scholarly journals 737 Inhibition of P21-activated kinase 4 (PAK4) reverts immune exclusion and restores anti-tumor immunity in the tumor microenvironment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A768-A768
Author(s):  
Yu 'Jerry' Zhou ◽  
Gina Chu ◽  
Eleanore Hendrickson ◽  
Johnni Gullo-Brown ◽  
Brandy Chavez ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Immune Cell ◽  
Mouse Tumor ◽  
Type I ◽  
List Type ◽  
P21 Activated Kinase ◽  
Immune Exclusion

BackgroundP21-activated kinase 4 (PAK4) is a serine/threonine protein kinase that is mostly expressed in tumor and stroma cells. PAK4 activates tumor WNT/β-catenin pathway and regulates cellular morphology, motility, EMT, cell proliferation and survival. Recent studies also showed that PAK4 can actively exclude T cells from tumors, suggesting that therapeutic inhibition of PAK4 can increase T cell infiltration in tumor microenvironment and overcome resistance to checkpoint inhibitor immunotherapy.1MethodsWe generated PAK4 knockout (KO) clones in human and mouse tumor cells to validate its biology in vitro and in vivo. We also performed pharmacological evaluation of PAK4 inhibition using Pfizer compounds (referred to as 'PAK4i compounds' below) for their potential tumor-intrinsic and immune-regulatory roles.ResultsNanostring, qPCR and RNASeq analysis showed that PAK4 depletion led to increase of cytokine expression in tumor, including conventional dendritic cell (cDC)- recruiting chemokine CCL4, and type I IFN / ISG pathway genes that are associated with MHC upregulation such as CXCL10. In addition, PAK4 KO sensitizes B16F10 tumors to anti-PD-1 treatment and increases infiltration of cDC and T cells in the tumor microenvironment.We also showed that small molecule PAK4i compounds induced more potent cancer cell growth inhibition over treated normal PBMCs. PAK4i compounds also increased immune-activating and decreased immune exclusion genes in B16F10 cells and tumor explants in vitro. Although PAK4 target engagement is demonstrated by CETSA assay, the compound potency on modulating PAK4 downstream Wnt/ β-catenin pathway is low, suggesting that the aforementioned phenotypic changes induced by PAK4i compounds may be partially attributed to other off-target effects.ConclusionsCollectively, our data suggests that genetic depletion or pharmacological inhibition of PAK4 may induce immune-activating cytokine production in tumor cells, revert immune cell exclusion in tumor microenvironment, and synergize with checkpoint blockade therapies. However, further optimization on these PAK4i compounds is needed to improve its specificity on modulating PAK4 enzyme activities.ReferenceAbril-Rodriguez G, Torrejon DY, Liu W, Zaretsky JM, Nowicki TS, Tsoi J, Puig-Saus C, Baselga-Carretero I, Medina E, Quist MJ, Garcia AJ, Senapedis W, Baloglu E, Kalbasi A, Cheung-Lau G, Berent-Maoz B, Comin-Anduix B, Hu-Lieskovan S, CWang CY, Grasso CS & Ribas A. PAK4 inhibition improves PD-1 blockade immunotherapy. Nat Cancer 2020;1:46–58.Ethics ApprovalAll animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Pfizer. Approved protocol # LAJ-2019-01347

scholarly journals 884 Engineered toxin body mediated depletion of TIGIT expressing immune cells for cancer immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A926-A926
Author(s):  
Elizabeth Saputra ◽  
Garrett Cornelison ◽  
Jennifer Mitchell ◽  
Karia Williams ◽  
Andrea Mendiola ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Cells ◽  
Immune Cell ◽  
Single Agent ◽  
Antibody Fragment ◽  
Cell Surface Receptor ◽  
Mouse Tumor ◽  
List Type ◽  
Proof Of Concept

BackgroundTIGIT (T cell immunoreceptor with Ig and ITIM domains) is an exciting novel target for immuno-oncology which functions as an immune checkpoint on multiple immune cell types including memory CD8+, CD4+ Treg, and memory CD4+ cells. TIGIT upregulation on tumor infiltrating lymphocytes (TILs) has been observed in multiple cancer types and contributes to an immunosuppressive tumor microenvironment (TME). Interestingly, TIGIT is commonly co-expressed with PD-1 on Tregs in the TME, tumor antigen specific CD8+ T cells and CD8+ TILs, leading to weakened anti-tumor immune responses.1–2 To date, TIGIT inhibiting monoclonal antibodies (mAb) have shown little activity as a monotherapy in clinical and preclinical studies. 3–4 Therefore, current clinical trials are now focused on combining TIGIT mAbs with known commercial PD-1 or PD-L1 mAbs. A TIGIT-specific engineered toxin body (ETB) represents a wholly new approach to targeting TIGIT expressing cells including those co-expressing TIGIT and PD-1.MethodsETBs targeting TIGIT were designed to deplete TIGIT-expressing TILs, including Tregs, directly in the TME. ETBs are proteins that consist of an antibody fragment genetically fused to a proprietary de-immunized (DI) form of the Shiga-like toxin A subunit (SLTA). These proteins are specific for a cell surface receptor, and function through triggering rapid internalization upon binding, followed by an enzymatic and irreversible termination of ribosomal protein synthesis resulting in cellular apoptosis. Here we provide proof of concept for ETBs as a novel modality for the depletion of TIGIT-expressing immune cells.ResultsTIGIT-targeting ETBs exhibit potent in vitro cytotoxicity of TIGIT over-expressing cell lines (IC50<1nM). These ETBs also lead to apoptotic depletion of ex vivo TIGIT-expressing regulatory T cells (Tregs) from healthy donors. In mixed culture assays, TIGIT ETBs increase the proliferation of TIGIT negative T cells by depleting TIGIT-expressing T cells.ConclusionsStudies to assess pharmacodynamics and efficacy of TIGIT targeting ETBs using a double knock-in (TIGIT and PD-1) mouse tumor model are ongoing, but these early proof of concept in vitro data support the hypothesis that ETBs can deplete TIGIT positive immune cell populations including those co-expressing PD-1. It is possible that targeted TIGIT inhibition through ETB-induced cell death could tip the balance towards tumor regression by eliminating this novel checkpoint (and TIGIT/PD-1 co-expression) at the level of the TME.ReferencesJinhua X, Ji W, Shouliang C, Liangfeng Z. Expression of immune checkpoints in T cells of esophageal cancer patients. Oncotarget 2016;7(39):1–10.Blessin NC, Simon R, Kluth M, Fischer K, et al. Patterns of TIGIT expression in lymphatic tissue, inflammation and cancer. Dis Markers 2019;2019:1–13.Johnston RJ, Comps-Agrar L, Hackney J, Yu X, et al. The immunoreceptor TIGIT regulates anti-tumor and antiviral CD8(+) T effector function. Cancer Cell 2014;26(6):923–927.Bendell JC, Bedrad P, Bang Y-J, LoRusso P, et al. Phase Ia/Ib dose-escalation study of the anti-TIGIT antibody Tiragolumab as a single agent and in combination with atezolizumab in patients with advanced solid tumors. Proceedings: AACR Annual Meeting 2020; April 27–28, 2020 and June 22–24, 2020; Philadelphia, PA.

705 Anti-tumor activity of CB-668, a potent, selective and orally bioavailable small-molecule inhibitor of the immuno-suppressive enzyme Interleukin 4 (IL-4)-Induced Gene 1 (IL4I1)

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A747-A747
Author(s):  
Andrew MacKinnon ◽  
Deepthi Bhupathi ◽  
Jason Chen ◽  
Tony Huang ◽  
Weiqun Li ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Tumor Microenvironment ◽  
Immune Cell ◽  
Interleukin 4 ◽  
Mouse Tumor ◽  
Syngeneic Mouse ◽  
Molecule Inhibitor ◽  
Anti Tumor Activity ◽  
Mouse Tumor Models

BackgroundTumors evade destruction by the immune system through multiple mechanisms including altering metabolism in the tumor microenvironment. Metabolic control of immune responses occurs through depletion of essential nutrients or accumulation of toxic metabolites that impair immune cell function and promote tumor growth. The secreted enzyme interleukin 4 (IL-4)-induced gene 1 (IL4I1) is an L-phenylalanine oxidase that catabolizes phenylalanine and produces phenyl-pyruvate and hydrogen peroxide. IL4I1 regulates several aspects of adaptive immunity in mice, including inhibition of cytotoxic T cells through its production of hydrogen peroxide (reviewed in1). In human tumors, IL4I1 expression is significantly elevated relative to normal tissues and is notably high in ovarian tumors and B cell lymphomas. Motivated by the hypothesis that IL4I1 is an immuno-metabolic enzyme that suppresses anti-tumor immunity, we discovered CB-668, the first known small-molecule inhibitor of IL4I1.MethodsIL4I1 enzymatic activity was measured using an HRP-coupled enzyme assay. RNA in-situ hybridization was carried out on the RNAScope platform. Syngeneic mouse tumor models were used to evaluate the anti-tumor activity of CB-668. The level of phenyl-pyruvate in tumor homogenates was measured by LC/MS.ResultsOur clinical candidate, CB-668 is a potent and selective non-competitive inhibitor of IL4I1 (IC50 = 15 nM). CB-668 has favorable in vitro ADME properties and showed low clearance and high oral bioavailability in rodents. Twice-daily oral administration of CB-668 was well-tolerated in mice and resulted in single-agent anti-tumor activity in the syngeneic mouse tumor models B16-F10, A20, and EG7. Oral CB-668 administration reduced the levels of phenyl-pyruvate in the tumor, consistent with inhibition of IL4I1 enzymatic activity. Anti-tumor activity of CB-668 was immune cell-mediated since efficacy was abrogated in CD8-depleted mice, and CB-668 treatment caused increased expression of pro-inflammatory immune genes in the tumor. Moreover, CB-668 had no direct anti-proliferative activity on tumor cells grown in vitro (IC50 > 50 µM). CB-668 also favorably combined with anti-PD-L1 therapy to reduce tumor growth in the B16-F10 tumor model.ConclusionsThese data support an immune-mediated anti-tumor effect of IL4I1 inhibition by CB-668, and suggest inhibition of IL4I1 represents a novel strategy for cancer immuno-therapy.ReferencesMolinier-Frenkel V, Prévost-Blondel A, and Castellano F. The IL4I1 Enzyme: A New Player in the Immunosuppressive Tumor Microenvironment. Cells 2019;8:1–9.

scholarly journals P01.02 TLR-mediated suppression of the CCL22-CCR4 axis as a new target for tumor immunotherapy

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A3.2-A4
Author(s):  
J Grün ◽  
I Piseddu ◽  
C Perleberg ◽  
N Röhrle ◽  
S Endres ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
Regulatory T Cells ◽  
Type I ◽  
List Type ◽  
Gm Csf

BackgroundUnmethylated CpG-DNA is a potent ligand for the endosomal Toll-like-receptor-9, important for the immune activation to pathogen-associated molecules.1 CpG and other TLR-ligands show effective immunotherapeutic capacities in cancer treatment by inducing an antitumorigenic immunity.2 They are able to reduce tumor progression by reduction of intratumoral secretion of the immunoregulating chemokine CCL223 and subsequent recruitment of immunosuppressive regulatory T cells (Treg), which express CCR4 the only so far known receptor for CCL22.4 Our recent work has shown that CCL22 secretion by dendritic cells (DC) in the lymph node, mediates tolerance by inducing DC-Treg contacts.5 Indeed, in the absence of CCL22, immune responses to vaccination were stronger and resulted in tumor rejection.6 Therefore, we are aiming to investigate the effects of TLR-ligands on systemic CCL22 levels, elucidating all involved mechanisms to identify new targets for cancer immunotherapy.Materials and MethodsT, B and CD11c+ DCs of wildtype (wt) and RAG1-/- mice were isolated from splenocytes by magnetic-activated cell sorting for in vitro assays. Different co-cultures were incubated with CpG and GM-CSF, known as an CCL22 inducer.5 For in vivo experiments, wt mice were treated with CpG, R484 or poly(I:C) alone and in combination with GM-CSF. CCL22-levels in a number of organs were analyzed.ResultsAnalyzing the different immune cell compartments in vitro, we found that DCs in whole splenocytes secrete CCL22 during culture while DC cultured alone showed no CCL22 secretion. When treated with CpG, CCL22-levels were reduced in splenocytes, while it was induced in DC culture alone. The same results were seen when RAG splenocytes, that lack functional B and T cells, were cultured with CpG. CpG treated B cells were able to suppress CCL22 secretion by DC unlike T cells alone. Co-cultures of T and B cells treated with CpG, however, induced the strongest CCL22 suppression in DC. In vivo, we could show that all TLR ligands tested reduced CCL22 in a number of organs significantly. Furthermore, CpG showed the strongest suppression of CCL22 even in the presence of the CCL22 inducer GM-CSF.5ConclusionsWe could show that B cells with T cells mediate CCL22 suppression by TLR ligands. The fact that CpG was able to reduce CCL22 levels even in the presence of the inducer GM-CSF demonstrates the potent CCL22 suppressive capacity of TLR ligands.ReferencesO’Neill LA, et al. The history of toll-like receptors – redefining innate immunity. Nat Rev Immunol 2013;13(6):453–60.Rothenfusser S, et al. Recent advances in immunostimulatory CpG oligonucleotides. Curr Opin Mol Ther 2003;5(2):98–106.Wang S, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A 2016;113(46): E7240–E7249.Rapp M, et al. CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes. J Exp Med 2019;216(5):1170–1181.Piseddu I, et al. Constitutive expression of CCL22 is mediated by T cell-derived GM-CSF. J Immunol 2020;205(8):2056–2065.Anz D, et al. Suppression of intratumoral CCL22 by type i interferon inhibits migration of regulatory T cells and blocks cancer progression. Cancer Res 2015;75(21):4483–93.Disclosure InformationJ. Grün: None. I. Piseddu: None. C. Perleberg: None. N. Röhrle: None. S. Endres: None. D. Anz: None.

In Vitro Expansion of Blood γδ T Cells in Patients with Myeloma/Lymphoma and Anti-Tumor Cytotoxicity of the Expanded γδ T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3895-3895
Author(s):  
Anri Saito ◽  
Miwako Narita ◽  
Norihiro Watanabe ◽  
Nozomi Tochiki ◽  
Yumi Hiroi ◽  
...  
Keyword(s):  
T Cells ◽  
Cytotoxic Activity ◽  
Tumor Cells ◽  
Γδ T Cells ◽  
Cellular Immunotherapy ◽  
Target Cells ◽  
Type I ◽  
Type I Ifn ◽  
Lymphoma Cells

Abstract In order to establish an efficient anti-tumor cellular immunotherapy using blood In order to establish an efficient anti-tumor cellular immunotherapy using blood γδ T cells, we investigated the in vitro expansion of γδ T cells in the patients with myeloma and lymphoma by the culture of PB-MNC with bisphosphonate and a low dose of IL-2 and we demonstrated the cytotoxic activity of the expanded γδ T cells against myeloma/lymphoma cells. Simultaneously we explored the potent methods for enhancing the anti-tumor cytotoxic activity of γδ T cells by both directions of activating the expanded γδ T cells and making target tumor cells sensitive to γδ T cells. For the activation of γδ T cells, expanded γδ T cells were exposed with type I IFN, monocyte-derived dendritic cells (mo-DC), or plasmacytoid dendritic cell like cell line PMDC05 (leukemia cell line established from CD4+ CD56+ acute leukemia in our laboratory) for 2 days. For the enhancement of sensitivity of target tumor cell to γδ T cells, we aimed to increase the content of IPP (the potent pyrophosphate antigen for γδ T cells) in tumor cells by decreasing the metabolic downstream of IPP. For decreasing the downstream of IPP, we tried to suppress FPP synthetase, which is involved in downstream metabolism of IPP, by using nitrogen-containing bisphosphonate. In addition, the expression of stress-induced molecules such as MICA/B on target tumor cells was evaluated in association with the level of cytotoxicity of γδ T cells against the tumor cells. Compared with normal control, the patients with myeloma (n=8) demonstrated decreased percentage and counts of PB γδ T cells. Patients with lymphoma (n=7) showed a wide range of values in PB γδ T cells, covering a normal range. Amplification rate of PB γδ T cells by culture with zoledronate and IL-2 varied markedly from patient to patient up to 120 times in myeloma and 90 times in lymphoma. Expanded γδ T cells generated in patients with myeloma/lymphoma were demonstrated to possess the cytotoxic activity against myeloma/lymphoma cells by 51Cr-release assay and CFSE-labeled target cell. The cytotoxic activity of expanded γδ T cells was enhanced by the exposure of γδ T cells with type I IFN (IFN-α and IFN-β). The activation of γδ T cells, which was evaluated by the elevation of CD69 expression, was observed by the exposure of γδ T cells with type I IFN, mo-DC, or PMDC05 for 2 days. The sensitivity of target myeloma/lymphoma cells to γδ T cells was enhanced by the exposure of the target cells to bisphosphonate such as zoledronate. The expression level of MICA/B on target tumor cells was demonstrated to be associated with the potency of cytotoxicity of γδ T cells against the tumor cells. The present study demonstrated that γδ T cells expanded from myeloma/lymphoma patient’s blood are cytotoxic to myeloma/lymphoma cells. There are two methods practically available for enhancing the cytotoxic activity of expanded γδ T cells against myeloma/lymphoma cells, one of which is activating γδ T cells and the other is elevating the sensitivity of target cells by using bisphosphonate.

scholarly journals CD16A-Specific Tetravalent Bispecific Immune Cell Engagers Potently Induce Antibody-Dependent Cellular Phagocytosis (ADCP) on Macrophages

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1111-1111
Author(s):  
Susanne Wingert ◽  
Uwe Reusch ◽  
Armin Beez ◽  
Jens Pahl ◽  
Adelheid Cerwenka ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Immune Cell ◽  
Major Constituent ◽  
Target Cells ◽  
M2 Macrophages ◽  
Macrophage Subtypes

Abstract Introduction Affimed has developed high affinity tetravalent bispecific immune cell engagers for redirected optimized cell killing (ROCK platform). Using anti-CD16A and anti-tumor target-specific antibody domains, the engagers activate NK cells to efficiently kill target cells. The most advanced ROCK-based immune cell engager, AFM13, targeting CD30 on tumor cells and CD16A on immune effectors, is currently being evaluated in several clinical trials to treat CD30-positive malignancies. Based on the fact that CD16A is not exclusively expressed on NK cells, but also on macrophages, we hypothesized that CD16A-specific immune cell engagers would also be able to activate CD16A expressing macrophages through antibody-dependent cellular phagocytosis (ADCP) contributing to anti-tumor response. Macrophages are an essential component of the innate immune system and are a major constituent of normal tissues. They can be broadly classified into different subtypes including M1 (classically activated, generally characterized as pro-inflammatory and immuno-supportive) and M2 (alternatively activated, primarily of an anti-inflammatory profile) subtypes. Those subtypes greatly differ in their phenotype and function and appear to be highly plastic. While M1 macrophages are generally considered to be tumoricidal, M2 macrophages are mostly tumorigenic, depending on their context within the tumor microenvironment. Therapeutic agents focusing on macrophages such as the CD47/SIRPa axis, CSF-1R antibodies and elimination of tumor-associated macrophages (TAMs) have recently come into focus in immuno-oncology. Methods Peripheral monocytes derived from primary human hematopoietic cells of healthy donors were used to generate various macrophage subtypes (unpolarized macrophages, M1, M2a, M2c) in vitro using well-defined cytokine cocktails. These subtypes were characterized phenotypically for their CD16A expression and a wide number of additional markers. Subsequently, they were used to investigate the ability of a number of different CD16A-specific immune cell engagers derived from Affimed´s ROCK platform and control antibodies in vitro to activate and induce ADCP of target cells by flow cytometry and microscopy. Results We demonstrated that all of the macrophage subtypes generated in this study expressed CD16A and mediated ADCP of tumor cells. In addition, we showed that ADCP of tumor cells by several CD16A-specific engagers was both fast and robust for all investigated macrophage subtypes. Specifically, ADCP was detected as early as 2 hours after co-incubation of tumor cells with M1 or M2 macrophages and CD16A-specific immune cell engagers. Using appropriate control antibodies, it was demonstrated that ADCP mediated by CD16A-specific immune cell engagers was selective and at least as potent as ADCP mediated by classical monoclonal antibodies pan-specific for Fc-gamma receptors. Summary and conclusion: We have demonstrated a new mechanism whereby Affimed´s CD16A-specific immune cell engaging antibodies eliminate tumor cells by ADCP, mediated by different subsets of macrophages. Our data suggest that these antibodies may have the potential to boost tumoricidal function within the tumor microenvironment. Future directions of leveraging innate immunity as a therapeutic option in immuno-oncology will be presented. Disclosures Wingert: Affimed: Employment. Reusch:Affimed: Employment. Beez:Affimed: Employment. Pahl:Affimed: Research Funding. Cerwenka:Affimed: Research Funding; Dragonfly Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment.

scholarly journals 122 A powerful, precise targeting system controlled by tumor deletions transforms CEA and MSLN CAR-T cells into tumor-selective agents

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A131-A131
Author(s):  
Agnes Hamburger ◽  
Han Xu ◽  
Yuta Ando ◽  
Grace Asuelime ◽  
Kristian Bolanos-Ibarra ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Loss Of Heterozygosity ◽  
Tumor Antigens ◽  
Allelic Loss ◽  
List Type ◽  
Mixed Cell ◽  
Normal Cells

BackgroundMesothelin (MSLN) and carcinoembryonic antigen (CEA) are classic tumor-associated antigens that are expressed in many solid tumors including the majority of lung, colorectal and pancreatic cancers. However, both MSLN and CEA are also expressed in vital normal organs. This normal expression creates risk of serious inflammation for CEA- or MSLN-directed therapeutics. To date all active CEA- or MSLN-targeted investigational therapeutics have been toxic when administered systemically.MethodsWe have developed a safety mechanism to protect normal tissues without abrogating sensitivity of cytotoxic T cells directed at MLSN(+) or CEA(+) tumors in a subset of patients with defined loss of heterozygosity (LOH) in their tumors (figure 1). This dual-receptor (Tmod< sup >TM</sup >) system exploits common LOH at the HLA locus in cancer cells, allowing T cells to recognize the difference between tumor and normal tissue.1 2 T cells engineered with specific Tmod constructs contain: (i) a MSLN- or CEA-activated CAR; and, (ii) an inhibitory receptor gated by HLA-A*02. HLA-A*02 binding blocks T cell cytotoxicity, even in the presence of MSLN or CEA. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When HLA-A*02 is absent from tumors selected for LOH, the CARs are predicted to mediate potent killing of the A*02(-) malignant cells.ResultsThe Tmod system robustly protects surrogate normal cells even in mixed-cell populations in vitro while mediating robust cytotoxicity of tumor cells in xenograft models (see example in figure 2). The MSLN CAR can also be paired with other blockers, supporting scalability of the approach to patients beyond HLA-A*02 heterozygotes.Abstract 122 Figure 1Illustration of the Tmod T cell engaging with tumor cells with somatic loss of HLA-A*02 and with normal cells.Abstract 122 Figure 2Bioluminescence measurements show the average difference between the size of the MSLN(+)A*02(+) ‘normal’ graft compared to the MSLN(+)A*02(-) tumor graft on the two flanks of mice after T cell infusion. Both tumor and normal grafts are destroyed by CAR-Ts (CAR-3 and M5 benchmark) while the MSLN Tmod cells kill the tumor but not the normal graft.ConclusionsThe Tmod mechanism may provide an alternative route to leverage solid-tumor antigens such as MSLN and CEA in safer, more effective ways than previously possible.ReferencesHamburger AE, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol 2020;128:298–310.Hwang MS, Mog BJ, Douglass J, et al. Targeting loss of heterozygosity for cancer-specific immunotherapy. Proc Natl Acad Sci U S A 2021;118(12):e2022410118.

500 P2RX7 agonist treatment boosts the ability of IL-12-activated CD8+ T cells to infiltrate and control murine melanoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A535-A535
Author(s):  
Kelsey Wanhainen ◽  
Stephen Jameson ◽  
Henrique Borges Da Silva
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Mitochondrial Respiration ◽  
Immune Cell ◽  
List Type ◽  
Memory Cd8 T Cells ◽  
And Control

BackgroundExtracellular adenosine triphosphate (eATP) is a ‘danger signal’ used to sense cellular damage, and recognized by purinergic receptors in mammals. Among those receptors, P2RX7 is preferentially expressed in immune cells. Notably, we recently discovered that P2RX7 is crucial for the generation and maintenance of long-lived tissue-resident and circulating memory CD8+ T cells.1 2 CD8+ T cell function is fundamental for tumor control, and therapies to harness protective CD8+ T cells that overcome exhaustion are currently in the limelight of anticancer strategies. Given our previous data, and the fact that eATP is abundantly present inside the melanoma microenvironment, we tested whether (a) P2RX7 is required for activated CD8+ T cells to infiltrate and control melanoma upon adoptive cell therapy, and (b) P2RX7 agonism can boost the anticancer capacity of CD8+ T cells.Methods(a) We in vitro-activated WT or P2rx7-/- CD8+ T cells (transgenic for the LCMV epitope gp33-P14 or for the ovalbumin SIINFEKL peptide-OTI) with anti-CD3/CD28/IL-2, ± IL-12, for 72h. Cells were adoptively transferred (single transfer of WT or P2rx7-/- cells) into mice with 7 days after subcutaneous transfer of B16 melanoma encoding gp33 or SIINFEKL. We tracked tumor growth until 60 days or at the appropriate endpoint. In some experiments, we sacrificed recipient mice 7 days after adoptive T cell transfer for immune cell phenotyping. Some parameters (cytokine production, mitochondrial respiration via Seahorse) were measured in in vitro-activated cells. (b) WT and P2rx7-/- cells were activated with anti-CD3/anti-CD28/IL-2, ± Bz-ATP, a P2RX7 agonist. Tumor growth was tracked over time until 60 days or at the appropriate endpoint.ResultsWT and P2RX7-deficient (P2rx7-/-) CD8+ T cells in the absence of IL-12 do not differ in tumor infiltration and/or control. However, P2rx7-/- CD8+ T cells activated in response to IL-12 tertiary stimulus do not control B16 melanomas as well as their WT counterparts. Phenotypically, IL-12-P2rx7-/- CD8+ T cells do not profoundly differ from IL-12-WT CD8+ T cells, except for diminished mitochondrial respiration levels in vitro, and diminished mitochondrial membrane potential (e.g. mitochondrial health) among tumor-infiltrating cells. Strikingly, Bz-ATP treatment increased the mitochondrial activity of WT CD8+ T cells in vitro and in vivo and led to increased B16 infiltration and control, in a P2RX7-dependent manner.ConclusionsWe are currently studying the mechanisms behind the ability of P2RX7 agonists to increase the antitumor function of CD8+ T cells; these are promising results that can lead to a new alternative in immune cell therapies against melanoma.AcknowledgementsWe would like to thank Jane Ding and Lily Qian for technical assistance, and Kristin Hogquist for scientific input.Ethics ApprovalThis study was approved by the IACUC board at the University of Minnesota (IACUC number A3456-01)ReferencesBorges da Silva H, Beura LK, Wang H, Hanse EA, Gore R, Scott MC, Walsh DA, Block KE, Fonseca R, Yan Y, Hippen KL, Blazar BR, Masopust D, Kelekar A, Vulchanova L, Hogquist KA, Jameson SC. The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells. Nature. 2018; 559(7713):264–268.Borges da Silva H, Peng C, Wang H, Wanhainen KM, Ma C, Lopez S, Khoruts A, Zhang N, Jameson SC. Extracellular ATP sensing via P2RX7 promotes CD8+ tissue-resident memory T cells by enhancing TGF-β sensitivity. Immunity 2020;53(1):158–171.

682 Antibody-mediated blockade of interleukin-10 receptor-alpha promotes the activation of immune cells from in vitro dissociated tumor samples

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A721-A721
Author(s):  
Alexey Berezhnoy ◽  
Kalpana Shah ◽  
Daorong Liu ◽  
Peter Lung ◽  
Vatana Long ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Expression Profiles ◽  
Interleukin 10 ◽  
Biologically Active ◽  
List Type ◽  
Receptor Alpha

BackgroundInterleukin-10 (IL-10) is a multifunctional cytokine that can mediate immune suppression or activation depending on the immunological context. Mouse studies have demonstrated that blockade of IL-10 enhances immune response against tumors and chronic viral infections;1 2 intriguingly, high concentrations of long-acting, pegylated IL-10 have also shown anti-tumor activity.3 Here we investigated IL-10 and IL-10 receptor-alpha (IL-10RA) expression profiles in normal and tumor tissues as well as the immunological effects of modulating the IL-10 pathway via antibody-mediated blockade of IL-10RA.MethodsIL-10 and IL-10RA mRNA are expressed by several tumors, including renal, lung, breast, and colon cancers. Fluorescent in-situ hybridization revealed that the majority of IL-10RA was expressed by CD3-negative tumor-infiltrating cells, localized in close proximity to T cells in the tumor microenvironment (TME). Immunohistochemistry studies confirmed expression of IL-10RA in the TME, while no expression was detected in healthy tissues. Furthermore, dissociated tumor cells produced biologically active levels of IL-10 in culture.ResultsMonoclonal antibodies (mAbs) against IL-10RA prevented IL-10 signaling and enhanced release of IL-12 by monocyte-derived dendritic cells activated with suboptimal LPS concentrations. The effect of IL-10RA blockade was greater than that observed with IL-10 neutralizing mAbs. In mixed lymphocyte reactions and superantigen-driven T-cell activation, IL-10RA blockade enhanced IL-2 secretion by T lymphocytes. Consistent with earlier observations in mouse models,4 the effect of IL-10RA blockade was nonredundant with blockade of the PD-1/PD-L1 axis, resulting in enhanced IL-2 and interferon-gamma secretion by T cells when both pathways were inhibited. Blockade of IL-10RA during CD3-redirected in vitro killing of tumor cells by PBMC induced IL-12 release as well as upregulation of CD86 and HLA-DR by CD3-negative cells. In in vitro dissociated tumor cells, IL-10RA blockade induced release of IL-2, interferon-gamma and other proinflammatory cytokines; additional PD-1/PD-L1 axis blockade further enhanced cytokine release.ConclusionsIn summary, antibody-mediated IL-10RA blockade can potentiate immune activation in the dissociated tumor cells and may be a valuable addition to cancer immunotherapies, including redirected T-cell killing and checkpoint blockade.ReferencesVicari A, et al. J. Exp. Med 2002;196:541–549.Ejrnaes M, et al. J Exp Med 2006;203:2461–72.Emmerich J, et al. Cancer Res 2012. 72:3570–3581.Brooks D, et al. Proc Natl Acad Sci U S A 2008;105:20428–33.

scholarly journals 778 Modulating tumor microenvironment with arginase-1 specific T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A813-A813
Author(s):  
Evelina Martinenaite ◽  
Mia Aaboe Jørgensen ◽  
Rasmus Erik Johansson Mortensen ◽  
Shamaila Munir Ahmad ◽  
Stine Emilie Weis-Banke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Memory T Cells ◽  
Ethics Committee ◽  
Mouse Tumor ◽  
Immune Modulators ◽  
Arginase 1 ◽  
Mouse Tumor Models

BackgroundIO112 is an immune modulatory cancer therapy under preclinical development to target arginase-1-expressing tumor cells and immune inhibitory myeloid cells, such as myeloid derived suppressor cells (MDSCs), and tumor associated macrophages (TAMs). Arginase-1 acts as a metabolic immune regulator at the tumor site by reducing availability of L-arginine to the infiltrating immune cells thus reducing T cell functionality and proliferation. Previously, we demonstrated that IO112 triggers activation of spontaneous CD4+ and CD8+ T-cell responses against arginase-1, found in both cancer patients and healthy individuals.1 These T cells are present in the memory T cell compartment, and are activated in arginase-1 inducing conditions, such as presence of TH2 cytokines IL-4 or IL-13 in vitro.2 3 In this study we aimed to explore the role of arginase-1-specific T cells as immune modulators in immune homeostasis and tumor microenvironment for the development of IO112 immunomodulatory therapy.MethodsHuman arginase-1-specific T cells were isolated and expanded for functional characterization of reactivity against arginase-1 expressing target cells as well as subsequent phenotyping of the targeted arginase-1 positive cells. Syngeneic C57BL/6 mouse tumor models were used to assess the therapeutic efficacy of IO112.ResultsWe show that arginase-1-specific memory T cells specifically recognize arginase-1 expressing cells, such as mRNA transfected autologous dendritic cells (DCs) and B cells as well as M2 polarized macrophages in vitro. In addition, activated arginase-1-specific T cells produce pro-inflammatory cytokines IFNγ and TNFα. Secretion of TH1 cytokines by these T cells suggests that they may act as potent immune modulators in the tumor microenvironment, since many arginase-1 expressing myeloid cells are not terminally differentiated and they can be re-polarized to an immunostimulatory, M1-like phenotype. We also observed that targeting of M2-polarized arginase-1 expressing monocytic leukemia cell line THP-1 with arginase-1-specific CD4+ T cells induces upregulation of PD-L1 on the THP-1 cells. Furthermore, we demonstrate anti-tumor activity of IO112 in syngeneic mouse tumor models (B16 and MC38), both as monotherapy and in combination with anti-PD-1 treatment. The therapeutic effect was associated with increased immune infiltration in the IO112-treated mice compared to the control.ConclusionsWe demonstrate that arginase-1 specific T cells can influence the polarization of arginase-1-expressing immune cells. Our study provides evidence that IO112 immune therapy against arginase-1 is an attractive way of modulating the immune suppressive tumor microenvironment for therapeutic benefit. With this rationale, we are currently undertaking Investigational New Drug (IND) application enabling studies to explore this approach in a clinical setting.ReferencesMartinenaite E, Mortensen REJ, Hansen M, Holmström MO, Ahmad SM, Jørgensen NGD, Met Ö, Donia M, Svane IM, Andersen MH. Frequent adaptive immune responses against arginase-1. Oncoimmunology 2018;7(3):e1404215.Martinenaite E, Ahmad SM, Svane IM, Andersen MH. Peripheral memory T cells specific for Arginase-1. Cell Mol Immunol 2019;16(8):718–719.Martinenaite E, Ahmad SM, Bendtsen SK, Jørgensen MA, Weis-Banke SE, Svane IM, Andersen MH. Arginase-1-based vaccination against the tumor microenvironment: the identification of an optimal T-cell epitope. Cancer Immunol Immunother 2019;68(11):1901–1907.Ethics ApprovalThis study was approved by the Scientific Ethics Committee for The Capital Region of Denmark and Danish Ethics Committee on experimental animal welfare.

scholarly journals 154 Context-dependent reversible modulation of cJUN expression by CAR T cells for cancer treatment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A164-A164
Author(s):  
Zhifen Yang ◽  
Francesco Marincola
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
List Type ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Context Dependent ◽  
Fadu Cells

BackgroundOverexpression of canonical AP-1 factor cJUN was shown to prevent CAR T cell exhaustion and improve anti-tumor potency in vivo (1). However, its clinical utilization is limited by potential for transformation and oncogenic risk. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-339) that delivers context-dependent upregulation of endogenous cJUN increasing CAR-T cell resistance to exhaustion.MethodsRB-339 is a CAR T cell engineered to conditionally turn on the transcription of the cJUN endogenous gene. The circuit includes a lentiviral construct encoding an anti-HER2 (4D5) single chain variable fragment, with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the promoter region of cJUN. A second construct encodes linker for activation of T cells, complexed to nuclease-deactivated/dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) via a TEV-cleavable linker (LdCV). Activation of CAR allows the release of dCas9 for nuclear localization and conditionally and reversibly induces the expression of cJUN. RB-339 was compared in vitro to control (cRB-339, lacking the cJUN sgRNA) and CAR-T cells engineered to constitutively express cJun.ResultsRB-339 induced cJUN upregulation upon stimulation with HER2-coated beads and this resulted in significantly elevated expression over a 6-day time course compared to the control cRB-339 (figure 1A-B). When HER2-coated beads were removed at day 3, cJUN expression returned to baseline parallel to cRB-339. The conditional upregulation of cJUN in RB-339 contrasted with the constitutive overexpression in the transgene carrying cells that was irrespective of antigen stimulation (figure 1C). Upon exposure to HER2+ FaDu cancer cells, RB-339 peaked at day 2 and declined afterwards when FaDu cells were killed at day 3; cJUN increased again at day 4 upon restimulation with FaDu cells at day 3 (figure 2). Such a dynamic induction of cJUN resulted in significantly enhanced CAR-T cells proliferation in RB-339 compared to the respective conventional CAR-T cells or cRB-339 (figure 3).Abstract 154 Figure 1Conditional upregulation of cJUN by RB-339 in vitro. RB-339 and its control cRB-339 were stimulated by HER2-coated or BSA-coated beads for either six days or three days followed by removal of beads at day 3 (figure 1A-B). Intracellular expression of cJUN was detected at indicated time points. Intracellular cJUN expression in overexpressed cJUN-CAR (figure 1C)Abstract 154 Figure 2Kinetics of cJUN upregulation in RB-339 upon exposure to HER2+ FaDu tumor cells. RB-339 and its control cRB-339 were stimulated by FaDu tumor cells for six days with or without restimulation at day 3Abstract 154 Figure 3Conditional upregulation of cJUN resulted in enhanced CAR-T proliferation in RB-339 in vitro after 6-day co-culture with FaDu tumor cells, compared to conventional HER2 CAR or cRB-339ConclusionsWe conclude that CAR-T engineered to conditionally express the canonical AP-1 factor cJUN increases expansion potential similarly to CAR-T cells engineered to constitutively express the cJun transgene. However, the context-dependent upregulation of cJUN limits the risk of oncogenic transformation. We are currently combining inducible and reversible cJUN and IL-12 upregulation for the generation of the next RB-339 product.ReferenceLynn RC, Weber EW, et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 2019; 576(7786):293–300.

Sign in / Sign up

Export Citation Format

Share Document