Adenosine 2A receptor and TIM3 suppress cytolytic killing of tumor cells via cytoskeletal polarization

Tumors generate an immune-suppressive environment that prevents effective killing of tumor cells by CD8+ cytotoxic T cells (CTL). It remains largely unclear upon which cell type and at which stage of the anti-tumor response mediators of suppression act. We have combined an in vivo tumor model with a matching in vitro reconstruction of the tumor microenvironment based on tumor spheroids to identify suppressors of anti-tumor immunity that directly act on interaction between CTL and tumor cells and to determine mechanisms of action. An adenosine 2A receptor antagonist, as enhanced by blockade of TIM3, slowed tumor growth in vivo. Engagement of the adenosine 2A receptor and TIM3 reduced tumor cell killing in spheroids, impaired CTL cytoskeletal polarization ex vivo and in vitro and inhibited CTL infiltration into tumors and spheroids. With this role in CTL killing, blocking A2AR and TIM3 may complement therapies that enhance T cell priming, e.g. anti-PD-1 and anti-CTLA-4.

Homogenous Multifunctional Microspheres Induce Ferroptosis to Promote the Anti-hepatocarcinoma Effect of Chemoembolization

Transcatheter arterial chemoembolization (TACE) is one of the main palliative therapies for advanced hepatocellular carcinoma (HCC), which is also regarded as a promising therapeutic strategy for cancer treatment. However, drug-loaded microspheres (DLMs), as commonly used clinical chemoembolization drugs, still have the problems of uneven particle size and unstable therapeutic efficacy. Herein, gelatin was used as the wall material of the microspheres, and homogenous gelatin microspheres co-loaded with adriamycin and Fe3O4 nanoparticles (ADM/Fe3O4-MS) were further prepared by a high-voltage electrospray technology. The introduction of Fe3O4 nanoparticles into DLMs not only provided excellent T2-weighted magnetic resonance imaging (MRI) properties, but also improved the anti-tumor effectiveness under microwave-induced hyperthermia. The results showed that ADM/Fe3O4-MS plus microwave irradiation had significantly better antitumor efficacy than the other types of microspheres at both cell and animal levels. Our study further confirmed that ferroptosis was involved in the anti-tumor process of ADM/Fe3O4-MS plus microwave irradiation, and ferroptosis marker GPX4 was significantly decreased and ACSL4 was significantly increased, and ferroptosis inhibitors could reverse the tumor cell killing effect caused by ADM/Fe3O4-MS to a certain extent. Our results confirmed that microwave mediated hyperthermia could amplify the antitumor efficacy of ADM/Fe3O4-MS by activating ferroptosis and the introduction of Fe3O4 nanoparticles can significantly improve TACE for HCC. This study confirmed that it was feasible to use uniform-sized gelatin microspheres co-loaded with Fe3O4 nanoparticles and adriamycin to enhance the efficacy of TACE for HCC.

Response to FEC Chemotherapy and Oncolytic HSV-1 Is Associated with Macrophage Polarization and Increased Expression of S100A8/A9 in Triple Negative Breast Cancer

The era of immunotherapy has seen an insurgence of novel therapies driving oncologic research and the clinical management of the disease. We have previously reported that a combination of chemotherapy (FEC) and oncolytic virotherapy (oHSV-1) can be used to sensitize otherwise non-responsive tumors to immune checkpoint blockade and that tumor-infiltrating B cells are required for the efficacy of our therapeutic regimen in a murine model of triple-negative breast cancer. In the studies herein, we have performed gene expression profiling using microarray analyses and have investigated the differential gene expression between tumors treated with FEC + oHSV-1 versus untreated tumors. In this work, we uncovered a therapeutically driven switch of the myeloid phenotype and a gene signature driving increased tumor cell killing.

DDRE-47. ASSESSMENT OF BRAIN PENETRANCE, BIODISTRIBUTION, AND EFFICACY OF PLATINUM (IV)-CONJUGATED FLUORINATED MACROCYCLIC CELL-PENETRATING PEPTIDES IN A MURINE GLIOBLASTOMA MODEL

INTRODUCTION Glioblastoma (GBM), an aggressive brain tumor with a poor prognosis, presents an average of 2% of patients surviving beyond 2 years after diagnosis. Therapies to effectively manage glioblastoma are hindered due to the presence of the blood-brain barrier (BBB). Previously, a cell-penetrating peptide, M13, was conjugated to a Pt(IV) cisplatin prodrug, via amide bond formation. The conjugated Pt(IV) releases active cisplatin upon intracellular reduction. Herein, we investigated the BBB-penetrance and biodistribution of M13 conjugated to Pt(IV), as well as its effectiveness against GBM in mouse models. METHODS M13 platinum-conjugate tumor cell killing capacity was assessed by luminescent cell viability assays in vitro. By using Inductively-Coupled Plasma Mass-Spectrometry for platinum detection, BBB penetration and bio-distribution studies were performed in a three-dimensional BBB spheroid in vitro model and in vivo in mouse brain, intracranial tumor, and peripheral organs. Dose-regime studies involved observations of symptomatology and weight variations after bi-weekly injections of platinum compounds at 2mg/kg and 5mg/kg. RESULTS The Pt(IV)-M13 conjugate possesses tumor cell killing effects similar to cisplatin when tested in GBM cell lines in vitro. Platinum increased by using Pt(IV)-M13 when compared to cisplatin in our in vitro BBB-spheroid model (20-fold, p-value=0.0033), in brain tissue (10-fold, p< 0.0001) and GBM tumor-bearing mice models (7.5-fold, p< 0.0001). Bio-distribution of platinum delivered by Pt(IV)-M13 in spleen, heart and blood was significantly different to cisplatin 5hrs. after intravenous injection (p< 0.001). Bi-weekly dose regimes of Pt(IV)-M13 are tolerable in nude mice without toxicity at a similar concentration to reported tolerable cisplatin doses at 5 mg/kg. Finally, Pt(IV)-M13 significantly increased survival in a murine glioblastoma xenograft model compared with controls (median 24 days vs. 29 days, p-value=0.0071). CONCLUSION Overall, our data support the further development of BBB-crossing peptide-drug conjugates for GBM treatment.

681 An ex vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess the phagocytic activity of tumor resident innate immune cells

BackgroundCD47 is an innate immune checkpoint receptor that is overexpressed on tumor cells and contributes to immune evasion through engagement of a myeloid-lineage inhibitory protein SIRPα. Blockade of the CD47-SIRPα interaction is proved to enhance the phagocytosis of cancer cells and to induce effective antitumor immune response. Here we developed a novel ex vivo platform using fresh patient tumor samples with intact stromal components and tumor immune microenvironment to assess the therapeutic activity of immunotherapeutic drugs targeting CD47-SIRPα signaling axis in combination with the human IgG1 αPD-L1 antibody avelumab.MethodsAll tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma using proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient's tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with phrodo-labeled avelumab alone or in combination with anti-CD47 or anti-SIRPα therapeutics.ResultsMultiparameter flow analysis demonstrated tumor binding of avelumab confirming drug penetration into the intact tumor stroma that is further corroborated by high content confocal analysis. Using our confocal-based tumor cell killing assay we were able to quantify drug-induced tumor cell killing ex vivo. We further documented the impact of anti-CD47 and anti-SIRPα therapeutics on phagocytosis of dead tumor cells by tumor resident macrophages and activation of innate and adaptive effector cells by flow cytometry and confocal imaging. Additionally, pHrodo-labeled bioparticles were used to corroborate treatment-mediated changes in the phagocytic activity of tumor resident macrophages.ConclusionsIn this comprehensive study we demonstrate that the 3D-EXplore ex vivo platform can be used to assess the efficacy of therapeutic blockade of CD47/SIRPα axis on stimulation of phagocytic process within an intact tumor immune microenvironment.

844 A trispecific ROR1 x CD3 T cell engager mediates in vitro tumor cell killing and in vivo tumor eradication

BackgroundReceptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed on a variety of difficult to treat solid and hematological malignancies. Several therapeutic concepts targeting ROR1 are currently in clinical studies, including antibody-drug conjugates (ADCs), chimeric antigen receptor engineered T cells, as well as a bispecific T cell engager. In contrast to ADCs, T cell engagers have the capacity to induce tumor cell depletion irrespective of tumor cell mitotic activity. For the therapy of ROR1 expressing tumors, we engineered a T cell engager with prolonged half-life to support convenient administration schemes.MethodsNM32-2668, a ROR1-targeting T cell engager with prolonged serum half-life was engineered by joining three humanized rabbit antibody variable region (Fv) fragments specific for ROR1, CD3ɛ, and serum albumin, into our tri-specific scMATCHTM3 format. Each Fv fragment was stabilized using the ʎ-capTM technology. NM32-2668 was tested in assays for specific tumor lysis, induction of T cell proliferation, and cytokine release. These studies were performed using human T cells co-cultured with tumor cell lines and human tumor samples expressing various levels of ROR1. In vivo xenograft mouse studies were conducted using a human mantle cell lymphoma model in NCG mice engrafted with human PBMCs.ResultsHere we report the design and the promising preclinical activity of the scMATCHTM3 ROR1/CD3/hSA T cell engager NM32-2668 in vitro and in vivo. Importantly, we demonstrate potent and specific cytotoxic activity in the sub-nanomolar range on tumor cell lines expressing different levels of ROR1. NM32-2668 also mediates ROR1 dependent T cell activation and cytokine release. We observe robust tumor cell killing activity of NM32-2668 over an extended time period and at multiple ratios of effectors to targets in a real time imaging-based cytotoxicity assay. This molecule also mediates T cell proliferation in response to target cell binding. NM32-2668 mediates in vitro lysis of CLL patient tumor cells, T cell activation, and cytokine release, with minimal IL-6 involvement. In an in vivo mantle cell lymphoma model (Jeko-1) engrafted with human PBMCs, we observe tumor regression and eradication.ConclusionsCollectively, these data demonstrate robust anti-tumor efficacy by NM32-2668, a scMATCHTM3 ROR1/CD3/hSA. Our results demonstrate that NM32-2668 promotes ROR1 dependent T cell activation and proliferation, as well as T cell-mediated tumor cell lysis. The activity of NM32-2668 has the potential to provide significant benefit to patients with ROR1+ malignancies on a convenient dosing schedule. We intend to rapidly progress NM32-2668 to clinical development.

191 GAPDH knock-in of high affinity CD16 in iPSC derived NK cells drives high-level expression and increased anti-tumor function

BackgroundNatural killer (NK) cells have emerged as an alternative cell type for clinical utility given the low propensity for graft-versus-host disease, thereby making NK cells a potential off-the-shelf cell therapy. One critical pathway NK cells use to target tumor cells is through expression of Fc gamma receptor III alpha (CD16). Antibodies that bind tumor antigens are recognized by CD16 on NK cells, promoting NK-mediated tumor cell killing. High-affinity CD16 variants in the human population correlate with better clinical outcome and anti-tumor response. One mechanism tumors use to evade NK cell recognition is through down-regulation of CD16 expression on the NK cell surface. After being activated, CD16 is cleaved by A Disintigrin and Metalloprotease-17 (ADAM-17). By using a highly-active engineered AsCas12a to knock-in high-affinity CD16 (hCD16KI) at the GAPDH locus, hCD16 is constitutively expressed, continuously replacing hCD16, thereby allowing for repeated ADCC mediated killing.Methods iPSCs were edited at the GAPDH locus with an engineered AsCas12a along with the CD16 donor construct. The bulk edited population was then plated at clonal density and single clones were selected and screened. iPSC clones were then differentiated into NK cells. A 3D tumor spheroid killing assay was used to demonstrate NK cell cytotoxicity against an ovarian cancer cell line (SKOV-3). In addition, a serial killing assay was used to better model NK cell serial killing.ResultsBi-allelic CD16KI iPSC clones were successfully generated. These iPSCs exhibited normal morphology and were able to differentiate into iNK cells. hCD16KI iNK cells showed normal differentiation and surface marker expression, such as CD45/CD56, compared to unedited iNK cells. CD16KI iNK cells demonstrated significantly increased cytotoxicity in the presence of antibody against tumor cells when compared with unedited iNK cells, as measured by reduction in tumor spheroid size in a 3D tumor spheroid killing assay. Importantly, enhanced surface expression of hCD16 on iNK cells after tumor exposure was detected, demonstrating the replenishment of cleaved hCD16. Notably, hCD16KI iNK cells demonstrated prolonged and enhanced tumor cell killing after being subjected to repeated tumor cell exposure in a serial killing assay.ConclusionsThis work demonstrates a powerful new method to drive high-level constitutive hCD16 expression on the surface of iNK cells through transgene knock-in at the GAPDH locus using an engineered AsCas12a. The high level constitutive hCD16 expression enhances ADCC of iNK cells and enables enhanced serial tumor killing and is expected to exert enhanced anti-tumor activity in the clinic.

P09.01 The use of FDA approved JAK, mTOR and Src inhibitors to regulate T cell-bispecific antibody-induced cytokine release while not preventing T cell cytotoxicity

BackgroundT cell bispecific antibodies (TCBs) are potent T cell engagers, harboring a 2+1 format with one binder to the CD3ε chain and two binders to specific tumor antigens. Crosslinking of CD3 with tumor antigens triggers T cell activation and proliferation, cytokine release and tumor cell killing. TCB treatment is sometimes associated with safety liabilities due to on-target on-tumor or on-target off-tumor cytotoxicity and cytokine release. Off-tumor activity of the TCB may occur if the targeted tumor antigens are expressed on healthy cells, which may potentially result in tissue damages and compromise the patient’s safety. Patients treated with TCBs may also experience a Cytokine Release Syndrome (CRS), characterized by fever, hypotension and respiratory deficiency and associated with the release of pro-inflammatory cytokines such as IL-6, TNF-α, IFN-γ, and IL-1β. Tyrosine kinases such as Src, mTOR and JAK1/2 are involved in downstream signaling pathways after engagement of the T cell receptor.Materials and Methods52 FDA approved kinase inhibitors were screened in the presence of T cells activated on CD3 coated plates, mimicking TCB stimulation. Src, mTOR and JAK inhibitors were selected based on their capacity to prevent both, cytokine release and T cell proliferation. Using an in vitro model of target cell killing by human peripheral blood mononuclear cells stimulated with TCBs, we validated the effects of mTOR, JAK and Src kinase inhibitors on TCB-induced T cell activation, tumor cell killing and cytokine release. In vivo, the effect of mTOR, JAK and Src kinase inhibitors on TCB-induced cytokine release was confirmed in humanized NOD scid gamma (NSG) mice engrafted with human hematopoietic stem cells and treated with CD19-TCB.ResultsIn line with previous reports for CAR-T cells, dasatinib (a src inhibitor) was found to fully switch off TCB-induced T cell functionality as well as the other src inhibitors bosutinib and ponatinib. In contrast, temsirolimus, sirolimus and everolimus (mTOR inhibitors) and ruxolitinib, baricitinib, tofacitinib, and fedratinib (JAK1/2 inhibitors) were found to more potently prevent TCB-induced cytokine release without blocking TCB-mediated target cell killing.ConclusionsThese results provide evidence that the mechanisms of TCB-dependent cytokine release and tumor cell killing can be uncoupled. The FDA-approved mTOR and JAK1/2 inhibitors could potentially be used to mitigate CRS whereas the Src inhibitor dasatinib could rather stand as a potential antidote for on-target off-tumor activity or high-grade CRS.Disclosure InformationG. Leclercq: A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. H. Haegel: A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. A. Schneider: A. Employment (full or part-time); Modest; Roche. A. Giusti: A. Employment (full or part-time); Modest; Roche. V. Pulko: A. Employment (full or part-time); Modest; Roche. A. Toso: A. Employment (full or part-time); Modest; Roche. T. Zimmermann: A. Employment (full or part-time); Modest; Roche. L. Green: A. Employment (full or part-time); Modest; Roche. N. Steinhoff: A. Employment (full or part-time); Modest; Roche. J. Sam: A. Employment (full or part-time); Modest; Roche. M. Bacac: A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. P. Umaña: A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. C. Klein: A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche.

Integration of Salmonella into Combination Cancer Therapy

Current modalities of cancer treatment have limitations related to poor target selectivity, resistance to treatment, and low response rates in patients. Accumulating evidence over the past few decades has demonstrated the capacity of several strains of bacteria to exert anti-tumor activities. Salmonella is the most extensively studied entity in bacterial-mediated cancer therapy, and has a good potential to induce direct tumor cell killing and manipulate the immune components of the tumor microenvironment in favor of tumor inhibition. In addition, Salmonella possesses some advantages over other approaches of cancer therapy, including high tumor specificity, deep tissue penetration, and engineering plasticity. These aspects underscore the potential of utilizing Salmonella in combination with other cancer therapeutics to improve treatment effectiveness. Herein, we describe the advantages that make Salmonella a good candidate for combination cancer therapy and summarize the findings of representative studies that aimed to investigate the therapeutic outcome of combination therapies involving Salmonella. We also highlight issues associated with their application in clinical use.

The NHance® Mutation-Equipped Anti-MET Antibody ARGX-111 Displays Increased Tissue Penetration and Anti-Tumor Activity in Advanced Cancer Patients

Dysregulation of MET signaling has been implicated in tumorigenesis and metastasis. ARGX-111 combines complete blockade of this pathway with enhanced tumor cell killing and was investigated in 24 patients with MET-positive advanced cancers in a phase 1b study at four dose levels (0.3–10 mg/kg). ARGX-111 was well tolerated up to 3 mg/kg (MTD). Anti-tumor activity was observed in nearly half of the patients (46%) with a mean duration of treatment of 12 weeks. NHance® mutations in the Fc of ARGX-111 increased affinity for the neonatal Fc receptor (FcRn) at acidic pH, stimulating transcytosis across FcRn-expressing cells and radiolabeled ARGX-111 accumulated in lymphoid tissues, bone and liver, organs expressing FcRn at high levels in a biodistribution study using human FcRn transgenic mice. In line with this, we observed, in a patient with MET-amplified (>10 copies) gastric cancer, diminished metabolic activity in multiple metastatic lesions in lymphoid and bone tissues by 18F-FDG-PET/CT after two infusions with 0.3 mg/kg ARGX-111. When escalated to 1 mg/kg, a partial response was reached. Furthermore, decreased numbers of CTC (75%) possibly by the enhanced tumor cell killing witnessed the modes of action of the drug, warranting further clinical investigation of ARGX-111.