Immune Agonists Research Articles

The Multi-Kinase Inhibitor Lucitanib Enhances the Antitumor Activity of Coinhibitory and Costimulatory Immune Pathway Modulators in Syngeneic Models.

Lucitanib is a multi-tyrosine kinase inhibitor whose targets are associated with angiogenesis and other key cancer and immune pathways. Its antiangiogenic properties are understood, but lucitanib's immunomodulatory activity is heretofore unknown. Lucitanib exhibited such activity in vivo, increasing CD3 + , CD8 + , and CD4 + T cells and decreasing dendritic cells and monocyte-derived suppressor cells in mouse spleens. Depletion of CD8 + T cells from syngeneic MC38 colon tumor-bearing mice reduced the antitumor efficacy of lucitanib and revealed a CD8 + T-cell-dependent component of lucitanib's activity. The combination of lucitanib and costimulatory immune pathway agonists targeting 4-1BB, glucocorticoid-induced TNFR (GITR), inducible T-cell co-stimulator (ICOS), or OX40 exhibited enhanced antitumor activity compared with each single agent in immunocompetent tumor models. Lucitanib combined with blockade of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein-1 (PD-1) coinhibitory immune pathways also showed enhanced antitumor activity over the single agents in multiple models. In CT26 tumors, lucitanib, alone or combined with anti-PD-1, reduced CD31 + vessels and depleted F4/80 + macrophages. Combination treatment also increased the number of intratumoral T cells. Gene expression in pathways associated with immune activity was upregulated by lucitanib in MC38 tumors and further potentiated by combination with anti-PD-1. Accordingly, lucitanib, alone or combined with anti-PD-1, increased intratumoral CD8 + T-cell abundance. Lucitanib's antitumor and pharmacodynamic activity, alone or combined with anti-PD-1, was not recapitulated by specific vascular endothelial growth factor receptor-2 (VEGFR2) inhibition. These data indicate that lucitanib can modulate vascular and immune components of the tumor microenvironment and cooperate with immunotherapy to enhance antitumor efficacy. They support the clinical development of lucitanib combined with immune pathway modulators to treat cancer.

Abstract LB211: Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) remains one of the major causes of cancer related mortality worldwide, yet clinical advances with checkpoint inhibitors suggest that an anti-tumor immune response can be harnessed to prolong survival in NSCLC patients. We recently described the identification of a tumor reactive natural killer (NK) cell population by its in vivo upregulation of PD-L1 in the presence of tumor, referred to here as TRACK-NK cells. PD-L1(+) NK cells are more potent than PD-L1(-) NK cells against tumor cell targets. Further, the administration of anti-PD-L1 antibody (atezolizumab) functions as an immune agonist on TRACK-NK cells resulting in significantly enhanced degranulation (P < 0.001) and secretion of IFN-gamma (P < 0.001) in the presence of tumor. Here we investigated the functional activity and product safety of allogeneic, off-the-shelf TRACK-NK cells retrovirally transduced to express soluble (s) IL-15 to treat NSCLC without or with the addition of atezolizumab. TRACK-NK cells derived from umbilical cord blood NK cells were expanded over 1,000-fold ex-vivo in 17 days with ~50% of the final product engineered to express sIL15 and cytokine-activated to express PD-L1. The phenotype of the final TRACK-NK cell product showed a significant increase in the expression of PD-L1, CD25 and CD69 when compared to non-transduced (P < 0.01) or NK cells transduced to express sIL15 without cytokine activation (s15 NK cells) (P < 0.01). Following cryopreservation, our thawed TRACK-NK cell product demonstrated high recovery (>85%) and viability (>80%). In a Real Time Cell Analysis (RTCA) in vitro cytotoxicity assay against a human NSCLC cell line (A549), TRACK-NK cells were 10 times more potent than non-transduced (NT) NK cells (P < 0.001) and 2 times more potent than s15 NK cells (P < 0.01). To assess the efficacy of our frozen, allogeneic off-the-shelf TRACK-NK cells in vivo, we utilized immunodeficient mice bearing human A549 metastatic NSCLC. Intravenous infusion of TRACK-NK cells resulted in significantly more suppression of luciferase-A549 NSCLC growth in mice compared to mice treated with NT NK cells (P < 0.05) or s15 NK cells (P < 0.01). In terms of dose response, 1 × 107 TRACK-NK cells per dose delivered every other day for a total of 4 doses showed significantly better tumor control compared to a dose of 5 × 106 TRACK-NK cells (P < 0.001) but similar control compared to 2 × 107 TRACK-NK cells. The combination of TRACK-NK cells plus atezolizumab showed better control of NSCLC growth in vivo compared to TRACK-NK cells alone (P < 0.01). Finally, we evaluated the safety of our TRACK-NK product in immunodeficient mice without tumor engraftment and did not observe any significant change in body weight, body temperature, liver enzymes, kidney function, or blood counts compared to mice treated with vehicle control. No cytokine release syndrome was observed. In summary, our studies using human allogeneic off-the-shelf TRACK-NK cells proved to be safe and non-toxic, demonstrated enhanced cytotoxicity against NSCLC in vitro and enhanced control of tumor growth in vivo, further improved with atezolizumab. As the intravenous infusion of activated NK cells naturally traffic to lung, our TRACK-NK platform will move forward to the clinic for the treatment of relapsed and refractory NSCLC patients. Citation Format: Ting Lu, Anthony G. Mansour, Rui Ma, Christian Bustillos, Zhiyao Li, Shoubao Ma, Zhenlong Li, Hanyu Chen, Kun-Yu Teng, Jianying Zhang, Michael A. Caligiuri, Jianhua Yu. Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of 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 LB211.

Abstract 5616: Tumor-targeted immune activation via a site-specific TLR7-agonist antibody-drug conjugate

Abstract Traditional antibody-drug conjugates (ADCs) selectively deliver cytotoxic payloads to tumor-associated antigen (TAA)-expressing tumor cells, thereby limiting healthy cell damage and toxicities associated with systemic administration. Analogously, a novel class of immune-stimulating antibody conjugates (ISACs) has recently emerged to achieve tumor-targeted activation of anti-tumor immune responses, avoiding dose-limiting immunotoxicities seen with the systemic immune agonist delivery. Most of the ADC or ISAC to date utilize random payload conjugation to native surface-exposed lysines or cysteines, and relatively labile linkage chemistries, leading to conjugation site and drug-to-antibody ratio (DAR) heterogeneity, and ADC/ISAC instability in systemic circulation. In particular, the presence of high-DAR species and linker instability can lead to off-tumor toxicity, thus limiting the therapeutic window. Here we describe the generation and preclinical characterization of a site-specific TLR7-agonist (TLR7a) ISAC targeting an undisclosed TAA1. To specify the conjugation site and DAR, a non-natural amino acid (nnAA), para-acetyl-L-phenylalanine (pAF) was genetically incorporated at defined sites within the anti-TAA1 antibody, providing an orthogonal chemical handle for covalent TLR7 agonist payload conjugation through a highly stable oxime bond. Using this site-specific, homogenous ISAC platform, we systematically screened different conjugate sites and TLR7 agonist payloads to obtain an optimized TAA1-TLR7a ISAC drug candidate. ISAC platform stability, homogeneity, and flexibility were critical, because conjugation site and payload structure dramatically influenced ISAC in vitro activity, PK profile and in vivo efficacy. Using in vitro co-culture assays with human immune cells and tumor cell lines, we showed that the optimized TAA1 ISAC can induce multiple anti-tumor immune mechanisms, including proinflammatory cytokine production, the myeloid cell activation markers induction and enhanced ADCC mediated tumor cell killing. The TAA1 ISAC is 100-fold more active than the free, unconjugated TLR7 agonist, and all activity is conditional on the presence of TAA1-expressing tumor cells. Finally, we demonstrated in vivo efficacy in both xenograft and syngeneic tumor models. In a syngeneic MC38-tumor model expressing human TAA1, ISAC treatment led to complete tumor regression and formation of immunologic memory. These results provide a strong rationale for site-specific TLR7 agonist ISAC as a next generation platform for tumor-targeted, innate immune agonist immunotherapy. Citation Format: Mingchao Kang, Sung-Ju Moon, Ji Young Kim, Andy Beck, Molly Allen, Jay Nelson, Keith Tatsukawa, Hon Tran, Manoj Pal, Michael Gray, Nick Knudsen, Lillian Skidmore, David Mills, Yingchun Lu, Ying Buechler, Sukumar Sakamuri, Shawn Zhang, Feng Tian. Tumor-targeted immune activation via a site-specific TLR7-agonist antibody-drug conjugate [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 5616.

Abstract 1391: PBMC humanized mouse model for ImmunoOncology drug evaluation

Abstract Since the first immune checkpoint blocker Ipilimumab was approved by US FDA in 2011, more and more immune therapeutic drugs popped up. Humanized PBMC reconstitution in immune deficient mice is becoming a good model for evaluating therapeutic antibodies, especially the BsAb with one arm of anti-CD3 that mediates immune cells and another arm targeting a tumor antigen. However, limited window of dosing regimen triggered by graft-versus-host-disease (GvHD) after 30-40 days of reconstitution, and insufficient tumor-infiltrated immune cells naturally from reconstituted circulatory system hindered the widely application of the model in immunotherapy in development of multiple immune checkpoint inhibitors or immune agonists. To overcome this, LIDE has developed a unique human PBMC-cancer cells co-inoculated model instead. In our platform, cancer-priming PBMCs were mixed with the fresh cancer cells in MatriGel, co-inoculated into NCG mice to form a huPBMC well-infiltrated tumor tissue for immunotherapy. Our platform has been successfully helped evaluate biological function of PD1, Tigit, PVRIG, CD73, CD47, CD38, CD40, GITR Antibodies, Tgfb1/PDL1 BsAb, DLL3/CD3 BsAb in multiple cancers, such as melanoma, breast cancer and lung cancer. Citation Format: Bin Xie, Hongkui Chen, Xin Hou, Danyi Wen. PBMC humanized mouse model for ImmunoOncology drug evaluation [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 1391.

Abstract 365: Intratumoral delivery of aluminum hydroxide-tethered IL-12 induces potent anti-tumor immune response

Abstract IL-12 is a potent cytokine that mediates anti-tumor immune response, but its clinical development has been hindered by systemic toxicity. Intratumoral (IT) delivery can improve the therapeutic window of cytokine drugs, but is in turn limited by rapid clearance of injected drug from the tumor which reduces efficacy, necessitates frequent administration, and increases systemic accumulation. Ankyra has developed a novel IT drug delivery platform for stable linking of engineered cytokines and other immune agonists to an aluminum hydroxide (Alhydrogel®) scaffold. Here, we describe the therapeutic and immune effects of Ankyra’s intratumoral IL-12 using relevant in vitro and in vivo murine models. Single-chain human IL-12 was genetically fused at its c-terminus to a proprietary, phosphorylated alum-binding peptide (IL-12-ABP) that forms a high affinity complex with Alhydrogel®. Human IL-12-ABP proteins were mixed with a 10-fold mass excess of Alhydrogel® to form the therapeutic complex, ANK-101. Since human IL-12 is not active in mice, a surrogate complex containing the mouse IL-12 sequence (mANK-101) was also generated. The functional potency of ANK-101 and mANK-101 was assessed in multiple cellular assays in comparison to their respective unmodified IL-12 controls. The therapeutic efficacy of mANK-101 was evaluated in syngeneic mouse tumor models including MC38, CT26, 4T1, and B16F10. The intratumoral retention of mANK-101 after a single IT dose was analyzed by IVIS imaging studies, and immune profiling of tumors was carried out by flow cytometry. Statistical analyses were done using Student’s t-test for comparison between groups and Kaplan-Meier method for survival. ANK-101 complexes retained IL-12 activity as demonstrated by concentration dependent increases in IFNγ production by anti-CD3 stimulated peripheral blood mononuclear cells. IVIS studies conducted in tumor bearing mice showed that the labeled mANK-101 complexes were retained in the tumor for >21 days while free mIL-12-ABP protein was cleared in <24 hours. One or two doses of IT administered mANK-101 induced tumor regressions in diverse syngeneic tumor models, including complete responses either alone or in combination with approved checkpoint blockers targeting PD-1 or CTLA-4 and other immunotherapies. Efficacious doses were well tolerated in mice with no significant weightloss compared to vehicle treated animals. Immune profiling of tumors 7 days post treatment showed an increased intratumoral CD8+/Treg ratio, and prolonged myeloid cell activation. In addition, IHC analysis of residual tumors post treatment showed increased infiltration of CD8+ T cells. In conclusion, Ankyra’s platform is a novel approach that expands the therapeutic window of IL-12, which will be tested in a phase I clinical trial and the platform has potential to extend the therapeutic effectiveness of other IT administered immunomodulatory drugs. Citation Format: Sailaja Battula, Gregory Papastoitsis, Howard L. Kaufman, Darrell J. Irvine, K. Dane Wittrup, Michael M. Schmidt. Intratumoral delivery of aluminum hydroxide-tethered IL-12 induces potent anti-tumor immune response [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 365.

Abstract LB218: A safe and highly potent anti-PD-1-IL-2c fusion that decouples efficacy and toxicity of IL-2 therapy

Abstract Interleukin 2 (IL-2) is a pivotal immune agonist for tumor immunotherapy that has produced impressive clinical efficacy in a number of cancer types. Nevertheless, its pleiotropic effect has led to prohibitory side effects and immune restraint. In contrast, the PD-1 blockade-based cancer immunotherapy has a good safety profile by targeting and sustaining the activity of tumor-antigen specific T cells within cancer tissues. To take full advantage of complementary antitumor activity of PD-1 monoclonal antibody (mAb) and IL-2 as well as supreme intratumoral T-cell-targeting property of PD-1, a bifunctional fusion protein composed of PD-1 mAb and IL-2 mutein (αPD-1-IL-2c) is designed. IL-2c is a chimeric molecule that contains a fragment from IL-15 and does not bind to IL-2 receptor alpha (IL-2Rα). αPD-1-IL-2c stimulated greater pSTAT5 activation and proliferation in PD-1+ T cells than PD-1- T cells. αPD-1-IL-2c also induced a much stronger anti-tumor immune response compared to PD-1 mAb, IL-2c, or PD-1 mAb in combination with IL-2c in multiple syngeneic models such as B16F10, CT26, and MC38. In CT26 and MC38 models, αPD-1-IL-2c achieved complete response starting from 0.3 mg/kg dosing. In B16F10 model, αPD-1-IL-2c showed greater than 70% tumor growth inhibition (TGI). Global gene expression profiling studies showed that PD-1-IL-2c treatment significantly elevated expression levels of Cd3d, Cd3e, Cd8a, Il2rα, Cxcr3, Cxcr6, Zap70, Lck, and Pdcd1 in tumor tissues compared to treatment with PD-1 mAbs or IL-2c, indicating a specific expansion and activation of T cell in the tumor microenvironment (TME). The safety profile of αPD-1-IL-2c was assessed and compared to WT IL-2 and a leading “No-α” αPD-1-IL-2 fusion protein (αPD-1-IL-2x). WT IL-2 and αPD-1-IL-2x induced significant body weight loss and severe splenomegaly when dosed at 1.25 mg/kg. In contrast, αPD-1-IL-2c dosed at 10 mg/kg did not cause appreciable body weight loss and only led to minimal splenomegaly. In summary, we have developed a safe and highly potent αPD-1-IL-2 mutein that achieves full PD-1 blockade and TME-specific T cell activation and expansion without appreciable toxicity. Citation Format: Fan Ye, Jianing Huang, Zoey Huang, Sandra Chen, Eric Liao, Ella Li, Allison Quach, Jenny Jiang, Hanna Lin, Michael Hua, Savannah Yung, Lili Cheng, Ziyang Zhong. A safe and highly potent anti-PD-1-IL-2c fusion that decouples efficacy and toxicity of IL-2 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 LB218.

A combined phase I/II study of a novel bicycle tumor-targeted immune cell agonist BT7480 in patients with nectin-4 associated advanced malignancies.

TPS2689 Background: BT7480 is a novel, first-in-class, Nectin-4/CD137 Bicycle tumor-targeted immune cell agonist ( Bicycle TICA) that co-ligates cluster of differentiation (CD)137 (on immune cells) and Nectin-4 (on tumor cells). BT7480 comprises 3 bicyclic peptides ( Bicycles), 1 that binds to Nectin-4 and 2 that bind to CD137. The Bicycles are conjugated via a branched trimeric polyethylene glycol linker. Co-ligation of Nectin-4 and CD137 by BT7480 is hypothesized to induce oligomerization and activation of CD137 resulting in a tumor-localized costimulatory signal that leads to an antitumor response in preclinical studies. Nectin-4 is reported to be highly expressed in numerous tumors with unmet medical need including bladder, pancreas, breast, ovary, esophagus, head and neck, stomach, and lung cancers. Many of these tumor types also contain CD137 expressing cells. BT7480 exhibited a favorable preclinical profile supporting initiation of a first-in-human study to investigate safety and efficacy in indications with evidence of Nectin-4 expression. Methods: BT7480-100 (NCT05163041) is a PhI/II study to evaluate safety and tolerability of BT7480 administered as an intravenous infusion QW in a 28-day cycle, and to determine a recommended Phase 2 dose to further explore efficacy and safety. Patients will be recruited with advanced solid tumors associated with Nectin-4 expression including urothelial; head and neck squamous cell; non-small cell lung; ovarian, breast, gastric or esophageal carcinoma after exhausting standard of care options. Patients must have available tumor tissue, acceptable hematologic and other critical organ function. Exclusion criteria include uncontrolled brain metastases, uncontrolled hypertension, significant history of autoimmune disease and prior CD137 targeted therapy. Pharmacokinetic and pharmacodynamic analyses will be performed to support dose escalation decisions and increase the understanding of safety and clinical activity signals observed during the study. Tumor response will be assessed per RECIST every 8 weeks. Cohorts 1 and 2 have been completed without DLT. Enrollment in cohort 3 began in January 2022. Clinical trial information: NCT05163041.

A phase 1/2 study of RTX-224, an engineered red blood cell expressing 4-1BB ligand and membrane-bound IL-12, for the treatment of patients with select advanced solid tumors.

TPS2680 Background: RTX-224 is a genetically engineered off-the-shelf, allogeneic red blood cell that expresses the costimulatory molecule 4-1BB ligand (4-1BBL) and cytokine interleukin-12 (IL-12) on the cell surface. The use of 4-1BB and IL-12 agonists for cancer immunotherapy has been limited due to systemic toxicities. Unlike agonist antibodies and recombinant cytokines, which are distributed systemically, Red Cell Therapeutics, such as RTX-224, are restricted to the vasculature and spleen, which may limit toxicities previously observed with agonist 4-1BB monoclonal antibodies and recombinant IL-12. RTX-224 is designed to be a broad immune agonist of both adaptive and innate responses that activates and expands effector and memory CD8+ and CD4+ T cells, cytotoxic natural killer (NK) cells and produces inflammatory cytokines and chemokines, leading to enhanced antigen presentation. In preclinical studies, the combined activation of both adaptive and innate immune responses by the murine surrogate of RTX-224 led to antitumor activity while the restricted biodistribution improved the safety profile. Methods: The RTX-224-01 study is a Phase 1/2, first-in-human, multi-center, dose-escalation and expansion study of RTX-224 in patients with relapsed or refractory urothelial cancer, squamous cell carcinoma of the head and neck, non-small cell lung cancer, triple negative breast cancer and cutaneous melanoma. Safety, tolerability, pharmacokinetics and pharmacodynamics and anti-tumor activity of RTX-224 will be assessed. Approximately 28 patients will be enrolled across dose level cohorts to identify the recommended Phase 2 dose (RP2D). The starting dose is 100 million (1x108) cells administered intravenously every 3 weeks (Q3W) and the dose will be escalated by half-log increments following a Bayesian logarithmic regression model (BLRM) with overdose control. Following RP2D selection, each of the 5 expansion cohorts will enroll approximately 20 patients. Pharmacodynamic and exploratory biomarker studies correlative to clinical response will be evaluated on peripheral blood and paired tumor biopsies. Multiple technologies will be employed to profile the innate and adaptive responses following RTX-224 treatment. The study is open and enrolling patients in Phase 1. Clinical trial information: NCT05219578.

Amelioration of systemic antitumor immune responses in cocktail therapy by immunomodulatory nanozymes.

Nanozymes that mimic natural enzyme–like activities have gradually emerged in cancer therapy. To overcome the bottlenecks of single-mode nanozymes, including “off-target” toxicity and ineffectiveness toward metastatic cancers, we designed magnetic nanoparticle–based multifunctional visualized immunomodulatory nanozymes. Besides the partial initiation of the prime immune response by intrinsic immunogenicity, as a smart drug delivery system with a temperature- and pH-sensitive dual response to the tumor microenvironment, these nanozymes released immune agonists to boost enhanced systemic immune response, eventually ameliorating the cancer immune microenvironment through many aspects: activating dendritic cells, improving the function of CD8+ T cells, and decreasing the population of myeloid-derived suppressor cells, which inhibited both primary and metastatic cancers. Mechanistically, these nanozymes regulated the reactive oxygen species–related Akt signaling pathway and consequently activated cell apoptosis–related signaling pathways, which provided a deeper understanding of the synergistic mechanism of multifunctional nanozymes. Our findings offer a promising imaging-guided cocktail therapy strategy through immunomodulatory nanozymes.

Probiotic Engineering and Targeted Sonoimmuno-Therapy Augmented by STING Agonist.

Tumor targeting and effective immunomodulation are of critical significance during tumor treatment by sonodynamic therapy (SDT). Herein, the probiotic engineering of the clinically approved sonosensitizer (hematoporphyrin monomethyl ether (HMME)) is reported onto the probiotic bacterium Bifidobacteria Longum (BiL) for sonosensitive bifidobacterium construction (HMME@BiL cells). Based on the hypoxic tropism feature of the strain, effective tumor‐targeted sonodynamic therapeutics can be achieved both in vitro and in vivo. To improve the immunological responses against tumor during sonodynamics, a recently‐developed stimulator of interferon genes immune agonist SR717 has been employed to improve the anti‐tumor immunity with prominent activities, eradicating both primary and metastatic tumors with high efficiency and satisfied biocompatibility. The present work provides a promising paradigm of microbiotic nanomedicine in a sophisticated sonoimmunotherapeutic strategy against malignant tumors.

An in house qPCR System to Evaluate Transcripts Stimulated by an Immune Innate Agonist (CIGB2020) during the Covid19 Pandemic Scenario

Background: A low cost and simple real-time PCR method to quantify different transcripts in human samples is a need in most developing countries. A quantitative real-time PCR assay to evaluate the relative RNA expression stimulation of specific transcripts during the Pandemic COVID19 scenario was developed at the Center for Genetic Engineering and Biotechnology in Havana, using a commercial amplification system with standards and specific primers that are produced in house. Methods: Oro-pharyngeal scrape samples from patients that were treated previously with an innate immune agonist, the CIGB2020, were employed to RNA purification, cDNA synthesis, and evaluation of different gene transcripts by the in house qPCR assay using different standard curves constructed with specific primers that amplify a pool of the own evaluated transcripts. Results: Preliminary results showed a typical standard curve from the in house qPCR system developed. A typical standard curve during the evaluation of the TLR3 gene transcript was described as example, and was calculated automatically by plotting the Ct values against each standard of known concentration, and the linear regression of this curve was also calculated as: M=-3.31641, B=42.73084, R=0.9516. It was also observed the influence of the CIGB2020 on the stimulation of the three toll like receptors from the innate immune system. Conclusions: This study demonstrated that the in house qPCR system is useful to quantify the relative induction of the expression of several genes transcripts considered as immunological markers of the immune innate system

A Novel Toll-Like Receptor 2 Agonist Protects Mice in a Prophylactic Treatment Model Against Challenge With Bacillus anthracis.

Current therapies for anthrax include the use of antibiotics (i.e., doxycycline, and ciprofloxacin), an anthrax vaccine (BioThrax) and Bacillus anthracis-specific, monoclonal antibody (mAb) (i.e., Raxibacumab and obiltoxaximab). In this study, we investigated the activity of immunomodulators, which potentiate inflammatory responses through innate immune receptors. The rationale for the use of innate immune receptor agonists as adjunctive immunomodulators for infectious diseases is based on the concept that augmentation of host defense should promote the antimicrobial mechanism of the host. Our aim was to explore the anti-B. anthracis effector function of Toll-like receptor (TLR) agonists using a mouse model. Amongst the six TLR ligands tested, Pam3CSK4 (TLR1/2 ligand) was the best at protecting mice from lethal challenge of B. anthracis. We then evaluated the activity of a novel TLR2 ligand, DA-98-WW07. DA-98-WW07 demonstrated enhanced protection in B. anthracis infected mice. The surviving mice that received DA-98-WW07 when re-challenged with B. anthracis 20 days post the first infection showed increased survival rate. Moreover, ciprofloxacin, when treated in adjunct with a suboptimal concentration of DA-98-WW07 demonstrated augmented activity in protecting mice from B. anthracis infection. Taken together, we report the prophylactic treatment potential of DA-98-WW07 for anthrax and the utility of immunomodulators in combination with an antibiotic to treat infections caused by the B. anthracis bacterium.

STimulator of INterferon Genes Agonism Accelerates Antitumor Activity in Poorly Immunogenic Tumors.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.

An injectable superior depot of Telratolimod inhibits post-surgical tumor recurrence and distant metastases

The clinical success of Toll-like receptor (TLR) agonists is based on their capacity to efficiently mobilize both innate and adaptive immunity. However, rapid distribution of TLR agonists into the systemic circulation may result in systemic cytokine storms. Telratolimod (Tel) is a TLR 7/8 agonist whose structure has a hydrophobic long chain that helps to prolong its release. Despite this, the phase I study of Tel showed cytokine release syndromes in 3/35 patients. Herein, we designed an injectable phase transition gel (PGE) that served as a superior drug depot for fatty acid-modified drugs. PGE further minimized the systemic drug exposure of Tel and the possible cytokine storms. In vivo studies demonstrated that Tel@PGE facilitated the recruitment of effector CD8+ T lymphocytes (T cells) and the polarization of myeloid-derived suppressor cells (MDSCs) and immunosuppressive M2-like macrophages to tumoricidal antigen-presenting cells. The reshaping of the tumor microenvironment (TME) by Tel@PGE elicited systematic immune responses to significantly prevent B16F10 or 4T-1 tumor postoperative recurrence and metastasis. Therefore, this platform of Tel is expected to provide a clinically available option for effective postoperative combined therapy. Statement of significanceA series of prodrugs or conjugates containing hydrophobic blocks were designed to achieve sustained release at the injection site by reducing the water solubility. However, this strategy sometimes failed short of expectations. Thus, we constructed a biocompatible and biodegradable injectable phase transition gel (PGE) with superior release properties that can be injected subcutaneously into the surgery site. In the long-lasting treatment, the melanoma and breast cancer immunotherapeutic effect significantly enhanced and the risk of cancer metastasis and relapse was reduced. Crucially, for some immune agonists, a superior release control can significantly reduce adverse effects which was decisive for the availability of the drugs.

Maximizing response to intratumoral immunotherapy in mice by tuning local retention

Direct injection of therapies into tumors has emerged as an administration route capable of achieving high local drug exposure and strong anti-tumor response. A diverse array of immune agonists ranging in size and target are under development as local immunotherapies. However, due to the relatively recent adoption of intratumoral administration, the pharmacokinetics of locally-injected biologics remains poorly defined, limiting rational design of tumor-localized immunotherapies. Here we define a pharmacokinetic framework for biologics injected intratumorally that can predict tumor exposure and effectiveness. We find empirically and computationally that extending the tumor exposure of locally-injected interleukin-2 by increasing molecular size and/or improving matrix-targeting affinity improves therapeutic efficacy in mice. By tracking the distribution of intratumorally-injected proteins using positron emission tomography, we observe size-dependent enhancement in tumor exposure occurs by slowing the rate of diffusive escape from the tumor and by increasing partitioning to an apparent viscous region of the tumor. In elucidating how molecular weight and matrix binding interplay to determine tumor exposure, our model can aid in the design of intratumoral therapies to exert maximal therapeutic effect.

CD40L-armed oncolytic herpes simplex virus suppresses pancreatic ductal adenocarcinoma by facilitating the tumor microenvironment favorable to cytotoxic T cell response in the syngeneic mouse model

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Despite the promising outcome of immune checkpoint inhibitors and agonist antibody therapies in different malignancies, PDAC exhibits high...

Antiviral Responses in Cancer: Boosting Antitumor Immunity Through Activation of Interferon Pathway in the Tumor Microenvironment.

In recent years, it became apparent that cancers either associated with viral infections or aberrantly expressing endogenous retroviral elements (EREs) are more immunogenic, exhibiting an intense intra-tumor immune cell infiltration characterized by a robust cytolytic apparatus. On the other hand, epigenetic regulation of EREs is crucial to maintain steady-state conditions and cell homeostasis. In line with this, epigenetic disruptions within steady-state cells can lead to cancer development and trigger the release of EREs into the cytoplasmic compartment. As such, detection of viral molecules by intracellular innate immune sensors leads to the production of type I and type III interferons that act to induce an antiviral state, thus restraining viral replication. This knowledge has recently gained momentum due to the possibility of triggering intratumoral activation of interferon responses, which could be used as an adjuvant to elicit strong anti-tumor immune responses that ultimately lead to a cascade of cytokine production. Accordingly, several therapeutic approaches are currently being tested using this rationale to improve responses to cancer immunotherapies. In this review, we discuss the immune mechanisms operating in viral infections, show evidence that exogenous viruses and endogenous retroviruses in cancer may enhance tumor immunogenicity, dissect the epigenetic control of EREs, and point to interferon pathway activation in the tumor milieu as a promising molecular predictive marker and immunotherapy target. Finally, we briefly discuss current strategies to modulate these responses within tumor tissues, including the clinical use of innate immune receptor agonists and DNA demethylating agents.

More fuel for the fire: Gut microbes and toxicity to immune agonist antibodies in cancer

Microbes in the gut impact response, resistance, and toxicity to numerous cancer therapies, though mechanisms remain incompletely understood. Blake et al. provide further evidence that gut microbes promote toxicity to immune-agonistic antibodies, with opportunities to target these in cancer treatment.1

Targeting acute myeloid leukemia through multimodal immunotherapeutic approaches

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a dismal prognosis. Immunotherapeutic approaches using single agent checkpoint inhibitors have thus far shown limited success. We hypothesized that successful adaptive anti-AML specific immune responses require additional modulation of innate immunity. DMXAA exposure resulted in modest apoptosis of C1498 AML cells with a subtle increase in PD-L1 expression and limited production of IL-6 and IFN-β. In contrast, DMXAA + anti-PD-1 ab, but not either agent alone, significantly decreased in vivo disease burden and prolonged overall survival in C1498 engrafted leukemic mice. Combination-treated mice demonstrated increased memory T-cells and mature dendritic cells, lower numbers of regulatory T-cells and evidence of leukemia apoptosis. Furthermore, these effects were associated with markedly increased serum levels of type I interferon (IFN) and IFN gamma. We demonstrate that combining an innate immune agonist with a checkpoint inhibitor synergistically improved anti-tumor activity in a preclinical AML model.

Immunotherapy in hematological malignancies: recent advances and open questions.

Over recent years, tremendous advances in immunotherapy approaches have been observed, generating significant clinical progress. Cancer immunotherapy has been shown, in different types of blood cancers, to improve the overall survival of patients. Immunotherapy treatment of hematopoietic malignancies is a newly growing field that has been accelerating over the past years. Several US FDA approved drugs and cell-based therapies are being exploited in the late stage of clinical trials. This review attempt to highlight and discuss the numerous innovative immunotherapy approaches of hematopoietic malignancy ranging from nonmyeloablative transplantation, T-cell immunotherapy, natural killercells and immune agonist to monoclonal antibodies and vaccination. In addition, a brief discussion on the future advances and accomplishments required to counterpart the current immunotherapeutic approaches for hematopoietic malignancies were also highlighted.