Efficacy and pharmacodynamic effect of anti-CD73 and anti-PD-L1 monoclonal antibodies in combination with cytotoxic therapy: observations from mouse tumor models.

CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.

A B7-H4-Targeting Antibody-Drug Conjugate Shows Antitumor Activity in PARPi and Platinum-Resistant Cancers with B7-H4 Expression.

PURPOSE: Platinum and PARP inhibitors (PARPi) demonstrate activity in breast and ovarian cancers, but drug resistance ultimately emerges. Here, we examine B7-H4 expression in primary and recurrent high-grade serous ovarian carcinoma (HGSOC) and the activity of a B7-H4-directed antibody-drug conjugate (B7-H4-ADC), using a pyrrolobenzodiazepine-dimer payload, in PARPi- and platinum-resistant HGSOC patient-derived xenograft (PDX) models. EXPERIMENTAL DESIGN: B7-H4 expression was quantified by flow cytometry and IHC. B7-H4-ADC efficacy was tested against multiple cell lines in vitro and PDX in vivo. The effect of B7-H4-ADC on cell cycle, DNA damage, and apoptosis was measured using flow cytometry. RESULTS: B7-H4 is overexpressed in 92% of HGSOC tumors at diagnosis (n = 12), persisted in recurrent matched samples after platinum treatment, and was expressed at similar levels across metastatic sites after acquired multi-drug resistance (n = 4). Treatment with B7-H4-ADC resulted in target-specific growth inhibition of multiple ovarian and breast cancer cell lines. In platinum- or PARPi-resistant ovarian cancer cells, B7-H4-ADC significantly decreased viability and colony formation while increasing cell-cycle arrest and DNA damage, ultimately leading to apoptosis. Single-dose B7-H4-ADC led to tumor regression in 65.5% of breast and ovarian PDX models (n = 29), with reduced activity in B7-H4 low or negative models. In PARPi and platinum-resistant HGSOC PDX models, scheduled B7-H4-ADC dosing led to sustained tumor regression and increased survival. CONCLUSIONS: These data support B7-H4 as an attractive ADC target for treatment of drug-resistant HGSOC and provide evidence for activity of an ADC with a DNA-damaging payload in this population. See related commentary by Veneziani et al., p. 1434.

Age-induced changes in anti-tumor immunity alter the tumor immune infiltrate and impact response to immuno-oncology treatments.

Introduction: Immuno-oncology (IO) research relies heavily on murine syngeneic tumor models. However, whilst the average age for a cancer diagnosis is 60 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that ageing has been shown to have a significant impact on the immune response. Methods: Using aged (60-72 weeks old) mice bearing CT26 tumors, we investigated the impact of ageing on tumor growth as well as the immune composition of the tumor and peripheral lymphoid organs. Results: We found many differences in the immune cell composition of both the tumor and tumor-draining lymph node between aged and young mice, such as a reduction in the naïve T cell population and a decreased intratumoral CD8/Treg ratio in aged animals. We hypothesized that these differences may contribute to impaired anti-cancer immune responses in aged mice and therefore assessed the anti-tumor efficacy of different IO therapies in aged mice, including both co-stimulation (using an anti-OX40 antibody) and immune checkpoint blockade (using anti-PD-L1 and anti-CTLA-4 antibodies). Whilst aged mice retained the capacity to generate anti-tumor immune responses, these were significantly attenuated when compared to the responses observed in young mice. Discussion: These differences highlight the importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.

CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

Small Gene Networks Delineate Immune Cell States and Characterize Immunotherapy Response in Melanoma.

Single-cell technologies have elucidated mechanisms responsible for immune checkpoint inhibitor (ICI) response, but are not amenable to a clinical diagnostic setting. In contrast, bulk RNA sequencing (RNA-seq) is now routine for research and clinical applications. Our workflow uses transcription factor (TF)-directed coexpression networks (regulons) inferred from single-cell RNA-seq data to deconvolute immune functional states from bulk RNA-seq data. Regulons preserve the phenotypic variation in CD45+ immune cells from metastatic melanoma samples (n = 19, discovery dataset) treated with ICIs, despite reducing dimensionality by >100-fold. Four cell states, termed exhausted T cells, monocyte lineage cells, memory T cells, and B cells were associated with therapy response, and were characterized by differentially active and cell state-specific regulons. Clustering of bulk RNA-seq melanoma samples from four independent studies (n = 209, validation dataset) according to regulon-inferred scores identified four groups with significantly different response outcomes (P < 0.001). An intercellular link was established between exhausted T cells and monocyte lineage cells, whereby their cell numbers were correlated, and exhausted T cells predicted prognosis as a function of monocyte lineage cell number. The ligand-receptor expression analysis suggested that monocyte lineage cells drive exhausted T cells into terminal exhaustion through programs that regulate antigen presentation, chronic inflammation, and negative costimulation. Together, our results demonstrate how regulon-based characterization of cell states provide robust and functionally informative markers that can deconvolve bulk RNA-seq data to identify ICI responders.

Targeting DNA damage response components induces enhanced STING-dependent type-I IFN response in ATM deficient cancer cells and drives dendritic cell activation.

The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example, ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However, additional antitumor benefits from targeting the DDR pathways, which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal, inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here, we demonstrate that ATM -/- FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However, STING deficiency in tumor cells does not prevent DC activation, suggesting that transactivation of the STING pathway occurs within DCs. Furthermore, depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells, indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary, in this study, we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation, which is enhanced in ATM-deficient cells.

Deep phenotyping of surface stimulatory and inhibitory co-receptors on cancer-resident T and NK cells reveals cell subsets within the tumor-reactive CTL population that are uniquely defined by NKG2A expression.

The field of Immuno-Oncology (IO) is evolving to utilise novel antibody backbones that can co-target multiple cell-surface stimulatory and inhibitory co-receptors (SICR). This approach necessitates a better understanding of SICR co-expression at the single-cell level on IO-relevant tumor-infiltrating leukocyte (TIL) cell types such as T and natural killer (NK) cells. Using high-dimensional flow cytometry we established a comprehensive SICR profile for tumor-resident T and NK cells across a range of human solid tumors where there is a clear need for improved immunotherapeutic intervention. Leveraging the power of our large flow panel, we performed deep-phenotyping of the critical CD8+CD39+ Cytotoxic T Lymphocyte (CTL) population that is enriched for tumor-reactive cytotoxic cells, revealing subsets that are differentiated by their SICR profile, including three that are uniquely defined by NKG2A expression. This study establishes a comprehensive SICR phenotype for human TIL T and NK cells, providing insights to guide the design and application of the next generation of IO molecules.

Recombinant Newcastle Disease Virus Immunotherapy Drives Oncolytic Effects and Durable Systemic Antitumor Immunity.

A recombinant Newcastle Disease Virus (NDV), encoding either a human (NDVhuGM-CSF, MEDI5395) or murine (NDVmuGM-CSF) GM-CSF transgene, combined broad oncolytic activity with the ability to significantly modulate genes related to immune functionality in human tumor cells. Replication in murine tumor lines was significantly diminished relative to human tumor cells. Nonetheless, intratumoral injection of NDVmuGM-CSF conferred antitumor effects in three syngeneic models in vivo; with efficacy further augmented by concomitant treatment with anti-PD-1/PD-L1 or T-cell agonists. Ex vivo immune profiling, including T-cell receptor sequencing, revealed profound immune-contexture changes consistent with priming and potentiation of adaptive immunity and tumor microenvironment (TME) reprogramming toward an immune-permissive state. CRISPR modifications rendered CT26 tumors significantly more permissive to NDV replication, and in this setting, NDVmuGM-CSF confers immune-mediated effects in the noninjected tumor in vivo Taken together, the data support the thesis that MEDI5395 primes and augments cell-mediated antitumor immunity and has significant utility as a combination partner with other immunomodulatory cancer treatments.

Novel non-terminal tumor sampling procedure using fine needle aspiration supports immuno-oncology biomarker discovery in preclinical mouse models.

BACKGROUND: Immuno-oncology therapies are now part of the standard of care for cancer in many indications. However, durable objective responses remain limited to a subset of patients. As such, there is a critical need to identify biomarkers that can predict or enrich for treatment response. So far, the majority of putative biomarkers consist of features of the tumor microenvironment (TME). However, in preclinical mouse models, the collection of tumor tissue for this type of analysis is a terminal procedure, obviating the ability to directly link potential biomarkers to long-term treatment outcomes. METHODS: To address this, we developed and validated a novel non-terminal tumor sampling method to enable biopsy of the TME in mouse models based on fine needle aspiration. RESULTS: We show that this technique enables repeated in-life sampling of subcutaneous flank tumors and yields sufficient material to support downstream analyses of tumor-infiltrating immune cells using methods such as flow cytometry and single-cell transcriptomics. Moreover, using this technique we demonstrate that we can link TME biomarkers to treatment response outcomes, which is not possible using the current method of terminal tumor sampling. CONCLUSION: Thus, this minimally invasive technique is an important refinement for the pharmacodynamic analysis of the TME facilitating paired evaluation of treatment response biomarkers with outcomes and reducing the number of animals used in preclinical research.

Design and Efficacy of a Monovalent Bispecific PD-1/CTLA4 Antibody That Enhances CTLA4 Blockade on PD-1+ Activated T Cells.

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1- T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995.

Activity of murine surrogate antibodies for durvalumab and tremelimumab lacking effector function and the ability to deplete regulatory T cells in mouse models of cancer.

Preclinical studies of PD-L1 and CTLA-4 blockade have relied heavily on mouse syngeneic tumor models with intact immune systems, which facilitate dissection of immunosuppressive mechanisms in the tumor microenvironment. Commercially developed monoclonal antibodies (mAbs) targeting human PD-L1, PD-1, and CTLA-4 may not demonstrate cross-reactive binding to their mouse orthologs, and surrogate anti-mouse antibodies are often used in their place to inhibit these immune checkpoints. In each case, multiple choices exist for surrogate antibodies, which differ with respect to species of origin, affinity, and effector function. To develop relevant murine surrogate antibodies for the anti-human PD-L1 mAb durvalumab and the anti-human CTLA-4 mAb tremelimumab, rat/mouse chimeric or fully murine mAbs engineered for reduced effector function were developed and compared with durvalumab and tremelimumab. Characterization included determination of target affinity, in vivo effector function, pharmacokinetic profile, and anti-tumor efficacy in mouse syngeneic tumor models. Results showed that anti-PD-L1 and anti-CTLA-4 murine surrogates with pharmacologic properties similar to those of durvalumab and tremelimumab demonstrated anti-tumor activity in a subset of commonly used mouse syngeneic tumor models. This activity was not entirely dependent on antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis effector function, or regulatory T-cell depletion, as antibodies engineered to lack these features showed activity in models historically sensitive to checkpoint inhibition, albeit at a significantly lower level than antibodies with intact effector function.

Intratumoral administration of the Toll-like receptor 7/8 agonist 3M-052 enhances interferon-driven tumor immunogenicity and suppresses metastatic spread in preclinical triple-negative breast cancer.

OBJECTIVES: Loss of tumor-inherent type I interferon (IFN) signalling has been closely linked to accelerated metastatic progression via decreased immunogenicity and antitumor immunity. Previous studies in murine models of triple-negative breast cancer (TNBC) demonstrate that systemic IFN inducers are effective antimetastatic agents, via sustained antitumor CD8+ T-cell responses. Repeated systemic dosing with recombinant IFNs or IFN inducers is associated with significant toxicities; hence, the use of alternate intratumoral agents is an active area of investigation. It is critical to investigate the impact of intratumoral agents on subsequent metastatic spread to predict clinical impact. METHODS: In this study, the local and systemic impact of the intratumoral Toll-like receptor (TLR) 7/8 agonist 3M-052 alone or in combination with anti-PD1 was evaluated in metastatic TNBC models. The IFN-α receptor (IFNAR1) blocking antibody, MAR1-5A3, along with immune-deficient mice and ex vivo assays are utilised to examine the key targets of this agent that are critical for an antimetastatic response. RESULTS: Single intratumoral administration of 3M-052 reduced mammary tumor growth, induced a T-cell-inflamed tumor microenvironment (TME) and reduced metastatic spread to lung. Metastasis suppression was reliant on IFN signalling and an antitumor immune response, in contrast to primary tumor growth inhibition, which was retained in NSG and CD8+ T-cell-depleted mice. 3M-052 action was demonstrated via dendritic cell activation and production of type I IFN and other pro-inflammatory cytokines to initiate a T-cell-inflamed TME and promote tumor cell antigen presentation. CONCLUSION: This work supports neoadjuvant TLR agonist-based immunotherapeutics as realistic options for immune activation in the TME and long-term metastatic protection in TNBC.

Structural and functional characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 function via a novel non-competitive mechanism of action.

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.

Intratumoral IL12 mRNA Therapy Promotes TH1 Transformation of the Tumor Microenvironment.

PURPOSE: While immune checkpoint inhibitors such as anti-PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials. EXPERIMENTAL DESIGN: We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects. RESULTS: A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell-dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti-PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti-PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression. CONCLUSIONS: These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity.See related commentary by Cirella et al., p. 6080.

Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.

Oncolytic Newcastle disease virus activation of the innate immune response and priming of antitumor adaptive responses in vitro.

Oncolytic virus (OV) therapy is an emerging approach with the potential to redefine treatment options across a range of cancer indications and in patients who remain resistant to existing standards of care, including immuno-oncology (IO) drugs. MEDI5395, a recombinant Newcastle disease virus (NDV), engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibits potent oncolytic activity. It was hypothesized that activation of immune cells by MEDI5395, coupled with its oncolytic activity, would enhance the priming of antitumor immunity. Using MEDI5395 and recombinant NDVs encoding fluorescent reporter genes, we demonstrated preferential virus uptake and non-productive infection in myeloid cells, including monocytes, macrophages, and dendritic cells (DCs). Infection resulted in immune-cell activation, with upregulation of cell surface activation markers (e.g., CD80, PD-L1, HLA-DR) and secretion of proinflammatory cytokines (IFN-α2a, IL-6, IL-8, TNF-α). Interestingly, in vitro M2-polarized macrophages were more permissive to virus infection than were M1-polarized macrophages. In a co-culture system, infected myeloid cells were effective virus vectors and mediated the transfer of infectious NDV particles to tumor cells, resulting in cell death. Furthermore, NDV-infected DCs stimulated greater proliferation of allogeneic T cells than uninfected DCs. Antigens released after NDV-induced tumor cell lysis were cross-presented by DCs and drove activation of tumor antigen-specific autologous T cells. MEDI5395 therefore exhibited potent immunostimulatory activity and an ability to enhance antigen-specific T-cell priming. This, coupled with its tumor-selective oncolytic capacity, underscores the promise of MEDI5395 as a multimodal therapeutic, with potential to both enhance current responding patient populations and elicit de novo responses in resistant patients.

Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies.

BACKGROUND: Immune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically "cold" tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated. METHODS: The response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models. RESULTS: Targeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models. CONCLUSION: Localized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.

A cell-engineered system to assess tumor cell sensitivity to CD8+ T cell-mediated cytotoxicity.

In vitro assays that evaluate CD8+ T cell-mediated cytotoxicity are important to aid in the development of novel therapeutic approaches to enhance anti-tumor immune responses. Here, we describe a novel cytotoxicity co-culture assay that circumvents the problem of highly variable allogeneic responses and obviates the constraints of HLA-restriction between effector and target cells. We show that this assay can be easily applied to a panel of tumor cell lines to provide additional insights into intrinsic drivers of sensitivity/resistance to T cell-mediated killing, and to evaluate the impact of targeted therapies on both tumor and T cell compartments.

Further Advances in Cancer Immunotherapy: Going Beyond Checkpoint Blockade.

Significant advances have been made to identify effective therapies that either restore or generate de novo a patient's immune response to cancer, so-called immunotherapy or immuno-oncology (IO) therapies. Some tumors overcome immune surveillance by promoting mechanisms to evade or suppress the immune system. This conference report highlights the clinical promise and current challenges of IO therapy, including the use of immune-checkpoint antagonist monoclonal antibodies. Furthermore, this report investigates advances in preclinical modeling of cancer immunobiology and how this is helping our understanding of which patients will receive clinical benefits from current immune-checkpoint treatment. Looking to the future, the report looks at emerging IO approaches, which aim to specifically target the tumor microenvironment. This includes the use of toll-like receptors (TLRs) agonists that link the activation of innate immune, cells to the priming of T cells and an adaptive memory anti-tumor immune response through to the reversal of local immunosuppression using adenosinergic and indoleamine 2,3-dioxygenase (IDO) inhibitors.

CSF1R+ Macrophages Sustain Pancreatic Tumor Growth through T Cell Suppression and Maintenance of Key Gene Programs that Define the Squamous Subtype.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to most therapies including single-agent immunotherapy and has a dense desmoplastic stroma, and most patients present with advanced metastatic disease. We reveal that macrophages are the dominant leukocyte population both in human PDAC stroma and autochthonous models, with an important functional contribution to the squamous subtype of human PDAC. We targeted macrophages in a genetic PDAC model using AZD7507, a potent selective inhibitor of CSF1R. AZD7507 caused shrinkage of established tumors and increased mouse survival in this difficult-to-treat model. Malignant cell proliferation diminished, with increased cell death and an enhanced T cell immune response. Loss of macrophages rewired other features of the TME, with global changes in gene expression akin to switching PDAC subtypes. These changes were markedly different to those elicited when neutrophils were targeted via CXCR2. These results suggest targeting the myeloid cell axis may be particularly efficacious in PDAC, especially with CSF1R inhibitors.