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Immune cell dysfunction within the tumor microenvironment (TME) undermines the control of cancer progression. Established tumors contain phenotypically distinct, tumor-specific natural killer (NK) cells; however, the temporal dynamics, mechanistic underpinning and functional significance of the NK cell compartment remains incompletely understood. Here, we use photo-labeling, combined with longitudinal transcriptomic and cellular analyses, to interrogate the fate of intratumoral NK cells. We reveal that NK cells rapidly lose effector functions and adopt a distinct phenotypic state with features associated with tissue residency. NK cell depletion from established tumors did not alter tumor growth, indicating that intratumoral NK cells cease to actively contribute to anti-tumor responses. IL-15 administration prevented loss of function and improved tumor control, generating intratumoral NK cells with both tissue-residency characteristics and enhanced effector function. Collectively, our data reveals the fate of NK cells after recruitment into tumors and provides insight into how their function may be revived. Natural killer (NK) cells control tumor growth through direct cytotoxicity and recruitment of other leukocytes. Here, using photoconversion-based labeling to track the fate of NK cells in vivo, the authors demonstrate that loss of NK cell function occurs very rapidly following their entry into tumors, but can be reversed by IL-15 administration.

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7 + DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7 + DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7 + DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7 + DCs co-localise with PD-1 + CD8 + T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7 + DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells. Recognition of tumour antigen induces dendritic cell activation and migration to the lymph node. Here, the authors use photoconvertible mice to demonstrate that some activated dendritic cells are retained in tumours and gradually lose function, but their ability to support local anti-tumour responses can be augmented by anti-PD-L1 blockade.

The presence of tertiary lymphoid structures (TLS) in the tumor microenvironment is associated with better clinical outcome in many cancers. In non-small cell lung cancer (NSCLC), we have previously showed that a high density of B cells within TLS (TLS-B cells) is positively correlated with tumor antigen-specific antibody responses and increased intratumor CD4 + T cell clonality. Here, we investigated the relationship between the presence of TLS-B cells and CD4 + T cell profile in NSCLC patients. The expression of immune-related genes and proteins on B cells and CD4 + T cells was analyzed according to their relationship to TLS-B density in a prospective cohort of 56 NSCLC patients. We observed that tumor-infiltrating T cells showed marked differences according to TLS-B cell presence, with higher percentages of naïve, central-memory, and activated CD4 + T cells and lower percentages of both immune checkpoint (ICP)-expressing CD4 + T cells and regulatory T cells (Tregs) in the TLS-B high tumors. A retrospective study of 538 untreated NSCLC patients showed that high TLS-B cell density was even able to counterbalance the deleterious impact of high Treg density on patient survival, and that TLS-B high Treg low patients had the best clinical outcomes. Overall, the correlation between the density of TLS-B high tumors with early differentiated, activated and non-regulatory CD4 + T cell cells suggest that B cells may play a central role in determining protective T cell responses in NSCLC patients.

Antibodies targeting T-cell inhibitory pathways, such as CTLA-4 and PD-1/PD-L1, are emerging as an important class of cancer therapeutics, and a next generation of immunomodulatory therapies targeting alternative inhibitory (e.g. TIM-3, LAG-3, B7-H4, B7-H3, VISTA, A2aR), as well as co-stimulatory (e.g. CD27, OX40, GITR, CD137), pathways are poised to join them. Most of these immunomodulatory antibodies are of IgG isotypes that have low, or no, binding to the Fc gamma receptors (FcγRs) that trigger cell-mediated cytotoxic effector functions such as antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). These isotypes were selected to minimise the risk of depleting the T cells upon which such antibodies depend for their mechanism of action. However, recent preclinical data highlight a potential role for FcγR engagement in the activity of such antibodies. Here we review the biology of the FcγRs and IgG isotypes in both humans and mice, detail the potential roles that FcγR interactions can play in the activity of monoclonal antibodies in general, and of immunomodulatory antibodies in particular, and discuss how preclinical studies on these interactions might be best interpreted and translated to a human setting.

Background Immune checkpoint therapies (ICTs) targeting the programmed cell death-1 (PD1)/programmed cell death ligand-1 (PD-L1) pathway have improved outcomes for patients with non-small cell lung cancer (NSCLC), particularly those with high PD-L1 expression. However, the predictive value of manual PD-L1 scoring is imperfect and alternative measures are needed. We report an automated image analysis solution to determine the predictive and prognostic values of the product of PD-L1+ cell and CD8+ tumor infiltrating lymphocyte (TIL) densities (CD8xPD-L1 signature) in baseline tumor biopsies. Methods Archival or fresh tumor biopsies were analyzed for PD-L1 and CD8 expression by immunohistochemistry. Samples were collected from 163 patients in Study 1108/NCT01693562, a Phase 1/2 trial to evaluate durvalumab across multiple tumor types, including NSCLC, and a separate cohort of 199 non-ICT- patients. Digital images were automatically scored for PD-L1+ and CD8+ cell densities using customized algorithms applied with Developer XD™ 2.7 software. Results For patients who received durvalumab, median overall survival (OS) was 21.0 months for CD8xPD-L1 signature-positive patients and 7.8 months for signature-negative patients ( p  = 0.00002). The CD8xPD-L1 signature provided greater stratification of OS than high densities of CD8+ cells, high densities of PD-L1+ cells, or manually assessed tumor cell PD-L1 expression ≥25%. The CD8xPD-L1 signature did not stratify OS in non-ICT patients, although a high density of CD8+ cells was associated with higher median OS (high: 67 months; low: 39.5 months, p  = 0.0009) in this group. Conclusions An automated CD8xPD-L1 signature may help to identify NSCLC patients with improved response to durvalumab therapy. Our data also support the prognostic value of CD8+ TILS in NSCLC patients who do not receive ICT. Trial registration ClinicalTrials.gov identifier: NCT01693562 . Study code: CD-ON-MEDI4736-1108. Interventional study (ongoing but not currently recruiting). Actual study start date: August 29, 2012. Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).

Immune checkpoint blockade has revolutionized the field of oncology, inducing durable anti-tumour immunity in solid tumours. In patients with advanced prostate cancer, immunotherapy treatments have largely failed 1 – 5 . Androgen deprivation therapy is classically administered in these patients to inhibit tumour cell growth, and we postulated that this therapy also affects tumour-associated T cells. Here we demonstrate that androgen receptor (AR) blockade sensitizes tumour-bearing hosts to effective checkpoint blockade by directly enhancing CD8 T cell function. Inhibition of AR activity in CD8 T cells prevented T cell exhaustion and improved responsiveness to PD-1 targeted therapy via increased IFNγ expression. AR bound directly to Ifng and eviction of AR with a small molecule significantly increased cytokine production in CD8 T cells. Together, our findings establish that T cell intrinsic AR activity represses IFNγ expression and represents a novel mechanism of immunotherapy resistance. . Androgen-receptor blockade can overcome immunotherapy resistance in prostate cancer by intrinsically enhancing T cell function and IFNγ responses. 

BackgroundAZD5863 is a bispecific T cell engager (TCE) with high affinity to CLDN18.2 and low affinity to cluster of differentiation 3 (CD3), designed to decrease its peripheral cytokine release potential, improve the therapeutic index, and maintain potent anti-tumor activity.MethodsAZD5863 was evaluated using CLDN18.2-expressing human cell lines alone or in co-cultures with human or cynomolgus monkey peripheral blood mononuclear cells to determine affinities, specificity, potency, and bystander killing activity. In vivo, AZD5863-mediated tumor growth inhibition and pharmacodynamics were evaluated in humanized mice or human CD3 transgenic mice implanted with CLDN18.2-expressing cancer cell lines.ResultsAZD5863 was shown to bind specifically to human and cynomolgus monkey CLDN18.2 and to CD3, with CLDN18.2 binding also conserved against the murine protein. AZD5863 mediated T cell-dependent anti-tumor activity against CLDN18.2-expressing lines, with potency significantly correlating with CLDN18.2 receptor density. Cytokine secretion induced by AZD5863, in vitro and in vivo, was lower compared with a CLDN18.2 TCE with higher affinity for CD3. AZD5863 mediated T cell-dependent bystander killing of CLDN18.2-negative cells in the presence of CLDN18.2-expressing cells, in a mechanism partly dependent on interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and Fas ligand. In vivo, AZD5863 treatment resulted in potent tumor control in pancreatic, gastric, and esophageal models and enhanced engraftment of immune populations in a humanized model.ConclusionsAZD5863 mediates potent anti-tumor activity in vitro and in vivo, while inducing limited levels of cytokines. This work improves our understanding of the mechanism of action of affinity balanced TCEs and informs the design of a phase 1 trial testing AZD5863 in gastric, pancreatic, and esophageal adenocarcinoma (NCT06005493).

Understanding resistance to antibody to programmed cell death protein 1 (PD-1; anti-PD-1) is crucial for the development of reversal strategies. In anti-PD-1-resistant models, simultaneous anti-PD-1 and vaccine therapy reversed resistance, while PD-1 blockade before antigen priming abolished therapeutic outcomes. This was due to induction of dysfunctional PD-1 + CD38 hi CD8 + cells by PD-1 blockade in suboptimally primed CD8 cell conditions induced by tumors. This results in erroneous T cell receptor signaling and unresponsiveness to antigenic restimulation. On the other hand, PD-1 blockade of optimally primed CD8 cells prevented the induction of dysfunctional CD8 cells, reversing resistance. Depleting PD-1 + CD38 hi CD8 + cells enhanced therapeutic outcomes. Furthermore, non-responding patients showed more PD-1 + CD38 + CD8 + cells in tumor and blood than responders. In conclusion, the status of CD8 + T cell priming is a major contributor to anti-PD-1 therapeutic resistance. PD-1 blockade in unprimed or suboptimally primed CD8 cells induces resistance through the induction of PD-1 + CD38 hi CD8 + cells that is reversed by optimal priming. PD-1 + CD38 hi CD8 + cells serve as a predictive and therapeutic biomarker for anti-PD-1 treatment. Sequencing of anti-PD-1 and vaccine is crucial for successful therapy. PD-1 blockade can enhance antitumor responses in a subset of cases. Khleif and colleagues demonstrate that PD-1 blockade in the context of suboptimal T cell activation engenders a state of non-responsiveness but not when there is strong stimulation by vaccination.

MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326-349 and 388-410, with critical residues F(326), T(328), N(333), V(388), G(389), P(390), E(392), I(408), and N(410). Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA.

Vesicular stomatitis virus encoding the IFNβ transgene (VSV-IFNβ) is a mediator of potent oncolytic activity and is undergoing clinical evaluation for the treatment of solid tumors. Emerging preclinical and clinical data suggest treatment of tumors with oncolytic viruses may sensitize tumors to checkpoint inhibitors and increase the anti-tumor immune response. New generations of immuno-oncology molecules including T cell agonists are entering clinical development and could be hypothesized to enhance the activity of oncolytic viruses, including VSV-IFNβ. Here, we show that VSV-IFNβ exhibits multiple mechanisms of action, including direct cell killing, stimulation of an innate immune response, recruitment of CD8 T cells, and depletion of T regulatory cells. Moreover, VSV-IFNβ promotes the establishment of a CD8 T cell response to endogenous tumor antigens. Our data demonstrate a significant enhancement of anti-tumor function for VSV-IFNβ when combined with checkpoint inhibitors, but not OX40 agonists. While the addition of checkpoint inhibitors to VSV-IFNβ generated robust tumor growth inhibition, it resulted in no increase in viral replication, transgene expression, or immunophenotypic changes beyond treatment with VSV-IFNβ alone. We hypothesize that tumor-specific T cells generated by VSV-IFNβ retain activity due to a lack of immune exhaustion when checkpoint inhibitors were used. Replication competent oncolytic viruses are targeted to specifically lyse tumor cells. They have been proposed as additions to immuno-oncology treatment regimens, hypothesized to exhibit complementary mechanisms of action. Durham et al. demonstrate that checkpoint inhibitors, but not a T cell agonist molecule combine with oncolytic VSV-IFNβ to enhance survival.