culture systems, mimicking tumor and TME, that our CARTEAM construct is superior in the elimination of both cancer cell line and CAF, in comparison to control constructs, including mesothelin targeting CAR T cells (meso CAR) and meso CAR T cells secreting an unspecific CD19 TEAM (mesoCD19). We also used acoustic force microscopy to evaluate the additive effect of the TEAM molecule secreted to binding to tumor cells by mesothelin CAR T cells. In vivo experiments of subcutaneously injected tumor cells admixed with CAFs, show superior tumor control when treated with CARTEAM in comparison to control constructs. Based on these data, we demonstrate both the effective in vitro elimination of CAFs and pancreatic cancer cells through the application of CARTEAM and control of pancreatic tumor growth in vivo. Our studies provide a deeper insight into a dual targeting strategy using a novel CAR T cell secreting a TEAM against pancreatic cancer and its tumor microenvironment. Citation Format: Marc Wehrli, Adam Kuo, Rebecca Larson, Irene Scarfò, Amanda Bouffard, Korneel Grauwet, Mark Leick, Andrea Schmidts, Stefanie Bailey, Tamina Kienka, Michael Kann, Sonika Vatsa, Harrison Silva, Kathleen Gallagher, Max Jan, Bryan Choi, David Ting, Marcela Maus. Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME) [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 569.

Abstract 3517: Pharmacologic inhibition of HPK1 kinase activity enhances immune cell activation and T cell mediated anti-tumor activity

Abstract Hematopoietic progenitor kinase 1 (HPK1, mitogen-activated kinase kinase kinase kinase 1 [MAP4K1]), a cytosolic STE20 ser/thr kinase from the germinal center family of kinases, regulates the function of diverse immune populations including T cells, B cells, and dendritic cells. In T cells, activated HPK1 regulates T cell receptor (TCR) signaling by phosphorylating the adaptor protein SLP76 on Ser376, which induces the association of SLP76 with 14-3-3 proteins. This SLP76/14-3-3 complex leads to its disassociation from the TCR signalosome, resulting in reduced downstream TCR signaling. The kinase activity of HPK1 is necessary for its regulatory function as mice expressing a mutant kinase-inactive HPK1 demonstrate enhanced T cell activity and superior tumor growth control compared to their WT counterparts. Here we describe a highly potent, selective and orally bioavailable small molecule inhibitor of HPK1 which displays sub-nanomolar HPK1 inhibition, good selectivity against MAP4K family members and oral bioavailability in preclinical animal models. Pharmacological inhibition of HPK1 kinase activity with Gilead’s HPK1 inhibitor compound demonstrates robust inhibition of pSLP76 S376 with concomitant enhancement in T cell activation, proliferation and effector function in in vitro assays. These immune enhancing effects translate into in vivo preclinical tumor models, where treatment with Gilead’s HPK1 inhibitor compound results in heightened T cell activation and effector function and enhanced tumor growth control both as a single agent and in combination with PD-1 blockade. In summary, our data show that Gilead’s HPK1 inhibitor compound is expected to be a selective, orally bioavailable HPK1 inhibitor with potential to combine with checkpoint inhibitors and other immuno-oncology agents. Citation Format: Jacob S. Lee, Pamela Toy, Gundula Min-Oo, Tony Lee, Patrick Salvo, JeanPhilippe Belzile, Sheila Clancy, Jonathan Nazareno, Mengshu Xu, Weimei Xing, Marilyn Giacomini, Rex Santos, Lance Stapleton, Adele Wang, Vladislava Juric, Scott A. Mitchell, Michelle Kuhne. Pharmacologic inhibition of HPK1 kinase activity enhances immune cell activation and T cell mediated anti-tumor activity [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 3517.

Abstract 5287: Aggregation of HER2 receptors on the tumor cell surface induced by a novel anti-HER2 bispecific antibody led to enhanced tumor cell growth inhibition

Abstract Trastuzumab, an anti-HER2 monoclonal antibody developed by Roche, is now a standard of care for treating HER2 overexpressing cancers. However, patients treated with trastuzumab generally develop resistance to the drug within a year after initiation of the treatment. Pertuzumab, another anti-HER2 monoclonal antibody developed by Roche, targets the domain 2 of the HER2 excellular domain (ECD) instead of the domain 4 to which trastuzumab binds and demonstrates enhanced antitumor activities when combined with trastuzumab. Here, we developed a tetravalent bispecific antibody (BsAb) which targets the domain 2 and 4 of the HER2 ECD simultaneously. Using time-lapse microscopy, we showed that the BsAb induced aggregation of HER2 on NCI-N87 tumor cells, possibly indicating that the receptors could be crosslinked by the BsAb intermolecularly. Furthermore, the tetravalent BsAb induced aggregation faster than did the bivalent BsAb. Importantly, the tetravalent BsAbs exhibited superior tumor cell growth inhibition to that of the two parental monoclonal antibodies alone or combined. In line with the aggregation rates, the tetravalent BsAb was also more potent in inhibiting tumor cell growth than that of the bivalent BsAb. Thus we provide a piece of evidence that the BsAbs targeting two distinct domains of HER2 simultaneously might induce the receptor aggregation which might associate with the enhanced inhibition of tumor cell growth. Citation Format: Lan Deng, Jie Zhao, Xiaoqing Meng, Aotian Xiao, Zhenping Zhu, Haomin Huang. Aggregation of HER2 receptors on the tumor cell surface induced by a novel anti-HER2 bispecific antibody led to enhanced tumor cell growth inhibition [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 5287.

Abstract 6027: Layered 3D co-cultured tumor model including vascularized stromal tissue may reflect drug sensitivities in vivo tumor

Abstract Background: Multiple 3D culture models have been reported as a superior tumor model than 2D culture. The importance of interaction between cancer and stromal cells has been widely recognized in tumor progression and resistance to treatment. Therefore, ex vivo model recapturing the tumor microenvironment is needed to evaluate the drug efficacy under the condition imitating the patient tumor tissue. Purpose: We developed a unique 3D co-cultured tumor model with stromal tissues containing a microvascular network. Here, we investigated drug sensitivity in conventional 2D culture, our 3D co-cultured model and in vivo tumor. Methods: Drug sensitivity and gene expression on our model were evaluated using patient-derived cancer cells (PDC) established from colorectal cancer (CRC) patients in JFCR. The characteristics were compared with those of conventional 2D cultured cells or patient-derived xenograft (PDX). Results: In our 3D model, the drug sensitivities tended to be decreased in comparison with those of 2D. However, some drugs presented potent efficacy in our 3D model rather than 2D, and such drugs showed significant tumor growth suppression or tumor regression in vivo. Transcriptome profiles of our 3D model showed relatively higher similarity to those of in vivo tumors than those of 2D. Conclusion: Our study proposed the unique 3D co-cultured tumor model which may enable more accurate evaluation of drug sensitivities reflecting the in vivo circumstances. Citation Format: Yuki Takahashi, Shiro Kitano, Eiji Shinozaki, Satoshi Nagayama, Ryohei Katayama, Naoya Fujita. Layered 3D co-cultured tumor model including vascularized stromal tissue may reflect drug sensitivities in vivo 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 6027.

Abstract 2814: Anti-CAIX Immune Restoring (IR) CAR-T cells display superior antitumor activity and reverse immunosuppressive TME in a humanized ccRCC orthotopic mouse model

Abstract Chimeric Antigen Receptor (CAR) T cell therapy is a new type of “living drug” that has proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. To date, there are five CAR-T products approved by FDA, four CD19 targeted CAR-T cells, and one targeting B-cell maturation antigen (BCMA). However, this success has not yet been transferred to solid tumors. A major challenge is the immunosuppressive tumor microenvironment (TME). Development of immunotherapies has traditionally been hampered by discrepancies observed between in vitro and in vivo studies and actual clinical trial outcomes. The lack of clinically relevant mouse models for human immunotherapy testing is often seen as the primary cause. Existing clear cell renal cell carcinoma (ccRCC) in vivo models poorly recapitulate the tumor microenvironment (TME). Here we report a ccRCC orthotopic humanized NSG-SGM3 mouse model (ccRCC-hNSG-SGM3) with reconstituted human lymphocytes and bearing human ccRCC skrc-59 cells under the kidney capsule. Human leukocyte antigen (HLA) matched CD34+ human stem cells were used for the humanization to reduce T cell alloreactivity against skrc-59 human ccRCC cells. Tumors were harvested, sorted for CD45+ tumor infiltrating leukocytes (TILs) and single cell RNA sequencing (scRNAseq) was performed to profile the TME in ccRCC-hNSG-SGM3. By comparing to patient data from prospective clinical trials of the anti-PD-1 monoclonal antibody (mAb) nivolumab in advanced ccRCC, the results demonstrated that CD45+ TILs from ccRCC-hNSG-SGM3 reconstitute most CD45+ cell types, including dendritic cells, exhausted CD8 T cells, and regulatory T cells (Tregs), that are observed in advanced ccRCC patient TME. Furthermore, we generated HLA matched Immune Restoring (IR) CAR-T cells which can secrete anti-PD-L1 monoclonal antibody (mAb) locally to restore active antitumor immunity, and we assessed the efficacy and safety of IR CAR-T cells in this model. Anti-CAIX CAR-T cells armored with anti-PD-L1 mAb showed superior efficacy in tumor regression and significantly decreased TIL exhaustion compared to irrelevant CAR or irrelevant payload in this model. In addition, in situ hybridization (ISH) results showed CAR-T cells infiltration in tumor but no CAR-T cells were observed in normal tissues. In summary, the ccRCC-hNSG-SGM3 system is able to model the advanced ccRCC TME and provides a powerful tool for ccRCC TME study and immunotherapy assessment. IR CAR-T cells exhibited superior tumor regression and reversed immunosuppressive TME in humanized mice. Citation Format: Yufei Wang, Alicia Buck, Marion Grimaud, Aedin Culhane, David Braun, Sreekumar Kodangattil, Cecile Razimbaud, Matthew Chang, Atef Fayed, Audrey Apollon, Gabriella Kastrunes, Luann Zerefa, Brandon Piel, Elena Ivanova, Dennis Bonal, Kristen Jones, Quang-De Nguyen, Zhu Zhu, Kevin Wei, Nicholas Hayden, Madison O'Donnell, Ying Huang, Rebecca Jenning, Miriam Ficial, Maura Aliezah Sticco-Ivins, Sabina Signoretti, Catherine Wu, Toni Choueiri, Jon Wee, Cloud Paweletz, David A. Barbie, Gordon Freeman, Wayne A. Marasco. Anti-CAIX Immune Restoring (IR) CAR-T cells display superior antitumor activity and reverse immunosuppressive TME in a humanized ccRCC orthotopic mouse 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 2814.

Abstract 3428: NI-2901, a CD47xPD-L1 bispecific antibody for dual immune checkpoint blockade with fine-tuned affinity to reduce erythrocyte binding and improve biodistribution

Abstract Blocking the CD47/SIRPα checkpoint has recently emerged as an effective approach to mobilize the myeloid cell compartment and to improve antitumor responses in the clinic. Preclinical models have demonstrated the synergistic benefit of combined CD47/SIRPα and PD-1/PD-L1 blockade. Combinations of monoclonal antibodies (mAbs) targeting these two checkpoint pathways are being explored in the clinic. CD47xPD-L1 bispecific antibodies (bsAbs) stand as an attractive alternative to mAb combinations, even more so as they provide a potential solution to improve the pharmacokinetic profile and safety issues faced by CD47 targeted-mAbs and SIRPα-Fc fusion proteins. CD47xPD-L1 bsAbs are expected to preferentially inhibit CD47 on PD-L1 expressing cells, displaying improved safety and pharmacokinetics, but also superior tumor microenvironment targeting capabilities. With the objective of finding the optimal CD47xPD-L1 bsAb, an array of bispecific antibodies (bsAbs) was generated associating a high affinity PD-L1 arm to CD47 arms with varying affinities. The CD47xPD-L1 bsAbs of human IgG4 isotype were generated using our fully human κλ body antibody platform. The candidate molecules were screened for binding and receptor-blocking activity and tested for their capacity to enhance T-cell activation and phagocytosis of tumor cells in the presence of anti-HER-2 mAb, trastuzumab. Selected bsAbs were also evaluated in a xenograft mouse model. The CD47xPD-L1 bsAbs demonstrated an effective blockade of the PD-1/PD-L1 interaction, being able to induce T-cell activation in vitro similar to the anti-PD-L1 clinical benchmark, atezolizumab. Consistent with their CD47 affinities, the bsAbs showed varying levels of CD47 blockade on PD-L1-negative cells and a low binding capacity to red blood cells. Nonetheless, trastuzumab-mediated phagocytosis of tumor cells expressing low levels of PD-L1 could be significantly enhanced by these bsAbs, confirming the PD-L1-independent activity of the CD47 blocking arms. The latter findings were corroborated in vivo using PD-L1-negative Raji cells in a xenograft mouse model. Selected bsAbs will be now tested for tolerability and pharmacokinetic profiles in human-CD47/human-SIRPα transgenic mice. Lead candidate(s) will be evaluated further for PK and safety attributes in non-human primates in early Q1, 2022. Citation Format: Xavier Chauchet, Sébastien Calloud, Margaux Legrand, Laura Cons, Laurence Chatel, Pauline Lloveras, Coline Burnet-Merlin, Louis Hellequin, Nicolas Bosson, Pauline Malinge, Nicolas Pleche, Jérémie Bourguignon, Guillemette Pontini, Christophe Guillamo, Ulla Ravn, Valéry Moine, Bruno Daubeuf, Yves Poitevin, Giovanni Magistrelli, Limin Shang, Walter Ferlin, Krzysztof Masternak. NI-2901, a CD47xPD-L1 bispecific antibody for dual immune checkpoint blockade with fine-tuned affinity to reduce erythrocyte binding and improve biodistribution [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 3428.

Abstract 2746: Preclinical activity and manufacturing feasibility of genetically modified PDCD-1 knockout (KO) tumor-infiltrating lymphocyte (TIL) cell therapy

Abstract Background: Adoptive cell therapy with autologous TIL has demonstrated an objective response rate (ORR) of 36% in the post-immune checkpoint inhibitor (ICI) setting in patients (pts) with advanced/unresectable melanoma (Sarnaik JCO 2021), while in ICI-naïve pts who received early-line combination of TIL and pembrolizumab, the ORR was 60%, with a 30% CR rate (O’Malley SITC 2021). Although effective, anti-PD-1 therapy is limited by poor penetration into the tumor, internalization, and endocytic clearance, in contrast with TIL, which overcome this inherent limitation. PDCD-1 gene inactivation (PD-1 KO) may enhance TIL cell therapy efficacy in the post-ICI setting and abrogate the need for systemic anti-PD-1 therapy in ICI-naïve pts. In preclinical studies, PD-1 KO TIL maintain robust effector function and phenotypic markers indicative of functional TIL (Ritthipichai ESMO 2020). We describe preclinical activity, clinical-scale manufacturing process development, and characterization of IOV-4001, an autologous PD-1 KO TIL cell product. Methods: hIL-2 NOG mice engrafted with melanoma tumor cells received adoptive transfer of autologous PD-1 KO TIL (developed with TALEN® gene editing technology in collaboration with Cellectis), mock TIL (electroporation without TALEN), mock TIL + anti-PD-1 antibody, or no adoptive transfer (n=14 each). Tumor size was measured 2×/wk for 39 days. A 22-day clinical-scale manufacturing process was established, including pre-rapid expansion protocol (pre-REP), activation, electroporation, resting, and REP, for the generation of PD-1 KO TIL. Final PD-1 KO TIL product was characterized for total viable cells (TVC), purity (% viability), identity (% CD45+CD3+), and function (PD-1 KO efficiency). Results: Day 39 mean ± SEM tumor size (mm2) for mice treated with PD-1 KO TIL (6 ± 2.8) showed superior tumor control relative to mock TIL (26 ± 8.5, P<0.05), mock TIL + anti-PD-1 (33 ± 8.8, P<0.01), and no adoptive TIL transfer (112 ± 8.4, P<0.0001). Product attributes from 6 clinical-scale manufacturing runs for PD-1 KO TIL were acceptable, with a median (range) TVC, purity, and identity of 8.3 × 109 (0.9×109-35.7×109), 94% (91%-99%), and 99% (98%-99%), respectively. Median (range) PD-1 KO efficiency was 48% (31%-84%). PD-1 KO TIL function and phenotype (differentiation, memory, activation, and exhaustion) were comparable to mock TIL. Conclusions: Anti-tumor activity of PD-1 KO TIL was superior to mock TIL suggesting that endogenous PD-1 inhibition may confer a functional advantage to the TIL over an antibody combination. PD-1 KO TIL clinical manufacturing was feasible and the TIL product quality attributes and phenotype were acceptable; importantly, lack of complete PD-1 KO may spare other PD-1-dependent in vivo cellular functions. Together, these data support clinical investigation of IOV-4001, an autologous PD-1 KO TIL cell therapy. Citation Format: Arvind Natarajan, Anand Veerapathran, Adrian Wells, Kenneth Onimus, Marcus Machin, Seth Wardell, Jamie L. Blauvelt, Madan Jagasia, Rafael Cubas. Preclinical activity and manufacturing feasibility of genetically modified PDCD-1 knockout (KO) tumor-infiltrating lymphocyte (TIL) cell 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 2746.

Pancoast Tumor Presenting as Neck Pain in the Chiropractic Office: A Case Report and Literature Review

Patient: Male, 59-year-old Final Diagnosis: Adenocarcinoma of the lung • pancoast tumor Symptoms: Cough • neck and shoulder pain and numbness Medication: — Clinical Procedure: Magnetic resonance imaging (MRI) of the cervical and thoracic spine Specialty: Infectious Diseases • Pulmonology • Radiology • Rehabilitation Objective: Rare disease Background:Pancoast tumors, also called superior sulcus tumors, are a rare type of cancer affecting the lung apex. These tumors can spread to the brachial plexus and spine and present with symptoms that appear to be of musculoskeletal origin.Case Report:A 59-year-old Asian man presented to a chiropractor in Hong Kong with a 1-month history of neck and shoulder pain and numbness that had been treated unsuccessfully with exercise, medications, and acupuncture. He had an active history of tuberculosis, which was currently treated with antibiotics, and a 50-pack-year history of smoking. Cervical magnetic resonance imaging (MRI) was performed urgently, revealing a small cervical disc herniation thought to correspond with radicular symptoms. However, as the patient did not respond to a brief trial of care, a thoracic MRI was urgently ordered, revealing a large superior sulcus tumor invading the upper to mid-thoracic spine. The patient was referred for medical care and received radiotherapy and chemotherapy with a positive outcome. A literature review identified 6 previously published cases in which a patient presented to a chiropractor with an undiagnosed Pancoast tumor. All patients had shoulder, spine, and/or upper extremity pain.Conclusions:Patients with a previously undiagnosed Pancoast tumor can present to chiropractors given that these tumors may invade the brachial plexus and spine, causing shoulder, spine, and/or upper extremity pain. Chiropractors should be aware of the clinical features and risk factors of Pancoast tumors to readily identify them and refer such patients for medical care.

Proton Pump Inhibitors Enhance the Antitumor Effect of Chemotherapy for Esophageal Squamous Cell Carcinoma.

Simple SummaryThe use of proton pump inhibitors (PPIs) as V-ATPase inhibitors has been reported to enhance the effectiveness of chemotherapy in some cancers. This study aimed to evaluate the effect of PPIs on 5-Fuorouracil (5-FU)-based therapy for advanced esophageal cancer based on in vitro experiments and a clinical study. In the present study, PPIs showed a dose-dependent antitumor effect on esophageal cancer cells and enhanced the sensitivity of esophageal cancer cells to 5-FU at sublethal concentrations. In the clinical setting, patients treated with oral PPIs showed a superior tumor response to 5-FU and better overall survival in comparison to the non-PPI group. These results indicate that PPIs can enhance chemosensitivity in esophageal cancer patients treated with 5-FU.Background: Vacuolar ATPase (V-ATPase) is involved in cancer development. The use of proton pump inhibitors (PPIs) as V-ATPase inhibitors has been reported to enhance the effectiveness of chemotherapy in certain cancers. This study aimed to evaluate the effect of PPIs on chemotherapy for esophageal cancer. Methods: To investigate the effects of PPIs on esophageal cancer cells, human KYSE50 and 70 cells were plated and 3 PPIs (lansoprazole, esomeprazole, vonoprazan) were added at various concentrations with 5-Fluorouracil (5-FU) to the corresponding cells for a cell viability assay. To investigate the effects of PPI treatment on patients undergoing 5-FU-based therapy in the clinical setting, we retrospectively analyzed the clinical outcomes and chemotherapy-related adverse events in 40 esophageal cancer patients who received 5-FU chemotherapy in our hospital between May 2013 and April 2017. Results: In the viability assays, all PPIs significantly enhanced the cytotoxic effect of 5-FU on the two esophageal cancer cell lines. In the clinical study, PPI-treated patients showed better overall survival (OS) than patients managed without PPI treatment. A multivariate analysis revealed that PPI treatment was independently associated with OS (p = 0.009, HR, 0.33; 95% CI, 0.12–0.76). Conclusions: PPI treatment may safely enhance chemosensitivity in esophageal cancer patients.

Favorable tumor uptake and nuclear transport of Auger electrons by nuclear targeting with 111In-trastuzumab in an intraperitoneal tumor mouse model.

The 111In-labeled anti-HER2 antibody trastuzumab modified with a nuclear-localizing sequence (NLS) peptide (111In-trastuzumab-NLS) is a radiopharmaceutical candidate for Auger electron radioimmunotherapy (AE-RIT). However, in-vivo action of 111In-trastuzumab-NLS is poorly understood in intraperitoneal tumors. We aimed to elucidate the nuclear targeting activity of 111In-trastuzumab-NLS in a mouse model of intraperitoneal tumors. Trastuzumab, trastuzumab-NLS-S with shorter NLS peptides, and trastuzumab-NLS-L with longer NLS peptides were tested in an intraperitoneal tumor xenograft. The AE-emitting radionuclide 111In was labeled with these antibodies. The cell-binding activity, nuclear importation, and cytotoxicity of those radiolabeled antibodies were examined in human cancer cell lines. Analyses of the biodistribution and in-vivo nuclear importation of 111In were conducted in a mouse model. The two111In-trastuzumab-NLS variants delivered the radionuclide into the nucleus more efficiently and had a comparable cytotoxicity to 111In-trastuzumab against human gastric cancer cells, although had a lower cell binding affinity. 111In-trastuzumab-NLS-L exhibited both a superior tumor uptake and in vivo nuclear transportation of the radionuclide than 111In-trastuzumab. Nuclear targeting using 111In-trastuzumab-NLS promotes a more efficient tumor cell uptake and subsequent nuclear translocation of the 111In AE-emitting radionuclide in vivo. This radio-immunoconjugate will likely be an effective agent for HER2-targeting by AE-RIT.

Sequence-dependent synergistic effect of aumolertinib-pemetrexed combined therapy on EGFR-mutant non-small-cell lung carcinoma with pre-clinical and clinical evidence

BackgroundInevitably developed resistance of the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) limited its clinical benefit on non-small cell lung cancer (NSCLC). Upfront combination therapy is promising to prevent this resistance. Compelling clinical evidence indicated the failure of third-generation EGFR TKIs combined with either immunotherapy or antiangiogenic agents. In comparison, combined treatment of third-generation EGFR TKIs and chemotherapy might be a favorable choice. Herein, we systematically analyzed and compared the effects of pemetrexed and a novel third-generation EGFR TKI aumolertinib combined in different sequences, subsequently revealed the potential mechanisms and proved the optimal combination schedule with clinical retrospective study.MethodsThree combination schedules involving pemetrexed and aumolertinib in different sequences were developed. Their inhibition effects on cell proliferation and metastasis were firstly compared upon three human NSCLC cell lines in vitro, by cell counting kit-8, colony formation, wound healing and transwell assays respectively. Further evaluation in vivo was proceeded upon H1975 and HCC827 xenograft model. Gene and protein expression were detected by Q-PCR and western blot. Drug concentration was determined by LC–MS/MS. VEGF secretion was determined by ELISA. Tumor vessel was visualized by immunofluorescence. Lastly, a clinical retrospective study was raised with 65 patients’ data.ResultsThe combination of pemetrexed and aumolertinib exhibited a sequence-dependent and EGFR mutant-dependent synergistic effect in vitro and in vivo. Only treatment with aumolertinib following pemetrexed (P-A) exhibited synergistic effect with stronger anti-tumor growth and anti-metastasis ability than monotherapy and also other combination sequences. This synergism could exclusively be observed in H1975 and HCC827 but not A549. Pathway analysis showed that P-A significantly enhanced the suppression of EGFR pathway. In addition, our results intriguingly found an obvious reduction of VEGF secretion and the accompanying normalization of the intratumor vessel, consequently increasing intratumoral accumulation of pemetrexed in P-A group. Finally, the clinical retrospective study verified the synergistic effect of P-A combination by significantly superior tumor response than aumolertinib monotherapy.ConclusionAumolertinib-pemetrexed combined therapy is promising for EGFR mutant NSCLC but only in right administration sequence. P-A could become an advantageous combination strategy in clinical with synergistic inhibition of tumor growth and metastasis.

Evaluating potential toxicities of cancer immunotherapies using autoimmune animal models.

Abstract During the past few decades, cancer immunotherapy has recorded breakthroughs involving therapeutic agents that mediate immune response against several tumor types. However, an important consideration with immunotherapy treatment in the clinic has been the emergence of immune related adverse events (irAEs), that could lead to frequent hospitalization, premature therapy cessation and fatalities, thus limiting the efficacy of these therapies. Additionally, despite the clinical benefits of combining therapies, it has been observed that severity of toxicity increases with combination of therapy. Although, these irAEs are commonly seen in patients receiving immunotherapy, such events have been rarely observed in preclinical mouse models due to their nature: inbred, resistant, and short study duration. Here, we report the use of EAE autoimmune mouse model to evaluate potential toxicities of a novel cancer immunotherapy: anti-CD3ɛ mono-Fab, that is effective as a single therapy and could synergize with checkpoint inhibitors for superior anti-metastatic tumor response. EAE model groups treated with combination therapies showed more disease severity recapitulating the higher grade of irAEs seen in patients receiving combination therapies. The data suggest that autoimmune mouse models could serve as an effective tool to study the severity of toxicity associated with immunotherapeutic agents, and to determine optimized combinatorial regimens for best therapeutic performance. Supported by NCI U01CA244314

Hydrogen sulfide signaling in promoting the anti-tumor T cell response

Abstract Adoptive cell therapy (ACT), a form of immunotherapy, continues to emerge as a novel therapeutic strategy for treating advanced malignancies. New approaches to improve ACT protocols are needed to enhance in vivo persistence of adoptively transferred tumor antigen-specific T cells and overcome tumor-induced immunosuppression and oxidative stress, which remain significant barriers to the clinical success of ACT. Here, we show that exogenous hydrogen sulfide (H2S), an important gaseous signaling molecule, promoted a central memory phenotype with enhanced antioxidant capacity in tumor antigen-specific T cells. In vitro, T cells treated with exogenous H2S upregulated key antioxidant enzymes and molecules associated with stemness while simultaneously downregulating immune checkpoint molecules. H2S-treated T cells also displayed an enhanced reactivity to tumor antigen, with an increase in production of pro-inflammatory cytokines as well as an increase in overall protein translation. H2S-treated T cells also displayed an enhanced ability to combat oxidative stress, with a reduced accumulation of reactive oxygen species and protection against hydrogen peroxide-induced cell death. In an in vivo murine model of melanoma, tumor-reactive T cells conditioned ex vivo with exogenous H2S demonstrated superior tumor control upon ACT. Even greater tumor control was observed when ACT-treated mice were injected systemically with an H2S donor compound. These data provide evidence that anti-tumor T cells exposed to H2S possess an enhanced metabolic and stem-like phenotype that allows them to persist in the immunosuppressive tumor microenvironment and exert greater tumor control upon adoptive transfer into tumor-bearing hosts. Supported by NIH R01 CA236379

Directing T cell alloreactivity against solid tumors through tumor antigen dependent TCR expression

Abstract A major challenge in T cell therapy against solid tumors is the heterogeneity of tumor antigen (TA) expression. Elimination of TA+ tumor cells often fails to suppress tumor growth due to the expansion of TA− tumor cells. To address this, we developed tumor-activated alloreactive T cells (TAATs) that display alloreactivities only in tumors. TAATs lack endogenous CD3ɛ expression and contain a tumor sensing mechanism that drives the expression of an exogenous copy of CD3ɛ in the presence of TA+ tumor cells. TAATs are therefore inactive in normal tissues due to retention of TCR/CD3 in the ER but become alloreactive in tumors. TAATs attack all HLA+ cells in tumors including tumor cells, stromal cells and cells that suppress immune functions. To generate TAATs, HLA-mismatched allogeneic T cells were stimulated with HLA class I+ breast cancer cells to expand alloreactive T cells. Endogenous CD3ɛ expression was then knocked out using CRISPR/Cas9. The T cells were also modified to express a Her2-specific synthetic Notch receptor that releases an intracellular domain containing a Gal4-VP64 transcription activator upon receptor engagement. In addition, an expression cassette with a CD3ɛ coding sequence driven by a promoter containing Gal4 binding sites was introduced to the T cells. When co-cultured with Her2+ cancer cells, TAATs restored TCR/CD3 expression and were able to degranulate and produce IFNγ in response to anti-CD3 stimulations. TAATs were also able to kill Her2+ cancer cells in co-cultures and kill Her2− cancer cells in the presence of Her2+ cancer cells. Compared with CAR T cells, TAATs may achieve superior tumor suppression through attacking a wider range of cells in tumors and by overcoming the immunosuppressive tumor microenvironment.

Conformational adjustment overcomes multiple drug-resistance mutants of tropomyosin receptor kinase

Mutation-induced resistance to targeted drug treatment poses a serious threat to successful chemotherapy. Multiple mutations underlying drug resistance remain a largely unsolved scientific issue. Tropomyosin receptor kinases (TRKs) are promising therapeutic targets for several malignant human cancers, but they have become less effective due to multiple resistance mutations. Thus, TRKs are representative cases to explore the problem of multiple resistance mutations. Here, we proposed a conformational adjustment strategy of drug design to overcome multiple resistance mutations in cancer treatments. A representative inhibitor, TIY-7, exhibited remarkable inhibitory activity against five TRK mutants, showing an IC50 value of 1.1 nM against the most severe mutant TRKA-G595R. Moreover, it displayed superior tumor growth inhibitory activity compared with the clinically used drug selitrectinib. These results validated our strategy to design a new inhibitor structure to overcome multiple resistance mutations.

To Optimize Radiotherapeutic Plans for Superior Tumor Coverage Predicts Malignant Glioma Prognosis and Normal Tissue Complication Probability.

Background: Radiotherapy (RT) provides a modern treatment to enhance the malignant glioma control rate. The purpose of our study was to determine the effect of tumor coverage on disease prognosis and to predict optimal RT plans to achieve a lower normal tissue complication probability (NTCP). Methods: Ten malignant-glioma patients with tumors adjacent to organs at risk (OARs) were collected. The patients were divided into two groups according to adequate coverage or not, and prognosis was analyzed. Then, using intensity-modulated radiation therapy (IMRT), volume-modulated arc therapy (VMAT), and helical tomotherapy (TOMO) to simulate new treatment plans for 10 patients, the advantages of these planning systems were revealed for subsequent prediction of NTCP. Results: The results of clinical analysis indicated that overall survival (p = 0.078) between the adequate and inadequate groups showed no differences, while the adequate group had better recurrence-free survival (p = 0.018) and progression-free survival (p = 0.009). TOMO had better CI (p < 0.001) and also predicted a lower total-irradiated dose to the normal brain (p = 0.001) and a lower NTCP (p = 0.027). Conclusions: The TOMO system provided optimal therapeutic planning, reducing NTCP and achieving better coverage. Combined with the clinical results, our findings suggest that TOMO can make malignant glioma patients close to OARs achieve better disease control.

Myeloid-Derived Suppressor Cells and Radiotherapy.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of pathologically activated, mostly immature, myeloid cells that exert robust immunosuppressive functions. MDSCs expand during oncogenesis and have been linked to accelerated disease progression and resistance to treatment in both preclinical tumor models and patients with cancer. Thus, MDSCs stand out as promising targets for the development of novel immunotherapeutic regimens with superior efficacy. Here, we summarize accumulating preclinical and clinical evidence indicating that MDSCs also hamper the efficacy of radiotherapy (RT), as we critically discuss the potential of MDSC-targeting strategies as tools to achieve superior immunotherapeutic tumor control by RT in the clinic.

CCR7 expression in CD19 chimeric antigen receptor-engineered natural killer cells improves migration toward CCL19-expressing lymphoma cells and increases tumor control in mice with human lymphoma

Background aimsChimeric antigen receptor (CAR) T-cell therapy can be associated with significant toxicities. CAR-engineered natural killer (NK) cells provide a safer alternative while maintaining anti-tumor effects. Activated NK (aNK) cells are a clinical-grade cellular product obtained from the NK-92 cell line that have demonstrated both safety and potent cytotoxicity toward a wide range of cancers in phase 1 trials. Genetically engineered variants of aNK cells expressing a high-affinity Fc receptor (haNK) or co-expressing a CAR (t-haNK) are currently in phase 1/2 clinical trials. A key factor in the efficacy of cellular immunotherapies is biodistribution and tumor infiltration, which affect the local effector:target ratio. The chemokines CCL19 and CCL21 can drive recruitment of CCR7 receptor-expressing immune cells to secondary lymphoid organs. MethodsSince NK-92 cells do not spontaneously express CCR7, clinical-grade aNK cells were transfected with a non-viral vector containing the CCR7 receptor, an anti-CD19 CAR and a high-affinity CD16 Fc receptor. ResultsCCR7-engineered CD19 t-haNK showed significant migration in vitro toward K562 cells engineered to secrete CCL19. This observation was confirmed in a NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse model in which subcutaneous tumors of CCL19-expressing K562 cells displayed a higher number of infiltrating CCR7_CD19 t-haNK cells than CCR7-negative CD19 t-haNK cells. In NSG mice inoculated either intravenously or subcutaneously with CCL19-secreting Raji cells, treatment with CCR7_CD19 t-haNK improved survival and tumor control compared with CD19 t-haNK or vehicle. ConclusionsExpression of CCR7 receptor by off-the-shelf t-haNK cells improves their homing toward lymph node chemokines both in vitro and in vivo, resulting in superior tumor control.

Design, synthesis, and preclinical evaluation of a novel bifunctional macrocyclic chelator for theranostics of cancers.

This study was to design and synthesize a novel bifunctional chelator, named Dar, primarily validated by conjugating to tumor targeting motifs, labeled with radiometals, and performed preclinical evaluation of tumor imaging and cancer therapy in murine tumor models. The designed Dar was synthesized and characterized by X-ray crystallography, 1H/13C NMR, and mass spectrometry. Dar-PSMA-617 was conjugated and radiolabeled with 68Ga, 177Lu, and 89Zr. The in vivo behavior of 68Ga/89Zr-labeled Dar-PSMA-617 were evaluated using micro-PET imaging and biodistribution from image quantitation and tissue radioactivity counting, with 68Ga/89Zr-labeled NOTA/DOTA/DFO-PSMA-617 analogs as controls, respectively. The [177Lu]-Dar-PSMA-617, with [177Lu]-DOTA-PSMA-617 as control, was evaluated in competitive cell uptake, tumor cell internalization, and efflux studies. The treatment efficacy of [177Lu]Lu-Dar-PSMA-617, with [177Lu]Lu-DOTA-PSMA-617 as control, was evaluated in PSMA-positive LNCaP tumor-bearing mice. In addition, the ability of Dar for radiolabeling nanobody was tested by conjugating Dar to KN035 nanobody. The resultant [89Zr]Zr-Dar-KN035 nanobody, with [89Zr]Zr-DFO-KN035 as control, was evaluated by micro-PET imaging and biodistribution in a mouse model bearing MC38&MC38-hPD-L1 colon cancer. 68Ga, 89Zr, and 177Lu-radiolabeled Dar-PSMA-617 complexes were able to be produced under mild condition with high radiochemical yield and purity successfully. [177Lu]Lu-Dar-PSMA-617 had higher cellular uptake yet similar internalization and efflux properties in LNCaP cells, as compared to [177Lu]Lu-DOTA-PSMA-617. Micro-PET images demonstrated significantly higher tumor uptake of [68Ga]Ga-Dar-PSMA-617, than that of the analog [68Ga]Ga-DOTA-PSMA-617. The tumor uptake values of [68Ga]Ga-Dar-PSMA-617 at multiple time points are comparable to that of [68Ga]Ga-NOTA-PSMA-617, although a higher and persistently prolonged kidney retention was resulted in during the study period. The Dar chelator can also successfully mediate the radiolabeling with 89Zr, while the resultant [89Zr]Zr-Dar-PSMA-617 demonstrated a similar biodistribution with [89Zr]Zr-DFO-PSMA-617 measured at 96h p.i. The treatment with [177Lu]Lu-Dar-PSMA-617 significantly inhibited the tumor growth, showing much better efficacy than that of [177Lu]Lu-DOTA-PSMA-617 at the same injected radioactivity and mass dose. Dar was covalently linked to KN035 nanobody and enabled radiolabeling with 89Zr in high yield and radiochemical purity at room temperature. The resultant [89Zr]Zr-Dar-KN035, with [89Zr]Zr-DFO-KN035 as control, demonstrated superior tumor uptake and detection capability in PET imaging studies. The Dar, as a novel bifunctional chelator for medicating the labeling of radiometals onto tumor targeting carriers, was successfully synthesized and chemically characterized. Test radiolabeling, on PSMA-617 and a nanobody as tool targeting molecule carriers, demonstrated the Dar has potential as a universal bifunctional chelator for radiolabeling various radiometals (at least 68Ga, 177Lu, and 89Zr tested) commonly used for clinical imaging and therapy. Using a novel Dar chelator results in altered in vivo behavior of the carriers even though labeled with the same nuclide. This capability makes Dar an alternative to the existing choices for radiolabeling new carrier molecules with various radiometals, especially the radiometals with large radius.